WO2018080308A2

WO2018080308A2 - Surface topographies for stimulating endothelialization

Info

Publication number: WO2018080308A2
Application number: PCT/NL2017/050701
Authority: WO
Inventors: Berendien Jacoba Papenburg; Jan De Boer; Godefridus Francicus Bernardus HULSHOF
Original assignee: Materiomics B.V.
Priority date: 2016-10-28
Filing date: 2017-10-30
Publication date: 2018-05-03
Also published as: WO2018080308A3; WO2018080308A8

Abstract

The invention pertains to surface topographies which can be used to modulate the morphology, proliferation, biochemical functioning, differentiation, attachment, migration, signaling, and/or cell death of an endothelial cell population by physical stimulation. Such topographies can be applied in vitro and in vivo to modulate cell behavior. Specific examples include vascular or lymphatic implants provided with a topography of the invention which increase endothelialization.

Description

Title: Surface topographies for stimulating endothelialization Introduction

It is known that cells can contact and interact with a surface. In nature, the direct physical interaction between cells and their environment is part of their normal physiological behavior. When cells are cultured out of their natural environment, for instance in a cell culture dish, or when cells are exposed to non- natural environments, such as is the case when exposed to medical implants such as hip implants or pacemakers, their physiology is typically not the same as in their original environment. Many efforts are put into optimizing these cellular responses in vitro and on implant surfaces.

In cell cultures, for example, cells can be placed on a surface while in a suitable culture medium, the goal being to have these cells to proliferate as natural as possible, and/or express specific biological properties such as differentiation, and/or responding to a specific chemical, physical or electrical stimulus. A conventional strategy to change cell behavior is to add a variety of hormones, chemical compounds, growth factors, enzymes, salts and the like, which may have the effect of an altered cell response, such as a change in proliferation or an induced differentiation. However, although such changes can be achieved on a surface, altered cell behavior in these conventional cultures is generally chemically induced, through interaction of the hormones, chemical compounds, growth factors, enzymes, salts and the like with the cell surface and/or cell interior. In such conventional cultures as well as on medical implants, potential micropatterns present on the surface generally are not taken into account.

It has recently been found that physical interaction affects cell behavior. For example, it has been shown that titanium-based implants featuring surface micro-roughness caused by chemical etching and/or mechanical blasting improves the mechanical strength of newly-formed tissues bridging the implant to the bone/dental tissue [Buser D et al. Int J Oral Maxillofac Implants 1998; 13(5):611- 619., Buser D et al. J Biomed Mater Res 1999;45(2):75-83.] .

The impact of topographical cues at cell level has been observed in different cell types. For example, it is shown that hepatocyte cell attachment, morphology and function were remarkably improved when cultured on surfaces featuring nano-topographies as compared to smooth substrates [You J et al. ACS Appl. Mater. Interfaces 2015; 5: 12299- 12308.].

The effect of surface topographies on endothelial cells has been also investigated previously. These studies show that the topographical cues of the substrate affects endothelial cell shape, cell proliferation, cell migration and cell functionality [US 20140314723A1., US 20140178920A1., Teo B et al. Acta

Biomaterialia 2012; 8:2941-2952., Lu J et al. TISSUE ENGINEERING: Part A 2012; 18: 1389-1396., Ding Y ACS Appl. Mater. Interfaces 2014; 6: 12062-12070., Sprague E et al. Circ Cardiovasc Interv. 2012 Aug l;5(4):499-507., Palmaz J et al. J Vase Interv Radiol. 1999 Apr; 10(4):439-44., Shen Y et al. Acta Biomaterialia 2009; 5: 3593-3604., Loya M et al. Acta Biomaterialia 2010; 6: 4589-4595.]

Endothelial cells make up the inner lining of blood vessels and lumen forming the interface between the circulating blood or lymph and the rest of the vessel wall. Endothelial cells are involved in many vascular functions, including the prevention of blood clotting at the vessel wall. Proper endothelialization, i.e. formation of a uniform endothelial layer, is highly important in neovascularization (regeneration of blood vessels) and on the surface of implanted medical devices that are in contact with circulating blood or lymph. For example, accelerated formation of the endothelial layer on vascular medical devices such as stents, grafts and stent grafts is highly beneficial for proper functionality and performance of these medical device. Cell migration as well as cell attachment and proliferation are the major processes that drive endothelialization of a surface, either on their own or in combination.

Description of Figures

Figure 1 a, b: Schematic top view of two adjacent protrusions comprising (a) one and (b) two protrusion elements. (Imaginary) sample lines indicate the method used for determining the average distance between the two adjacent protrusions.

Figure 1 c, d: 3D schematic top view of 2x2 pattern of protrusions, containing (c) one and (d) two protrusion elements. Protrusions, valley walls, valley wall sections and valley wall punctures are indicated. Figure 2: Topographies El - E20 stimulate overall endothelialization. Images show visualization of endothelial cell migration through nano-bead uptake by the cells cultured on substrates featuring selected surface topographies, nonpatterned (NP) substrates and control substrates with basic topographies (B3, B4). Accumulation of the nano-beads in the cells, which is a measure for migration, are indicated by the white arrows.

Figure 3: Topographies El - E20 stimulate overall endothelialization. Graph shows topographies El-20 steering endothelial cell migration, attachment and proliferation. Analyzed for (a) nano-bead uptake, (b) the number of attached cells, (c) the number of EdU-positive cells and (d) the percentage of EdU-positive cells over all cells on the surface. NP control levels are indicated by the dashed line, basic topographies (B3, B4) are included for comparison.

Detailed description

It is an object of the invention to provide a surface topography for modulating endothelial cells in vitro or in vivo, as well as objects provided with such surface. Modulation is defined as altering the physiological state of the cells by altering morphology, proliferation, biochemical functioning, differentiation, attachment, migration, signaling, and/or cell death of an endothelial cell population by physical stimulation through the surface topography. Preferably, in the present context, modulation is defined as modifying morphology, proliferation, biochemical functioning, differentiation, attachment, and/or migration of endothelial cells, in vivo or in vitro. In most preferred embodiments, modulation means enhancing attachment, enhancing proliferation and increasing migration, in particular enhancing migration and/or enhancing proliferation, in vivo or in vitro. In a preferred embodiment, modulation means enhancing migration. In another preferred embodiment, modulation means enhancing proliferation. In yet another embodiment, modulation means enhancing attachment. Modulation of endothelial cells thus means the modulation of morphology, proliferation, biochemical functioning, differentiation, attachment, migration, signaling, and/or cell death of an endothelial cell population. Modulation of an endothelial cell population results in stimulated endothelialization. This also results in enhanced growth of blood and/or lymph vessels, preferably at the site of the surface topography. The invention furthermore provides methods to modulate endothelial cells, as well as an object provided with a surface topography as defined below for use in modulating endothelial cells. Also, the invention provides a surface topography as defined below for the preparation of an object for use in the treatment of various (blood or lymphatic) vessel conditions. Furthermore, the invention provides for a surface topography as defined herein, for the preparation of a medicament for the treatment of various vessel conditions.

Endothelial cells, in this context, are defined as the cells found in the inner layer of blood vessels or lymphatic vessels, and are well-known in the art. The inner layer of blood vessels or lymphatic vessels comprises endothelial cells, which in the case of blood vessels are known as vascular endothelial cells, and in the case of lymphatic vessels are known as lymphatic endothelial cells. Both types are comprised in the term "endothelial cells" as used herein. The inner layer of blood vessels and lymphatic vessels comprising endothelial cells are also known as the endothelium (or endothelium layer).

The endothelium in the blood vessels and lymphatic vessels is involved in several functions. The endothelium forms an interface between the circulating blood or lymph and the vessel wall, and acts as a selective barrier between the blood or lymph in the vessels and vessel wall. Thus, the endothelium affects among others the quantity and type of fluid passing to and from the vessel fluid to and from the other side of the vessel wall. The endothelium is also involved in hormone trafficking and background muscle activity, as well as in formation of new (blood or lymphatic) vessels (angiogenesis). In addition, a major function of endothelial cells in blood vessels is to provide a non-thrombogenic surface to avoid blood clotting.

Endothelial cells, in the present context, refers to primary endothelial cells as present in the blood or lymphatic vessels, as well as to progenitor endothelial cells, endothelial-like cells, and progenitor endothelial-like cells.

Endothelial-like cells are cells which are not, and will not become, primary endothelial cells, but which have largely similar biophysical and biochemical function. Endothelial-like cells include for example immortalized endothelial cell lines such as HMEC-1, as well as endothelial cells derived from any type of stem cell, such as from induced pluripotent stem cells (iPSCs). In preferred

embodiments, endothelial cells are primary endothelial cells or endothelial-like cells, and most preferably, endothelial cells are primary endothelial cells (herein also referred to as "primary cells").

Endothelial cells as used herein may be from human or animal origin, and are preferably mammalian cells, in particular cells from human, monkey, bovine, pig, rat or mouse. Most preferably, the cells are human endothelial cells.

Endothelialization is the formation of an endothelium on the surface of implanted medical devices. This is crucial for maintaining tissue functioning when it comes to the interactions with blood or lymph. Stimulating one or more of the parameters involved in endothelialization, including attachment, proliferation and migration of endothelial cells to or over the surface of the implant or tissue allows for (accelerated) formation of an endothelium and thus stimulates

endothelialization.

A group of surface topographies has been identified that is able to modulate endothelial cells, in particular by promoting attachment, proliferation and/or migration of endothelial cells, in vivo or in vitro. In the below, the word "cell population" is to be understood as referring to an endothelial cell population.

Similarly, the word "cell" refers to an endothelial cell as defined above.

The identified topographies allow for higher cell motility and migration capacity compared to surfaces without such topography (nonpatterned surface) or with basic (square, circular, triangular and the like) topographies. Higher motility results in increased presence of endothelial cells at the surface with topography.

Furthermore, the identified topographies stimulate cell attachment compared to nonpatterned surfaces or surfaces with basic topographies. Increased attachment results in anchoring of migrating endothelial cells to the surface topography, thus also increasing the presence of endothelial cells at the location of the surface topography.

Furthermore, the identified surface topographies do not negatively affect endothelial cell proliferation. Surface topographies of the invention either increase the cell proliferation capacity or maintain a natural proliferation capacity, in comparison to a nonpatterned surface or a surface with a basic topography.

The experiments show that surface topographies of the invention stimulate endothelialization by modulation of endothelial cells as defined above, preferably by triggering migration and/or attachment and/or proliferation of endothelial cells.

The invention pertains to a surface topography, as well as to an object comprising a surface part provided with one or more of such topographies. The surface topography is formed by the presence of regularly spaced protrusions on the surface part, and can be defined by two alternative definitions. In one definition, the surface topography can be defined by the average distance between adjacent protrusions, the top surface area of the protrusions, and the coverage of the surface part, and furthermore by the length and width of the protrusions (the "protrusion '-definition). In another definition, the topography can be defined by the valleys which are located between the protrusions (the "valley" -definition). These definitions function as two alternative numerical representations of a topography.

T\ie "protrusion" definition

The present invention provides an object, comprising a surface part provided with one or more topographies capable of modulating the morphology, proliferation, biochemical functioning, differentiation, attachment, migration, signaling, and/or cell death of a cell population by physical stimulation, and wherein the topography comprises a surface provided with a regular pattern of protrusions, which protrusions comprise one or more protrusion elements, which protrusion elements are defined as surface portions elevated above the surface having a top surface area and a circumferential side face connecting the top surface area with the surface, wherein each protrusion element has a maximum height of between 0.5 and 50 μm above the surface, and wherein

a) the average distance between adjacent protrusions is between 0 and 50 μm; b) the top surface area of the protrusion is between 1 and 6000 μm²; and c) the protrusions cover between 3 and 90 % of the surface.

Preferably, the object of the invention has a length and width

rotrusions or protrusion elements as follows:

a) if the protrusion comprises one protrusion element: the length of the protrusion, defined as the length of the longest straight- line fitting within the circumference of the top surface area parallel to the surface, is 0.01 - 100 μm;

the width of the protrusion, defined as the length of the longest straight- line fitting within the circumference of the top surface area parallel to the surface, perpendicular to the length, is 0.01 - ΙΟΟμm;

b) if the protrusion comprises more than one protrusion element: the length of each protrusion element, defined as the length of the longest straight-line fitting within the circumference of the top surface area parallel to the surface, is 0.01 - 100 μm;

The width of each protrusion element, defined as the length of the longest straight-line fitting within the circumference of the top surface area parallel to the surface perpendicular to the length, is 0.01 - 100 μm;

The average distance between two circumferences of adjacent protrusion elements of the same protrusion is 0 - 50 μm.

The topography comprises a surface provided with a regular pattern of protrusions. Protrusions are comprised of one or more protrusion elements, which elements are surface portions elevated above the surface on which the protrusions are formed, i.e. a surface surrounding the relevant protrusion or protrusion element. Each protrusion element is defined by an elevated surface portion and by a top surface area, as well as by a circumferential side face which connects the top surface area with the surface. The elevated surface portion is the total of the top surface area and the circumferential side face. One protrusion may be defined as a single protrusion element, but one protrusion may also comprise multiple protrusion elements. In one preferred embodiment, a protrusion comprises a single protrusion element. In another preferred embodiment, a protrusion comprises at least two protrusion elements.

As such, protrusions are small bumps or groups of bumps on the surface. The protrusions define between them a three-dimensional network of valleys in the surface of the object. It has been found that a regular pattern of protrusions with a specific size, shape and configuration results in a topography that is capable of modulating the cell response, most notably morphology, proliferation, biochemical functioning, differentiation, attachment, migration, signaling, and/or cell death of a cell population by physical stimulation.

The regular pattern can be defined by an imaginary grid of intersecting gridlines which can be laid over the surface, which gridlines define a pattern of unit cells, such that each unit cell comprises a maximum of one protrusion.

Because the grid of intersecting gridlines need not physically be present, but preferably is an imaginary way to define the regular pattern, the unit cells may have any shape. Preferably, a unit cell has a rectangular, especially square, trapezoid, triangular or hexagonal shape.

A regular pattern in this context means that any section of the topography which consists of m unit cells, for instance in a n X n arrangement, is repetitively present in sections adjacent to that section. Preferably, m and n are 1, but m may be 4 - 16 and n may be 2 - 4, in particular in case of unit cells comprising differently shaped or differently oriented protrusions, or in case of triangular, hexagonal or trapezoid unit cells.

A unit cell may for instance have a surface area of between 1 and 10000 μm², preferably between 1 and 2500 μm², more preferably between 25 and 2000 μm², more preferably between 100 and 1000 μm². In case of square or rectangular unit cells, the unit cell may have length X width dimensions of (1-100) μm X (1- 100) μm, preferably (1-50) μm X (1-50) μm. In highly preferred

embodiments, unit cells are square and have length X width dimensions of 5 μm X 5 μm to 45 X 45 μm, preferably 5 X 5 to 30 μm X 30 μm such as for example 5 μm X 5 μm, 10 μm X 10 μm, 15 μm X 15 μm, 20 μm X 20 μm, 25 μm X 25 μm or 28 μm X 28 μm.

The unit cells defined by the pattern of intersecting gridlines each comprise a maximum of a single protrusion, which protrusion may be comprised of multiple protrusion elements as defined above. Preferably, each unit cell comprises a protrusion of equal dimensions, i.e., each unit cell comprises protrusions identical to one another. The dimensions of a protrusion are defined as the total

characterizing features, including for instance the height of the protrusion, the number and relative position of protrusion elements inside the unit cell, the length and width of each protrusion element, the angle of the circumferential side face relative to the surface, and the shape of the top surface area of each protrusion element.

The height of a protrusion, or at least of a protrusion element, is between 0.5 and 50 μm above the surface. That is, the height of each protrusion element may be between 0.5 and 50 μm above the surface, even though in case a protrusion comprises multiple protrusion elements, the height of various protrusion elements in the protrusion may be different within the mentioned boundaries. Preferably, however, the height of each protrusion element is the same. The height is measured as a maximum height of the top surface area of a protrusion or protrusion element above the surface, especially above a surrounding surface area within a unit cell.

Further preferably, the height of a protrusion may be between 1 and 45, preferably between 2 and 40 μm, more preferably between 3 and 35 more preferably between 4 and 30 μm, even more preferably between 4 and 28 μm.

The number and relative position of protrusion elements inside a unit cell may vary from one unit cell to another as long as a regular pattern of protrusions is obtained. That is, two or more different unit cells comprising the same or different protrusions may be defined, which unit cells are placed in a regular pattern, for example alternate regularly on the surface. In case of triangular or trapezoid unit cells with protrusions of equal dimensions, this may result in a regular pattern of the same protrusions which have different orientations, for example alternatingly opposite orientation on the surface. In case of two different square unit cells each having a single, but different protrusion, an alternating pattern of said unit cells results in a regular pattern of rows of said protrusions, which rows are either oriented parallel to the edges of the unit cells, or parallel to the diagonal of the unit cell. Alternatively, square unit cells all having the same protrusion may be oriented differently on the surface, so as to obtain a surface in which the

protrusions are regularly distributed with varying orientation.

Countless ways of obtaining a regular pattern of protrusions by applying the concept of unit cells may be devised, and can be used in the present invention. Preferably however, square, rectangular and hexagonal unit cells are all oriented in the same direction, while triangular unit cells are alternatingly oriented.

Further preferably, all unit cells contain an identical protrusion. The length of each protrusion (or in case of protrusions comprising multiple protrusion elements, the length of each protrusion element), is defined as the length of the longest straight-line fitting within a circumference of the top surface area of the protrusion or protrusion element, parallel to the surface.

In case of protrusions comprising only a single protrusion element, the length of a protrusion is 0.01 - 100 μm. Preferably, the length is 0.5 - 50 μm, more preferably 1 - 45, more preferably 2 - 40 μm.

In case of protrusions comprising multiple protrusion elements, the length of a protrusion element is 0.01 - 100 μm. Preferably, the length is 0.1 - 45 μm, more preferably 0.5 - 40 μm, even more preferably 1 - 35 μm.

The width of each protrusion (or in case of protrusions comprising multiple protrusion elements, the width of each protrusion element), is defined as the length of the longest straight-line fitting within the circumference of the top surface area of the protrusion or protrusion element parallel to the surface, perpendicular to the length.

In case of protrusions comprising only a single protrusion element, the width of a protrusion is 0.01 - 100 μm. Preferably, the width is 0.5 - 50 μm, more preferably 1 - 45 μm, more preferably 2 - 40 μm.

In case of protrusions comprising multiple protrusion elements, the width of a protrusion element is 0.01 - 100 μm. Preferably, the width is 0.1 - 45 μm, more preferably 0.5 - 40 μm, even more preferably 1 - 35 μm.

Furthermore, in case of protrusions comprising multiple protrusion elements, the number and relative position of protrusion elements inside the unit cell is an important feature of the dimensions of the protrusion.

The average distance between adjacent protrusion elements of the same protrusion is determined by dividing the sides of the protrusion elements which face each other into equal line segments of about 0.4 μm, and determining the distance of each line segment of the first protrusion element to the facing line segment of the adjacent protrusion element (see figure). The average of all distances is the average distance between the protrusion elements. Of course, when a protrusion comprises more than two protrusion elements, the average distance of one protrusion element to a second protrusion element of the same protrusion may be different from the average distance of the same protrusion element to a third protrusion element of the same protrusion. In case the angle between the surface and the circumferential side face of the protrusion element is not 90°, the average distance between adjacent protrusions is determined at half the height of the protrusion. The same type of calculation is applied for determining the average distance between two facing protrusions, as has been exemplified in figure la and lb. "Facing" in this context means that the distance between adjacent protrusions is determined on the basis of line segments stretching between those protrusions, over the length of the protrusion which is the shortest.

Generally, the average distance between two protrusions is 0 - 50 μm, preferably 0.5 - 40 μm, even more preferably 1 - 30 μm, even more preferably 2 - 20 μm. The average distance between two protrusion elements in the same protrusion is similarly defined. In a much preferred embodiment, if the average distance between two adjacent protrusions in one direction is 0, then the average distance between adjacent protrusions in the direction perpendicular to that distance should be larger than 0. In an even more preferred embodiment, the average distance between adjacent protrusions in any direction is larger than 0.

Also, the surface topography can be defined by determining the shortest and the longest distance between facing protrusions. The shortest distance is defined by the shortest straight line as defined above for the calculation of average distance which can be drawn between two adjacent protrusions or protrusion elements. The shortest distance is preferably 0 - 50 μm, more preferably 0 - 40 μm, more preferably 0 - 20 μm, and more preferably 0 - 15 μm. In further preferred embodiments, the shortest distance is 1 - 50 μm, preferably 2 - 40 μm, more preferably 3 - 30 μm, even more preferably 4 -20 μm.

The longest distance is defined as the longest straight line as defined above for the calculation of average distance which can be drawn between two adjacent protrusions or protrusion elements. The longest distance can be 0 - 50 μm, preferably 0.5 - 45 μm, more preferably 1 - 40 μm and more preferably 5 - 35 μm.

The circumferential side face of the protrusion elements may have any angle between 0 and 180° with the surface at the position where the side face intersects with the surface. An angle of 90° is defined as normal to the surface, and an angle between 0° and 90° is defined as the situation where the top surface area is larger than the elevated portion of the surface, so that the top surface area at least partially covers the surface. An angle between 90° and 180° is defined as the situation wherein the top surface area is smaller than the elevated portion of the surface. The elevated portion of the surface is defined as the circumference of the protrusion at the point where it intersects the surface. As such, angles between 0° and 90° lead to protrusion elements which have a top surface area which hangs over the surface, whereas angles between 90° and 180° lead to protrusion elements which rise gradually toward the top surface area.

Preferably, the circumferential side face of at least a portion of the protrusion or protrusion elements, preferably the whole protrusion or protrusion element, has an angle between 45° and 135° with the surface, more preferably between 60° and 120°, even more preferably between 75° and 115°, and even more preferably between 80° and 100°. Most preferably, the circumferential side face of at least a portion of the protrusion or protrusion elements, preferably the whole protrusion or protrusion element, extends substantially normal to the surface at the position where the side face intersects with the surface, such as at an angle of 88 - 92°. In a further preferred embodiment, all protrusion elements have about the same angle with the surface at the position where the side face intersects with the surface.

The shape of the top surface area of each protrusion element is also an important feature of the dimensions of the protrusion, and may also be referred to as the shape of the protrusion (or protrusion element). This is because this shape defines the shape of the valley walls where cells of adjacent tissue may grow to provide fixation. The shape of the circumference of the top surface area is determined parallel to the surface. As such, it is a top-view of the protrusion element, which may have any geometrical form. In some embodiments, the shape may comprise a circular, oval, triangular, square, rectangular, trapezoid, pentagonal, hexagonal, heptagonal or octagonal shape. Such generally known shapes will be referred to as basic shapes herein. A basic shape is symmetric, i.e. has at least one symmetry axis. In much preferred embodiments, the shape may comprise a combination of basic shapes.

In some embodiments, the shape of the top surface area of each protrusion /protrusion element may not be a single geometric shape, such as a square, triangle, circle, octagonal, 5-pointed star, hexagonal, 3-pointed star, half moon, or a circle with a corner taken out ("pacman"). In some embodiments the shape may not be circular, oval, or a shape such as a polygon, triangle, rectangle, square, hexagon, star, parallelogram.

In some embodiments, protrusions may have a shape as shown in table 1. In other embodiments, the topography does not comprise a protrusion shaped as shown in Table 1. In one embodiment, a topography does not have shape 1 from table 1. In another embodiment, a topography does not have shape 2 from table 1. In another embodiment, a topography does not have shape 3 from table 1. In yet other embodiments, a topography does not have, independently, shape 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, or 32 from table 1. In some embodiments, the topography does not comprise a selection of the topographies in table 1, and in further embodiments, the

topography does not comprise all of the shapes in table 1.

In a preferred embodiment, the top surface area is a surface positioned substantially parallel to the surface, although the top surface area may be slightly concave or convex, such as for instance slightly dome-shaped.

Particularly preferred protrusions (or protrusion elements) comprise overlapping or adjacent combinations of basic shapes, preferably interconnected by one or more bridging or overlapping portions, so as to obtain a complex shape. It has been found that the effect of modulating the cell response, most notably morphology, proliferation, biochemical functioning, differentiation, attachment, migration, signaling, and/or cell death of a cell population by physical stimulation by protrusions with specifically defined complex shapes is influenced not only by the dimensions of the protrusion, but also by the shape of the protrusion (or protrusion elements) alone. For protrusions of similar height, weight, length and width, but of different shape, there is a distinct effect on the cell response of the physically stimulating surface part. The individual effect of the shape of

protrusions on modulating morphology, proliferation, biochemical functioning, differentiation, attachment, and/or migration of endothelial cells of a cell population by physical stimulation will be further elaborated upon in the examples.

In case the protrusion (or protrusion element) comprises a combination of basic shapes, a complex shape can be obtained. Complex shapes may comprise overlapping or adjacent circular, oval, triangular, square, rectangular, trapezoid, pentagonal, hexagonal, heptagonal or octagonal shapes. In case of adjacent basic shapes, the adjacent basic shapes are preferably connected by an overlapping (bridging) portion, so that the top-view of the basic shape has a larger surface area than the surface area of the basic shapes alone. The increased surface area relative to the separate shapes is preferably located between the basic shapes, effectively increasing the width of any point of contact between the basic shapes.

Further preferably, the circumferential side face of a protrusion (or protrusion element) is a continuous side face, which may be a connected series of straight line portions, connected by angles. Alternatively, the straight line segments and/or the angles can be smoothly curved, so as to obtain a smoothly curved circumferential side face.

A smoothly curved circumferential side face in this context means that the circumferential side face only has rounded corners. Corners may stem from their presence in a shape, such as the corners in squares, triangles and hexagons, but such corners may also stem from the contact point between two adjacent shapes in a protrusion element, or from the presence of an overlapping (or bridging) portion between two basic shapes in a protrusion element. That is, corners in this sense refers to corners when the protrusion is seen with a top -view. When all corners in the circumferential side face of a protrusion element are rounded, a smoothly curved circumferential side face is obtained.

In preferred embodiments, a protrusion has a complex shape. A complex shape is a combination of basic shapes, in which the basic shapes are present as single protrusion elements, or as overlapping or adjacent basic shapes. A complex shape is generally non-symmetric, i.e. has no symmetry axis. A complex shape is further defined by the amount of corners in the circumferential side face. A corner, in this respect, may be a straight or rounded corner, and can be defined as any change in direction in the circumferential side face (top-view). Corners which take the shape of a line with a length of 5 % of the length of the circumferential side face will be dubbed a "bent". Straight lines in the circumferential side face will be called "straights".

A corner can be narrow or wide. Narrow corners are corners with an angle of less than 90 °. That is, narrow corners result in a "spike" in the shape, which spike is either directed inward or outward, relative to the protrusion. Wide corners are corners in which protrusion's circumferential side face displays an angle of more than 90 °. Straight corners are corners with an angle of 90 °.

Complex shapes are defined as shapes having at least one wide corner, wherein preferably, the quantity of narrow corners is not the same as the quantity of wide corners. Alternatively, complex shapes comprise at least one bent

(preferably at least two or at least three), and at least one corner (preferably at least two, more preferably at least three corners). In further alternative preferred embodiments, complex shapes comprise both at least one bent and at least one straight. In further preferred embodiments, a complex shape is a shape comprising more than two straights, preferably more than three, more preferably more than four, in which at least 75 % (preferably at least 85 %, more preferably at least 95 %) of all straights are of different length. In other preferred embodiments, complex shapes comprise at least 3 bents.

An adjacent (or facing) protrusion to a protrusion in a particular unit cell is a protrusion which is present in a unit cell which shares a side with that particular unit cell. Adjacent unit cells are similarly defined. Unit cells which only share a corner with the particular unit cell are not adjacent. Given a regular pattern of protrusions, one protrusion is for example adjacent to three protrusions in case of triangular unit cells, to four protrusions in case of trapezoid, square or rectangular unit cells, and to six protrusions in case of hexagonal unit cells.

The top surface area of the protrusion is defined as the surface area of the protrusion at its most elevated circumference. It follows from the above that if a protrusion comprises multiple protrusion elements, that the top surface area of the protrusion is the sum of all surface areas of all protrusion elements of the protrusion, each determined at the most elevated circumference of the protrusion element. The top surface area of a protrusion may be determined by for instance counting the number of pixels (as 0.4 μm X 0.4 μm elements) which make up the top surface area of the protrusion (or protrusion element).

The top surface area of the protrusion is between 1 and 6000 μm², preferably 10 and 3000 μm², more preferably between 20 and 1500 μm², more preferably between 30 and 1000 μm², even more preferably between 35 and

700 μm².

The coverage of the protrusions is defined as the percentage of top surface area relative to the full area which is covered by protrusions. Thus, the coverage is the sum of all top surface area, divided by the total area of the surface topography, times 100 %. This is the same as calculating which percentage of a unit cell comprises the top surface area of the protrusion.

The protrusions cover between 3 and 90 % of the surface part, preferably between 5 and 85 %, more preferably between 10 and 80 % of the surface part, more preferably between 20 and 78 %, more preferably between 25 and 75 %. Thus, a protrusion covers between 3 and 90 % of a unit cell, preferably between 5 and 85 % of a unit cell, more preferably between 10 and 80 % of a unit cell, more preferably between 20 and 78 % of a unit cell, more preferably between 25 and 75 % of a unit cell.

The "valley" definition

As has been mentioned, the regular pattern of protrusions at the same time defines a pattern of valleys. Thus, the surface topography of the invention may alternatively be defined by a surface part for modulating morphology, proliferation, biochemical functioning, differentiation, attachment, migration, signaling, and/or cell death of a cell population by physical stimulation, comprising a top surface area provided with a regular pattern of valleys defined by a regular pattern of protrusions, which valleys comprise a valley bottom, a first valley wall and a second valley wall, which first valley wall comprises first valley wall sections and optionally first valley wall punctures, and which second valley wall comprises second valley wall sections and optionally second valley wall punctures, wherein said first and second valley wall sections are defined by a side of a protrusion adjacent to the valley, and wherein said first and second valley wall punctures are defined as portions of the valley wall where a line perpendicular to the valley and parallel to the valley bottom does not contact the protrusion adjacent to the valley, and wherein

a) the valley wall has a height, defined as the distance normal to the valley bottom from the valley bottom to the top surface area, of 0.5 - 50 μm;

b) the valley wall profile of the first and second valley wall is independently between 0 and 40 μm.

c) the length of the first and second valley wall sections is independently

between 0.01 - 100 μm.

d) the length of the first and second valley wall punctures, if any, is

independently between 0 and 50 μm.

e) the average width of the valley is between 0 and 50 μm.

The top surface area has been defined above, and is the area defined by all top surface areas of the protrusions. Between the protrusions, a series of valleys is defined. The valleys are defined by two valley walls, which valley walls comprise those sections of the circumferential side face of the protrusions which are adjacent to the valley. The valley wall is the hypothetical straight-line average of those parts of the circumferential side face of the protrusions which are adjacent to the valley, in the direction of the valley. See figure lc and Id.

Thus, a valley wall comprises valley wall sections, wherein a valley wall section is defined by a side of the protrusion (i.e. one side of a protrusion's circumferential side face) adjacent to the valley. Valley wall sections are thus hypothetical straight lines on the valley wall, of a length defined by the presence of a protrusion. In case of touching protrusions in one direction (i.e. in cases where the average distance between adjacent protrusions in one direction of the regular pattern is 0), the valley wall in that direction comprises only valley wall sections, and no valley wall punctures.

However, in the preferred embodiment where the protrusions are individually spaced protrusions, i.e. with a spacing to all adjacent protrusions such that the average distance in all directions is larger than 0, a valley wall further comprises valley wall punctures.

Valley wall punctures are defined as portions of the valley wall where a line perpendicular to the valley and parallel to the valley bottom does not contact a protrusion adjacent to the valley. As such, it is a portion of the valley wall where there is no valley wall section, i.e. the point in a valley wall where no protrusion is adjacent to the valley. See figure lc and Id.

Depending on the shape of the protrusions, the first and second valley wall defining a valley may be the same or different, and have the same or different valley wall profile.

The valley bottom is defined as the surface which is located between the protrusions, at the farthest possible distance from the top surface area. The valley bottom may have some slight curvature due to processing, in which case the valley bottom extends from the point where the first valley wall rises to the point where the second valley wall rises. As such, it can also be defined by the average distance between the protrusions which form the valley.

In line with the height of the protrusions, a valley wall has a height, defined as the distance normal to the valley bottom from the valley bottom to the top surface area, of 0.5 - 50 μm. Preferably, the height of the valley wall is between 1 and 45 μm, more preferably between 2 and 40 μm, more preferably between 3 and 35 μm, more preferably between 4 and 30 μm, even more preferably between 4 and 28 μm.

A valley wall is defined by the shape of the protrusions adjacent to the valley. As these shapes may be irregular, a valley wall is also defined by the valley wall profile. The valley wall profile, in this respect, is defined as the deviation in the direction normal to the valley wall, and is calculated on the basis of the valley wall sections only. Any valley wall puncture in the valley wall is excluded from the calculation.

The valley wall profile is expressed as the arithmetic mean of the distance of each point of a valley wall section from the valley wall. It is

conventionally expressed as

wherein R_a is the valley wall profile, and wherein n is the number of data points and wherein | yi I is a positive number which indicates the distance to the valley wall segment, normal to the valley wall, i.e. normal to the average surface of the valley wall section. The size of n is normally dependent on the resolution of the device used to measure R_a, which can for instance be 0.4 μm, in which case n is the length of the valley wall section in μm. R_a can suitably be determined by

mathematical analysis of top view images of the surface topography.

The valley wall profile is between 0 and 40 μm, preferably 0.1 and 30 μm, more preferably between 0.5 and 25 μm, even more preferably between 0.8 and 20. A valley wall profile of 0 indicates that all protrusions which define the valley wall have a straight line parallel to the valley, such as may for instance occur in the case of square or rectangular protrusions. Of course, depending on the shape of the protrusions, the valley wall profile of the first and second valley wall may be different. The word "profile", or valley wall profile, as used here, can be equated with the term "roughness", although the term "roughness" is usually applied for the surface roughness of a surface, which is determined by the deviations of the average surface in vertical direction (for a horizontal surface), normal to the surface. In the present case, the valley wall profile is the roughness of the valley wall, i.e. the roughness of a vertical surface comprised on a horizontally oriented topography, which is determined by the deviation of the average valley wall in a direction parallel to the surface topography, and normal to the valley wall.

The length of valley wall sections can be the same as the length of a protrusion in the case when the longest straight-line fitting within the

circumference of the top surface area parallel to the surface is parallel to the valley wall. In other cases, a valley wall section is shorter than the length of the protrusion (or protrusion element). As such, the length of valley wall section is 0.01 - 100 μm, preferably, the length of a valley wall section is 0.05 - 50 μm, more preferably 0.1 - 40 μm, even more preferably 1 - 35 μm. Depending on the shape of the protrusions which define the valley, the length of valley wall sections in the two valley walls which define a valley may be the same or different. The length of the valley wall punctures is generally between 0 and 50 μm, preferably 0.5 - 40 μm, more preferably 1 - 30 μm, even more preferably between 2 and 20 μm. Valley wall punctures themselves define a valley, which is oriented perpendicular to the valley of interest. Depending on the shape of the protrusions which define the valley, the length of valley wall punctures in the first and second valley wall may be the same or different.

The average width of the valley is between 0 and 50 μm. The average width of a valley is defined in line with the average distance between protrusions, or protrusion elements, and is calculated by dividing valley wall segments on opposing sides of the valley in line segments of about 0.4 μm, and determining the distance between each line segment of a valley wall section on one side of the valley and the facing line segment of the valley wall section of the adjacent protrusion on the other side of the (same) valley, and then averaging those distances. The average valley width is the average distance between two adjacent protrusions.

The average width of the valley is between 0-50, preferably between 0.5 and 40 μm, more preferably between 1 and 30 μm, more preferably 2 and 20 μm.

Alternatively, the valley width may be defined by the shortest and longest distance between facing protrusions on opposite sides of the same valley, in line with the shortest and longest distance between facing protrusions defined above.

In a much preferred embodiment, the surface topography comprises individually spaced protrusions, which do not touch adjacent protrusions. Thus the distance between protrusions, i.e. the valley width, is larger than 0, and each valley wall comprises valley wall punctures. In this embodiment, a regular pattern of crossing valleys is obtained. In this embodiment, each valley wall comprises valley wall sections and valley wall punctures.

Because it is preferred that the side face of at least a number of the protrusions, or protrusion elements, extends substantially normal to the surface at the position where the side face intersects with the surface, it is also preferred that the valley wall is substantially normal relative to the valley bottom. The angle that a valley wall can have relative to the valley bottom is defined in line with the angle that the circumferential side face may have with the surface. Thus, in case of a topography comprising only a single type of protrusion comprising a single protrusion element, all valley wall sections have the same profile (which is defined by the circumferential side face of the protrusion adjacent to the valley). In this case, the valley wall sections alternate with valley wall punctures which punctures all have equal length.

In case of topographies comprising protrusions which comprise multiple protrusion elements, or in case of topographies comprising differentially shaped or differentially oriented protrusions, a single protrusion defines multiple valley wall sections which may have a different profile, and multiple valley wall punctures, which may be of non-equal length. In this case, the valley wall sections alternate with valley wall punctures in a regular order.

The object.

The topography as defined above is preferably present on an object having a surface part comprising a metal, polymeric, composite or ceramic material. The surface part may be an exterior or interior surface part of the object. An exterior surface part is a surface part which can be freely contacted by cells which are located at the outside of the object, when the cells are brought into contact with the object. An exterior or interior surface part may include holes, pores or wells.

Suitable materials are known in the art, and include any material which is suitable for the application at hand. That is, for application of a topography of the invention on for instance a culture plate, a material suitable to construct a culture plate should be used. Similarly, for application of a topography of the invention on a vascular implant, a material suitable to construct a vascular implant should be used. The skilled person knows what type of material is suitable for what type of application, and is capable of choosing an appropriate material accordingly.

Any material may be used to make the object, but preferably, the object, or at least the surface part thereof which is provided with the topography, is made of a ceramic, glass, metal, polymeric material or composite materials. Most preferably, the object comprises a surface part comprising carbon such as graphite, graphene, or LTI carbon. Alternatively, a metal alloy can be used, such as an alloy comprising one or more elements selected from the group of silver, cobalt, iron, gold, platinum, zinc and titanium, as well as one or more elements selected from the group of aluminum, carbon, chromium, copper, iridium, manganese, magnesium, mercury, molybdenum, nickel, niobium, neodymium, palladium, tantalum, vanadium, tungsten, and zirconium. Preferred alloys are Nitinol, a cobalt-chromium alloy, a Co/Cr/Mo alloy, as well as micro- galvanic alloys such as a Zn/Ag, Mg/Zn, Mg/Fe, Mg/Ni, or Mg/Cu, ferromagnetic alloys such as Mg/Al/Nd. Also preferred are conductive polymers, preferably polypyrrole, polyaniline and poly(3,4-ethylenedioxythiophene), polyurethane, polytetrafluoroethylene (Teflon), and polyethylene terephthalate, and biodegradable polymers like polyesters such as polylactic acid, polyethylene glycol (PEG or PEO) and polyglycolic acid, polyether ether ketone (PEEK), and composites of these materials.

An object comprising one (or more) topographies according to the invention can be made by any technique known for creating specifically shaped microstructures onto specific materials. The skilled person knows what types of techniques are suitable for what type of material. Examples of suitable techniques are 3D printing, laser printing, writing, layer-by-layer coating, electrospinning, deposition techniques, spraying and sputtering, stamping, (hot) pressing, (hot) embossing, (nano) imprinting, (injection) molding, casting, supercritical fluid-based processes, etching, laser machining, laser cutting and ablation, (precision) electrochemical machining, (precision) electrochemical gridding and (precision) electrical discharge machining, etc.

The object of the invention is preferably made by laser-machining, precision-electro(chemical) technologies, precision-electro assembly, engraving, printing, coating, stamping, (injection) molding, (hot) embossing, or etching the topography into or onto the object. Alternatively, the object may be formed with the topography in a single process, such as by injection molding, pressing and hot- embossing. Suitable techniques to obtain an object provided with a topography as described are well-known in the art.

Printing may be achieved by employing 3D printing, laser printing and writing surface protrusions on a surface part of a metallic, polymeric, ceramic, composite or other substrate, as is known in the art.

Coating may be achieved by producing the protrusion on a substrate and then coat the surface part using spraying, sputtering, layer by layer coating, electrospinning, or deposition from solutions. Stamping may be achieved by first producing a mold containing the negative form of the protrusions and then later stamp that mold onto a surface part by using techniques such as pressing, hot press, embossing, hot embossing, imprinting and nanoimprinting. Other techniques including injection molding, processing from a melt, or casting is also possible.

Etching may be achieved by using different solvents in the form of liquid and/or vapor, such as acids and/or organic solvents, as well as a suitable mold, such as to remove selective portions of the surface part defined by the mold design to obtain the surface topography. Laser machining, engraving and ablation

techniques may also be used to carve the topographies into the surface of a suitable material. Precision-electro(chemical) technologies could include (precision) electrochemical machining, (precision) electrochemical gridding, (precision) electrical discharge machining.

In a much preferred embodiment, the topography comprising a regular pattern of protrusions is a non-separable part of the object. This may be achieved by stamping, printing, etching, coating the topography onto the object or by using other methods mentioned above, for example by forming the object in a single process such as by injection molding.

The surface between the protrusions (valley bottom), the circumferential side face (the valley wall sections) and/or the top surface area of the protrusions may be smooth or substantially smooth, i.e. having a roughness of about 0 (i.e. 0 ± 0.01 μm).

In an optional embodiment however, the surface between the protrusions and/or the top surface area and/or the circumferential side face may have a roughness, commonly abbreviated R_a, of 0.01 μm - 10 μm, preferably 0.05 - 8 μm, even more preferably 0.1 - 5 μm. Also, the roughness may be 0.2 - 20 or 0.15 - 3 μm. Roughness, in this respect, is the surface roughness obtained by techniques such as etching, blasting or brushing the surface part prior to or after forming of the topography. The valley wall profile, abbreviated and calculated by the same formula, can thus be seen as the roughness of the valley wall normal to that surface.

The roughness can be determined using atomic force microscopy analysis. A randomly roughened surface part may be obtained by conventional etching, brushing, blasting or the like, of the surface topography comprising a regular pattern of protrusions. Alternatively, a flat surface part, such as a surface part of an implant, may be roughened first, where after the surface topography of the invention is formed.

It is essential that any randomly roughened surface part does still comprise the protrusions of the invention, so that a randomly roughened surface part may not have a roughness which is larger than the height and/or width of the protrusions, so that protrusions with the sizes indicated elsewhere remain present.

The topography of the invention is a surface topography consisting of a multitude of regularly spaced protrusions. Thus, unit cells comprising a protrusion are distributed over the surface part so as to provide a single topography, as defined above. The quantity of unit cells in a single topography is preferably at least 50, more preferably at least 100, even more preferably at least 200, even more preferably at least 500, even more preferably at least 1000. Topographies which extend over a considerable surface part can be of high potential influence on the functioning of large cell collections, such as specific tissue. This is deemed appropriate for in vitro use, such as the growth of cell cultures or tissue, or in vivo use, such as the creation of implants.

A topography of the invention is capable of modulating the cell response, most notably modulating morphology, proliferation, biochemical functioning, differentiation, attachment, migration, signaling, and/or cell death of an

endothelial cell population by physical stimulation. That is, the topography of the invention is capable of altering the behavior of one or more living endothelial cells by inducing changes in morphology, proliferation, biochemical functioning, differentiation, attachment, migration, signaling, and/or cell death. The behavior is altered by physical stimulation.

Physical stimulation in this context means that the signals which induce the change in behavior are transferred by among others shape, hardness and size of the surroundings where the cells are located, i.e. the topography. The

"surroundings" of the cell(s) directly modulate the cell(s). Physical stimulation means that the surface topography itself alters the cell response on various aspects, among which cell morphology, proliferation, biochemical functioning, differentiation, attachment, migration, signaling, and/or cell death, and that this response is different for different topographies. By changing a surface topography in contact with a cell or cell population, the cell's morphology, proliferation, biochemical functioning, differentiation, attachment, migration, signaling, and/or cell death can be influenced, all other parameters kept equal.

The physical stimulation which is responsible for the altered behavior is a separate effect from chemical stimulation to alter behavior. In chemical stimulation, the onset of the signals received by the cell to alter behavior is chemically induced through an added compound or through surface chemistry (e.g. choice of bulk material, coating, or surface-functionalization) and is subsequently transferred by signaling molecules which interact with receptors on the cell surface or which enter the cell to interact with receptor molecules within the cell. The mechanism behind the presently observed physical stimulation to alter behavior is still not fully understood, but the examples show that shape of the surface topography, rather than chemical stimulation, are causing the onset of the observed modulation of cell morphology, proliferation, biochemical functioning, differentiation, attachment, migration, signaling, and/or cell death of a cell population. So, the physical stimulation of cells is independent of the chemistry of the surface and/or environment. The altered cell physiological behavior initiated by the physical stimulus of a surface topography might affect the cell's response to and interaction with signaling molecules and receptor molecules.

In the Examples, data are shown which display the effects of various topographies-featuring poly(lactic acid) materials on modulation of endothelial cells by physical stimulation, while the same material without topographies did not similarly modulate the cell behavior.

Modulation of endothelial cells includes stimulating endothelial-like behavior, which can be derived from stimulated morphology, stimulated

differentiation and biochemical functioning, stimulated cell attachment, stimulated proliferation, and stimulated migration and motility of endothelial cells. One or more of these features stimulate the formation of an endothelium layer

(endothelium).

Stimulated morphology can be ascertained by imaging the cells through different techniques including immunofluorescent labeling and scanning electron microscopy. Stimulated differentiation and biochemical functioning can be

ascertained by evaluation of functional biomarkers of endothelial cells including CD31, Vascular endothelial cadherin (VE-cadherin), vascular endothelial growth factor (VEGF), etc. at gene or protein level.

The expression of cell markers can be evaluated at gene- and protein- level using various techniques such as qPCR, ELISA, immunofluorescent staining, imaging, western blot and fluorescent- activated cell sorting (FACS), etc.

Stimulated attachment and proliferation can be ascertained by (1) Measuring the number of cells on a surface, through DNA-assay levels, imaging, counting, or otherwise, shortly after seeding (normally within 24 hrs) which indicates cell attachment to that surface and (2) by counting the number of viable cells over time, quantifying the DNA content or quantifying the number of cells over time or label cells that have or are replicating, e.g. by a EdU-positive cell assay. EdU or 5-ethynyl-2'-deoxyuridine is a biomolecule that accumulates in the DNA of the cells during DNA replication and can be visualized by

immunofluorescent labeling.

Stimulated migration can be ascertained by migration assays such as live or time laps imaging or quantifying the migration over a surface through labeling migrating cells through e.g. a nano-bead uptake assay where the surface is, prior to adding the cells, coated with nano-beads that are taken up by cells when they adhere to that specific part of the surface. The more a cell migrates, the more surface that cell will have covered, the more nano-beads that will be taken up by that cell, which is subsequently measured.

In one embodiment, endothelial cells are modulated in vivo. In this embodiment, an object of the invention can be a vascular or lymphatic implant, such as a cardiovascular implant, part of a cardiovascular implant, a lymphatic vessel implant or part of a lymphatic vessel implant and includes but is not limited to implants such as an artificial vessel, vascular graft, stent, AAA stent, stent graft, cardiac valve, cardiac pump, cardiac rhythm prevention product, total heart implant (artificial heart), pacemaker including (leadless) stimulator, blood pump, aneurism implants (coil), transcatheter pacing system, (intravascular) catheter, 2D or 3D scaffold for tissue regeneration, sensor. In another embodiment, endothelial cells are modulated in vitro. In this embodiment, an object of the invention can be a culture dish such as culture flask, plate, dish, slide, bottle, chamber or bag, microfluidic device, 2D or 3D scaffold, chips, (micro)fluidic devices, biore actors, membranes, etc. comprising a surface for culturing of endothelial cells.

Suitable culture media for this embodiment include any medium suitable for culturing endothelial cells, as known in the art. Such media generally comprise water, and furthermore may comprise glucose, proteins, amino acids, vitamins, penicillin, antibiotics, and inorganic salts and whole blood or derivatives thereof. In other applications, the cells may be cultured in other liquids such as phosphate buffered saline (PBS), water, or on a gel such as Matrigel or agarose gel, on a coating such as collagen, laminin, fibronectin, fibrinogen, or polyamine or be embedded into a biomaterial such as a hydrogel or polymer carrier. Cells may be fixed using fixation solution and analyzed under wet or dry conditions.

For the purpose of clarity and a concise description features are described herein as part of the same or separate embodiments, however, it will be

appreciated that the scope of the invention may include embodiments having combinations of all or some of the features described. In particular, features described under the general description may be part of the specific embodiments.

The invention will now be illustrated by the following, non-limiting examples.

Materials and Methods Sample fabrication

In the example, Poly(lactic acid) substrates featuring surface

topographies are used. Each topography is analyzed for 2 sets of parameters (1: migration assay and 2: attachment and proliferation assay), for both sets the number of substrates for each topography is n=24.

For polymer substrates to be provided with a topography, the following procedure is followed:

1. Fabricate silicon wafers (mold) holding the inverse topography design through photolithography using a chromium mask. 2. Coat the silicon wafer with perfluorodecyl trichlorosilane (FOTS, ABCR).

3. Replicate the topography design into intermediate molds of

polydimethylsiloxane (PDMS Sylgard 184¾), Dow Corning) and OrmoStamp (Micro Resist Technology GmbH).

4. Hot-emboss the topography into the polymer sheet using the intermediate molds at 140 °C (all samples demolding at 80 °C).

5. 02 gas plasma (standard treatment for cell culture substrates).

6. All substrates were sterilized, with 70% ethanol, washed with PBS and wetted with cell medium

7. Half of the substrates (1^st set) were subsequently coated with fluorescently- labeled nano-beads (Fluospheres (fluorescent nano-beads, Life Technologies)) to analyze the motility of the endothelial cells in a migration assay. The substrates were again washed with PBS and protected from light. Cells were cultured on the topography-featuring substrates, and the influence of the selected topographies was compared to non-patterned (flat) surfaces and control basic topographies.

Endothelial migration was determined using a fluorescent nano-bead uptake assay in which, prior to adding the cells, the substrate was coated with a homogeneous layer of fluorescently-labeled nano-beads. Cells migrating over the surface will take up these nano-beads so the more a cell migrates, the more nano- beads it will take up which is being measured. Endothelial proliferation was assessed using an EdU incorporation assay. EdU (5-ethynyl-2^'-deoxyuridine) is an alkyne -containing thymidine analog that incorporates in DNA during DNA replication and can be detected by a chemical reaction using a fluorescent dye. The number of cells positive for EdU indicates cells that are or have been replicating, and when given in a percentage over all cells present, is a good indication for overall proliferation. Especially considering the early time point (18 hrs) only those surfaces that allow cells to immediately start proliferating, important for rapid endothelialization, will have higher levels of EdU-positive cells. The overall number of cells by itself is a good measure for cell attachment, especially in such early time point (18 hrs.) and taking into account proliferation. Culturing human coronary endothelial cells

Human coronary endothelial cells (Lonza) were we cultured in complete EGM2-MV medium (Lonza) to reach approx. 70% confluency. After trypsinization, cells were seeded on the topographies; the first set of substrates (migration assay) were seeded with a cell density of 6000 cells/cm² and the second set of substrates (attachment and proliferation assay) with a cell density of 10000 cells/cm². For the 1^st set of substrates, a 10 μΜ EdU solution (Click-iT® EdU Alexa Fluor® 647 Imaging Kit, Thermo Fisher) was added to the cell medium (50:50). The cultures were kept at 37°C in 5% CO₂ with regular medium refreshment.

Analysis

The cultures were harvested after 18 hours of culture, washed with PBS, fixed with 3.7% formaldehyde, permeabilized with 0.1% Triton X-100 and treated with 2% BSA for blocking of non-specific binding sites. The cell body was labeled with CellMask™ Deep Red (L^f set of substrates) or Orange (2^nd set of substrates, ThermoFisher). The 2^nd set of substrates were then labeled with EdU labeling solution and DAPI (Sigma Aldrich). The stained topographies were mounted onto glass slides.

Bright-field and fluorescence images were acquired with a Nikon slide scanner and visually inspected for Quality control. Image quality verification and analysis was performed using custom designed MATLAB scripts and CellProfiler pipelines. Stimulated endothelialization is defined based on: high levels of nano- beads accumulated inside the cells reflecting strong motility of the cells over the surface, and total number of cells attached and total number of EdU-positive cells reflecting stimulation of cellular proliferation in a short time.

Results

All topographies identified as being successful in steering

endothelialization had either higher bead uptake compared to the nonpatterned control (NP), up to 60% higher (for El- 15) or bead uptake level similar to NP (E16- E20) (Figure 2, Figure 3a). When compared to basic topographies, topographies of the invention had higher bead uptake, from 30% higher to even up to 130% higher (Table 2, Figure 3a). For example, the bead uptake on surface El was 230%) of that on basic surface B3 and B4 (Figure 3a). Cell attachment to the topographies of the invention was generally higher than to the non-patterned control (Figure 3b).

Regarding proliferation, topographies of the invention El- 15 had similar levels of EdU-positive cells (54-67%) compared to the nonpatterned control (68%) and slightly higher compared to basic topographies (50%) while topographies E16-E20 showed significantly increased number of EdU-positive cells (up to 80%>) (Figure 3c- d).

The surface topographies of the invention stimulate endothelialization by triggering migration and/or proliferation processes. Surface topographies E1-E15 have a more dominant effect through the migration process in endothelial cells while surface topographies E16-E20 affect the proliferation process more dominantly. In strong contrast, the basic shapes do not stimulate

endothelialization. Even though some basic shapes slightly improve one function, at the same time they reduce other functions leading to an overall lower level of endothelialization. For example, basic shape B4 has similar levels of EdU-positive cells compared to the nonpatterned control, but reduces migration by 30%. And basic shape B3 reduces both migration and the number of EdU-positive cells, both with 30% compared to the nonpatterned control. Conclusions

To stimulate endothelialization of implanted medical devices, endothelial cells need to quickly migrate from the out linings of the surrounding blood vessels or lumen over the surface and subsequently attach and proliferate to cover the surface. Simulating one of those three parameters (migration, attachment, and proliferation), while keeping the other parameters stable or stimulate those too, allows more rapid endothelialization of that surface. We have identified surface topographies that promote endothelialization both in vivo and in vitro through either attachment, proliferation or migration, or a combination of those. The surface topographies that perform the best in improving endothelialization, through either stimulating endothelial cell attachment, - proliferation or - migration, or a combination of those, while maintaining the levels of the other parameters, have protrusions with a top surface area of 1-6000 and preferably 30- 1000 μm², surface coverage of 3-90% and preferably 10-80 % and average distance between protrusions of 0-50 μm and preferably 1-30 μm. In more preferred embodiments, the ranges are as defined in the general text above.

Claims

1. An object, comprising a surface part provided with one or more topographies capable of modulating the morphology, proliferation, biochemical functioning, differentiation, attachment, migration, signaling, and/or cell death of an endothelial cell population by physical stimulation, and wherein the topography comprises a surface provided with a regular pattern of protrusions, which protrusions comprise one or more protrusion elements, which protrusion elements are defined as surface portions elevated above the surface having a top surface area and a circumferential side face connecting the top surface area with the surface, wherein each protrusion element has a maximum height of between 0.5 and 50 μm above the surface, and wherein

a) the average distance between adjacent protrusions is between 0 and 50 μm; b) the top surface area of the protrusion is between 1 and 6000 μm- and c) the protrusions cover between 3 and 90 % of the surface.

2. An object according to claim 1, wherein

if the protrusion comprises one protrusion element:

the length of the protrusion, defined as the length of the longest straight- line fitting within the circumference of the top surface area parallel to the surface, is 0.01 - 100 μm;

the width of the protrusion, defined as the length of the longest straight-line fitting within the circumference of the top surface area parallel to the surface, perpendicular to the length, is 0.01 - 100 μm;

if the protrusion comprises more than one protrusion element:

the length of each protrusion element, defined as the length of the longest straight-line fitting within the circumference of the top surface area parallel to the surface, is 0.01 - 100 μm;

the width of each protrusion element, defined as the length of the longest straight-line fitting within the circumference of the top surface area parallel to the surface perpendicular to the length, is 0.01 - 100 μm; c) the average distance between two circumferences of adjacent protrusion elements of the same protrusion is 0 - 50 μm.

3. An object according to claims 1 or 2, wherein the surface part comprises a metal, polymeric, composite or ceramic material.

4. An object according to any of claims 1 - 3, wherein the regular pattern of protrusions is defined by an imaginary grid of intersecting gridlines which can be laid over the surface, which gridlines define a pattern of unit cells, such that each unit cell comprises a maximum of one protrusion.

5. An object according to any of claims 1 - 4 for modulating the

morphology, proliferation, biochemical functioning, differentiation, attachment, migration, signaling, and/or cell death of the endothelial cell population by physical stimulation, comprising on its surface part one to ten different topographies, and preferably one topography.

6. An object according to claim 5 for use in stimulating endothelialization.

7. An object according to claim 5 or 6 for use as a vascular or lymphatic implant.

8. An object for use according to claim 7, wherein the object increases attachment, proliferation and/or migration of endothelial cells.

9. An object for use according to claim 7 or 8, wherein the topography is defined by

a) the average distance between adjacent protrusions is 1 - 30 μm; and b) the top surface area of the protrusion is between 30 and 1000 μm²; and c) the protrusions cover between 10 and 80 % of the surface.

10. An object as defined in claims 1 - 9 comprising one or more topographies capable of modulating the morphology, proliferation, biochemical functioning, differentiation, attachment, migration, signaling, and/or cell death of an

endothelial cell population by physical stimulation, wherein the topography can be alternatively defined as comprising a top surface area provided with a regular pattern of valleys defined by a regular pattern of protrusions, which valleys comprise a valley bottom, a first valley wall and a second valley wall, which first valley wall comprises first valley wall sections and optionally first valley wall punctures, and which second valley wall comprises second valley wall sections and optionally second valley wall punctures, wherein said first and second valley wall sections are defined by a side of a protrusion adjacent to the valley, and wherein said first and second valley wall punctures are defined as portions of the valley wall where a line perpendicular to the valley and parallel to the valley bottom does not contact the protrusion adjacent to the valley, and wherein

b) the valley wall profile of the first and second valley wall is independently 0 -

40 μm.

c) the length of the first and second valley wall sections is independently 0.01 - 100 μm.

d) the length of the first and second valley wall punctures, if any, is

independently between 0 and 50 μm.

e) the average width of the valley is between 0 and 50 μm.

11. A method to modulate the morphology, proliferation, biochemical functioning, differentiation, attachment, migration, signaling, and/or cell death of an endothelial cell population by physical stimulation, comprising

1) contacting the one or more endothelial cells in a suitable medium with a surface part provided with a regular pattern of protrusions, the regular pattern of protrusions defined by an imaginary grid of intersecting gridlines which can be laid over the surface part, which gridlines define a pattern of unit cells, such that each unit cell comprises a maximum of one protrusion, which protrusions comprise one or more protrusion elements, which protrusion elements are defined as surface portions elevated above the surface having a top surface area and a circumferential side face connecting the top surface area with the surface, wherein each protrusion element has a maximum height of between 0.5 and 50 μm above the surface, and wherein

a) the average distance between adjacent protrusions is between 0 and 50 μm;

b) the top surface area of the protrusion is between 1 and 6000 μm^2; and c) the protrusions cover between 3 and 90 % of the surface;

2) allowing the cells to respond to the surface.

12. A method according to claim 11, wherein the topography is defined by a) the average distance between adjacent protrusions is between 1 - 30 μm; b) the top surface area of the protrusions is between 30 - 1000 μm²; and c) the protrusions cover 10 - 80 % of the surface part.

13. A method according to claim 11 or 12, wherein modulation of the endothelial cell population occurs in vitro.