BACKGROUND

Disclosed herein are surfaces for controlled bioadhesion, methods of manufacture thereof and articles comprising the same.

Articles used in hospitals such as door handles, bed railings, walls in hospitals, medical devices, and the like that are frequently contacted by patients as a result of which, they become saturated with bacteria and virus that can transmit diseases to other healthy patients. It is desirable to create surfaces, where such bacteria and viruses can be immobilized so that the transmission of diseases from one person to another can be minimized or prevented.

The prevention of transmission of diseases would negate the investment of substantial amounts of money for the development of drugs, some of which have adverse impacts on patients and induce drug resistant strains of organisms.

Bio fouling is the unwanted accumulation of organic and inorganic matter of biological origin on surfaces. For example, in the marine environment biofouling is the result of marine organisms settling, attaching, and growing on submerged marine surfaces. The biofouling process is initiated within minutes of a surface being submerged in a marine environment by the absorption of dissolved organic materials which result in the formation of a conditioning film. Once the conditioning film is deposited, bacteria (e.g. unicellular algae) colonize the surface within hours of submersion. The resulting biofilm produced from the colonization of the bacteria is referred to as micro fouling or slime and can reach thicknesses on the order of 500 μιη.

Biofouling is estimated to cost the U.S. Navy alone over $1 billion per year by increasing the hydrodynamic drag of naval vessels. This in turn decreases the range, speed, and maneuverability of naval vessels and increases the fuel consumption by up to 30-40%. Thus, biofouling weakens the national defense. Moreover, biofouling is also a major economical burden on commercial shipping, recreational craft, as well as civil structures, bridges, and power generating facilities. Additionally, biofouling is one mechanism by which invasive species are transported around the globe.

It is therefore desirable to create surfaces where the growth of organisms (e.g., bacteria, viruses, and the like) can be controlled. SUMMARY

Textured surfaces comprising hydrogels can be used to selectively modify surfaces for bioadhesion. In one embodiment, the textured surface comprising the hydrogels can be used to resist or minimize the bioadhesion of organic materials such as proteins, antibodies, and the like. In another embodiment, the textured surface comprising hydrogels can be reacted with molecules having reactive functionalities that can selectively bond to proteins, antibodies, and the like. Thus, by texturing a surface and by disposing reactive functionalities on certain features of the texture, bioadhesion can be restricted to certain regions of the surface. In other words, bioadhesion can be controlled to those regions of the surface where it is desired while being minimized or reduced in other regions where it is not desired.

Disclosed herein is an article comprising a first plurality of spaced features; the spaced features arranged in a plurality of groupings; the groupings of features comprising repeat units; the spaced features within a grouping being spaced apart at an average distance of about 1 nanometer to about 500 micrometers; each feature having a surface that is substantially parallel to a surface on a neighboring feature; each feature comprising a hydro gel.

Disclosed herein too is an article comprising a first plurality of spaced features; the spaced features arranged in a plurality of groupings; the groupings of features comprising repeat units; the spaced features within a grouping being spaced apart at an average distance of about 1 nanometer to about 500 micrometers; each feature having a surface that is substantially parallel to a surface on a neighboring feature; each feature having a hydrogel disposed thereon.

Disclosed herein too is a method comprising disposing a pattern upon a base surface; the pattern comprising a first plurality of spaced features; the spaced features arranged in a plurality of groupings; the groupings of features comprising repeat units; the spaced features within a grouping being spaced apart at an average distance of about 1 nanometer to about 500 micrometers; each feature having a surface that is substantially parallel to a surface on a neighboring feature; wherein each feature comprises a hydrogel.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 A is a depiction of a textured surface that comprises hydrogels;

Figure IB is a depiction of a textured surface that comprises hydrogels. The surface contains features that are protruded into the base surface; Figure 2A is a depiction of another textured surface that comprises hydrogels;

Figure 2B is a depiction of another textured surface that comprises hydrogels;

Figure 2C is a depiction of another textured surface that comprises hydrogels;

Figure 2D is a depiction of another textured surface that comprises hydrogels; Figure 3 is a depiction of a textures surface that comprises hydrogels;

Figure 4 is a graphical depiction of 3 separate assays showing that PEGDMA, PEGDMA-co-GMA and PEGDMA-co-HEMA consistently reduced the attachment of spores of Ulva compared to smooth PDMSe. The total average percent reduction for PEGDMA versus PDMSe is 55%, PEGDMA-co-GMA versus PDMSe is 87% and for PEGDMA-co-HEMA versus PDMSe it is 85%;

Figure 5 is a graphical depiction of 3 separate assays showing that the initial attachment density of Navicula was lowest on PEGDMA-co-GMA which was significantly lower than initial attachment densities on PEGDMA, PDMSe and PEGDMA- co-HEMA (a = 0.05, p<0);

Figure 6 is a graphical depiction of 3 separate assays showing that the initial attachment density of cells of C. marina was reduced on the hydrogels compared to a smooth PDMSe standard (up to 62%) with lowest densities on PEGDMA-co-GMA and PEGDMA-co-HEMA;

Figure 7 shows that the highest degree of orientation was observed on the +1CH2X2 topography for HCAECs cultured for 24 hours; and

Figure 8 shows that the highest degree of orientation was observed on +lSK2x2_n4 for HCASMCs after 24 hours and 7 days.

DETAILED DESCRIPTION

As used herein, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. The endpoints of all ranges directed to the same component or property are inclusive of the endpoint and independently combinable, except when the modifier "between" is used. The modifier "about" used in connection with a quantity is inclusive of the stated value and has the meaning dictated by the context (e.g., includes the degree of error associated with measurement of the particular quantity). A "combination" is inclusive of blends, mixtures, alloys, reaction products, and the like.

In general, the compositions or methods can alternatively comprise, consist of, or consist essentially of, any appropriate components or steps disclosed. The invention can additionally, or alternatively, be formulated so as to be devoid, or substantially free, of any components, materials, ingredients, adjuvants, or species, or steps used in the prior art compositions or that are otherwise not necessary to the achievement of the function and/or objectives of the present claims.

Disclosed herein are base surfaces that comprise hydrogels to from a surface that can be used to control bioadhesion (hereinafter a "bioadhesive surface"). The hydrogels are disposed upon the base surface and can be used to control or manipulate bioadhesion. The base surface can be textured or untextured prior to disposing the hydrogels on the surface. The bioadhesive surface can be used to trap certain bacteria or viruses. The bioadhesive surface may alternatively be used to trap certain bacteria or viruses while at the same time allowing other bacteria or viruses to escape. The bioadhesive surface may also be used to prevent some bacteria or viruses from settling on the surface and forming colonies where they can breed and multiply.

The bioadhesive surface can be used to control bioadhesion. The term "control" is used to imply that bioadhesion can be completely eliminated or alternatively can be increased. Depending upon the texture of the base surface and the hydrogel, the bioadhesive surface can be used to eliminate bioadhesion, grow a certain type of bioorganism while excluding other bioorganisms, or serve as a template for the growth of certain types of cell cultures.

In one embodiment, the texturing of the bioadhesive surface can be used to grow cells with preferred orientations. Depending upon the texture of the base surface, certain preferred cell morphologies can be grown on a particular surface. In one embodiment, the base morphology can be used to influence the settling, growth and morphology of certain cells. Examples of such cells are endothiliel cells and smooth muscle cells. In one embodiment, the texture of the surface can influence the phenotypic expression of cells.

A textured surface (also referred to as a patterned surface that comprises patterns) can comprise a plurality of spaced features. The spaced features can be disposed on the base surface (i.e., they are raised features) or are protruded into the base surface. The features may be arranged in a plurality of groupings. In one embodiment, the groupings of features are arranged with respect to one another so as to define a tortuous pathway. In another embodiment, the groupings of features are arranged with respect to one another to define only linear pathways. In another embodiment, the base surface is untextured prior to disposing the hydrogels on the surface. The base surface can then be textured by disposing the hydrogels on selected regions.

In yet another embodiment, the base surface is untextured prior to disposing the hydrogels on the surface. The hydrogels are disposed upon the entire base surface thereby not producing any particular texturing of the surface.

Hydrogels are a network of polymer chains that are water-insoluble, sometimes found as a colloidal gel in which water is the dispersion medium. Hydrogels are highly absorbent (they can contain over 99% water) natural or synthetic polymers. Hydrogels also possess a degree of flexibility very similar to natural tissue, due to their significant water content. The hydrogels can comprise a homopolymer, a copolymer, a block copolymer, an alternating copolymer, an alternating block copolymer, a random copolymer, a random block copolymer, a graft copolymer, a star block copolymer, an ionomer, a dendrimer, or a combination comprising at least one of the foregoing polymers.

The hydrogels can be amorphous or crystalline and can be hydrogen bonded to neighboring units. The can comprise an ionic charge. The ionic charge can bond one polymer molecule to a neighboring molecule. The hydrogels can be neutral, catatonic, anionic or ampholytic. In one embodiment, the hydrogel can be an ionotropic hydrogel that comprises polyanions. The polyanions can have multivalent cations. In yet another embodiment, the hydrogel can comprise a complex coacervate or a polyion complex hydrogel. The hydrogels can be natural polymers (e.g., bio-polymers), synthetic polymers, or combinations of natural polymers or synthetic polymers.

Examples of hydrogels are proteins, dextran, chitosan, collagen, dextran sulfate, agarose, alginate, collagen, fibrin, gelatin, hyaluronic acid (HA), grafted starch, methyl cellulose, carboxymethyl cellulose, hydroxypropyl cellulose, natural gums (e.g., alginates, xanthan gum, locust bean gum and the like), or the like, or a combination comprising at least one of the foregoing polymers.

Examples of synthetic hydrogels are poly(acrylic acid), poly (methacrylic acid), poly(acrylamides), poly( vinyl ethers), maleic anhydride copolymers with vinyl ethers and alp ha-ole fins, poly(vinyl pyrrolidone), poly(vinylmorpholinone), poly(vinyl alcohol), polyalkylene glycols, polysiloxanes, or the like, or combinations thereof; copolymers of polyalkylene glycols with poly(acrylic acid), poly (methacrylic acid), poly(acrylamides), poly(vinyl ethers), maleic anhydride copolymers with vinyl ethers and alpha-olefms, poly(vinyl pyrrolidone), poly(vinylmorpholinone), poly(vinyl alcohol), polysiloxanes, or the like, or combinations thereof; alkali metal and ammonium salts of poly(acrylic acid), poly (methacrylic acid), poly(acrylamides), poly(vinyl ethers), maleic anhydride copolymers with vinyl ethers and alp ha-ole fins, poly( vinyl pyrrolidone), poly(vinylmorpholinone), poly(vinyl alcohol), polysiloxanes, and mixtures and copolymers thereof. Mixtures of natural and wholly or partially synthetic absorbent polymers can also be disposed upon the surfaces.

Exemplary hydrogels are polyacrylate and polymethacrylates gels, specifically copolymers of polyalkylene glycols with polyacrylates and polymethacrylates, and more specifically polyethylene glycol dimethacrylate (PEGDMA), polyethylene glycol dimethacrylate-co-glycidyl methacrylate (PEGDMA-co-GMA) and polyethylene glycol dimethacrylate-co -hydroxy ethyl methacrylate (PEGDMA-co-HEMA). In one embodiment, the hydrogel comprises crosslmked polyethylene glycol dimethacrylate (PEGDMA), crosslmked polyethylene glycol dimethacrylate-co-glycidyl methacrylate (PEGDMA-co-GMA) and crosslmked polyethylene glycol dimethacrylate-co- hydro xyethyl methacrylate (PEGDMA-co-HEMA).

Various cross-linked agents may be used to crosslink the aforementioned monomers and polymers. Ammonium persulfate may be used to crosslink the polyethylene glycol dimethacrylate (PEGDMA), polyethylene glycol dimethacrylate-co- glycidyl methacrylate (PEGDMA-co-GMA) and polyethylene glycol dimethacrylate-co- hydroxyethyl methacrylate (PEGDMA-co-HEMA). Ascorbic acid can be used as an initiator. The hydrogels can be crosslinked using electromagnetic radiation, heat, or other forms of energy.

The hydrogels can be chemically reacted to the base substrate. In one embodiment, the hydrogels are covalently bonded to the base substrate. In another embodiment, the hydrogels are ionically bonded to the base substrate. In yet another embodiment, the hydrogels form a semi-interpenetrating or an interpenetrating network with polymer chains on the base substrate. In yet another embodiment, mechanical roughning of the base substrate can be used to dispose the hydrogel on the base substrate. The mechanical roughening can take the forms of a rough surface. In one embodiment, the rough surface can include hooks, loops, and other equivalent mechanical configurations that can enable the hydrogel to be affixed to the base surface.

As noted above, the surface can be textured or non-textured. Figure 1A and IB are depictions of one embodiment of a textured surface prior to disposing a hydrogel on the surface. Surface layer comprises a plurality of features 111 which are attached to and project out from a base surface 130. The base surface 130 can be a roofing material, the inner surface of a water inlet pipe for a power or water treatment plant, an implantable medical device or material, such as a breast implant, a catheter or a heart valve. Each of the features 111 have at least a width of about 0.5 nanometers to about 100 micrometers, specifically about 1 nanometer to about 50 micrometers, and more specifically about 10 nanometers to about 10 micrometers. In an exemplary embodiment, the width of the features can be about 1 micrometer to about 5 micrometers.

The spacing between the features can be about 0.5 nanometers to about 100 micrometers, specifically about 1 nanometer to about 50 micrometers, and more specifically about 10 nanometers to about 10 micrometers. In an exemplary embodiment, the spacing between the features can be about 1 micrometer to about 5 micrometers.

The height of the features above the base surface can be about 0.5 nanometers to about 100 micrometers, specifically about 1 nanometer to about 50 micrometers, and more specifically about 10 nanometers to about 10 micrometers. In an exemplary embodiment, the height of the features can be about 0.1 nanometers to about 5 micrometers. Additonal feature spacings, widths and heights are provided below.

Figure IB is a scanned optical profilometry image of a pattern having a plurality of features 161 projecting into a base surface 180, according to another embodiment of the invention. Features 161 comprise indented void volumes into base surface 180. Although not shown, a surface can include regions having raised features 111 shown in Figure 1A together with regions having indented features 161 shown in Figure IB.

Figure 2A - 2D illustrate some exemplary architectural patterns (unit cells) that can be used with the invention. Figure 2A shows a riblet pattern fabricated from PDMS elastomer having features spaced about 2 μιη apart on a silicon wafer. The features were formed using conventional photolithographic processing. Figure 2B shows a star/clover pattern, Figure 2C a gradient pattern, while Figure 2D shows a triangle/circle pattern.

In one embodiment, the hydrogel can be disposed on the raised features or on the protruded features seen in the Figures 1A through 2D. In another embodiment, the hydrogel can be disposed on the base surface in the spaces between the raised or the protruded features. In yet another embodiment, the raised features are themselves hydrogels. In yet another embodiment, some of the raised features comprise a first hydrogel while other raised features comprise a second hydrogel. In yet another embodiment, the raised or protruded features comprise a first hydrogel, while the spaces between the raised or protruded features comprise a second hydrogel. The patterns depicted in the Figures 1A through 2D have at least one tortuous pathway when viewed along the base surface. In one embodiment, when viewed in a second direction, the pathway between the features may be non-linear and non-sinusoidal. In other words, the pathway can be non-linear and aperiodic. In another embodiment, the pathway between the features may be linear but of a varying thickness. The plurality of spaced features may be projected outwards from a surface or projected into the surface. In one embodiment, the plurality of spaced features may have the same chemical composition as the surface. In another embodiment, the plurality of spaced features may have a different chemical composition from the surface.

In one embodiment, an article having a surface topography for resisting bioadhesion of organisms comprises a base article having a surface. The composition of the surface and/or the base article comprises a polymer, a metal or an alloy, a ceramic. Combinations of polymers, metals and ceramics may also be used in the surface or the base article. The surface having a topography comprising a plurality of patterns; each pattern being defined by a plurality of spaced apart features attached to or projected into the base article. The plurality of features each have at least one neighboring feature having a substantially different geometry. The average first feature spacing between the adjacent features is about 0.5 nanometers to about 500 nanometers in at least a portion of the surface, wherein said plurality of spaced apart features are represented by a periodic function. It is to be noted that each of the features of the plurality of features are separated from each other and do not contact one another.

In one embodiment, the surface is monolithically integrated with the base article, wherein a composition of the base article is the same as the composition of the surface. In another embodiment, the surface comprises a coating layer disposed on the base article. In yet another embodiment, the composition of the coating layer is different from the composition of the base article. In one embodiment, the polymer comprises a non- electrically conducting polymer.

In another embodiment, the topography provides an average roughness factor (R) of from 2 to 50. The base surface may comprise an elastomer that has an elastic modulus of about 10 kPa to about 10 MPa.

As noted above, the pattern is separated from a neighboring pattern by a tortuous pathway. The tortuous pathway may be represented by a periodic function. The periodic functions may be different for each tortuous pathway. In one embodiment, the patterns can be separated from one another by tortuous pathways that can be represented by two or more periodic functions. The periodic functions may comprise a sinusoidal wave. In an exemplary embodiment, the periodic function may comprise two or more sinusoidal waves. It is desirable for the path to be tortuous, unobstructed and periodic, with a periodicity that does not vary from one part of the path to another part of the path.

In one embodiment, the tortuous path separates one pattern from another neighboring pattern, where each pattern comprises a plurality of individual features that are different in size or geometry from one another. The neighboring patterns are identical with one another or different from one another.

In another embodiment, when a plurality of different tortuous pathways are represented by a plurality of periodic functions respectively, the respective periodic functions may be separated by a fixed phase difference. In yet another embodiment, when a plurality of different tortuous pathways are represented by a plurality of periodic functions respectively, the respective periodic functions may be separated by a variable phase difference.

In one embodiment, the plurality of spaced apart features have a substantially planar top surface. In another embodiment, a multi-element plateau layer can be disposed on a portion of the surface, wherein a spacing distance between elements of said plateau layer provide a second feature spacing; the second feature spacing being substantially different when compared to the first feature spacing.

In one embodiment, the pattern comprises a coating layer disposed on said base article. In other words, the coating layer comprises the pattern and is disposed on the base article.

In another embodiment, an article having a surface topography for controlling (e.g., resisting or facilitating) the bioadhesion of organisms, comprises a base article having a surface; wherein the composition of the surface comprises a polymer, a ceramic or a metal. The surface has a topography comprising a pattern defined by a plurality of spaced apart features attached to or projected into the base article. The surface comprises the hydrogel. The hydrogel may have reactive functionalities disposed on it. The reactive functionalities may be covalently bonded or ionically bonded to the hydrogel. In one embodiment, the reactive functionalities may interact with the hydrogel via polar bonds or via Vander Waals interactions.

The plurality of features each have at least one nanometer or micrometer sized dimension and have at least one neighboring feature having a substantially different geometry. The features are separated from each other and the average feature spacing is about 1 nanometer to about 500 micrometers. The topography is numerically representable using at least one periodic function; the periodic function being representable by a pathway situated substantially between a plurality of patterns of the spaced apart features.

In one embodiment, the first feature spacing is between 0.5 micrometers (μιη) and

5 μιη in at least a portion of the surface. In another embodiment, the first feature spacing is between 15 and 60 μιη in at least a portion of said surface. As noted above, the periodic function comprises two different sinusoidal waves. In one embodiment, the topography resembles the topography of shark-skin (e.g., a Sharklet). In another embodiment, the pattern comprises at least one multi-element plateau layer disposed on a portion of the surface, wherein a spacing distance between elements of the plateau layer provides a second feature spacing; the second feature spacing being substantially different when compared to said first feature spacing.

In yet another embodiment, an article having a surface topography for resisting bioadhesion of organisms, comprises a base article having a surface. The surface has a topography that comprises a pattern defined by a plurality of spaced apart features attached to or projected into the base article. The plurality of features comprises at least one feature having a substantially different geometry. The features are separated from each other. One of these features that is a part of the pattern is shared by a neighboring pattern. The plurality of spaced apart features has at least one microscale dimension. The neighboring patterns are separated from each other by a tortuous pathway. The tortuous pathway has at least two or more directions.

In another embodiment, an article comprises a plurality of spaced features. The features are arranged in a plurality of groupings; the groupings of features comprise repeat units. The spaced features within a grouping are spaced apart at an average distance of about 0.5 to about 200 micrometers. The groupings of features are arranged with respect to one another so as to define a tortuous pathway, the groupings have patterns of features wherein one or more features are shared between groupings. These are generally referred to as shared features. The plurality of spaced features extend outwardly from a surface.

In one embodiment, a sum of a number of features shared by two neighboring groupings is equal to an odd number. In another embodiment, a sum of a number of features shared by two neighboring groupings is equal to an even number.

In one embodiment, the plurality of spaced features has a similar chemical composition to the surface. In another embodiment, the plurality of spaced features has a different chemical composition from that of the surface. In one embodiment, the features have similar geometries, while in another embodiment the features can have different geometries. As will be detailed below, the groupings show dilational symmetry.

The plurality of spaced features is applied to the surface in the form of a coating and can comprise an organic polymer, a ceramic or a metal. As noted above, the groupings of features are arranged with respect to one another so as to define a linear pathway or a plurality of channels. The tortuous pathway is defined by a sinusoidal function.

In one embodiment, the features are periodic. In another embodiment, the features are aperiodic. The features have a roughness factor (R) of about 2 to about 20.

An occasional feature may lie in the otherwise tortuous pathway. In one embodiment, a tangent to the tortuous pathway will always intersect a single separated feature of the pattern. In one embodiment, a frequency of intersection between the tangent to the tortuous pathway and the spaced feature is periodic. In another embodiment, a frequency of intersection between a tangent to the tortuous pathway and a spaced feature is aperiodic. In another embodiment, a frequency of intersection between a tangent to the tortuous pathway and a shared feature is periodic. In another embodiment, a frequency of intersection between a tangent to the tortuous pathway and the shared spaced feature is aperiodic.

It is generally desirable for the groupings of features to comprise at least one repeat unit and to share at least one common feature. In one embodiment, the smallest feature in each repeat unit is shared by two adjacent repeat units or by two adjacent groups of features. The sharing of the feature by two or more groups of patterns results in the formation of the tortuous pathway. The number of features in a given pattern can be odd or even. In one embodiment, if the total number of features in a given pattern are equal to an odd number, then the number of shared features are generally equal to an odd number. In another embodiment, if the total number of features in a given pattern are equal to an even number, then the number of features in the given pattern are equal to an even number.

The spaced features can have variety of geometries and can exist in one, two or three dimensions or any dimensions therebetween. The spaced features can have similar geometries with different dimensions or can have different geometries with different dimensions. The geometries can be regular (e.g., described by Euclidean mathematics) or irregular (e.g., described by non-Euclidean mathematics). Euclidean mathematics describes those structures whose mass is directly proportional to a characteristic dimension of the spaced feature raised to an integer power (e.g., a first power, a second power or a third power). In one embodiment, the geometries can comprise shapes that are described by Euclidean mathematics such as, for example, lines, triangles, circles, quadrilaterals, polygons, spheres, cubes, fullerenes, or combinations of such geometries.

In one embodiment, a repeat unit can be combined with a neighboring repeat unit so as to produce a combination of spaced apart features that have a geometry that is described by Euclidean mathematics. The respective repeat units can be combined to produce different geometries. Similarly, 3 or more neighboring repeat units can be combined to produce a rhombohedral, while six repeat units can be combined to produce a hexagon. Thus repeat units may be combined to produce structures whose geometries can be described by Euclidean mathematics.

In one embodiment, the spaced features can have irregular geometries that can be described by non-Euclidean mathematics. Non-Euclidean mathematics is generally used to describe those structures whose mass is directly proportional to a characteristic dimension of the spaced feature raised to a fractional power (e.g., fractional powers such as 1.34, 2.75, 3.53, or the like). Examples of geometries that can be described by non- Euclidean mathematics include fractals and other irregularly shaped spaced features.

In one embodiment, spaced features whose geometries can be described by Euclidean mathematics may be combined to produce features whose geometries can be described by non-Euclidean mathematics. In other words, the groupings of features can have dilational symmetry. The fractal dimension can be measured perpendicular to the surface upon which the features are disposed or may be measured parallel to the surface upon which the features are disposed. The fractal dimensions are measured in the inter- topographical gaps.

In one embodiment, the fractal dimensions can have fractional powers of about 1.00 to about 3.00, specifically about 1.25 to about 2.25, more specifically about 1.35 to about 1.85 in a plane measured parallel to the surface upon which the features are disposed. In another embodiment, the fractal dimensions can have fractional powers of about 1.00 to about 3.00, specifically about 1.25 to about 2.25, more specifically about 1.35 to about 1.85 in a plane measured perpendicular to the surface upon which the features are disposed. In yet another embodiment, the fractal dimensions can have fractional powers of about 3.00 to about 4.00, specifically about 3.25 to about 3.95, more specifically about 3.35 to about 3.85 in a plane measured perpendicular to the surface upon which the features are disposed. In other words, the tortuous pathway or the surface of each feature may be textured with features similar to those of the pattern (albeit on a smaller scale), thus creating micro -tortuous pathways and nano-tortuous pathways within the tortuous pathway itself.

In another embodiment, the spaced features may have multiple fractal dimensions in a direction parallel to the surface upon which the features are disposed. The spaced features may be arranged to have 2 or more fractal dimensions, specifically 3 or more dimensions, specifically 4 or more dimensions in a direction parallel to the surface upon which the features are disposed. The features have 3 different fractal dimensions in a plane parallel to the surface upon which the features are disposed. The fractal dimensions created by the features in a direction from the top to the bottom of the micrograph are 1.444 and 1.519 respectively, while the fractal dimension created by the features in a direction from left to right have dimensions of 1.557. The presence of the texture having multiple fractal dimensions prevents bioadhesion of algae, bacteria, virus, and other organisms.

In yet another embodiment, the spaced features may have multiple fractal dimensions in a direction perpendicular to the surface upon which the features are disposed. The spaced features may be arranged to have 2 or more fractal dimensions, specifically 3 or more dimensions, specifically 4 or more dimensions in a direction parallel to the surface upon which the features are disposed.

As will be noted below, the tortuous pathway may be defined by a sinusoidal function, a spline function, a polynomial function, or the like. The tortuous pathway generally exists between a plurality of groupings of spaced features and may occasionally be interrupted by the existence of a feature or by contact between two features. The frequency of the intersection between the tortuous pathway and the spaced feature may be periodic or aperiodic. In one embodiment, the tortuous pathway may have a periodicity to it. In another embodiment, the tortuous pathway may be aperiodic. In one embodiment, two or more separate tortuous pathways never intersect one another.

The tortuous pathway can have a length that extends over the entire length of the surface upon which the pattern is disposed, if the features that act as obstructions in the tortuous pathway are by-passed. The width of the tortuous pathway as measured between two adjacent features of two adjacent patterns are about 10 nanometers to about 500 micrometers, specifically about 20 nanometers to about 300 micrometers, specifically about 50 nanometers to about 100 micrometers, and more specifically about 100 nanometers to about 10 micrometers.

The spaced features have linear pathways or channels between them. In one embodiment, the spaced features can have a plurality of linear pathways or a plurality of channels between them.

The spaced features can be periodic or aperiodic. In a similar manner, the patterns can be periodic or aperiodic.

As noted above, the spaced features can have different dimensions (sizes). The average size of the spaced features can be nanoscale (e.g., they can be less than 100 nanometers) or greater than or equal to about 100 nanometers. In one embodiment, the spaced features can have average dimensions of 1 nanometer to 500 micrometers, specifically about 10 nanometers to about 200 micrometers, and more specifically about 50 nanometers to about 100 micrometers.

In another embodiment, the average periodicity between the spaced features can be about 1 nanometer to about 500 micrometers. In one embodiment, the periodicity between the spaced features can be about 2, 5, 10, 20, 50, 100 or 200 nanometers. In another embodiment, the average periodicity between the spaced features can be about 2, 5, 10, 20, 50, 100 or 200 nanometers. In another embodiment, the periodicity can be about 0.1, 0.2, 0.5, 1, 5, 10, 20, 50, 100, 200, 300, 400 or 450 micrometers. In yet another embodiment, the average periodicity can be about 0.1, 0.2, 0.5, 1, 5, 10, 20, 50, 100, 200, 300, 400 or 450 micrometers.

In one embodiment, the spaced features can have dimensions of 1 nanometer to 500 micrometers, specifically about 10 nanometers to about 200 micrometers, and more specifically about 50 nanometers to about 100 micrometers.

In another embodiment, the periodicity between the spaced features can be about 1 nanometer to about 500 micrometers. In one embodiment, the periodicity between the spaced features can be up to about 2, 5, 10, 20, 50, 100 or 200 nanometers. In another embodiment, the periodicity between the spaced features can be about 2, 5, 10, 20, 50, 100 or 200 nanometers. In another embodiment, the periodicity can be up to about 0.1, 0.2, 0.5, 1, 5, 10, 20, 50, 100, 200, 300, 400 or 450 micrometers. In yet another embodiment, the periodicity can be up to about 0.1, 0.2, 0.5, 1, 5, 10, 20, 50, 100, 200, 300, 400 or 450 micrometers. In one embodiment, each feature of a pattern has at least one neighboring feature that has a different geometry (e.g., size or shape). A feature of a pattern is a single element. Each feature of a pattern has at least 2, 3, 4, 5, or 6 neighboring features that have a different geometry from the feature. In one embodiment, there are at least 2 or more different features that form the pattern. In another embodiment, there are at least 3 or more different features that form the pattern. In yet another embodiment, there are at least 4 or more different features that form the pattern. In yet another embodiment, there are at least 5 or more different features that form the pattern.

In another embodiment, at least two identical features of the pattern have at least one neighboring feature that has a different geometry (e.g., size or shape). A feature of a pattern is a single element. In one embodiment, two identical features of the pattern have at least 2, 3, 4, 5, or 6 neighboring features that have a different geometry from the identical features. In another embodiment, three identical features of the pattern have at least 2, 3, 4, 5, or 6 neighboring features that have a different geometry from the identical features.

In another embodiment, each pattern has at least one or more neighboring patterns that have a different size or shape. In other words, a first pattern can have a second neighboring pattern that while comprising the same features as the first pattern can have a different shape from the first pattern. In yet another embodiment, each pattern has at least two or more neighboring patterns that have a different size or shape. In yet another embodiment, each pattern has at least three or more neighboring patterns that have a different size or shape. In yet another embodiment, each pattern has at least four or more neighboring patterns that have a different size or shape.

In one embodiment, the features may have a tortuous unobstructed periodic path disposed between features (or protruions into the base surface) that are identical with one another. The Figure 3 depicts a pattern where the neighboring features are identical and neighboring patterns are therefore identical with one another. The neighboring patterns do not comprise features that are different from one another. The hydrogel may be disposed upon the features or in the spaces between the features.

As noted above the chemical composition of the spaced features can be different from the surface. The spaced features and the surfaces from which these features are projected or projected into can also comprise organic polymers or inorganic materials.

Organic polymers used in the spaced features and/or the base surface can be may be selected from a wide variety of thermoplastic polymers, blend of thermoplastic polymers, thermosetting polymers, or blends of thermoplastic polymers with thermosetting polymers. The organic polymer may also be a blend of polymers, copolymers, terpolymers, or combinations comprising at least one of the foregoing organic polymers. The organic polymer can also be an oligomer, a homopolymer, a copolymer, a block copolymer, an alternating block copolymer, a random polymer, a random copolymer, a random block copolymer, a graft copolymer, a star block copolymer, a dendrimer, a polyelectrolyte (polymers that have some repeat groups that contain electrolytes), a polyampholyte (a polyelectrolyte having both cationic and anionic repeat groups), an ionomer, or the like, or a combination comprising at last one of the foregoing organic polymers.

Examples of the organic polymers are polyacetals, polyolefms, polyacrylics, polycarbonates, polystyrenes, polyesters, polyamides, polyamideimides, polyarylates, polyarylsulfones, polyethersulfones, polyphenylene sulfides, polyvinyl chlorides, polysulfones, polyimides, polyetherimides, polytetrafluoroethylenes, poly etherketones, polyether etherketones, polyether ketone ketones, polybenzoxazoles, polyphthalides, polyacetals, polyanhydrides, polyvinyl ethers, polyvinyl thioethers, polyvinyl alcohols, polyvinyl ketones, polyvinyl halides, polyvinyl nitriles, polyvinyl esters, polysulfonates, polysulfides, polythio esters, polysulfones, polysulfonamides, polyureas, polyphosphazenes, polysilazanes, styrene acrylonitrile, acrylonitrile-butadiene-styrene (ABS), polyethylene terephthalate, polybutylene terephthalate, polyurethane, ethylene propylene diene rubber (EPR), polytetrafluoroethylene, perfluoroelastomers, fluorinated ethylene propylene, perfluoroalkoxyethylene, polychlorotrifluoroethylene, polyvinylidene fluoride, polysiloxanes, or the like, or a combination comprising at least one of the foregoing organic polymers.

Examples of polyelectrolytes are polystyrene sulfonic acid, polyacrylic acid, pectin, carageenan, alginates, carboxymethylcellulose, polyvinylpyrrolidone, or the like, or a combination comprising at least one of the foregoing polyelectrolytes.

Examples of thermosetting polymers suitable for use in the base surface include epoxy polymers, unsaturated polyester polymers, polyimide polymers, bismaleimide polymers, bismaleimide triazine polymers, cyanate ester polymers, vinyl polymers, benzoxazine polymers, benzocyclobutene polymers, acrylics, alkyds, phenol- formaldehyde polymers, novo lacs, resoles, melamine-formaldehyde polymers, urea- formaldehyde polymers, hydroxymethylfurans, isocyanates, diallyl phthalate, triallyl cyanurate, triallyl isocyanurate, unsaturated polyesterimides, or the like, or a combination comprising at least one of the foregoing thermosetting polymers.

Examples of blends of thermoplastic polymers include acrylonitrile-butadiene- styrene/nylon, polycarbonate/acrylonitrile-butadiene-styrene, acrylonitrile butadiene styrene/polyvinyl chloride, polyphenylene ether/polystyrene, polyphenylene ether/nylon, polysulfone/acrylonitrile-butadiene-styrene, polycarbonate/thermoplastic urethane, polycarbonate/polyethylene terephthalate, polycarbonate/polybutylene terephthalate, thermoplastic elastomer alloys, nylon/elastomers, polyester/elastomers, polyethylene terephthalate/polybutylene terephthalate, acetal/elastomer, styrene- maleicanhydride/acrylonitrile-butadiene-styrene, polyether etherketone/polyethersulfone, polyether etherketone/polyetherimide polyethylene/nylon, polyethylene/polyacetal, or the like.

Polymers that can be used for the pattern or the substrate include biodegradable materials. Suitable examples of biodegradable polymers are as polylactic-glycolic acid (PLGA), poly-capro lactone (PCL), copolymers of polylactic-glycolic acid and poly- caprolactone (PCL-PLGA copolymer), polyhydroxy-butyrate -valerate (PHBV), polyortho ester (POE), polyethylene oxide-butylene terephthalate (PEO-PBTP), poly-D,L- lactic acid-/?-dioxanone-polyethylene glycol block copolymer (PLA-DX-PEG), or the like, or combinations comprising at least one of the foregoing biodegradable polymers. The biodegradable polymers upon undergoing degradation can be consumed by the body without any undesirable side effects.

Polyelectrolytes and hydrogels are preferred for the surface. Examples of hydrogels are acrylamides, methacrylamides, N-vinylimidazole, poly(ethylene glycol)- dimethacrylate (PEGDMA), poly(ethylene glycol)-dimethacrylate-co-glycidyl dimethacrylate, poly(ethylene glycol)-dimethacrylate-co-hexaethylmethacrylate, and the like, and a combination comprising at least one of the foregoing hydrogels or polyelectrolytes.

As noted above, the textured surface may be reacted with reactive species that have functional groups. It is desirable for the reactive species to have at least two functional groups. One of the functional groups can react with the textured surface, while the other functional group can react with a biological entity. Examples of biological entities are proteins, antibodies, enzymes, enzyme, a glycoprotein, a cytokine, a mixture of proteins, or a combination comprising at least one of the foregoing proteins. The reactive functionalities that can bond with the biological entity are epoxy groups, ethylenically unsaturated functionalities (e.g., vinyl groups), thiol groups, SO ₃ H, COOH, NH ₂ , OH, R'CHOH, CHO, CN, COC1, COSH, SH, COOR', SR.', S1R ₃ ', Si-(OR') _y - R'(3- _y ), R", A1R ₂ ', halide, thiophene, ethylenically unsaturated functionalities, epoxide functionalities, or the like, wherein R' is hydrogen, alkyl, aryl, cycloalkyl, or araalkyl, cycloaryl, poly(alkylether), or the like, R" is fluoroalkyl, fluoroaryl, fluorocycloalkyl, fluoroaralkyl, cycloaryl, or the like.

In one embodiment, the pattern can comprise a polymeric resin that is blended with a biologically active agent to form a drug coating. The biologically active agent is then gradually released from the pattern, which simply acts as a carrier. When the polymeric resin is physically blended (i.e., not covalently bonded) with the biologically active agent, the release of the biologically active agent from the drug coating is diffusion controlled. It is generally desirable for the pattern to comprise an amount of about 5 weight percent (wt%) to about 90 wt% of the biologically active agent based on the total weight of the drug coating. Within this range, it is generally desirable to have the biologically active agent present in an amount of greater than or equal to about 10, preferably greater than or equal to about 20, and more preferably greater than or equal to about 30 wt% based on the total weight of the drug coating. Within this range it is generally desirable to have the biologically active agent present in an amount of less than or equal to about 75, preferably less than or equal to about 70, and more preferably less than or equal to about 65 wt% based on the total weight of the drug coating. The drug coating may be optionally coated with an additional surface coating if desired. When an additional surface coating is used, the release of the biologically active agent is interfacially controlled. The drug coating may be disposed only on the surface of the features or alternatively on the surface of the tortuous pathway.

In another exemplary embodiment, the biologically active agent may be covalently bonded with a biodegradable polymer to form the drug coating. The rate of release is then controlled by the rate of degradation of the biodegradable polymer. Suitable examples of biodegradable polymers are provided above. Within this range, it is generally desirable to have the biologically active agent present in an amount of greater than or equal to about 10, preferably greater than or equal to about 20, and more preferably greater than or equal to about 30 wt% based on the total weight of the drug coating. Within this range, it is also generally desirable to have the biologically active agent present in an amount of less than or equal to about 75, preferably less than or equal to about 70, and more preferably less than or equal to about 65 wt%, based on the total weight of the drug coating.

When the pattern is used in a medical device, the drug coating may be coated onto the medical device in a variety of ways. In one embodiment, the drug coating may be dissolved in a solvent such as water, acetone, alcohols such ethanol, isopropanol, methanol, toluene, dimethylformamide, dimethylacetamide, hexane, and the like, and coated onto the medical device in the form of the pattern. In another embodiment, a monomer may be covalently bonded with the biologically active agent and then polymerized to form the drug coating, which is then applied onto the medical device in the form of the pattern. In yet another embodiment, the polymeric resin may first be applied as a coating (in the form of the pattern) onto the medical device, following which the coated device is immersed into the biologically active agent, thus permitting diffusion into the coating to form the drug coating.

In one embodiment, a biologically active agent can be added to the pattern. Te biologically active agent can be disposed upon the surface of the pattern or can be included in the pattern (e.g., mixed with the material forming the pattern). It may also be desirable to have two or more biologically active agents dispersed in a single drug coating layer. Alternatively, it may be desirable to have two or more layers of the drug coating coated upon the medical device. Various methods of coating may be employed to coat the medical device such as spin coating, electrostatic painting, dip-coating, painting with a brush, and the like, and combinations comprising at least one of the foregoing methods of coating.

Various types of biologically active agents may be used in the drug coating, which is used to coat the medical device. The coatings on the medical device may be used to deliver therapeutic and pharmaceutically biologically active agents including anti- analgesic agents, anti-arrhythmic agents, anti-bacterial agents, anti-cholinergic agents, anti-coagulant agents, anti-convulsant agents, anti-depressant agents, anti-diabetic agents, anti-diuretic agents, anti-fungal agents, anti-hypertensive agents, anti-inflammatory agents, anti-malarial agents, anti-neoplastic agents, anti-nootropic agents, anti-Parkinson agents, anti-retroviral agents, anti-tuberculosis agents, anti-tussive agents, anti-ulcerative agents, anti-viral agents, or the like, or a combination comprising at least one of the foregoing therapeutic and pharmaceutically biologically active agents.

Examples of other suitable therapeutic and pharmaceutically biologically active agents are anti-pro liferative/antimito tic agents including natural products such as vinca alkaloids (e.g., vinblastine, vincristine, and vinorelbine), paclitaxel, epidipodophyllotoxins (e.g., etoposide, teniposide), antibiotics (e.g., dactinomycin, actinomycin D, daunorubicin, doxorubicin, penicillin V, penicillin G, ampicillin, amoxicillin, cephalosporin, tetracycline, doxycycline, minocycline, demeclocycline, erythromycin, aminoglycoside antibiotics, polypeptide antibiotics, nystatin, griseofulvin, and idarubicin), anthracyclines, mitoxantrone, bleomycins, plicamycin, mithramycin and mitomycin, enzymes (L- asparaginase, which systemically metabolizes L-asparagine and deprives cells which do not have the capacity to synthesize their own asparagine), antiplatelet agents such as G(GP) Ilb/IIIa inhibitors and vitronectin receptor antagonists, anti-pro liferative/antimito tic alkylating agents such as nitrogen mustards (e.g., mechlorethamine, cyclophosphamide and analogs, melphalan, chlorambucil), ethylenimines and methylmelamines (e.g., hexamethylmelamine and thiotepa), alkyl sulfonates- busulfan, nitrosoureas (e.g., carmustine (BCNU) and analogs, streptozocin), trazenes—dacarbazinine (DTIC), antiproliferative/antimitotic antimetabolites such as folic acid analogs (e.g., methotrexate), pyrimidine analogs (e.g., fluoro uracil, floxuridine, cytarabine), purine analogs and related inhibitors (e.g., mercaptopurine, thioguanine, pentostatin and 2- chlorodeoxyadenosine {cladribine}), platinum coordination complexes (e.g., cisplatin, carboplatin), procarbazine, hydroxyurea, mitotane, aminoglutethimide, hormones (e.g., estrogen), anti-coagulants (e.g., heparin, synthetic heparin salts and other inhibitors of thrombin), fibrinolytic agents (e.g., tissue plasminogen activator, streptokinase and urokinase), aspirin, dipyridamole, ticlopidine, clopidogrel, abciximab, antimigratory, antisecretory (e.g., breveldin), antiinflammatory: such as adrenocortical steroids (e.g., Cortisol, cortisone, fludrocortisone, prednisone, prednisolone, 6a-methylprednisolone, triamcinolone, betamethasone, and dexamethasone), non-steroidal agents (e.g., salicylic acid derivatives such as aspirin, para- aminophenol derivatives such as acetominophen, indole and indene acetic acids (e.g., indomethacin, sulindac, etodalac), heteroaryl acetic acids (e.g., tolmetin, diclofenac, ketorolac), arylpropionic acids (e.g., ibuprofen and derivatives), anthranilic acids (e.g., mefenamic acid, meclofenamic acid), enolic acids (e.g., piroxicam, tenoxicam, phenylbutazone, oxyphenthatrazone), nabumetone, gold compounds (e.g., auranofin, aurothioglucose, gold sodium thiomalate), immunosuppressives (e.g., cyclosporine, tacrolimus (FK-506), sirolimus (e.g., rapamycin, azathioprine, mycophenolate mofetil), angiogenic agents such as vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), angiotensin receptor blockers, nitric oxide donors, anti-sense oligionucleotides and combinations thereof, cell cycle inhibitors, mTOR inhibitors, and growth factor receptor signal transduction kinase inhibitors, retenoids, cyclin/CDK inhibitors, HMG co-enzyme reductase inhibitors (statins) or protease inhibitors.

The biologically active agents may also include cancer inhibitors. Suitable examples of cancer inhibitors are (-)-Ci-Cdpl, (-)-Ci-Cdp2, (-)-epigallocatechin gallate, (+)-Cbi-Cdpi2, (+)-Ci-Cdp2, 10-Deacetylbaccatin Iii, 4-demethoxy daunorubicin, 5- azacytidine/5-aza-2'-deoxycytidine, 5 -fluoro uracil, 5-iminodoxorubicin hydrochloride, 6- mercaptopurine, aclarubicin, acodazole, actinomycin D, adenine phosphate, adenosine, aderbasib, adozelesin; U-73,975, afeletecan, alemtuzumab, alitreninoin, alosetron HC1, alphitolic acid, altretamine, alvespimycin, ambazone, ametantrone, amifostine, aminoglutethimide, amsacrine HC1, amsilarotene, amygdalin, anagrelide, anastrozole, anaxirone, ancitabine, annomontacin, annomuricin A, (C19/C20-Erythro), annomuricin B, (ClO/Cl l,C19/C20-Erythro), annomuricin C, (All Threo) annomuricin E, annonacin, annonacin-10-One, annonacin-A-One, annonidin B, annonin VI, annosquamosin A, annosquamosin B, antramycin, apaziquone, argimesna, aristoforin, arsenic trioxide, artemisinin, ascomycin, asparaginase, atosiban, atrimustine, axitinib, azasetron HC1, azatepa, azathioprine, azotomycin, bafetinib, balamapimod, banoxantrone, batabulin, batimastat, Bbr-34384, becatecarin, belotecan, benaxibine, bendamustine, benzodepa, berubicin, betulin, betulinic acid, betulinic aldehyde, bevacizumab, bexarotene, bicalutamide, bietaserpine, biricodar, bisantrene, bistramid A; bistratene A, bizelesin, bleomycin, bleomycin A2 [Sulfate], bleomycin A5, bleomycin Sulfate, bortezomib, bosentan, bosutinib, brequinar sodium, brequinar, bropirimine, brostallicin, budotitane, bullatacin, buserelin, busulfan, cabazitaxel, calcium folinate, calcium levofolinate, calusterone, camptothecin, canertinib, canfosfamide, cantharidin, capecitabine, caracemide, carbetimer, carboplatin, carboprost (carboprost tromethamine), carboquone, carfilzomib, carglumic acid, carmofur, carmustine, carzelesin, cedefingol, cemadotin, cetuximab, cevipabulin, chlorambucil, chlormethine (mechlorethamine), chlorotamoxifen, chlorotrianisene, cioteronel, cisplatin, cladribine, clanfenur, clofarabine, clofazimine, clomifene citrate, cordycepin, corosolic acid, crisnatol, curcumin, cyclocytidine, cyclophosphamide, cytarabine, cytidine, D-aminolevulinic acid, dacarbazine, damsin, daniquidone, danusertib, daporinad, darinaparsin, dasatinib, daunoblastin, daunorubicin/daunomycin, decitabine, deferasirox, deforolimus, demecolcine, denibulin, detorubicin, dexniguldipine, dexormaplatin, dezaguanine, dianhydrodulcitolum, dibrospidium chloride, dienogest, diflomotecan, dinalin, disermolide, docetaxel, dofequidar, dolasetron mesylate, dovitinib, doxifluridine, doxorubicin, dromostanolone, duazomycin, duocarmycin, dynemicin, ecomustine, edatrexate, edotecarin, edotreotide, eflornithine, elacridar, eacytarabine, elesclomol, elinafide, elomotecan, elsamitrucin, emitefur, enloplatin, enocitabine, enpromate, entecavir, entinostat, entricitabine, enzastaurin, epirubicin, eptaloprost, eribulin, erlotinib, Esorubicin, estramustine, etalocib, etanidazole, etoglucid, etoposide, exatecan, exemestane, exisulind, fadrozole, fazarabine, fiacitabine, floxuridine, fludarabine, fluoxymesterone, flurocitabine, flutamide, formestane, forodesine, fosfluridine tidoxil, fosquidone, fostriecin, fotemustine, fotretamine, fulvestrant, fumagillin, galarubicin, galocitabine, gefitinib, gemcitabine, gemtuzumab ozogamicin, geroquinol, gigantetronenin, gigantetroneninone, gimatecan, gimeracil, gloxazone, glufosfamide, goniothalamicin, goniothalamicinone, goserelin, granisetron HCl, gusperimus, hexarelin, homoharringtonine, hydrocamptothecine, hydroxycarbamide, hydroxyurea, hypericin, ibandronate sodium, ibandronic acid, idarubicin HCl, idronoxil, ifosfamide, ilmofosine, imatinib, imatinib mesylate, imexon, improsulfan, incadronate, indibulin, indisulam, inolitazone, inproquone, intiquinatine, intoplicine, iobenguane, irinotecan hydrochloride, irofulven, irsogladine, ispinesib, ixabepilone, ketotrexate, L-alanosine, laniquidar, lapatinib ditosylate, laromustine, larotaxel, ledoxantrone, lenalidomide, lentinan, lestaurtinib, letrozole, leuprolide acetate, leuprorelin, lexacalcitol, liarozole, lobaplatin, lomustine, lonafarnib, lonidamine, losoxantrone, Ly-83583, lysipressin, mafosfamide, mannomustine, mannosulfan, marimastat, marinomycin A, masitinib, maslinic acid, masoprocol, mechlorethamine, medorubicin, megestrol, mepitiostane, mercaptopurine, mesna, methotrexate, methyl amino levulinate, metomidate, metoprine, meturedepa, miboplatin, midostaurin, mifamurtide, milataxel, miproxifene, miriplatin, misonidazole, mitindomide, mitoflaxone, mitoguazone, mitomycin, mitonafide, mitoquidone, mitotane, mitoxantrone, mitozolomide, mivobulin, mizoribine, mofarotene, mopidamol, motesanib, motexafin, mubritinib, muricapentocin, muricatacin, mustine HCl, mycophenolate mofetil, mycophenolic acid, nedaplatin, nelzarabine, nemorubicin, neocuproine, neptamustine, neratinib, nigericin, nilotinib, nilutamide, nimustine, ninopterin, nitracrine, nogalamycin, nolatrexed, norcantharidine, nor-dihydroguaiaretic acid, nortopixantrone, novembichin, obatoclax, octreotide, olaparib, oleanolic aldehyde, omacetaxine mepesuccinate, ombrabulin, omtripolide, ondansetron HCl, ortataxel, oteracil, oteracil potassium, oxaliplatin, oxisuran, oxophenarsine, paclitaxel ceribate, palifosfamide, palonosetron, pamidronate disodium, pamidronic acid, panitumumab, panobinostat, patubilone, pazelliptine, pazopanib, pegaspargase, peldesine, pelitinib, pelitrexol, pemetrexed disodium, pentostatin, peplomycin, peretinoin, perfosfamide, perifosine, pibrozelesin hydrobromide, picoplatin, pinafide, piposulfan, pirarubicin, pirfenidone, piritrexim, piroxantrone, pixantrone, plevitrexed, plicamycin, plitidepsin, plomestane, podophyllotoxin, pomalidomide, porfimer sodium, pralatrexate, prinomastat, procarbazine HC1, propamidine, prospidium chloride, pumitepa, puromycin, pyrazofurin, ouarfloxin, raltegravir, raltitrexed, ramosetron HC1, ranimustine, retaspimycin, retelliptine, riboprine, ritrosulfan, rituximab, roflumilast, romidepsin, ropidoxuridine, roquinimex, rosabulin, rubitecan, sabarubicin, safingol, salirasib, sapacitabine, saracatinib, sardomozide, satrap latin, sebrip latin, seliciclib, semaxanib; SU-5416, semustine, sermorelin, simotaxel, simtrazene, sitagliptin, sizofiran, soblitodin, sobuzoxane, sodium phenylbutyrate, sorafenib, sparfosic acid, sparsomycin, spiroplatin, squalamine, squamocin, streptonigrin, streptovarycin, streptozocin, sufosfamide, sulofenur, sunitinib, swainsonine, tacedinaline, tafluposide, talabostat, talisomycin, tallimustine, talotrexin, taltobulin, tamoxifen citrate, tandutinib, tanespimycin, tariquidar, tasidotin, tasisulam, tauromustine, tegafur, tegafur- uracil, telantinib, teloxantrone, temozolomide, teniposide, tenuazonic acid, terameprocol, teriparatide, tesetaxel, testolactone, tezacitabine, thiamiprine, thioguanine, thiotepa, thymopoietin, tiazofurine, tilomisole, tilorone, timcodar, timonacic, tioguanine, tirapazamine, tocladesine, tomudex, topotecan hydrochloride, toremifene citrate, tosedostat, tositumomab, toxipantrone, trastuzumab, trenimon, tretinoin, triciribine, trilostane, trimetrexate, triplatin tetranitrate, triptolide, triptorelin, trofosfamide, tropisetron HC1, tubulozole, tylophorin, U-67786, U-68415, U-71184, U-76074, U-78057, ubenimex, uramustine, uredepa, urethane, uridine, ursolic acid, ursolic aldehyde, vadimezan, valrubicin, valspodar, vandetanib, vapreotide, vatalanib; PTK-787, verteporfin, vildagliptin, vinblastine sulfate, vincristine, vindesine, vinepidine, vinflunine, vinformide, vinfosiltine, vinleucinol, vinleurosine, vinorelbine [Base], vinorelbine tartrate, vintriptol, vinzolidine, voriconazole, vorinostat, vorozole, wilforlide A, xanthomycin A, zalcitabine, zenip latin, zilascorb, zinostatin, zoledronic acid, zorubicin, zosuquidar, or the like, or a combination comprising at least one of the foregoing cancer inhibitors.

Algaecides, herbicides, repellents and antifouling agents may also be disposed upon the pattern or blended in with the material used to form the pattern. Examples of suitable algaecides are 2,2-dibromo-3-nitrilopropionamide (DNP), methylene bis- thiocyanate (MBT), 5-chloro-2-methyl-4-isothiazolin-3-one/2-methyl-4-isothiazol in-3- one (CMI), tetrahydro-3,5-dimethyl-2H,l,3,5-thiadiazine-2-thione (TDD), sodium dimethyldithiocarbamate/sodium ethylene bis dithio carbamate (SDT), alkyl dimethylbenzyl amonium chloride family, poly[oxyethylene (dimethyliminio) ethylene (dimethyliminio) ethylene dichloride, copper sulfate, or the like, or a combination comprising at least one of the foregoing algaecides. Examples of suitable repellents are zosteric acid, or a combination comprising zosteric acid.

In one embodiment, the biologically active agents may be encapsulated in microballoons and incorporated into the pattern as part of the drug coating. In another embodiment, the biologically active agents may be encapsulated in microballoons and incorporated on the surface of the pattern or incorporated into the channels that form the tortuous pathway. The microballoons may serve to release the drugs gradually over a period of time. In other words the pattern can serve as a time-release coating for the drugs. In one embodiment, the pattern can release drugs after being subjected to a stress.

The inorganic materials used in the spaced features and/or the surface can comprise ceramics and/or metals. The inorganic materials can comprise inorganic oxides, inorganic carbides, inorganic nitrides, inorganic hydroxides, inorganic oxides having hydroxide coatings, inorganic carbonitrides, inorganic oxynitrides, inorganic borides, inorganic borocarbides, or the like, or a combination comprising at least one of the foregoing inorganic materials. Examples of suitable inorganic materials are metal oxides, metal carbides, metal nitrides, metal hydroxides, metal oxides having hydroxide coatings, metal carbonitrides, metal oxynitrides, metal borides, metal borocarbides, or the like, or a combination comprising at least one of the foregoing inorganic materials.

Examples of suitable inorganic oxides include silica (Si0 ₂ ), alumina (A1 ₂ 0 ₃ ), titania (Ti0 ₂ ), zirconia (Zr0 ₂ ), ceria (Ce0 ₂ ), manganese oxide (Mn0 ₂ ), zinc oxide (ZnO), iron oxides (e.g., FeO, y-Fe ₂ 0 ₃ , Fe ₃ 0 ₄ , or the like), calcium oxide (CaO), manganese dioxide (Mn0 ₂ and Mn ₃ 0 ₄ ), or combinations comprising at least one of the foregoing inorganic oxides. Examples of inorganic carbides include silicon carbide (SiC), titanium carbide (TiC), tantalum carbide (TaC), tungsten carbide (WC), hafnium carbide (HfC), or the like, or a combination comprising at least one of the foregoing carbides. Examples of suitable nitrides include silicon nitrides (Si ₃ N ₄ ), titanium nitride (Ti ), or the like, or a combination comprising at least one of the foregoing. Examples of suitable borides are lanthanum bo ride (LaB ₆ ), chromium borides (CrB and CrB ₂ ), molybdenum borides (MoB ₂ , Mo ₂ B ₅ and MoB), tungsten boride (W ₂ B ₅ ), or the like, or combinations comprising at least one of the foregoing borides. Exemplary inorganic substrates are those that comprise naturally occurring or synthetically prepared silica and/or alumina. Metals used in the spaced features and/or the surface can be transition metals, alkali metals, alkaline earth metals, rare earth metals, or the like, or a combination comprising at least one of the foregoing metals. Examples of metals are iron, copper, aluminum, tin, tungsten, chromium, gold, silver, titanium, or a combination comprising at least one of the foregoing metals.

The pattern can be aligned such that linear channels between respective features in a pattern can be arranged to be perpendicular and/or parallel to an average direction of fluid flow, when the pattern is disposed on a surface that contacts a flowing fluid. In one embodiment, the pattern may be disposed on the substrate so that the linear channels between respective features in a pattern can be arranged to occupy an angle of 1 to about 360 degrees, specifically about 5 to about 270 degrees, and more specifically about 10 to about 200 degrees, and more specifically about 20 to about 200 degrees with respect to the average direction of fluid flow.

In one embodiment, the pattern can be dynamically modified during use. In other words, the surface can comprise a material such as, for example, a shape memory alloy, a shape memory polymer, a magnetorheological fluid, an electrorheological fluid, and the like, that can be activated when desired to either inhibit or to facilitate bioadhesion.

In an exemplary embodiment, when the pattern comprises an organic polymer, the organic polymer can be filled with an electrically conducting filler thereby making the surface electrically conductive. By passing an electrical signal to the surface, the pattern can be heated and consequently the dimensions of the features and those of the patterns can be changed during use. In another embodiment, the pattern can comprise magnetic particles, which can be activated by a magnetic field.

Although not required to practice the present invention, Applicants not seeking to be bound by the mechanism believed to be operable to explain the efficacy of the present invention, provide the following. The efficacy of surfaces according to the invention is likely to be due to physically interfering with the settlement and adhesion of microorganisms, such as algae, bacteria and barnacles. Properly spaced features (such as "ribs") formed on or formed in the surface can be effective for organisms from small bacteria (<1 μιη, such as 200 to 500 nm), to large tube worms (>200 μιη, such as 200 to 500 μιη), provided the feature spacing scales with the organism size. Specifically, bioadhesion is retarded when the specific width of closely packed, yet dissimilar features (e.g. ribs) in the pattern is too narrow to support settlement on top, yet the ribs are too closely packed to allow settlement in between. However, a feature spacing too small is believed to make the surface look flat to the settling organism, i.e. like the base surface, and thus ineffective. Accordingly, a feature spacing that scales with 25 to 75% of the settling organism's smallest physical dimension has been found to be generally effective to resist bioadhesion. Various different surface topographies can be combined into a hierarchical multi-level surface structure to provide a plurality of spacing dimensions to deter the settlement and adhesion of multiple organisms having multiple and wide ranging sizes simultaneously, such as algae, spores and barnacles.

Topographies according to the invention can generally be applied to a wide variety of surfaces for a wide variety of desired applications. Applications for inhibiting bioadhesion using the invention described in more detail below include base articles used in marine environments or biomedical or other applications which may be exposed to contamination by biological organisms, such as roofs on buildings, water inlet pipes in power plants, catheters, cosmetic implants, and heart valves. As described below, surfaces according to the invention can be formed on a variety of devices and over large areas, if required by the application.

Barnacle cyprids are known to be generally elliptically shaped have a nominal length of about 100 μιη, and a nominal width of about 30 μιη. Algae are also generally elliptically shaped and have a nominal length of about 7 μιη, and a nominal width of about 2 μιη, while spores are generally elliptically shaped have a nominal length of about 5 μιη, and a nominal width of about 1.5 μιη. Features according to the invention are generally raised surfaces (volumes) which emerge from a base level to provide a first feature spacing, or in the case of hierarchical multi-level surface structures according to the invention also include the a second feature spacing being the spacing distance between neighboring plateaus, which themselves preferably include raised features thereon or features projected into the base article.

As noted above, if the feature spacing is smaller than the smaller dimension of the organism or cell, it has been found that the growth is generally retarded, such between 0.25 and 0.75 of the smaller dimension of the cell or organism. A feature spacing of about ½ the smaller dimension of a given organism to be repelled has been found to be near optimum. For example, for an algae spore 2 to 5 μιη in width, to retard adhesion, a feature spacing of from about 0.5 to 3.75 μιη, preferably 0.75 to 2 μιη is used. For example, to repel barnacles 20 to 50 μιη in width, a feature spacing of between 5 and 200 μιη, preferably 10 to 100 μιη, has been found to be effective. For repelling both barnacles and spores, a hierarchical multi-level surface structure according to the invention can include a raised surfaces (volumes) which emerge from or are projected into a base level having a feature spacing of about 2 μηι, and a plurality of striped plateau regions spaced 20 μηι apart, the plateau regions also including raised surfaces (volumes) which emerge from or are projected into the plateau having a spacing of about 2 μιη. One or more additional plateau regions can be used to repel additional organisms having other sizes. The additional plateau regions can be aligned (parallel) with the first plateau, or oriented at various other angles.

Although generally described for deterring bioadhesion, the invention can also be used to encourage bioadhesion, such as for bone growth. Feature dimensions of at least equal to about the size of the larger dimension of bioorganism or cells to be attached have been found to be effective for this purpose.

Although the surface is generally described herein as being an entirely polymeric, the coating can include non-polymeric elements that contribute to the viscoelastic and topographical properties. A "feature" as used herein is defined a volume (L, W and H) that projects out the base plane of the base material or an indented volume (L, W and H) which projects into the base material. The claimed architecture is not limited to any specific length. For example, two ridges of an infinite length parallel to one another would define a channel in between. In contrast, by reducing the overall lengths of the ridges one can form individual pillars. Although the surface is generally described as a coating, which is generally a different material as compared to the base article, as noted above, the invention includes embodiments where the coating and base layer are formed from the same material, such as provided by a monolithic design, which can be obtainable by micromolding.

In the case of a surface coating, the coating can comprise a non-electrically conductive material, defined as having an electrical conductivity of less than l x lO ^"6 S/cm at room temperature. The coating layer can comprise elastomers, rubbers, polyurethanes and polysulfones. The elastic modulus of the coating layer can be between 10 kPa and 10 MPa. In the case of 10 to 100 kPa materials, the coating can comprise hydrogels such as polyacrylic acid and thermo sensitive hydrogels such as poly isopropylacrylamide. The coating layer can be various thicknesses, such as 1 μιη to 10 mm, preferably being between 100 μι ίο 1 mm.

Each of the features have at least one microscale dimension. In some embodiments, the top surface of the features are generally substantially planar. Although feature spacing has been found to be the most important design parameter, feature dimensions can also be significant. In a preferred embodiment of the invention, each of the features includes at least one neighboring feature having a "substantially different geometry". "Substantially different geometry" refers to at least one dimension being at least 10%, more preferably 50% and most preferably at least 100% larger than the smaller comparative dimension. The feature length or width is generally used to provide the substantial difference.

The feature spacing in a given pattern should generally be consistent. Studies by the present Inventors have indicated that small variations in micrometer scale spacing of the ribs that compose the surface features have demonstrated that less than 1 μιη changes (10% or less than the nominal spacing) can significantly degrade coating performance. For example, the consistency of a 2 μιη nominal spacing should be within ±0.2 μιη for best retardation of Ulva settlement.

The composition of the patterned coating layer may also provide surface elastic properties, which also can provide some bioadhesion control. In a preferred embodiment when bioadhesion is desired to be minimized, the coated surface distributes stress to several surrounding features when stress is applied to one of the features by an organism to be repelled from the surface.

The roughness factor (R) is a measure of surface roughness. R is defined herein as the ratio of actual surface area (Ra _Ct ) to the geometric surface area (R _geo ); R=Ract/R _g eo). An example is provided for a 1 cm ² piece of material. If the sample is completely flat, the actual surface area and geometric surface area would both be 1 cm ² . However if the flat surface was roughened by patterning, such as using photolithography and selective etching, the resulting actual surface area becomes much greater that the original geometric surface area due to the additional surface area provided by the sidewalls of the features generated. For example, if by roughening the exposed surface area becomes twice the surface area of the original flat surface, the R value would thus be 2.

The typography generally provides a roughness factor (R) of at least 2. It is believed that the effectiveness of a patterned coating according to the invention will improve with increasing pattern roughness above an R value of about 2, and then likely level off upon reaching some higher value of R. In a preferred embodiment, the roughness factor (R) is at least 4, such as 5, 6, 7, 8, 9, 10 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25 or 30. Assuming deeper and more closely spaced features can be provided, R values can be higher than 30.

As noted above, the patterned surface layer may also provide surface elastic properties which can influence the degree of bioadhesion directly, an in some cases, also modulate surface chemistry of the surface layer. It is believed that a low elastic modulus of the patterned coating layer tends to retard bioadhesion, while a high elastic modulus tends to promote bioadhesion. A low elastic modulus is generally from about 10 kPa and 10 MPa, while a high elastic modulus is generally at least 1 GPa.

The patterned surface can be formed or applied using a number of techniques, which generally depend on the area to be covered. For small area polymer layer applications, such as on the order of square millimeters, or less, techniques such as conventional photolithography, wet and dry etching, and ink-jet printing can be used to form a desired polymer pattern. When larger area layers are required, such as on the order of square centimeters, or more, spray, dipcoat, hand paint or a variant of the well known "applique" method be used. These larger area techniques would effectively join a plurality of smaller regions configured as described above to provide a polymer pattern over a large area region, such as the region near and beneath the waterline of a ship.

A paper by Xia et al entitled "Soft Lithography" discloses a variety of techniques that may be suitable for forming comparatively large area surfaces according to the invention. Xia et al. is incorporated by reference into the present application. These techniques include microcontact printing, replica molding, microtransfer molding, micromolding in capillaries, and solvent-assisted micromolding, which can all generally be used to apply or form topographies according to the invention to surfaces. This surface topography according to the invention can thus be applied to devices as either a printed patterned, adhesive coating containing the topography, or applied directly to the surface of the device through micromolding.

Another tool that can be used is the Anvik HexScan® 1010 SDE micro lithography system which is a commercially available system manufactured by Anvik Corporation, Hawthorne, N.Y. 10532. Such a tool could be used to produce surface topographies according to the invention over a large area very quickly. It has a 1 micron resolution which can produce our smallest pattern at a speed of approximately 90 panels (10" by 14") per hour.

In one embodiment, the hierarchical surface architecture comprises a first plurality of spaced features upon which are disposed a second plurality of spaced features. A hierarchical surface architecture comprising a first plurality of spaced features upon which are disposed a second plurality of spaced features. The spaced features of the first plurality of spaced features arranged in a plurality of groupings. The groupings of the first plurality of spaced features comprise repeat units. The spaced features within a grouping being spaced apart at an average distance of about 1 nanometer to about 500 micrometers. Each feature may have a surface that is substantially parallel to a surface on a neighboring feature. In one embodiment, each feature may have a surface that is not parallel to a surface on a neighboring feature. Each feature is separated from the neighboring features. The groupings of features are arranged with respect to one another so as to define a tortuous pathway; the plurality of spaced features provide the article with an engineered roughness index of about 5 to about 30.

The second plurality of spaced features are disposed upon the first plurality of spaced features. The spaced features of the second plurality of spaced features are arranged in a plurality of groupings. The groupings of features of the second plurality of spaced features comprise repeat units. The spaced features within a grouping being spaced apart at an average distance of about 1 nanometer to about 50 micrometers; specifically at an average distance of about 10 nanometer to about 30 micrometers; and more specifically at an average distance of about 20 nanometer to about 10 micrometers. The groupings of features in the second plurality of spaced features are arranged with respect to one another so as to define a tortuous pathway; the plurality of spaced features providing the article with an engineered roughness index of about 5 to about 30. The second plurality of spaced features may have disposed thereon a third plurality of spaced features, and so on.

In one embodiment, the raised features can comprise the aforementioned hydrogels. The hydrogels can be disposed upon selected regions of the base surface by methods involving micropatterning or nanopatterning, where portions of the base surface are covered by a mask, while the exposed surfaces are coated with the hydrogel. The hydrogel may then be reacted to the base surface.

In another embodiment, the base surface may first be treated to create certain reactive regions. A hydrogel is then disposed upon the entire surface to react with the reactive regions. After a period of time, the unreacted hydrogel is removed to create a pattern on the base surface. As noted above, the hydrogel may be covalently bonded across the entire surface to form a biocontrolled adhesive surface.

The following examples which are not meant to be limiting demonstrate the compositions, methods and articles described herein.

EXAMPLE

Example 1 This example was conducted to demonstrate the the effects of disposing a hydrogel on a surface. Bioadsorption tests using a variety of different species were conducted to determine the effects of disposing a hydrogel on a polydimethylsiloxane surface.

PEGDMA-polyethylene glycol dimethacrylate (having number average molecular wegith <Mn> = 1 kg mol ^"1 ) - a tetrafunctional polyethylene glycol macro monomer - was purchased from Polysciences Inc. (Warrington, PA). 2-hydroxyethyl methacrylate (HEMA) 98% stabilized was purchased from Acros Organics (Geel, Belgium). Glycidyl methacrylate (GMA) >97%, ascorbic acid (AA) 99+%, and ammonium persulfate were purchased from Sigma- Aldrich (Milwaukee, WI). Methacryloxypropyltriethoxysilane was purchased from Gelest Inc. (Morrisville, PA). Ultra-pure water was produced by a Barnstead Nanopure Ultra Pure Water System (Waltham, MA). The base material for standards was a platinum-catalyzed PDMSe (Silastic® T2; Dow Corning Corporation).

PEGDMA, PEGDMA-co-GMA, and PEGDMA-co-HEMA hydrogels were produced using a thermally activated polymerization. Aqueous solutions were prepared by combining 25 wt% PEGDMA (<Mn> = 1 kg mol-1) used as is, 0.5 wt % ammonium persulfate and ascorbic acid as chemical initiators, and ultra-pure water to balance. To create a functionalized PEGDMA hydrogel 5 wt% of GMA or HEMA was added to the aqueous solution.

The hydrogels were either produced as free standing films or coatings attached to 76 x 22 mm silanated microscope glass slides. Glass slides were pretreated with 0.5%> methacryloxypropyltriethoxysilane (MPS) in a 95% ethanol/water solution for 10 min, rinsed thoroughly with 95% ethanol, and dried at 120°C for 15 min. All prepolymer compounds were combined in a glass beaker and stirred until a solution was achieved, i.e., the PEGDMA was dissolved. The prepolymer solution was then poured into two centrifuge tubes and centrifuged for 10 minutes at 3300 RPM. The centrifuged prepolymer solution was pipetted into a mold. The mold contained a polydimethylsiloxane (PDMSe) gasket with an opening (2.5 cm x 7.6 cm x 2 mm) for a pre -treated glass slide. The gasket was placed on top of a glass plate (12.7 cm x 12.7 cm x 0.32 cm) and the pretreated slide was fitted into the opening in the gasket. A microtopographically modified silicon wafer was placed on top of the PDMSe spacer, with the topography facing down, to create engineered microtopographies similar to those described in the Figure 1A. Smooth samples were cast against a second glass plate. The mold was assembled by adding a second glass plate on the back of the silicon wafer and clamping with 3-2 inch binder clips. The entire assembly was heated to 45°C for 45 minutes. Hydrogel-coated slides were removed from the assembly by peeling. Two topographies, continuous channels 2.6 μιη tall, 2 μιη wide and spaced by 2 μιη (+2.6CH2x2) and the Sharklet AF™ pattern (similar to those shown in the Figure 1A) 2.8 um tall, 2 μιη wide and spaced by 2 μιη (+2.8SK2x2) were created with this process.

To create PDMSe smooth standards and topographically modified surfaces the elastomer was prepared by mixing 10 parts by weight of resin and 1 part by weight curing agent. The mixture was stirred by hand for 5 minutes and degassed under vacuum (28-30 in Hg) for 30 minutes to remove bubbles. An allyltrimethoxysilane-coupling agent was applied to clean glass microscope slides (0.5 wt% in 95% ethanol/water solution) and heated for 10 minutes at 120 °C. The Silastic® T2 was then placed in contact with the treated slides in a mold consisting of two glass plates and aluminum spacers. The elastomer was polymerized at ambient for 24 hours. Topographically modified PDMSe samples were prepared in a two-step casting process previously described (Carman, et al. 2006).

The samples were exposed to a number of different biological attachment assays.

Ulva linza.

A total of four Ulva linza attachment assays were performed. Six replicates of two topographies, +2.6CH2x2 and +2.8SK2x2, and smooth surfaces created in PEGDMA-co-

HEMA and PDMSe were attached to glass slides and provided for analysis in the fourth assay. Zoospores were obtained from fertile plants of Ulva linza collected from Llantwit

Major (Wales) and prepared for attachment assays as described previously (Callow, et al.

1997). Briefly, each sample was immersed in 10 mL of a spore suspension containing 1.5 x 106 spores mL ^"1 and incubated in the dark for 45 minutes.

Attached spores on hydrogel and PDMSe slides were counted using a Zeiss epifluorescence microscope with a lOx objective while the samples were still wet. Thirty counts were taken from each of the three replicates.

Navicula incerta

Cells of Navicula incerta were cultured in F/2 medium contained in 250 mL conical flasks until cells reached the logarithmic growth phase, approximately 3 days. Cells were washed 3 times in fresh medium before harvesting and diluting to give a suspension with a chlorophyll a content of approximately 0.25 μg mL ^"1 (Holland, et al. 2004). Six replicates of each hydrogel composition and PDMSe attached to glass slides were placed in Quadriperm dishes to which 10 mL of the diatom suspension were added. Cells were allowed to attach at ambient (~20°C) on laboratory benches for 2 hours. Samples were exposed to a submerged wash in seawater to remove cells which had not attached (the underwater immersion process avoided passing the samples through the air- water interface).

Three replicates were counted wet using an image analysis system attached to a fluorescence microscope. Counts were made for 30 fields of view (0.064 mm ² ) on each sample. The remaining three replicate samples were exposed to a shear stress of 45 Pa in a water channel (Schultz et al. 2000). The number of cells remaining attached was counted using the image analysis method described above.

Cobetia marina (C. marina)

Cultures of Cobetia marina (ATCC 25374) (Baumann et al. 1983) were grown in marine broth contained in 100 mL conical flasks, at 18°C on an orbital shaker at 60 rpm overnight. Cells were harvested by centrifugation (8000 rpm for 1 min) and washed 2 times in sterile (0.22 μιη filtered) Tropic MarinTM ASW to remove any residual marine broth. The cells were resuspended in sterile ASW and briefly sonicated to aid dispersion. The suspension was diluted to an absorbance of 0.3 at 600 nm. Six replicates of each hydrogel composition and PDMSe attached to glass slides were placed in Quadriperm dishes to which 10 mL of the suspended bacteria were added. The dishes were incubated at ambient (~20°C) on the laboratory bench for 2 hours. After incubation, the slides were washed gently in seawater to remove unattached bacteria.

Three replicates were stained with crystal violet (0.01% in seawater) and counted under a 20x objective while still wet. Counts were made for 30 fields of view (2500 μιη ² ) on each sample. The remaining three replicates with attached bacteria were exposed to a shear stress of 50 Pa in a water channel. The number of cells remaining attached was counted as described above.

The cell density per mm ² was calculated for each count (n = 90). The mean cell densities were compared using one-way analysis of variance (ANOVA) and Tukey's test for multiple comparisons.

Results of 3 separate assays showed that PEGDMA, PEGDMA-co-GMA and PEGDMA-co-HEMA consistently reduced the attachment of spores of Ulva linza compared to smooth PDMSe. The total average percent reduction for PEGDMA versus PDMSe is 55%, PEGDMA-co-GMA versus PDMSe is 87% and for PEGDMA-co-HEMA versus PDMSe it is 85%>. The results are shown in the Figure 4.

Diatom cells, unlike spores of Ulva linza, are not motile in the water column. The cells come into contact with a surface by gravity and water currents so at the end of a 2 hour incubation period, approximately the same number of diatoms will be in contact with all test surfaces. Differences in the density of attached cells of Navicula were quantified following a gentle underwater washing, which washed away cells that were not attached to the surface. The initial attachment density was lowest on PEGDMA-co-GMA which was significantly lower than initial attachment densities on PEGDMA, PDMSe and PEGDMA- co-HEMA (a = 0.05, p<0). The results are shown in the Figure 5. Initial attachment densities on PDMSe and PEGDMA-co-HEMA were not statistically different (a = 0.05). Exposure to a shear stress of 45 Pa in the water channel caused removal of 77% or more of diatom cells to be removed from all hydrogel surfaces (Figure 5). No cells were removed from the smooth PDMSe surface. The total percent reduction after removal for PEGDMA- co-GMA versus PDMSe was 95%.

The initial attachment density of cells of C. marina was reduced on the hydrogels compared to a smooth PDMSe standard (up to 62%) with lowest densities on PEGDMA- co-GMA and PEGDMA-co-HEMA. The results are shown in the Figure 6. Initial attachment densities of C. marina were not statistically different among the three hydrogel compositions but all hydrogels significantly reduced attachment versus PDMSe (a = 0.05, p < 0). Exposure to a 50 Pa shear stress in a water channel caused 44% and 45% removal from PEGDMA-co-GMA and PEGDMA-co-HEMA, respectively (Figure 6). There was no statistically significant removal of C. marina from PDMSe or PEGDMA. The cell density on PEGDMA-co-HEMA was 77% less, after exposure to a 50 Pa shear stress, than that on PDMSe.

Based on the results from all biological attachment assays, PEGDMA-co-HEMA was selected as the substrate for further testing. The channels (+2.6CH2x2) and Sharklet AF™ (+2.8SK2x2) topographies were replicated in PEGDMA-co-HEMA and PDMSe and tested with the standard Ulva zoospore attachment assay. The initial spore attachment density was reduced on both PDMSe +2.6CH2x2 and +2.8SK2x2 versus smooth. Smooth PEGDMA-co-HEMA reduced spore attachment by an average of 75% compared to smooth PDMSe. Topographies produced in PEGDMA-co-HEMA reduced Ulva attachment by an average of 82% for +2.6CH2x2 and 93% for +2.8SK2x2 compared to smooth PDMSe.

Example 2

This example was conducted to demonstrate increasing orientation of human cells in channels in the patterned surfaces. The aim of this work is to create a cell culture substrate for small-diameter vascular graft applications that has the potential to re- endothelialize in vivo and/or control smooth muscle cells (SMC) phenotype to reduce neointimal hyperplasia and thrombosis. It was hypothesized that a combination of surface chemistry and topography on a graft surface would capture circulating endothiliel progenitor cells EPCs in the peripheral blood and promote their differentiation into endothelial cells (ECs) to create a continuous tissue layer within the lumen of the graft.

PEGDMA (<Mn> = 1 kg/mo 1) was purchased from Polysciences Inc. (Warrington, PA). 2-hydroxyethyl methacrylate 98% stabilized was purchased from Acros Organics (Geel, Belgium). Glycidyl methacrylate >97%, ascorbic acid (AA) 99+%, ammonium persulfate, and albumin from bovine serum were purchased from Sigma- Aldrich (Milwaukee, WI). Methacryloxypropyltriethoxysilane (MPS) was purchased from Gelest Inc. (Morrisville, PA). Ultrapure water was produced by a Barnstead Nanopure UltraPure Water System (Waltham, MA). The base material for standards was a platinum-catalyzed PDMSe (Silastic® T2; Dow Corning Corporation).

PEGDMA, PEGDMA-co-GMA, and PEGDMA-co-HEMA hydrogels were produced using a thermally activated polymerization process. Aqueous solutions were prepared by combining 25wt% PEGDMA (<Mn> = 1 kg mol-1), 0.5wt % ammonium persulfate and ascorbic acid as chemical initiators, and ultrapure water to balance. To create a functionalized PEGDMA hydrogel 5 wt% of GMA or HEMA was added to the aqueous solution.

The hydrogels were either produced as free standing films or attached to 76 x 22 mm microscope glass slides during the curing process by way of a silane coupling agent as described in the Example 1. To produce free standing films all components of the prepolymer solution were combined in a glass beaker and stirred until the PEGDMA was dissolved. The prepolymer solution was then poured into two centrifuge tubes and centrifuged for 10 minutes at 3300 RPM. A pipette was used to fill a mold made of two glass plates and a PDMSe gasket with the centrifuged prepolymer solution. A topographically modified silicon wafer was added to the mold to create patterned samples. The mold was then placed in an oven to cure for 45 minutes at 45°C. Hydrogels were stored in deionized water after curing.

Smooth standards and topographically modified PDMSe standards were also produced. The elastomer was prepared by mixing 10 parts by weight of resin and 1 part by weight curing agent. The mixture was stirred by hand for 5 minutes and degassed under vacuum (28-30 in Hg) for 30 minutes to remove bubbles. An allyltrimethoxysilane- coupling agent was applied to clean glass microscope slides (0.5 wt% in 95% ethanol/water solution) and polymerized for 10 minutes at 120°C. The Silastic® T2 was then placed in contact with the treated slides in a mold consisting of two glass plates and aluminum spacers. The elastomer was polymerized at ambient for 24 hours. Topographically modified PDMSe samples were prepared in a two step casting process previously described (Carman, et al. 2006).

Hydrogels were attached to glass slides by way of a silane coupling agent. To prepare a solution of MPS, 30 mL of 190 proof ethanol was pipetted into a polypropylene cup. While mixing with a stir bar 1 to 2 drops of glacial acetic acid was added to adjust the pH of the solution to approximately 4.5-5.5. Then 0.17 mL of MPS was added to the solution and allowed to react for 5 minutes. Glass slides were cleaned by holding with forceps and passing through a flame 4 to 5 times. Slides were allowed to cool on an aluminum tray covered with Kimwipes ^® . A plastic transfer pipette was used to cover glass slides with the MPS solution. The MPS solution was allowed to react on the glass slides for 2 to 3 minutes. The slides were then rinsed with 190 proof ethanol and placed in the oven to dry at 120°C for 10 minutes. After the slides were treated with MPS, a slide was placed into a mold consisting of two glass plates and a PDMSe gasket. A topographically modified silicon wafer was placed in this mold to make slides with topographically modified hydrogel. Hydrogel solution was pipetted into the mold and the filled mold was placed into an oven at 45°C for 45 minutes to cure and attach to the glass slide.

Fibronectin was grafted to hydrogel surfaces by reacting the epoxide ring of the GMA with amine nucleophiles on the protein in an alkaline buffer (Volcker et al, 2001). To make the buffer a 0.2 M solution of anhydrous sodium carbonate was prepared by combining 2.12 g of sodium carbonate with 100 mL of deionized water. Then a 0.2 M solution of sodium bicarbonate was prepared by combining 1.68 g of sodium bicarbonate with 100 mL of deionized water. A carbonate-bicarbonate buffer was made by combining 4 mL of the sodium carbonate solution with 6 mL of sodium bicarbonate solution and bringing the volume up to 200 mL with deionized water. The pH of this solution was measured to be 9.8 at 24°C. This buffer was used to make a solution of 50 μg mL ^"1 Fn in buffer. The surface to be grafted was then covered with the Fn buffer solution and incubated at 37°C with 5% C0 ₂ for one hour. After incubation the Fn solution was removed from the surface and the surface was rinsed three times with phosphate buffered saline (PBS). Free-standing films of PEGDMA, PEGDMA-co-GMA and PDMSe were produced using the method described in the polymer synthesis section. Smooth and topographically modified surfaces were tested. The topographies tested included the n-series of Sharklet topographies and channels with a height of 1 μιη and width and spacing of 2 μιη. The n- series is a group of topographies designed to have an increasing number of unique features (n) arranged in the Sharklet pattern.

Porcine vascular endothelial cells (PVECs) from a primary culture provided by Dr. Edward Block's laboratory were seeded onto smooth and +1CH2X2 and +lSK2x2_4 topographies created in PEGDMA-graft-Fn and PEGDMA-co-GMA-graft-Fn at 5x104 cells mL-1. The PVECs were cultured on the hydrogels for 24 hour. Cells were fixed with 10% formalin for 5 minutes and stained with crystal violet. Three transmitted light micrographs were taken per sample at a magnification of 400x using a Zeiss Axioplan 2 Microscope with a digital camera. The number of cells per field of view was counted and the average number of cells mm-2 was reported as an indication of PVEC attachment to each hydrogel surface.

Two types of human cells were provided by Dr. Mark Segal's laboratory: human coronary artery endothelial cells (HCAECs) and human aortic smooth muscle cells (HASMCs). Topographies with a height of 1 μιη including the n-series and channels were cast in PDMSe and PEGDMA-co-GMA and prepared as described above. Smooth surfaces and empty tissue culture polystyrene (TCPS) wells were used as standards and controls in these assays. Each cell type was seeded on to an identical plate full of samples at 5x104 or 2.5x104 cells per well and placed into an incubator at 37°C with 5% C02. Samples were imaged after 24 hour and 7 days using an inverted phase contrast microscope. Cell morphology was observed as an indication of cell attachment and response to the surfaces. Three phase contrast images were taken at lOx magnification for each combination of chemistry and topography.

Phase contrast images were analyzed with ImageJ software. Images of topographically modified surfaces were first rotated until the channels between features were oriented horizontally. The boundaries of HCAECs and HCASMCs were outlined using the freeform select tool and added to the region of interest manager. The projected area (S) and perimeter (L) of each cell were measured. ImageJ was also used to fit an ellipse to each cell and the angle between the major axis of each fitted ellipse and the direction of the channels in each topography was measured. Cell morphology was quantified by calculating the cell shape index (CSI) (Sarkar et al. 2006a, Cao, et al. 2010). Elongated cells will have a CSI approaching 0 while cells with a circular shape will have a CSI closer to 1. Orientation was quantified by tallying the number of cells within ranges of 10 degrees and plotting the percentage of cells in each range (Sarkar, et al. 2006a). The mean angle of cell orientation relative to the channels on each topography was calculated using circular statistical methods to calculate the rectangular coordinates of the mean angle (Zar 1984).

Since the dispersion of the data was very small compared with the period of 360°, linear statistics were used to calculate 95% confidence limits and run an ANOVA with Tukey-Kramer Test for multiple comparisons (Batschelet 1981). A Kolmogorov-Smirnov test of each group showed that the mean angle data was not normally distributed. Therefore, the data was transformed by taking the square root. The 95% confidence limits and ANOVA were performed on the transformed data. The mean angle for every topography was plotted in bar charts with error bars representing 95% confidence limits and horizontal bars represent results of the Tukey-Kramer Test (a=0.05).

Cell morphology was quantified by calculating the CSI index for each cell. The

CSI ranges from 0 (elongated, linear cells) to 1 (circular shaped cells). After 24 hours HCAECs showed no statistically significant elongation compared to a smooth PDMSe standard and a TCPS control. The average CSI for HCASMCs after 24 hours was lowest on +lSK2x2_n4, however this value was only statistically different from +lSK2x2_n2 and the TCPS control (Tukey test <x=0.05).

The average CSI for HCAECs cultured for 7 days was lowest on +1CH2X2, which was not statistically different from +lSK2x2_n5, +lSK2x2_n4 and +lSK2x2_nl (Figure 6-20). Elongation was highest on +lSK2x2_n3 and +lSK2x2_n4 for HCASMCs after 7 d. The average CSI for these surfaces was not statistically different from the TCPS control (Figure 6-20) (Tukey test a=0.05). All topographies increased HCASMC elongation after 7 d compared to smooth PDMSe (Tukey test a=0.05).

Both HCAECs and HCASMCs showed increasing orientation along the direction of the channels in the topographies after 24 hours with increasing number of features (n) when cultured on the n-series Sharklet AF™ topographies. The highest degree of orientation was observed on the +1CH2X2 topography for HCAECs cultured for 24 hours. Cells cultured on the smooth PDMSe standard and TCPS control did not show preferential orientation, i.e., the mean angles on these topographies were approximately 45°. After 7 days the highest degree of orientation was observed on +lSK2x2_n5 for HCAECs (See Figure 7). More HCAECs were orientated along the direction of the channels in each topography after 7 days than after 24 hours. The mean angles were lower on topographically modified surfaces after 7 days. The highest degree of orientation was observed on +lSK2x2_n4 for HCASMCs after 24 hours and 7 days (See Figure 8).

While the invention has been described in detail in connection with a number of embodiments, the invention is not limited to such disclosed embodiments. Rather, the invention can be modified to incorporate any number of variations, alterations, substitutions or equivalent arrangements not heretofore described, but which are commensurate with the scope of the invention. Additionally, while various embodiments of the invention have been described, it is to be understood that aspects of the invention may include only some of the described embodiments. Accordingly, the invention is not to be seen as limited by the foregoing description, but is only limited by the scope of the appended claims.