Surface topography for changing the physiological functions of living cells

Abstract

To provide surface topographies which have been found to alter the physiological state of cells; to provide objects that provide at least one such topography that can be used in vivo or in vitro to alter cell behavior; and to provide methods to alter cell behavior using such objects.SOLUTION: 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 a cell population by physical stimulation. Such topographies can be applied in vitro and in vivo to modulate cell behavior. Specific examples include implants provided with a topography of the invention which regulates the immune response, or an implant which increases osteogenesis. The invention furthermore pertains to objects which are used in vitro to modulate cell behavior.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present invention relates to surface topographies that have been found to change the physiological state of cells without being limited to the morphology, proliferation, biochemical function, differentiation, adhesion, migration, signal transduction, and / or cell death of cell populations by physical stimuli. The present invention further provides an article comprising at least one such topography that can be used in vivo or in vitro to change cell behavior, and a method of changing cell behavior using such an article. behavior.

Background Art

[0002] Cells are known to interact in contact with surfaces. In nature, direct physical interactions between cells and their environment are part of their normal physiological behavior. For example, when culturing cells outside of their natural environment, such as in a cell culture dish, or when exposed to a medical implant such as a hip joint implant or pacemaker, their physiological functions are typically not the same as in their original environment. Many efforts have been made to optimize these cell responses on in vitro and implant surfaces.

[0003] For example, in cell culture, for the purpose of being able to grow as naturally as possible and / or exhibit distinct biological characteristics such as differentiation and / or respond to distinct chemical, physical, or electrical stimuli, cells can be placed on the surface of an appropriate culture medium. Conventional strategies for changing cell behavior have been to add various hormones, chemicals, growth factors, enzymes, salts, etc., which could have an impact on changed cell responses such as proliferation or induced differentiation. However, although such changes are made at the surface, in such conventional cultures, the changed cell behavior is generally chemically induced through the interaction of hormones, chemicals, growth factors, enzymes, salts, etc. on the cell surface and / or inside the cell. In such conventional cultures and medical implants, the potential micro-patterns present on the surface are generally not considered.

[0004] It has recently been found that physical interactions affect cell behavior. For example, titanium-based implants characterized by minute surface roughness caused by chemical etching and / or mechanical blasting have been shown to improve the mechanical strength of newly formed tissue bridging the implant to bone / dental tissue (Non-Patent Document 1).

[0005] The influence of topographical cues at the cell level has been observed in different cell types. For example, it has been shown that culturing on surfaces characterized by nanotopography significantly improves hepatocyte adhesion, morphology, and function compared to smooth substrates (Non-Patent Document 2).

Prior Art Documents

Non-Patent Documents

[0006]

Non-Patent Document 1

Non-Patent Document 2

Brief Description of the Drawings

[0007]

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Mode for Carrying Out the Invention

[0008] Detailed Description of the Invention General Embodiment: Topography for Regulating Cell Response The present invention relates to an object having a surface topography and a surface portion having one or more of such topographies. The surface topography is formed by the presence of protrusions regularly spaced on the surface portion 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, as well as the coverage of the surface portion, and further the length and width of the protrusions (definition of "protrusion"). In another definition, the topography can be defined by the valleys (definition of "valley") located between the protrusions. These definitions function as two alternative numerical representations of the topography.

[0009] Definition of "protrusion" The present invention provides an object comprising a surface portion having one or more topographies capable of regulating the morphology, proliferation, biochemical function, differentiation, adhesion, migration, signal transduction, and / or cell death of a cell population by physical stimuli. The topography comprises a surface having a regular pattern of protrusions, the protrusions comprising one or more protrusion elements, the protrusion elements being defined as a surface portion raised on a surface having a top surface area and a peripheral side surface connecting the top surface area to the surface, and having a maximum height of 0.5 to 50 μm on the surface of each protrusion element, a) The average distance between adjacent protrusions is 0 to 50 μm, b) The area of the top surface area of the protrusion is 1 to 6000 μm 2 and c) The protrusions cover 3 to 90% of the surface.

[0010] Preferably, the object of the present invention has the following lengths and widths of the protrusions or protrusion elements. a) When the protrusion comprises one protrusion element, the length of the protrusion defined as the length of the longest linear fitting within the periphery of the top surface area parallel to the surface is 0.01 to 100 μm, the width of the protrusion defined as the length of the longest linear fitting within the periphery of the top surface area perpendicular to the length and parallel to the surface is 0.01 to 100 μm, b) When the protrusion includes a plurality of protrusion elements, The length of each protrusion element defined as the length of the longest straight line fitting within the perimeter of the top surface region parallel to the surface is from 0.01 to 100 μm, The width of each protrusion element, which is perpendicular to the length and defined as the length of the longest straight line fitting within the perimeter of the top surface region parallel to the surface, is from 0.01 to 100 μm, The average distance between two perimeters of adjacent protrusion elements of the same protrusion is from 0 to 50 μm.

[0011] This topography comprises a surface having a regular pattern of protrusions. The protrusions are composed of one or more protrusion elements, and the element is a surface portion raised above the surface on which the protrusion is formed, i.e., the surface surrounding the associated protrusion or protrusion element. Each protrusion element is defined by a raised surface portion, a top surface region, and a surrounding side surface connecting the top surface region to the surface. The raised surface portion is the sum of the top surface region and the surrounding side surface. One protrusion may be defined as a single protrusion element, but one protrusion may also comprise a plurality of protrusion elements. In one preferred embodiment, the protrusion has a single protrusion element. In another preferred embodiment, the protrusion comprises at least two protrusion elements. Thus defined.

[0012] Thus, the protrusions are small bumps or groups of bumps on the surface. The protrusions define a three-dimensional network of valleys on the surface of the object. A regular pattern of protrusions having a particular size, shape, and morphology has been found to result in a topography that can regulate cell responses, particularly the morphology, proliferation, biochemical function, differentiation, adhesion, migration, signaling, and / or cell death of a cell population in response to physical stimuli.

[0013] A regular pattern can be defined by a grid of intersecting grid lines that can be placed on a surface and that define a pattern of unit cells such that each unit cell has at most one protrusion. The grid of intersecting grid lines need not physically exist, but since it is preferably a virtual way of defining a regular pattern, the unit cells can have any shape. Preferably, the unit cells have a rectangular, particularly square, trapezoidal, triangular or hexagonal shape.

[0014] In this context, a regular pattern means that any section of a topography consisting of m unit cells, for example an n×n arrangement, repeatedly exists in a plurality of sections adjacent to that section. Preferably, m and n are 1, but particularly in the case of unit cells having different shapes or differently oriented protrusions, or in the case of triangular, hexagonal or trapezoidal unit cells, m can be from 4 to 16 and n can be from 2 to 4.

[0015] The unit cells can have a surface area of, for example, 1 to 10000 μm 2 , preferably 1 to 2500 μm 2 , more preferably 25 to 2000 μm 2 , even more preferably 100 to 1000 μm 2 . In the case of square or rectangular unit cells, the unit cells can have length×width dimensions of (1 to 100) μm×(1 to 100) μm, preferably (1 to 50) μm×(1 to 50) μm. In a highly preferred embodiment, the unit cells are square and are 5 μm×5 μm to 45 μm×45 μm, preferably 5 μm×5 to 30 μm×30 μm, for example 5 μm×5 μm, 10 μm×10 μm, 15 μm×15 μm, 20 μm×20 μm, 25 μm×25 μm or 28 μm×28 μm.

[0016] Unit cells defined by the pattern of intersecting grid lines each have at most a single protrusion, which may be composed of a plurality of protrusion elements as defined above. Preferably, each unit cell has a protrusion of equal dimensions, i.e., each unit cell has the same protrusion as the others. The dimensions of the protrusion include, for example, the height of the protrusion, the number and relative positions of the protrusion elements within the unit cell, the length and width of each protrusion element, the angle of the surrounding side surface with respect to the surface, and the shape of the top surface region of each protrusion element.

[0017] The height of the protrusion, or at least the height of the protrusion element, is between 0.5 and 50 μm from the surface. That is, when the protrusion has a plurality of protrusion elements, the heights of the various protrusion elements of the protrusion may be different within the above-mentioned boundaries, but the height of each protrusion element can be between 0.5 and 50 μm on the surface. However, preferably, the height of each protrusion element is the same. The height is measured as the maximum height of the top surface region of the protrusion or protrusion element on the surface, particularly on the surrounding surface region within the unit cell.

[0018] More preferably, the height of the protrusion is 1 to 45, preferably 2 to 40 μm, more preferably 3 to 35, more preferably 4 to 30 μm, even more preferably 5 to 28 μm, even more preferably 6 to 28 μm.

[0019] The number and relative positions of the protrusion elements within a unit cell may differ from unit cell to unit cell as long as a regular protrusion pattern is obtained. That is, two or more different unit cells, including the same or different protrusions, can be defined, and these unit cells are in a regular pattern, for example, arranged alternately on the surface. In the case of triangular or trapezoidal unit cells with protrusions of equal dimensions, this may result in a regular pattern of the same protrusions with different orientations, for example, alternating opposite orientations on the surface. In the case of two different square unit cells with a single different protrusion, the alternating pattern of the unit cells results in a regular pattern of the columns of the protrusions, and the columns are arranged parallel to the edges of the unit cells or parallel to the diagonals of the unit cells. Alternatively, square unit cells with the same protrusions may be differently oriented on the surface to obtain a surface where the protrusions are regularly distributed in different orientations.

[0020] Innumerable ways of devising a regular pattern of protrusions by applying the concept of a unit cell for each can be conceived and can be used in the present invention. However, square, rectangular, and hexagonal unit cells are all oriented in the same direction, while triangular unit cells are preferably oriented alternately. More preferably, all unit cells contain the same protrusions.

[0021] The length of each protrusion (or, in the case of a protrusion containing a plurality of protrusion elements, the length of each protrusion element) is parallel to the surface and is defined as the length of the longest straight-line fitting within the perimeter of the top surface region of the protrusion or protrusion element.

[0022] In the case of a protrusion containing only a single protrusion element, the length of the protrusion is 0.01 to 100 μm. Preferably, the length is 0.5 to 50 μm, more preferably 1 to 40 μm.

[0023] In the case of a protrusion containing a plurality of protrusion elements, the length of the protrusion elements is 0.01 to 100 μm. Preferably, the length is 0.1 to 45 μm, more preferably 0.5 to 40 μm, and even more preferably 1 to 30 μm.

[0024] The width of each protrusion (or, in the case of a protrusion including a plurality of protrusion elements, the width of each protrusion element) is defined as the length of the longest straight line fitting within the perimeter of the top surface region of the protrusion or protrusion element that is parallel to the surface and perpendicular to the length.

[0025] In the case of a protrusion including only a single protrusion element, the width of the protrusion is 0.01 to 100 μm. Preferably, the width is 0.5 to 50 μm, more preferably 1 to 40 μm.

[0026] In the case of a protrusion including a plurality of protrusion elements, the width of the protrusion element is 0.01 to 100 μm. Preferably, the width is 0.1 to 45 μm, more preferably 0.5 to 40 μm, even more preferably 1 to 30 μm.

[0027] Furthermore, in the case of a protrusion including a plurality of protrusion elements, the number and relative positions of the protrusion elements within a unit cell are important features of the dimensions of the protrusion.

[0028] The average distance between adjacent protrusion elements of the same protrusion is determined by dividing the side surfaces of the facing protrusion elements into equal line segments of about 0.4 μm and determining the distance from each line segment of the first protrusion element to the line segment of the adjacent facing protrusion element (see figure). The average of all the distances is the average distance between the protrusion elements. Needless to say, when the protrusion has three or more protrusion elements, the average distance of one protrusion element with respect to the second protrusion element of the same protrusion may be different from the average distance to the third protrusion element of the same protrusion element of the same protrusion. When the angle between the surface and the peripheral side surface of the protrusion element is not 90°, the average distance between adjacent protrusions is determined by half of the height of the protrusion. The same type of calculation is applied to determine the average distance between two facing protrusions, as illustrated in FIGS. 1a and 1b. In this context, "facing" means that the distance between adjacent protrusions is determined over the length of the shortest protrusion based on the line segment extending between those protrusions. In this context, "facing" means that the distance between adjacent protrusions is determined over the length of the shortest protrusion based on the line segment extending between those protrusions.

[0029] Generally, the average distance between two protrusions is from 0 to 50 μm, preferably from 0.5 to 40 μm, more preferably from 1 to 30 μm, and even more preferably from 2 to 25 μm. The average distance between two protrusion elements within the same protrusion is defined similarly. In a more preferred embodiment, when the average distance between two adjacent protrusions in one direction is 0, the average distance between adjacent protrusions in the direction perpendicular to that distance must be greater than 0. In an even more preferred embodiment, the average distance between adjacent protrusions in any direction is greater than 0.

[0030] Also, the surface topography can be defined by determining the shortest and longest distances between facing protrusions. The shortest distance is defined by the shortest straight line defined above for the calculation of the average distance that can be drawn between two adjacent protrusions or protrusion elements. The shortest distance is preferably from 0 to 50 μm, more preferably from 0 to 40 μm, more preferably from 0 to 20 μm, and even more preferably from 0 to 10 μm. In a further preferred embodiment, the shortest distance is from 1 to 50 μm, preferably from 2 to 40 μm, more preferably from 3 to 30 μm, and even more preferably from 4 to 20 μm.

[0031] The longest distance is defined as the longest straight line defined above for the calculation of the average distance that can be drawn between two adjacent protrusions or protrusion elements. The longest distance can be from 0 to 50 μm, preferably from 0.5 to 40 μm, and more preferably from 1 to 35 μm. In a further preferred embodiment, the longest distance is from 2 to 50 μm, preferably from 3 to 40 μm, more preferably from 4 to 30 μm, and even more preferably from 5 to 20 μm.

[0032] The circumferential side surface of the protruding element can have an arbitrary angle between 0° and 180° with the surface at the position where the side surface intersects the surface. An angle of 90° is defined as perpendicular to the surface, and an angle between 0° and 90° is defined as a situation where the top surface region is larger than the raised portion of the surface, so that the top surface region at least partially covers the surface. An angle between 90° and 180° is defined as a situation where the top surface region is smaller than the raised portion of the surface. The raised portion of the surface is defined as the periphery of the protrusion at the point where it intersects the surface. Thus, an angle between 0° and 90° results in a protruding element having a top surface region hanging on the surface, and an angle between 90° and 180° results in a protruding element gradually rising towards the top surface region.

[0033] Preferably, at least a part of the protrusion or protruding element, preferably the entire circumferential side surface of the protrusion or protruding element, has an angle of 45° to 135°, more preferably 60° to 120°, even more preferably 75° to 115°, and even more preferably 80° to 100° with respect to the surface. Most preferably, at least a part of the protrusion or protruding element, preferably the entire circumferential side surface of the protrusion or protruding element, extends substantially perpendicular to the surface at the position where the side surface intersects the surface at an angle, for example, of 88 to 92°. In a further preferred embodiment, all protruding elements have substantially the same angle with the surface at the position where the side surface intersects the surface.

[0034] The shape of the top surface region of each protruding element is also an important feature of the dimensions of the protrusion and is also referred to as the shape of the protrusion (or protruding element). This is because this shape defines the shape of the valley walls on which adjacent tissue cells grow to provide fixation. The shape around the top surface region is determined parallel to the surface. Thus, it is a plan view of the protruding element and can have any geometric shape. In some embodiments, the shape can include a circle, an ellipse, a triangle, a square, a rectangle, a trapezoid, a pentagon, a hexagon, a heptagon, or an octagon. Such generally known shapes are herein referred to as basic shapes. In further embodiments, the shape can include combinations of basic shapes.

[0035] In some embodiments, the shape of the top surface region of each protrusion / protrusion element may not be a single geometric shape, such as a square, triangle, circle, octagon, pentagram, hexagon, triangular star, crescent moon, or a circular shape with a removed corner ("Pac-Man"). In some embodiments, the shape may not be a circular, oval, or polygonal shape, such as a triangle, rectangle, square, hexagon, star, parallelogram, etc.

[0036] In some embodiments, the protrusions can have the shapes shown in Table 1. In other embodiments, the topography does not include protrusions of the shapes shown in Table 1. In one embodiment, the topography does not have Shape 1 of Table 1. In another embodiment, the topography does not have Shape 2 of Table 1. In another embodiment, the topography does not have Shape 3 of Table 1. In still other embodiments, the topography does not independently have 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 of Table 1. In some embodiments, the topography does not include the selection of the topographies of Table 1, and in further embodiments, the topography does not include all the shapes of Table 1.

[0037] [Table 1]

[0038] In a preferred embodiment, the top surface region is a surface disposed substantially parallel to the surface, but the top surface region may be slightly concave or convex, such as slightly dome-shaped, for example.

[0039] Particularly preferred protrusions (or protrusion elements) preferably comprise overlapping or adjacent combinations of basic shapes interconnected by one or more crosslinks or overlapping portions to obtain a complex shape. The effect of physical stimulation by protrusions having a well-defined complex shape on the cellular responses of a cell population, particularly morphology, proliferation, biochemical function, differentiation, adhesion, migration, signaling, and / or cell death, is affected not only by the dimensions of the protrusions but also by the shape of the protrusions (or protrusion elements). For protrusions having similar height, weight, length, and width but different shapes, there is a clear effect on the cellular responses of the physically stimulating surface portion. The individual effects of the shape of the protrusions on the regulation of the morphology, proliferation, biochemical function, differentiation, adhesion, migration, signaling, and / or cell death of a cell population by physical stimulation are further detailed in the examples.

[0040] When the protrusion (or protrusion element) comprises a combination of basic shapes, a complex shape can be obtained. The complex shape can comprise overlapping or adjacent circular, elliptical, triangular, square, rectangular, trapezoidal, pentagonal, hexagonal, heptagonal, or octagonal shapes. In the case of adjacent basic shapes, the adjacent basic shapes are preferably connected by overlapping (crosslinked) portions, so that the plan view of the basic shapes has a surface area larger than the surface area of the basic shapes alone. The increased surface area for the distinct shapes is preferably located between the basic shapes and effectively increases any width of the contact points between the basic shapes.

[0041] More preferably, the peripheral side surface of the protrusion (or protrusion element) can be a continuous side surface that can be a series of straight portions connected by angles. Alternatively, the straight portions and / or angles can be smoothly curved to obtain a smoothly curved peripheral side surface.

[0042] In this context, a smoothly curved peripheral side simply means that the peripheral side has rounded corners. The corners can arise from the geometric presence of corners such as those of a square, triangle, and hexagon, but such corners can also arise from the junctions between two adjacent shapes of the protruding element, or from the presence of overlapping (or bridging) portions between two basic shapes of the protruding element. That is, the corners in this sense refer to the corners when the protrusion is viewed from the plane. When all the corners of the peripheral side of the protruding element are rounded, a smoothly curved peripheral side is obtained.

[0043] In a preferred embodiment, the protrusion has a complex shape. The complex shape is a combination of basic shapes where the basic shapes exist as a single protruding element, or overlap, or exist as adjacent basic shapes. The complex shape is further defined by the number of corners of the peripheral side. In this regard, the corners can be straight or rounded, and can be defined as any change in any direction in the peripheral side (plan view). A corner having the shape of a line with a length of 5% of the length of the peripheral side is called "curved". A straight line of the peripheral side is called "straight".

[0044] The corners can be narrow or wide. A narrow corner is a corner having an angle of less than 90°. That is, when the corner becomes narrow, a "spike" in the shape occurs, and the spike can be either inward or outward with respect to the protrusion. A wide corner is a corner where the peripheral side of the protrusion forms an angle of more than 90°. A straight corner is a corner having an angle of 90°.

[0045] A complex shape is defined as a shape having at least one wide corner, preferably, the number of narrow corners is not the same as the number of wide corners. Alternatively, a complex shape comprises at least one (preferably at least two or at least three) curvature and at least one corner (preferably at least two, more preferably at least three corners). In a further alternative preferred embodiment, a complex shape comprises both at least one curvature and at least one straight line. In a more preferred embodiment, a complex shape is a shape including three or more straight lines, preferably four or more, more preferably five or more straight lines, and at least 75% (preferably at least 85%, more preferably at least 95% of the straight lines) of all the straight lines have different lengths. In other preferred embodiments, a complex shape comprises at least three curvatures.

[0046] The protrusions adjacent to (or facing) the protrusion of a specific unit cell are the protrusions present within the unit cell sharing a side with that specific unit cell. Adjacent unit cells are defined similarly. A unit cell that only shares a corner with a specific unit cell is not adjacent. Given a regular pattern of protrusions, one protrusion is adjacent to, for example, three protrusions in the case of a triangular unit cell, four protrusions in the case of a trapezoidal, square or rectangular unit cell, and six protrusions in the case of a hexagonal unit cell.

[0047] The top surface area of a protrusion is defined as the surface area of the protrusion at its most elevated perimeter. From the above, when a protrusion comprises a plurality of protrusion elements, the top surface area of the protrusion is the sum of the total surface areas of all the protrusion elements of the protrusion, each determined at the most elevated perimeter of the protrusion element. The area of the top surface area of a protrusion can be determined, for example, by counting the number of pixels (0.4 μm × 0.4 μm elements) constituting the top surface area of the protrusion (or protrusion element).

[0048] The area of the top surface area of a protrusion is 1 - 6000 μm 2 , preferably 10 - 3000 μm 2 ​, more preferably 20 to 1500 μm 2 , more preferably 25 to 1000 μm 2 , even more preferably 30 to 750 μm 2 is.

[0049] The coverage rate of the protrusions is defined as the ratio of the top surface area to the total area covered by the protrusions. Therefore, the coverage rate is the % value obtained by multiplying 100 times the sum of the areas of all the top surface areas divided by the total surface topography area. This is the same as calculating what percentage of a unit cell has the top surface area of the protrusions.

[0050] The protrusions cover 3 to 90% of the surface part, preferably 5 to 80% of the surface part, more preferably 10 to 75% of the surface part, more preferably 20 to 70% of the surface part, more preferably 30 to 65% of the surface part. Therefore, the protrusions cover 3 to 90% of the unit cell, preferably 5 to 80% of the unit cell, more preferably 10 to 75% of the unit cell, more preferably 20 to 70% of the unit cell, more preferably 30 to 65% of the unit cell.

[0051] Definition of "valley" As described above, the regular pattern of the protrusions simultaneously defines the pattern of the valleys. Therefore, the surface topography of the present invention is defined by the surface part for regulating the morphology, growth, biochemical function, differentiation, adhesion, migration, signal transduction, and / or cell death of a cell population by physical stimulation, and the topography is defined as including a top surface area having a regular pattern of valleys, the valleys including a valley bottom, a first valley wall, and a second valley wall, the first valley wall including a first valley wall section and, if necessary, a puncture of the first valley wall, the second valley wall including a second valley wall section and, if necessary, a puncture of the second valley wall, the first and second valley wall sections being defined by the side surfaces of the protrusions adjacent to the valleys, and the punctures of the first and second valley walls being defined as the portions of the valley walls where a line perpendicular to the valley and parallel to the valley bottom does not contact the protrusions adjacent to the valley. a) The valley walls have a height of 0.5 to 50 μm, defined as the distance perpendicular to the valley bottom from the valley bottom to the top surface region. b) The profiles of the first and second valley walls are independently 0 to 40 μm. c) The lengths of the first and second valley wall sections are independently 0.01 to 100 μm. d) If there are punctures in the first and second valley walls, their lengths are independently 0 to 50 μm. e) The average width of the valley is 0 to 50 μm.

[0052] The top surface region is defined above and is the region defined by all the top surface regions of the protrusions. A series of valleys are defined between the protrusions. The valleys are defined by two valley walls, and the valley walls comprise portions of the peripheral side surfaces of the protrusions adjacent to the valleys. The valley walls are the virtual straight-line averages along the valley direction of the portions of the peripheral side surfaces of the protrusions adjacent to the valleys. Refer to FIGS. 1c and 1d.

[0053] Thus, the valley walls comprise valley wall sections, and the valley wall sections are defined by the side surfaces of the protrusions adjacent to the valleys (i.e., one side surface of the peripheral side surface of the protrusion). Therefore, the valley wall section is a virtual straight line of a length defined by the presence of the protrusion on the valley wall. In the case of protrusions that contact in one direction (i.e., when the average distance between adjacent protrusions in one direction of a regular pattern is 0), the valley wall in that direction comprises only the valley wall section and does not include the puncture of the valley wall.

[0054] However, in a preferred embodiment where the protrusions are individually spaced protrusions, i.e., all adjacent protrusions have an average distance greater than 0 in all directions, the valley walls further comprise punctures of the valley walls.

[0055] The puncture of the valley wall is defined as the portion where a line perpendicular to the valley wall and parallel to the valley bottom does not contact the protrusion adjacent to the valley. Therefore, it is the portion of the valley wall where there is no valley wall section, i.e., the portion where there is no protrusion adjacent to the valley. Refer to FIGS. 1c and 1d.

[0056] Depending on the shape of the protrusions, the first and second valley walls that define the valleys may be the same or different, and may have the same or different valley wall contours.

[0057] The bottom of the valley is defined as the surface located between the protrusions and as far as possible from the top surface region. The bottom of the valley may have a slight curvature due to processing, in which case the bottom of the valley extends from the point where the first valley wall rises to the point where the second valley wall rises. Thus, it can also be defined by the average distance between the protrusions forming the valley.

[0058] Along the height of the protrusion, the valley walls have a height of 0.5 to 50 μm, defined as the distance perpendicular to the bottom of the valley from the bottom of the valley to the top surface region. Preferably, the height of the valley walls is 1 to 45 μm, more preferably 2 to 40 μm, more preferably 3 to 35 μm, more preferably 4 to 30 μm, even more preferably 5 to 28 μm, and even more preferably 6 to 28 μm.

[0059] The valley walls are defined by the shape of the protrusions adjacent to the valleys. Since these shapes are irregular, the valley walls are also defined by the contour of the valley walls. In this regard, the contour of the valley walls is defined as the unevenness in the direction perpendicular to the valley walls and is calculated based only on the valley wall section. The punctures of the valley walls in the valley walls are excluded from the calculation.

[0060] The contour of the valley walls is expressed as the arithmetic mean of the distances of each point of the valley wall section from the valley walls. Conventionally,

Number

[0061] The contour of the valley wall is from 0 to 40 μm, preferably from 0.1 to 30 μm, more preferably from 0.5 to 15 μm, and even more preferably from 0.8 to 10 μm. When the contour of the valley wall is 0, it indicates that all the protrusions defining the valley wall have a straight line parallel to the valley, as can occur, for example, in the case of square or rectangular protrusions. Of course, depending on the shape of the protrusions, the contours of the first and second valley walls may be different. Although the term "roughness" is usually applied to the surface roughness of a surface, the term "contour" or the contour of the valley wall used here can be regarded as equivalent to the term "roughness" and is determined by the unevenness of the average surface in the vertical direction perpendicular to the surface (perpendicular to the horizontal plane). In this case, the contour of the valley wall is the roughness of the valley wall, i.e., the roughness of the vertical surface provided on the horizontally arranged topography, which is perpendicular to the valley wall and is determined by the unevenness of the average valley wall in the direction parallel to the surface topography.

[0062] The length of the valley wall section may be the same as the length of the protrusion when the longest straight line fitting within the perimeter of the top surface area parallel to the surface is parallel to the valley wall. In other cases, the valley wall section is shorter than the length of the protrusion (or protrusion element). Thus, the length of the valley wall section is from 0.01 to 100 μm, preferably the length of the valley wall section is from 0.05 to 50 μm, and more preferably from 0.1 to 40 μm. Depending on the shape of the protrusions defining the valley, the lengths of the valley wall sections of the two valley walls defining the valley may be the same or different.

[0063] The length of the puncture of the valley wall is generally 0 to 50 μm, preferably 0.5 to 40 μm, more preferably 1 to 30 μm, and even more preferably 2 to 25 μm. The puncture of the valley wall itself defines the valley, and it is arranged perpendicular to the valley of interest. Depending on the shape of the protrusion that defines the valley, the lengths of the punctures of the valley wall in the first and second valley walls may be the same or different.

[0064] The average width of the valley is 0 to 50 μm. The average width of the valley is defined along the average distance between the protrusions or protrusion elements. The valley wall segment on the side facing the valley is divided into line segments of about 0.4 μm, and the distance between each line segment of the valley wall section on one side of the valley and the line segment facing the adjacent protrusion on the opposite side of the (same) valley is measured, and the average is calculated. The average valley width is the average distance between two adjacent protrusions.

[0065] The average width of the valley is preferably 0.5 to 40 μm, more preferably 1 to 30 μm, and even more preferably 2 to 25 μm.

[0066] Alternatively, the valley width may be defined by the shortest and longest distances between the opposing protrusions defined above and the shortest and longest distances between the opposing protrusions on the opposite side of the same valley.

[0067] In a more preferred embodiment, the surface topography comprises individually spaced protrusions that do not contact adjacent protrusions. Therefore, the distance between the protrusions, i.e., the valley width, is greater than 0, and each valley wall comprises a puncture of the valley wall. In this embodiment, a regular pattern of intersecting valleys is obtained. In this embodiment, each valley wall comprises a valley wall section and a puncture of the valley wall.

[0068] At least, it is preferable that the sides of a large number of protrusions or protrusion elements extend substantially perpendicular to the surface at the position where the sides intersect the surface, so it is also preferable that the valley wall is substantially perpendicular to the bottom of the valley. The angle that the valley wall can have with respect to the bottom of the valley is defined to be consistent with the angle that the surrounding side can have with respect to the surface.

[0069] Thus, in the case of a topography that includes only a single type of protrusion including a single protrusion element , all valley wall sections have the same profile (defined by the peripheral side surface of the protrusion adjacent to the valley). In this case, the valley wall sections are arranged alternately with the punctures of the valley wall, and all the punctures have the same length.

[0070] In the case of a topography comprising protrusions including a plurality of protrusion elements, or in the case of a topography including protrusions protruding in different shapes or different directions, a single protrusion defines a plurality of valley wall sections having different profiles and defines a plurality of valley wall sections having punctures of the valley walls that can be of unequal length. In this case, the valley wall sections alternate with the punctures of the valley wall in a regular order.

[0071] object The topography defined above is preferably present on an object having a surface portion including a metal, polymer, composite material or ceramic material. The surface portion may be an outer surface portion or an inner surface portion of the object. The outer surface portion is a surface portion that is located outside the object and can be freely contacted by cells when the cells contact the object. The outer surface portion or the inner surface portion can include holes, pores or depressions.

[0072] Suitable materials are known in the art and include any material suitable for the intended application. That is, to apply the topography of the present invention to, for example, a reaction apparatus, a material suitable for constructing the reaction apparatus should be used. Similarly, to apply the topography of the present invention on an implant, a material suitable for constructing the implant should be used. Those skilled in the art know which type of material is suitable for which type of application and can select an appropriate material accordingly. Examples of suitable materials are titanium (e.g., hard (orthopedic or dental) medical implants), polyurethane (e.g., abdominal meshes and cosmetic implants), and polystyrene (e.g., in vitro culture products).

[0073] The object containing one or more topographies according to the invention can be produced by any known technique for creating specially shaped microstructures on a particular material. The skilled person knows which type of technique is suitable for which 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, etching, laser machining, laser cutting and ablation, (precision) electrochemical machining, (precision) electrochemical gridding, and (precision) electrical discharge machining.

[0074] The articles of the invention are preferably produced by laser machining, precision engineering, engraving, printing, coating, stamping, or etching a topography onto the article. Alternatively, the article may be formed with the topography in a single process, such as injection molding. Suitable techniques for obtaining articles with the described topographies are well known in the art.

[0075] Printing can be accomplished by 3D printing, laser printing, and writing surface protrusions onto the surface of a metal, polymeric, ceramic, composite, or other substrate, as known in the art.

[0076] Coating can be achieved by creating protrusions on a substrate and then coating the surface portion using spraying, sputtering, layer-by-layer coating, electrospinning, or precipitation from solution.

[0077] Stamping involves first producing a mold containing negative projections, then pressing, pressing, and This can be achieved by stamping the mould onto the surface using techniques such as hot pressing, embossing, hot embossing, imprinting and nanoimprinting. Other techniques including injection moulding, processing from the melt or casting are also possible.

[0078] Etching can also be achieved not only by using a suitable mold, but also by using different solvents in the form of liquids and / or vapors such as acids and / or organic solvents to remove a part of the surface portion not protected by the mold in order to obtain a surface topography. Topography can also be engraved on the surface of a suitable material using laser processing, engraving and ablation techniques. Precision techniques can include (precision) electrochemical machining, (precision) electrochemical grinding, (precision) electrical discharge machining.

[0079] In a more preferred embodiment, the topography including regularly patterned protrusions is an inseparable part of the object. This can be achieved by stamping, printing, etching, coating the topography on the object, or using other methods described above, for example, by forming the object in a single process such as injection molding.

[0080] The surface between the protrusions (valley bottoms), the surrounding side surfaces (valley wall sections) and / or the top surface area of the protrusions is smooth or substantially smooth, i.e., it may have a roughness of about 0 (i.e., 0 ± 0.01 μm).

[0081] However, in any embodiment, the surface between the protrusions and / or the top surface area and / or the surrounding side surfaces is 0.01 μm to 10 μm, preferably 0.05 to 8 μm, even more preferably 0.1 to 5 μm. Also, the roughness may be 0.2 to 20 or 0.15 to 3 μm. In this regard, the roughness is the surface roughness obtained by techniques such as etching, blasting or brushing of the surface portion before or after the formation of the topography. Therefore, the contour of the valley wall simplified and calculated by the same formula can be regarded as the roughness of the valley wall perpendicular to its surface.

[0082] The roughness can be determined using atomic force microscopy analysis. A randomly roughened surface portion can be obtained by conventional etching, brushing, blasting, etc. of a surface topography including protrusions of a regular pattern. Alternatively, a flat surface portion such as the surface portion of an implant may be first roughened after the surface topography of the present invention is formed.

[0083] Since it is essential that the randomly roughened surface portion still contains the protrusions of the present invention, the randomly roughened surface portion may not have a roughness greater than the height and / or width of the protrusions so that the protrusions having the size shown somewhere in the specification remain present.

[0084] The topography of the present invention is a surface topography composed of a large number of protrusions arranged at regular intervals. Therefore, the unit cells including the protrusions are distributed over the surface portion so as to provide a single topography as described above. The number 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, and even more preferably at least 1000. A topography extending over a considerable surface portion may have a great influence on a large cell collection function such as a specific tissue. This is considered appropriate for in vitro use, for example, cell culture or tissue growth, or in vivo use such as the creation of an implant.

[0085] The topography of the present invention can regulate cell reactions, particularly, cell population morphogenesis, proliferation, biochemical functions, differentiation, adhesion, migration, signal transduction, and / or cell death by physical stimulation. That is, the topography of the present invention can change the behavior of one or more living cells by inducing changes in morphology, proliferation, biochemical functions, differentiation, adhesion, migration, signal transduction, and / or cell death. The behavior changes by physical stimulation.

[0086] In this context, physical stimuli mean that signals inducing behavioral changes are transferred, among other things, by the shape, hardness and size of the surroundings where the cells are located, i.e., the topography. The "surroundings" of the cells directly regulate the cells. Physical stimuli mean that the surface topography itself changes the cell response in various aspects such as cell morphology, proliferation, biochemical function, differentiation, adhesion, migration, signal transduction, and / or cell death, and this response varies with different topographies. By changing the surface topography in contact with a cell or cell population, cell morphology, proliferation, biochemical function, differentiation, adhesion, migration, signal transduction, and / or cell death are affected while all other parameters are maintained equivalently.

[0087] As an example, cell culture on a single topography with specific parameters can show slight growth, and by changing the topography to a different set of parameters, it can lead to cell culture showing high growth under otherwise equivalent conditions.

[0088] Cells of a cell population are located between the protrusions of the topography, i.e., valleys, and / or at the tops of the protrusions, i.e., the top surface regions of the topography. This maximizes the physical stimuli of the cell population by the topography.

[0089] Physical stimuli involved in the changed behavior are a different effect from chemical stimuli that change behavior. In chemical stimuli, the beginning of the signal received by the cell to change behavior is chemically induced by the added compound or surface chemistry (e.g., the choice of bulk material, coating, or surface functionalization), and then it is transferred by signaling molecules that interact with receptors on the cell surface or signaling molecules that enter the cell to interact with intracellular receptor molecules. The mechanism of physical stimuli that change behavior currently observed is not yet fully understood, but examples show that the shape of surface topography rather than chemical stimuli causes the initiation of the observed regulation of cell morphology, proliferation, biochemical function, differentiation, adhesion, migration, signaling, and / or cell death in a cell population. Therefore, physical stimuli of cells are independent of the chemistry of the surface and / or environment. The changed cell physiological behavior initiated by physical stimuli of surface topography may affect the cell response to and interaction with signaling molecules and receptor molecules.

[0090] In Examples 1 - 7, the data show the effect of materials characterized by various topographies including titanium, polyurethane, and polystyrene on the morphology, proliferation, biochemical function, differentiation, adhesion, migration, signaling, and / or cell death in a cell population by physical stimulation of different cell types, while the same material without topography did not regulate cell behavior similarly.

[0091] Regulation of morphology includes changes in cell shape, such as spreading or elongation, flattening or circularity, changes in nuclear shape, changes in cell or nuclear size, cell surface area, cell length, cell perimeter, cell aspect ratio and eccentricity, adhesion points, and the presence of pseudopods and organization of the cytoskeleton, i.e., organization of actin fibers. In some embodiments, the surface topography of the present invention does not regulate cell morphology, particularly the cell morphology of human mesenchymal stem cells (hMSCs).

[0092] The cytoskeleton of a cell consists of a network of interconnected fibers and tubules, forming the cytoplasmic environment. Actin microfibers are the main filaments in the cytoskeleton of a cell. The reorganization of actin fibers within the cytoskeleton affects the shape and behavior of the cell.

[0093] Pseudopods are extensions of the cytoplasm of a cell and are formed by the cell through the reorganization of actin fibers. Pseudopods are involved in cell functions such as cell movement and nutrient uptake. Focal adhesions are cell structures used for cell attachment to a substrate or the extracellular matrix (ECM). Mechanical forces and chemical signals are transferred to the cell through focal adhesions from the substrate or ECM or other cells. Therefore, the shape of a cell seems to affect cell functions, especially through the reorientation of pseudopods and / or focal adhesions.

[0094] Morphological changes of cells are usually observed by bright-field or immunofluorescence microscopy using immunofluorescence (antibody) labeling or histological staining to visualize the cytoplasm, nucleus, or specific proteins within the cell. Different image analysis software can be used to quantify morphological observations such as CellProfiler.

[0095] The regulation of proliferation includes the following parameters, namely, cell number, growth rate (the ratio of the number of existing cells at a specific time point to the initial number of cells attached or seeded on the substrate, or the time required for doubling of the cell population), the number of cell colonies and the number of cells within such colonies, the culture density of the cell layer on the substrate, and the increase or decrease in proliferation or mitosis indicated by cell-cell binding.

[0096] Proliferation parameters can be measured by imaging cell survival or the final stage, regardless of the presence or absence of staining of cell nuclei and / or cytoplasm and / or cell membrane components, and by quantifying the number of cells using appropriate image analysis software such as CellProfiler. Other techniques such as lysing the cells, measuring the amount of DNA in the lysate, measuring the metabolic activity of the cells, separating the cells from the substrate, and counting them using a manual or automated cell counter can also be used to quantify cell proliferation.

[0097] In some embodiments, the surface topography of the present invention does not induce the regulation of the proliferation of human induced pluripotent stem cells (iPSCs) in particular.

[0098] Regulation of biochemical functions includes all chemical processes and reactions that occur within or in relation to cells. These processes include biosynthesis and cell metabolism, protein synthesis, protein transcription and protein secretion, enzyme reactions and enzyme expression, biochemical transport via membrane channels and receptors, cell signaling, and the immune response of cells.

[0099] Regulation of differentiation includes differentiation into specific cell types, maintenance of differentiated or undifferentiated phenotypes, maintenance of specific functions, dedifferentiation from differentiated cell types, and redifferentiation into another cell type.

[0100] The change of stem cells or progenitor cells into more differentiated cell types is called differentiation. Differentiated cells express specific protein markers and functions. Differentiated cells maintain the same phenotype, lose their differentiated phenotype, change into undifferentiated cells (dedifferentiation), or change into another differentiated cell type (redifferentiation).

[0101] Various known molecular biology techniques are used to analyze cell biochemical functions and differentiation states. For example, cell differentiation is determined using (combined) immunofluorescent labeling (antibody labeling) of differentiation-specific proteins in combination with fluorescence microscopy or fluorescence-activated cell sorting (FACS). It can be characterized by imaging the cells. Other techniques can be used to quantify the expression of specific cell type markers at the protein or gene level, including quantitative polymerase chain reaction (qPCR) or enzyme-linked immunosorbent assay (ELISA).

[0102] The regulation of cell adhesion includes an increase or decrease in the number of cells attached to the substrate, the formation of tight junctions between cells, the formation of focal adhesions, the secretion of proteins involved in cell adhesion, the detachment of cells from the surface, the separation of cells from each other, and the strength of cell attachment to the surface, etc.

[0103] The regulation of cell adhesion can be measured by immunofluorescent labeling (bright field, fluorescence or scanning electron) microscopy techniques of cytoplasmic or focal adhesion proteins and imaging of cells, lysis of cells and measurement of the amount of DNA in the lysate, measurement of the metabolic activity of cells, detachment of cells from the substrate, and counting of cells using a manual or automatic cell counter, etc. The regulation of cell adhesion is important in any application involving adherent cells. Cell adhesion is one of the major steps in cell interactions with biomaterials and basically affects all future interactions between cells and biomaterials. For example, a biomaterial that promotes the adhesion of macrophages can affect the level of encapsulation by fibrous tissue. A biomaterial with lower platelet adhesion can suppress blood clotting.

[0104] The regulation of cell migration includes the (microscopic) movement of cells in one area towards different areas, or the movement of cells migrating to different areas.

[0105] The regulation of cell migration can be measured by live imaging, time-lapse microscopy, fluorescent labeling, etc. The regulation of cell migration can affect all states of tissue development such as embryo and adult tissue formation, tissue regeneration and turnover, development of therapeutic strategies, healing of wounds and defects, and immune responses.

[0106] The regulation of cell signaling involves adjusting any physical or chemical interactions between cells (cell-cell interactions), gene expression, protein production, and secretion.

[0107] The regulation of cell signaling can be measured by quantifying biomarkers expressed or secreted from live cells using techniques such as immunofluorescence staining, qPCR, ELISA, Western blot, etc.

[0108] The regulation of cell signaling can affect all processes that govern cell fate and responses to foreign biomaterials, including morphology, proliferation, biochemical function, differentiation, attachment, migration, and cell death.

[0109] The regulation of cell death includes increases or decreases in apoptosis (programmed cell death), necrosis, mitotic catastrophe, or cell component death (autophagy).

[0110] The regulation of cell death, such as apoptosis, can be measured by viability assays, cell counting, immunofluorescence staining of apoptosis markers, etc.

[0111] The regulation of cell death, such as apoptosis or necrosis, can affect systems developed to support the culture and growth of live cells or to develop therapeutic methods for disease and cancer treatment.

[0112] In a preferred embodiment, the regulation of cell morphology, proliferation, biochemical function, differentiation, attachment, migration, signaling, and / or cell death of a cell population by physical stimuli is cell morphology, proliferation , including the regulation of biochemical functions, differentiation, and adhesion. In preferred embodiments, the regulation of cell response includes the regulation of cell morphology. In other preferred embodiments, the regulation of cell response includes the regulation of cell proliferation. In other preferred embodiments, the regulation of cell response includes the regulation of the biochemical functions of cells. In other preferred embodiments, the regulation of cell response includes the regulation of cell differentiation. In other preferred embodiments, the regulation of cell response includes the regulation of cell adhesion.

[0113] In some embodiments, the regulation of cell morphology, proliferation, biochemical function, differentiation, adhesion, migration, signaling, and / or cell death of a cell population by physical stimuli does not include physically stimulating human mesenchymal stem cells (hMSCs) to regulate differentiation, most notably inhibiting adipogenic differentiation. In other embodiments, such regulation does not include physically stimulating kidney epithelial cells by regulating cell adhesion, proliferation, and morphology, or by regulating cell morphology, adhesion, and function. In still other embodiments, such stimulation does not include physically stimulating induced pluripotent stem cells (iPSCs) to maintain pluripotent proliferation in vitro.

[0114] The cell population that can be used in the topography of the present invention is not particularly limited. The cell population comprises one or more living cells in a suitable medium as needed. The cell population may comprise a single cell type, but can also comprise multiple cell types in cell co-culture or biological tissue, etc.

[0115] The cell population is a population of eukaryotic cells such as those derived from humans, plants, animals, protists, yeast, and fungi. The cells can be from any plant, microorganism, or animal and can be of any type.

[0116] In some embodiments, the cells are not human iPSCs. In other embodiments, the cells are not hMSCs.

[0117] Mammalian cells, particularly cells of humans, monkeys, cows, pigs, rats or mice, are preferred. More preferably, the mammalian cells include mesenchymal stem cells, adipose-derived stem cells, osteoblasts, and immune response-related cells, preferably contained in macrophages and hepatocytes.

[0118] Cells can be obtained by known methods such as isolation from the tissue of a living or dead donor, cell culture, differentiating cell types with higher efficacy, dedifferentiating from cell types with a more advanced differentiation state, or redifferentiating from cell types with different differentiation states. Cell isolation or cytodiagnosis from tissue involves digestion of the extracellular matrix of cells by one or more enzymes, such as collagenase. In most cases, since the tissue consists of different cell types or cell populations, generally the next step after cell isolation includes purification of the cells. Other techniques can be used to obtain cells according to the cell type. For example, red blood cells can be obtained by centrifuging whole blood and collecting the separated layers containing different types of cells.

[0119] The substances of the present invention can be used together with a suitable culture medium, as is known in the art. Such a culture medium contains water and may further contain proteins such as fetal bovine serum (FBS) and growth factors, amino acids, vitamins, glucose, inorganic salts, and antibiotics. For applying the substances of the present invention in vitro, the use of a suitable culture medium is preferred. Under other conditions, the cells can be cultured on gels such as Matrigel or agarose gel, on coatings such as collagen, laminin, fibronectin, fibrinogen, or polyamine, in other liquids such as phosphate-buffered saline (PBS), water, or embedded in biomaterials such as hydrogels or polymer carriers. The cells can be fixed using a fixing solution and analyzed under wet or dry conditions.

[0120] The present invention further provides a method for regulating the morphology, proliferation, biochemical function, differentiation, adhesion, migration, signal transduction, and / or cell death of a cell population by physical stimuli, 1) contacting one or more cells in a suitable medium with a surface portion having a regular pattern of protrusions, the regular pattern of protrusions being defined by a grid of intersecting grid lines that can be placed on the surface portion, wherein the grid lines define a pattern of unit cells, each unit cell having at most one protrusion, the protrusion having one or more protrusion elements, the protrusion elements being defined as a surface portion raised above the surface having a top surface region and a peripheral side surface connecting the top surface region to the surface, each protrusion element having a maximum height of 0.5 to 50 μm on the surface, a) the average distance between adjacent protrusions is 0 to 50 μm, b) the area of the top surface region of the protrusion is 1 to 6000 μm 2 and c) the protrusions cover 3 to 90% of the surface, the step of, 2) enabling the cells to respond to the surface, and a method comprising the steps of.

[0121] The method of the present invention can be adapted to each of the specific applications described below. The method of the present invention can be an in vivo method, but is preferably an in vitro method.

[0122] The article of the present invention can be used in vivo or in vitro. In one embodiment, the article of the present invention modulates the morphology, proliferation, biochemical function, differentiation, adhesion, migration, signal transduction, and / or cell death of a cell population by physical stimulation in vitro. In vitro use includes, for example, culturing cells on a surface portion having the topography defined above. In this embodiment, the cells cultured on or in the article of the present invention are physically stimulated, directly or indirectly, to change their behavior.

[0123] For example, the article may be an article for cell culture having a surface for cell culture, such as a culture flask, plate, dish, slide, bottle, chamber or bag, and the surface is provided with the topography defined above.

[0124] The topography for articles for in vitro use can be of various sizes. For example, a culture flask has a topographic feature surface with a surface area of 5 to 10,000 cm 2 , preferably 10 to 5,000 cm 2 , more preferably 15 to 1,000 cm 2 , and the topographic feature surface present in the well of a culture plate usually has a surface area of 0.01 to 10 cm per well. 2

[0125] The present invention further relates to the use of the article of the present invention for regulating in vitro the morphology, proliferation, biochemical function, differentiation, adhesion, migration, signal transduction, and / or cell death of a cell population by physical stimulation. The article can be adapted for use for each specific application described below.

[0126] In another embodiment, the article of the present invention is adapted for in vivo use. Such an embodiment is preferably an implant having a topography for regulating cell biochemical function, cell adhesion or differentiation by physical stimulation, as described above.

[0127] In one embodiment of in vivo use, the present invention includes a method of treating a patient by applying the article defined above for regulating the morphology, proliferation, biochemical function, differentiation, adhesion, migration, signal transduction, and / or cell death of a cell population by physical stimulation. Such a method can be based on an article having a topography for each specific application purpose described below.

[0128] In another embodiment of in vivo use, the present invention relates to the use of the above-defined substances in the regulation of the morphology, proliferation, biochemical function, differentiation, adhesion, migration, signal transduction, and / or cell death of cell populations by physical stimuli. The substances may be for use in any of the specific applications described below.

[0129] In a further embodiment of in vivo use, the present invention relates to the use of the above-defined substances in the regulation of the morphology, proliferation, biochemical function, differentiation, adhesion, migration, signal transduction, and / or cell death of cell populations by physical stimuli. This substance may be for use in any of the specific applications described below.

[0130] In yet another preferred embodiment of in vivo use, the present invention relates to the use of the above-defined substances in the manufacture of agents for the regulation of the morphology, proliferation, biochemical function, differentiation, adhesion, migration, signal transduction, and / or cell death of cell populations by physical stimuli. The agents can target any of the specific applications described below.

[0131] All of the aspects and parameters generally defined above for the present invention can be combined with other aspects and parameters defined above. Further, each of the generally defined aspects and parameters defined above applies to each of the specific applications defined below, unless that aspect or parameter is specifically described for the application purpose using different terminology or parameters than those used above. In such a case, the aspects and parameters defined for the following specific application purposes supersede the general definition of the same aspect or parameter defined above.

[0132] Embodiments for Specific Applications of Cell Responses In one embodiment, the object of the present invention has, on its surface portion, preferably one, but not exceeding 10, different topographies. That is, in this embodiment, the object of the present invention has at most 10, preferably at most 9, more preferably at most 8, more preferably at most 7, more preferably at most 6, more preferably at most 5, more preferably at most 4, more preferably at most 3, more preferably at most 2, and most preferably one topography. In this embodiment, the topography can have a length, width or diameter of 1 μm to 10 m. The surface area of such a topography can be 1 μm 2 ~100 m 2 and can be.

[0133] In this embodiment, for a specific application, one (or at most a few) selected topographies are selected to promote cell response by physical stimulation. The topography can have any shape. Applications include, but are not limited to, objects for cell culture such as culture ware or culture platforms (e.g., chips or microfluidic devices), bioreactors, membranes or implants. Those skilled in the art can conceive of countless ways to apply the topographies of the present invention.

[0134] The advantages of the present invention are not limited to the precise regulation of cell response, physical stimulation without the need to use growth factors or (protein) coatings, the flexibility of the approach applied to different materials and products, but there are no leaching or layer defects, and no batch-to-batch differences.

[0135] In another embodiment for a specific application of cell response, the object of the present invention is adapted for in vivo use. Such use can comprise an object having a topography that is implanted or temporarily inserted into the body. In this case, the object is preferably an implant. Accordingly, the present invention further relates to an object as defined above for use as an implant. as defined.

[0136] The implant may be a temporary implant or a permanent implant. A temporary implant is an implant that remains in the body for a limited duration, known in the art, and can be removed from the body after serving its purpose or can slowly degrade within the body. A permanent implant is, in principle, an implant that should remain in a given position indefinitely, but is susceptible to the effects of lifespan factors (e.g., wear) of the implant in question.

[0137] The present invention further relates to a method of treating a subject in need of an implant using an implant comprising the topography defined above. The implant of the present invention can be, for example, an orthopedic or dental implant, a liver implant, or an implant for controlling an immune response. Other implants of the present invention can be muscle implants, nervous system implants, spinal cord stimulation implants, electroacoustic implants, deep brain stimulators, pacemaker implants, cosmetic implants, breast reconstruction implants, electrical stimulation implants including (abdominal) mesh implants. Such implants require appropriate regulation of the immune response.

[0138] In an embodiment of the present invention that is an implant, it is a clear advantage that the implant with the surface topography of the present invention regulates cells to provide a desired response. This enables, for example, regulated ingrowth of surrounding tissue, regulation of differentiation of surrounding cells, or regulated cell attachment. In the case of orthopedic or dental implants, the advantage is that it can increase new bone formation / ingrowth that results in better fixation of the implant within or onto bone or dental tissue.

[0139] Alternatively, this results in advantages such as, for example, enabling a reduction in the immune response to the implant and reducing problematic encapsulation of the implant or rejection of the implant by fibrous tissue. Alternatively still, this enables an increase in the adhesion and proliferation of endothelial cells, which has the advantage of, for example, having a functional monolayer of endothelial cells on the surface of the implant to avoid blood clotting and thrombosis. Alternatively still, this enables an increase in proliferation and maintenance of the hepatocyte phenotype, which has the advantage of increasing the long-term survival rate and functionality of the cells.

[0140] The implant of the present invention can be produced by any of the above techniques, as is known in the art. Examples of suitable techniques for providing to those having topography are described above.

[0141] For each of the specific application purposes defined below, a topography comprising protrusions having a specific shape is described as a "hit" topography. These topographies are shown in the table in the Examples section. Each aspect and parameter listed under the above general description and / or under the specific application purposes described below can be applied to each hit topography, unless otherwise stated.

[0142] Hit topographies reflect the protrusion shape that has the optimal effect for their application purpose. However, for each application purpose, it is reasonable to assume that the effect of the topography is the same for topographies having a shape similar to that of the hit topography. Whether the shapes are similar is essentially determined by visual comparison. As is known in the art, the center point of the protrusion can be numerically represented by mathematically defining it. The distance from that center point to a specific point on the surrounding side surface is called the radius facing that specific point. The shape is obtained by aligning the center points of the compared shapes to match the shape of the hit topography as closely as possible When similar shapes are superimposed by orienting the shape as such, the radius of the similar shape deviates from the radius of the hit topography by 10% or less, preferably 5% or less, more preferably 2.5% or less, and even more preferably 1% or less at any point on the peripheral side surface.

[0143] Hepatocytes In one embodiment, the substance of the present invention can be a substance for regulating hepatocytes. In this context, hepatocytes are defined as the main cells found in liver tissue, are involved in most of the biochemical functions of the liver, and as described above, can be derived from any human or animal, including rhesus monkeys, mice, etc. Hepatocytes in this context also refer to hepatocyte progenitor cells and hepatocyte-like cells (immortalized cell lines such as Hep G2).

[0144] In this embodiment, the topography is a) The average distance between adjacent protrusions is 0 to 50 μm, preferably 0.5 to 40 μm, more preferably 1 to 30 μm, and even more preferably 3 to 25 μm, b) The area of the top surface region of the protrusion is 1 to 6000 μm 2 , preferably 10 to 3000 μm 2 , preferably 15 to 1500 μm 2 , more preferably 17 to 1000 μm 2 , even more preferably 20 to 250 μm 2 , c) The protrusions cover 3 to 90% of the surface part, preferably 5 to 50%, more preferably 7 to 40%, and even more preferably 8 to 35%.

[0145] Furthermore, the topography can be defined by the length of the protrusion / protrusion element being 0.01 to 100 μm, preferably 0.5 to 50 μm, more preferably 1 to 40 μm, and even more preferably 2 to 35 μm, and the width of the protrusion / protrusion element being 0.01 to 100 μm, preferably 0.5 to 50 μm, more preferably 1 to 35 μm, and even more preferably 4 to 23 μm.

[0146] Alternatively, the substance for regulating hepatocytes of the present invention is a) The height of the valley wall, defined as the distance from the bottom of the valley to the top surface region, the perpendicular distance to the bottom of the valley, is 0.5 to 50 μm, preferably 1 to 40 μm, more preferably 2 to 35 μm, even more preferably 4 to 30 μm, and even more preferably 5 to 28 μm. b) The profiles of the first and second valley walls are, independently, 0 to 40 μm, preferably 0.1 to 35 μm, more preferably 0.5 to 30 μm, and even more preferably 1 to 20 μm. c) The lengths of the first and second valley wall sections are, independently, 0.01 to 100 μm, preferably 0.1 to 50 μm, more preferably 1 to 40 μm, and even more preferably 2 to 32 μm. d) If there are punctures in the first and second valley walls, their lengths are, independently, 0 to 50, preferably 0.2 to 40, more preferably 0.5 to 35 μm. e) The average width of the valley is 0 to 50 μm, preferably 0.5 to 40 μm, more preferably 1 to 30 μm, and even more preferably 3 to 25 μm.

[0147] Accordingly, the present invention also relates to a method for regulating the morphology, proliferation, biochemical function, differentiation, adhesion, migration, signal transduction, and / or cell death of a cell population, wherein the living cells are hepatocytes, the topography is as defined above, and by the physical stimuli defined above.

[0148] Any material is used to create an object, but preferably, the object, or at least its surface portion having topography, is made of glass, metal, or a polymer material, and even more preferably a polymer material. Most preferably, the object includes a surface portion having a hydrogel such as polystyrene, polypropylene, collagen, laminin, polyamine, and Matrigel, and an agarose gel.

[0149] The culture medium suitable for this embodiment includes, but is not limited to, one or more components such as water, glucose, protein, amino acids, vitamins, penicillin, antibiotics, and inorganic salts.

[0150] The regulation of hepatocytes includes the stimulation of hepatocyte-like morphology, the proliferation of hepatocytes in short time and / or more cycles, the long-term maintenance of hepatocytes in culture while retaining the phenotype and hepatocyte-specific functionality, the stimulation of stem cell adhesion at early and late points, and the prevention of early cell death. This functionality can be confirmed by the evaluation of functional biomarkers of hepatocytes, including detoxification markers such as cytochrome protein (CYP), albumin, E-cadherin, CD81, etc. The expression of these markers can be evaluated at the gene and protein levels using various techniques such as qPCR, ELISA, immunofluorescence staining, Western blot, and fluorescence-activated cell sorting (FACS).

[0151] A group of surface topographies has been identified that can support the in vitro culture of functional hepatocytes over a long period while retaining their phenotypes. These topographies were embedded in a polystyrene (PS) substrate and used with or without the addition of a collagen coating, and the interaction with hepatocytes was investigated using conventional cell culture techniques.

[0152] Samples featuring the selected topography enabled higher cell attachment compared to samples without such surface topography. Hepatocytes of rhesus monkey and human origin could be maintained in in vitro culture for at least one month when cultured on topography-embedded substrates with both PS with a collagen coating on the surface and PS without a collagen coating on the surface. This is in contrast to the current gold standard, i.e., the collagen sandwich culture (a double layer of collagen in which hepatocytes are cultured), which can support the in vitro culture of hepatocytes for a maximum of 8 - 10 days. Hepatocytes cultured on substrates embedded with topography were characterized for functionality and were found to be fully functional, i.e., the appropriate expression of hepatocyte-specific markers including more than 10 detoxification and metabolic activity markers. Hepatocytes cultured on topography-embedded substrates without a collagen coating expressed equivalent or higher levels of the analyzed hepatocyte-specific markers compared to topography-embedded substrates with a collagen coating, demonstrating that the selected topography obviates the need for a collagen coating that is expensive, cumbersome, and prone to failure. Similarly, regardless of the presence or absence of a collagen coating, hepatocytes seeded on topography-embedded substrates were successfully infected with Plasmodium falciparum and maintained in culture for one month, indicating the effectiveness of the surface topography in supporting the in vitro culture of hepatocytes under pathological conditions.

[0153] In another embodiment, the hepatocytes are regulated in vivo. In this embodiment, the article of the present invention can be a liver implant. In this embodiment, the average distance, the area of the top surface region, the coverage rate, the length and width, which are parameters in the definition of "protrusion", and the height of the valley wall, the contour of the valley wall, the section of the valley wall and the length of the puncture, and the average width of the valley are as defined above for the in vitro culture of hepatocytes.

[0154] The liver implant according to the present invention can have at least one surface part with a defined surface topography and can be a thing capable of providing hepatocytes. Preferably, the implant further comprises means for retaining hepatocytes on the surface part having the topography.

[0155] Suitable means for retaining hepatocytes on the surface part having the topography include, for example, a hydrogel layer, a polymer layer, a protein coating, and the like. Preferably, such a layer allows the diffusion of nutrients and other essential components for the cells to function in vivo to reach the hepatocytes on the thing, and / or results in better cell adhesion to the surface.

[0156] The liver implant can comprise a plurality of, for example, up to 10 surface topographies on the surface part to regulate the biological responses of hepatocytes and other types of cells involved in the function of the liver.

[0157] The liver implant can also be a 3D scaffold, preferably made of a material as defined above, porous, and include one or more surface topographies on the surface part, providing a suitable environment for hepatocyte growth and tissue formation.

[0158] The advantage of the liver implant having the topography defined above is to restore or enhance the function of the liver, including the metabolism and detoxification of biochemical compounds produced by the body or introduced into the body such as drugs.

[0159] Further uses of the topography of the present invention for regulating hepatocytes include, for example, artificial livers, 3D scaffolds for liver regeneration, drug screening platforms, and biosensors. These devices can be manufactured using different techniques as described above and can be provided with 1 to 10 regulatory surface topographies on the surface portion. For in vivo use, the article can be introduced into the body with or without cells, such as cells from a donor, autologous cells cultured in an experimental environment, or cells from different sources.

[0160] Bone formation In this embodiment, the article of the present invention is an orthopedic or dental implant that regulates the cell response of a cell population, most notably morphology, proliferation, biochemical function, differentiation, adhesion, migration, signaling, and / or cell death, by physical stimuli being the regulation of the biochemical functions, differentiation, and adhesion of bone formation. The cells whose cell response is regulated in this embodiment are preferably osteoblasts.

[0161] This embodiment relates to articles such as medical devices, preferably implants, that stimulate bone formation for transplantation into or onto human or animal bone or teeth, which are artificial replacements for natural tissues. The implant, which is a biomedical implant, is preferably implanted into or onto human or animal bone or teeth. In one preferred embodiment, the implant is implanted into human bone or teeth. In another preferred embodiment, the implant is implanted into animal bone or teeth.

[0162] Bone is a natural mineralized structure synthesized by osteoblasts and has the function of providing strength and support to the body of vertebrates. Bone also firmly supports muscle attachment to enable body movement.

[0163] Bone is generally said to exist in two types. Cortical bone tissue is the hard outer layer of bone and has a smooth, white, and shiny appearance. It consists of a high-density network of microscopic columns where osteoblasts and mineralized tissue are present.

[0164] Spongy bone, also known as cancellous bone, is found inside bones and consists of a more or less porous network of bone tissue. In the spaces between the bone tissues, bone marrow and stem cells are present along with blood vessels to supply nutrients to the living cells inside the bone.

[0165] Teeth are small structures made of highly mineralized tissues found in the jaws of humans and other vertebrates and play important roles in chewing and speaking. Teeth have two parts, the inner and outer sides of the gums called the root and the crown respectively. The tissues of the teeth include dentin, which is the layer under the enamel made from living cells in a hard inorganic matrix, the hardest and outermost layer of the teeth, enamel, which is mainly composed of calcium phosphate, the softer internal structure of the teeth containing living cells, blood vessels and nerves, cementum, a layer that firmly connects the roots of the teeth to the gums and jaws, and finally the periodontal ligament that is also involved in holding the teeth in the jawbone.

[0166] Bone tissue and tooth tissue, like most other tissue types, are continuously formed and decomposed. Due to the mineralization environment, the rate of formation and decomposition of bone and tooth tissues is relatively slow compared to most other tissue types. Therefore, when defects occur, the regeneration of bone or tooth tissue takes longer than the regeneration of other tissue types. Furthermore, when large segments of bone or teeth are defective or missing, regeneration may not be possible.

[0167] In such cases, the implant according to the present invention may be implanted into or onto the bone or tooth. This affects the restoration of the natural function of the body part. Preferably, in the case of a bone implant, the implant is implanted at least partially within the cortical bone. A dental implant is preferably implanted into the jawbone or tooth tissue. The implant of the present invention according to its specific topography defined below stimulates bone formation and, together with it, stimulates the fixation of the implant into the bone or tooth by in-growth of the implant tissue.

[0168] Intra-tissue growth in this context is understood to mean the process of bone or tooth formation on and / or within the implant, preferably at the interface where the natural bone or tooth tissue and the implant bind. When an implant is implanted into or onto natural bone or teeth, there is a small gap at this interface, which is filled over time by natural bone or tooth tissue formed by cells such as osteoblasts, ameloblasts, odontoblasts or cementoblasts present in the natural bone or tooth tissue within the gap at the interface with the implant. Thus, intra-tissue growth is the growth of natural bone or tooth tissue onto or within the implant, fixing the implant onto or within the bone or tooth tissue into which the implant is implanted.

[0169] The topography of the present invention stimulates bone formation and preferably, together therewith, increases the fixation of the implant within or onto bone or tooth tissue. The increase in fixation force means that the process of bone formation in the gap is accelerated and / or the strength with which the implant is fixed into or onto the bone or tooth is increased. Also, the increase in fixation force can mean that the fixation between the surrounding bone or tooth tissue and the implant, brought about by the intra-tissue growth of the natural tissue, can be maintained longer to avoid or delay replacement of the implant.

[0170] The increase in bone formation is measured by various methods such as, for example, analyzing the amounts of alkaline phosphatase enzyme (ALP), osteocalcin (OC), osteopontin (OP) and / or bone sialoprotein (BSP) present in stem cells growing on the topography in vitro.

[0171] The fixation force can be measured by performing in vivo tests before measuring the force required to pull the implant out of the implant site.

[0172] The topography of the present invention is present in objects such as medical devices for stimulating bone formation, preferably in implants that are implanted into or onto bone or teeth at locations where ingrowth of tissue is desired. Preferably, all positions of the implant that would benefit from increased fixation to natural bone or tooth tissue are covered by the topography of the present invention.

[0173] In a preferred embodiment, the device of the present invention is implanted into or onto human or animal bone, preferably into or onto human bone. In this case, the implant is referred to as an orthopedic implant.

[0174] In an alternative preferred embodiment, the device of the present invention is implanted into or onto the jawbone or teeth of a human or animal, preferably into or onto the jawbone or teeth of a human. In this case, the implant is referred to as a dental implant.

[0175] In this embodiment, the topography present in the object of the present invention can be defined as follows: a) The average distance between adjacent protrusions is from 0 to 50 μm, preferably from 0.5 to 40 μm, even more preferably from 1 to 30 μm, even more preferably from 2 to 25 μm; b) The area of the top surface region of the protrusion is from 1 to 6000 μm 2 , preferably from 10 to 3000 μm 2 , more preferably from 20 to 1500 μm 2 , more preferably from 25 to 1000 μm 2 , even more preferably from 30 to 750 μm 2 ; and c) The protrusions cover from 3 to 80% of the surface portion, preferably from 10 to 75% of the surface portion, more preferably from 20 to 70% of the surface portion, more preferably from 30 to 65% of the surface portion.

[0176] In addition, the length of the protrusion in a topography where the protrusion comprises only one protrusion element is 0.01 to 100 μm, preferably 0.5 to 50 μm, more preferably 1 to 40 μm, and the length of the protrusion in a topography where the protrusion comprises a plurality of protrusion elements can be 0.01 to 100 μm, preferably 0.1 to 45 μm, more preferably 0.5 to 40 μm, and even more preferably 1 to 30 μm.

[0177] The width of the protrusion in a topography where the protrusion comprises only one protrusion element is 0.01 to 100 μm, preferably 0.5 to 50 μm, more preferably 1 to 40 μm, and the width of the protrusion in a topography where the protrusion comprises a plurality of protrusion elements can be 0.01 to 100 μm, preferably 0.1 to 45 μm, more preferably 0.5 to 40 μm, and even more preferably 1 to 30 μm.

[0178] Alternatively, an orthopedic or dental implant can be defined as an article having at least one topography, a) The height of the valley wall is 0.5 to 50 μm, preferably 1 to 40 μm, more preferably 2 to 35 μm, more preferably 4 to 30 μm, and even more preferably 5 to 28 μm, b) The contour of the valley wall is 0 to 40 μm, preferably 0.1 to 30 μm, more preferably 0.5 to 15 μm, and even more preferably 0.8 to 10 μm, c) The length of the valley wall section is 0.01 to 100 μm, preferably 0.05 to 50 μm, and more preferably 0.1 to 40 μm, d) The length of the valley wall puncture is 0 to 50 μm, preferably 0.5 to 40 μm, more preferably 1 to 30 μm, and even more preferably 2 to 25 μm, e) The average width of the valley is 0 to 50 μm, preferably 0.5 to 40 μm, more preferably 1 to 30 μm, and more preferably 2 to 25 μm.

[0179] The advantage of the orthopedic or dental implant according to the present invention is to improve the fixation of the implant to the surrounding natural (bone and tooth) tissues.

[0180] Orthopedic implants are medical devices manufactured to replace a missing joint or bone or to support damaged bone. Examples of suitable orthopedic implants that can benefit from including the surface topography as presently described include knee replacement implants, femur components and tibial stem plates, (total) hip replacement implant femoral stems and acetabular shells, elbow and finger implant stems and hinges, craniofacial implants, skull implants, shoulder implants, ankle implants, nails, screws, staples, (external) fixation implants such as rods and plates, spinal cages, spinal plates, pedicle screws and rods used in injectable spinal reconstruction, and cervical plates. Preferably, the orthopedic implants include knee replacement implants, femur components and tibial stem plates, (total) hip replacement implant femoral stems and acetabular shells, and implants used in injectable spinal reconstruction including spinal cages, spinal plates, pedicle screws and rods and cervical vertebra plates.

[0181]

[0182] Dental implants are surgical components that come into contact with the bone of the jaw or skull to support dental prostheses such as crowns, bridges, dentures, facial prostheses, or function as orthodontic anchors. Examples of suitable dental implants that can benefit from including the presently described topography include jaw and craniofacial implants, dental plates and frameworks, dental base screws and post screws, crown implants. Preferably, the dental implants are dental plates and frameworks, dental base screws or post screws.

[0183] The materials for regulating the above-described bone-forming biochemical functions, differentiation, and adhesion can be made by any known method for the manufacture of implants. Such methods are generally described above and are known in the art.

[0184] Immune control mechanism In another embodiment, the regulation of the morphology, proliferation, biochemical function, differentiation, adhesion, migration, signal transduction, and / or cell death of a cell population by physical stimuli includes an immune control mechanism. The immune control mechanism in this context means regulating the immune response to foreign substances transplanted into or present in the body, i.e., foreign body reactions. The immune control mechanism includes controlling the formation of foreign body giant cells (FBGCs), inflammatory reactions, macrophage involvement, fibrosis, and encapsulation of foreign bodies. Thus, the present invention further provides a substance for the immune control mechanism of immune cells, and the topography is as defined above.

[0185] The immune response has an immediate response (at the early stage) followed by a more defined and stable final stage. At the early stage, the acute reaction is very important in controlling the acceptance of foreign substances (biocompatibility) and is also an important driver for the (chronic) immune activity level at the final stage. The first reaction is thought to need to be at an appropriate but controlled level in order to allow for appropriate but minimal encapsulation and an inactive (non-inflammatory) final stage. However, the exact mechanism is not yet fully understood. In a preferred embodiment, three classes of immune control topographies can be defined: (1) combining a low immune response at the early stage with a low response at the later stage, hereinafter referred to as L / L, which means reducing the immune response. (2) Combining a high immune response at the early stage with a high response at the later stage, hereinafter referred to as H / H, which means increasing the immune response. (3) Combining a high immune response at the early stage with a low response at the later stage, hereinafter referred to as H / L.

[0186] In a preferred embodiment, the L / L type of topography for regulating the immune response can be defined as follows: a) The average distance between adjacent protrusions is 0 to 50 μm, preferably 0.5 to 40 μm, more preferably 1 to 20 μm, and even more preferably 2 to 12 μm, and b) The area of the top surface region of the protrusion is 1 to 6000 μm2 Preferably 10 to 3000 μm 2 More preferably 15 to 1500 μm 2 Even more preferably 20 to 1000 μm 2 Even more preferably 20 to 200 μm 2 Most preferably 25 to 200 μm 2 and c) The protrusion covers 3 to 90%, preferably 5 to 80%, more preferably 10 to 75%, and even more preferably 26 to 50% of the surface portion.

[0187] Also, the length of the protrusion in a topography where the protrusion has only one protrusion element is 0.01 to 100 μm, preferably 0.5 to 50 μm, more preferably 1 to 50 μm, and even more preferably 2 to 40 μm, and the length of the protrusion in a topography where the protrusion has a plurality of protrusion elements can be 0.01 to 100 μm, preferably 0.1 to 45 μm, more preferably 0.5 to 40 μm, and even more preferably 1 to 30 μm.

[0188] The width of the protrusion in a topography where the protrusion has only one protrusion element is 0.01 to 100 μm, preferably 0.5 to 50 μm, more preferably 1 to 50 μm, and even more preferably 2 to 40 μm, and the width of the protrusion in a topography where the protrusion has a plurality of protrusion elements can be 0.01 to 100 μm, preferably 0.1 to 45 μm, more preferably 0.5 to 40 μm, and even more preferably 1 to 30 μm.

[0189] Alternatively, the L / L type immunomodulatory implant can be defined as an article having at least one topography, a) The height of the valley wall is 0.5 to 50 μm, preferably 1 to 40 μm, more preferably 2 to 35 μm, even more preferably 4 to 30 μm, and even more preferably 5 to 28 μm, b) The contour of the valley wall is 0 to 40 μm, preferably 0.1 to 35 μm, more preferably 0.5 to 30 μm, and even more preferably 1 to 20 μm, c) The length of the valley wall section is from 0.01 to 100 μm, preferably from 0.1 to 50 μm, more preferably from 1 to 40 μm, and even more preferably from 2 to 32 μm, d) The length of the puncture of the valley wall is from 0 to 50 μm, preferably from 0.2 to 40 μm, more preferably from 0.5 to 35 μm, e) The average width of the valleys is from 0 to 50 μm, preferably from 0.5 to 40 μm, more preferably from 1 to 20 μm, and even more preferably from 2 to 12 μm.

[0190] Such topography can reduce the immune response to foreign bodies and preferably can minimize the encapsulating tissue and inflammatory activity around the foreign bodies.

[0191] Furthermore, the article of the present invention is used to stimulate an immune response to a foreign body and preferably can be used to enhance the controlled encapsulation of the foreign body.

[0192] The topography that can be used in this embodiment is an H / H type topography, which can be defined as follows: a) The average distance between adjacent protrusions is from 0 to 50 μm, preferably from 0.5 to 40 μm, more preferably from 2 to 20 μm, and even more preferably from 5 to 12 μm, and b) The area of the top surface region of the protrusion is from 1 to 6000 μm 2 preferably from 10 to 3000 μm 2 more preferably from 20 to 1000 μm 2 more preferably from 25 to 250 μm 2 even more preferably from 20 to 70 μm 2 and c) The protrusions cover 3 to 90% of the surface part, preferably 5 to 50%, more preferably 10 to 40%, and even more preferably 15 to 25%.

[0193] In addition, the length of the protrusion in the topography where the protrusion comprises only one protrusion element is 0.01 to 100 μm, preferably 0.5 to 50 μm, more preferably 1 to 50 μm, and even more preferably 2 to 40 μm. In the topography where the protrusion comprises a plurality of protrusion elements, the length of the protrusion can be 0.01 to 100 μm, preferably 0.1 to 45 μm, more preferably 0.5 to 40 μm, and even more preferably 1 to 30 μm.

[0194] The width of the protrusion in the topography where the protrusion comprises only one protrusion element is 0.01 to 100 μm, preferably 0.5 to 50 μm, more preferably 1 to 50 μm, and even more preferably 2 to 40 μm. The width of the protrusion in the topography where the protrusion comprises a plurality of protrusion elements can be 0.01 to 100 μm, preferably 0.1 to 45 μm, more preferably 0.5 to 40 μm, and even more preferably 1 to 30 μm.

[0195] Alternatively, the H / H type immune control implant can be defined as an article having at least one topography, a) the height of the valley wall is 0.5 to 50 μm, preferably 1 to 40 μm, more preferably 2 to 35 μm, more preferably 4 to 30 μm, and even more preferably 5 to 28 μm, b) the contour of the valley wall is 0 to 40 μm, preferably 0.1 to 35 μm, more preferably 0.5 to 30 μm, and even more preferably 1 to 20 μm, c) the length of the valley wall section is 0.01 to 100 μm, preferably 0.1 to 50 μm, more preferably 1 to 40 μm, and even more preferably 2 to 32 μm, d) the length of the puncture of the valley wall is 0 to 50 μm, preferably 0.5 to 40 μm, more preferably 1 to 30 μm, and even more preferably 2 to 25 μm, e) the average width of the valley is 0 to 50 μm, preferably 0.5 to 40 μm, more preferably 2 to 20 μm, and even more preferably 5 to 12 μm.

[0196] In addition, the object of the present invention can be used to stimulate an early immune response against a foreign object in order to enable a reduced final-stage immune response, and preferably can minimize the encapsulating tissue and inflammatory activity around the foreign object at the final stage.

[0197] The topography that can be used in this embodiment has an H / L type of topography, which can be defined as follows: a) The average distance between adjacent protrusions is 0 to 50 μm, preferably 0.5 to 40 μm, more preferably 5 to 30 μm, and even more preferably 12.1 to 20 μm, and b) The area of the top surface region of the protrusion is 1 to 6000 μm 2 , preferably 10 to 3000 μm 2 , more preferably 20 to 1000 μm 2 , more preferably 25 to 250 μm 2 , even more preferably 25 to 65 μm 2 and c) The protrusion covers 3 to 90% of the surface part, preferably 4 to 50%, more preferably 5 to 30%, and even more preferably 5 to 10%.

[0198] In addition, the length of the protrusion in a topography having only one protrusion element is 0.01 to 100 μm, preferably 0.5 to 50 μm, more preferably 1 to 50 μm, and even more preferably 2 to 40 μm. The length of the protrusion in a topography having a plurality of protrusion elements can be 0.01 to 100 μm, preferably 0.1 to 45 μm, more preferably 0.5 to 40 μm, and even more preferably 1 to 30 μm.

[0199] The width of the protrusion in a topography having only one protrusion element is 0.01 to 100 μm, preferably 0.5 to 50 μm, more preferably 1 to 50 μm, and even more preferably 2 to 40 μm. The width of the protrusion in a topography having a plurality of protrusion elements can be 0.01 to 100 μm, preferably 0.1 to 45 μm, more preferably 0.5 to 40 μm, and even more preferably 1 to 30 μm.

[0200] Alternatively, an H / L type immunomodulatory implant can be defined as a substance having at least one topography, a) The height of the valley wall is 0.5 to 50 μm, preferably 1 to 40 μm, more preferably 2 to 35 μm, even more preferably 4 to 30 μm, and still more preferably 5 to 28 μm, b) The contour of the valley wall is 0 to 40 μm, preferably 0.1 to 35 μm, more preferably 0.5 to 30 μm, even more preferably 1 to 20 μm, c) The length of the valley wall section is 0.01 to 100 μm, preferably 0.1 to 50 μm, more preferably 1 to 40 μm, even more preferably 2 to 32 μm, d) The length of the valley wall puncture is 0 to 50 μm, preferably 0.5 to 40 μm, more preferably 1 to 30 μm, even more preferably 2 to 25 μm, e) The average width of the valley is 0 to 50 μm, preferably 0.5 to 40 μm, more preferably 5 to 30 μm, even more preferably 12.1 to 20 μm.

[0201] Immune cells, also known as white blood cells, are cells of the immune system that defend the body against foreign substances, including bacteria, viruses, and other external objects such as biomaterials implanted in the body. These cells include macrophages, monocytes, B cells, T cells, megakaryocytes, basophils, neutrophils, eosinophils, dendritic cells, lymphocytes, mast cells, natural killer cells, foreign body giant cells (FBGCs), and other multinucleated cells. Immune cells can be found in the blood or other tissues and are obtained from a variety of sources, including humans or animals, as is well known.

[0202] The human body's (immune) reaction to foreign objects such as transplanted biomaterials is called the foreign body reaction (FBR). Immune cells induce the foreign body reaction. The foreign body reaction can lead to the formation of a fibrous tissue capsule around the foreign object. This process, known as encapsulation, is the final and chronic stage of the inflammatory reaction to the foreign object and the surrounding wound tissue. This process occurs naturally to protect the body and initiate the wound healing process, but it can have undesirable effects on transplanted biomaterials and may impair their function. Therefore, the regulation of the foreign body reaction to transplanted biomaterials or materials used in vivo is an important issue in the design of biomaterials.

[0203] For this purpose, several approaches have been developed that mainly focus on the chemical level, including changes in the design of biomaterials and the physicochemical properties of biomaterials. Here, it is shown that by patterning the surface of a biomaterial with a specific topography, the reaction of immune cells to foreign objects can be regulated (stimulated or downregulated) regardless of the chemical nature of the biomaterial.

[0204] As described above, any material can be used to achieve the object of the present invention.

[0205] The regulation of immune cells includes affecting morphology, stimulating or suppressing adhesion and proliferation, stimulating or suppressing the fusion of mononuclear cells into multinuclear cells, maintaining or changing the immune cell phenotype (cell differentiation), affecting the function of immune cells, affecting the activation of the immune reaction, stimulating or preventing cell death, controlling cell migration, and affecting the regulation of fibrous capsule formation at the implant site.

[0206] For example, an appropriate surface topography can regulate immune cell migration by stimulating or preventing the arrival of macrophages from the blood to the tissue surrounding the implant site.

[0207] As another example, topography can stimulate the adhesion of macrophages to foreign objects and induce macrophage fusion and the formation of FBGCs. Conversely, other surface topographies can prevent macrophage fusion and reduce FBGC formation. Alternatively, macrophage adhesion can be stimulated while FBGC formation is inhibited, or vice versa.

[0208] The regulation of the immune control mechanism can be traced by labeling immune cells attached to the surface or migrating to the implant site and performing immunofluorescence staining, and then can be traced by imaging techniques. Furthermore, other techniques such as FACS can be used to detect surface markers expressed in immune cells, i.e., to detect the cell type. Furthermore, techniques such as immunofluorescence staining, qPCR, ELISA, Western blot, microarray, histology, and electron microscopy images can be used to analyze the differentiation and biological functions of immune cells.

[0209] The regulation of the immune response can also be analyzed in vivo in various animal models such as mice, rats, rabbits, etc., and the implanted material and surrounding tissue can be analyzed by (immuno)histological techniques (immunofluorescence staining, colorimetric staining), electron microscopy imaging, qPCR and RNA analysis, cytokine release analysis, mechanical testing, etc.

[0210] The regulation of immune cells is used in vitro as defined above using culture utensils such as flasks, plates, bags, Petri dishes, dishes, containers, etc., such as bioreactors, chips, (drug) screening platforms and / or biomolecule (vaccine / protein / antibody) production platforms.

[0211] In a preferred embodiment, the topography strongly affects the response of monocytes / macrophages to the polyurethane (PU) surface. The surface topography can substantially affect macrophage adhesion. The effects on cells include not only limitations on the number of attached cells but also changes in the morphological properties of cells such as cell area. Furthermore, these surfaces affect macrophage fusion and thus strongly affect the formation of foreign body giant cells. These effects were observed in an in vitro environment 10 days after culturing monocytes on a PU substrate characterized by the selected surface topography. The in vitro application of these immunoreactive topographies includes, but is not limited to, in vitro culture models or in vitro disease models for studying or analyzing the immune response or related physiology of cells. This early immune response is important for implant acceptance and promotes implant acceptance in the later stages of the immune response.

[0212] However, preferably, the modulation of the immune response is applied in vivo. The topography of the present invention can be provided to any long-term or chronically implanted device, including but not limited to biosensors and bioelectrodes, cosmetic implants such as breast implants, bone implants such as small bone implants, vascular implants (vascular grafts, vascular access), dialysis access, cochlear implants, and cardiovascular implants, to modulate the immune response.

[0213] In vivo, the PU substrate characterized by the topography affected the early and late FBR in a subcutaneous mouse model. Different surfaces were able to induce FBR ranging from very mild to strong. Infiltration of immune cells into the surrounding tissue (measurement of the level of inflammatory activity), the presence of macrophages and the formation of FBGCs, the formation of a fibrous capsule around the implant, and the encapsulation of the implant at the interface were some of the FBR-related parameters affected by the presence of different surface topographies.

[0214] The thickness of the fibrous capsule around the PU implant changed when different surface topographies were introduced onto the surface of the implant. In the late (final) stage of FBR, some surface topographies had a much lower amount of fibrous capsule, or were equal to or even lower than a wound (pseudo) where no foreign body was actually implanted, compared to the unpatterned control substrate and the silicone elastomer control material. These surface topographies clearly reduced the immune response and prevented encapsulation of the implant, thereby improving the functionality of the implant and may improve the patient's outcome.

[0215] For purposes of clarity and concise description, features are described herein as part of the same or separate embodiments, but the scope of the invention may include embodiments having combinations of all or some of the described features. In particular, features described under a general description may be part of a particular embodiment.

[0216] The present invention is illustrated by the following non-limiting examples.

[0217] Examples Materials and methods for all examples Sample fabrication In the examples, metal and polymer substrates are used. For the complete metal samples featuring individual topographies, i.e., titanium (Ti), the following procedure is applied to create protrusions with a height of 10 μm. 1. Annealed Ti plates (99.6% purity) are polished (roughness of approximately 0 - 0.01 μm) and cleaned. 2. 900 nm of SiOx (plasma enhanced chemical vapor deposition, Oxford Plasmalab system 80), lithography (positive photoresist OIR 906 - 12, Arch Chemical, Inc) and hard bake (T = 120 °C, 60 s) are deposited. 3. Etching the SiOx layer: Perform directional reactive ion etching (Adixen AMS 100 SE) for 4.5 minutes using a C4F8 flow rate of 20 sccm, a He flow rate of 150 sccm, a CH4 flow rate of 15 sccm, inductively coupled plasma (2800 W), capacitively coupled plasma (350 W) (T = -10 °C, 2 minutes). 4. Remove the photoresist with Oxygen plasma (Tepla 300E). 5. Etch the Ti substrate: Alternately perform an etching step (a combination of Cl2 / BCl3 / Ar plasma, 45 seconds) and an oxidation step (O2 plasma, Oxford Plasmalab system 100, 15 seconds; O2 flow rate 30 sccm, 500 W ICP, 15 W CCP and 30 pressure of 33 mTorr).

[0218] For polymer substrates, follow the following procedure for both substrates containing various different topographies and substrates holding one individual topography. 1. Fabricate a silicon wafer (mold) that holds the inverse topography design by photolithography using a chromium mask. 2. Coat the silicon wafer with perfluorodecyltrichlorosilane (FOTS, ABCR). 3. Optionally, replicate the topography design in an intermediate mold of polydimethylsiloxane (PDMS Sylgard 184 (registered trademark), Dow Corning) and OrmoStamp (Micro Resist Technology GmbH). 4. Hot emboss the topography onto the polymer sample using the silicon wafer or the intermediate mold (all samples are demolded at 78 °C). 5. Option: Sputter coat with the metal of interest. 6. Treat with O2 gas plasma (standard treatment for cell culture substrates).

[0219]

Table 2

[0220] Cell culture and analysis In in vitro examples, cells of interest were cultured on a substrate adapted to allow comparison between various topographies and a substrate retaining one individual topography. This was done according to the following procedure. 1. Sterilize (70% ethanol) and moisten the substrate (cell culture medium, minimum 24 hours). 2. Optionally: Incubate at room temperature, wash 3 times with PBS, and perform collagen coating with 0.02 M acetic acid, 100 μl / well, fresh 20 μg / ml collagen I (rat-tail, VWR) for 1 hour. 3. Seed the cells of interest (see Table 3 for details) and culture at 37 °C, 5% CO2 while changing the medium every 2 - 3 days.

[0221]

Table 3

[0222] 4. After collection, fix the samples with 4% paraformaldehyde and fluorescently label the cells (see Table 4 for details).

[0223]

Table 4

[0224] 5. Image the comparative topography using an automated slide scanner that captures images of each TopoUnit, or (II) capture 10 randomly selected images for a substrate characterized by topography. Evaluate the obtained images for both appropriate culture quality (cell number, distribution) and image quality. 6. Analyze the images using MATLAB® scripts and CellProfiler [Carpenter AE et al. Genome Biology 2006;7:R100, Hulsman M et al. Acta Biomaterialia 2015;5(15):29, Unadkat H et al. PNAS 2011,108:16565] (see Table 5 for details of the analysis parameters).

[0225]

Table 5

[0226] 7. In vitro validation and secondary screening using various methods Malaria infection (Example 2): On day 2, add 50,000 Plasmodium cynomolgi sporozoites / well (in 130 μl) and include HSP70 (Heat shock protein 70) during the labeling for visualizing P. sporozoites. Manually quantify the number of infected cells and calculate the infection efficiency (number of infected cells / total number of cells). qPCR (Examples 3-4): mRNA was isolated using the Trisol protocol, i.e., cells were lysed in Trisol and the lysate containing mRNA was purified. Two replicates of the lysate were pooled and three measurements were made per pool (6 biological replicates). For a specific gene, RNA isolated using the iScript kit (Bio-Rad) according to the manufacturer's protocol was synthesized into cDNA and diluted with water for quantitative real-time PCR (qPCR, Bio-Rad) using Sybr green I master mix (Invitrogen) and primers (Sigma) (Table 6). Gene expression was normalized to the housekeeping gene GAPDH level (ΔCT method), and then to the level of the same marker in the collagen sandwich on day 3 to show fold induction (ΔΔCT method).

[0227]

Table 6

[0228] 8. Validation in vivo using various animal models

[0229] Rabbit femoral model for confirmation of osteochondral integration (Example 5) Complete titanium topography feature samples (coin type, 6.25 mm in diameter, 1.95 mm thick) were implanted into 24 female New Zealand white rabbits together with controls to evaluate new bone formation and osseointegration. The samples were first sonicated in acetone for 1 hour and in IPA for 1 hour and sterilized using an autoclave. The procedures for the animal study were approved by the local ethics committee.

[0230] The transplantation was performed under general anesthesia and aseptic conditions. After sedation by intravenous application of pentobarbital sodium (3.0 mg / kg body weight), the animals were shaved at the surgical site and sterilized with iodine and 70% ethanol (EtOH). At the proximal part of the femur, an incision was made down to and including the underlying periosteum for 5 cm. The periosteum was removed from the surface. Two holes were drilled, the implant was inserted into the holes, and held in place by a mesh plate. The subcutaneous layer was repositioned and sutured with 4-0 silk sutures. The animals were sacrificed at 4 or 8 weeks later, and the transplanted femurs were removed. The explanted samples were then fixed in 10% neutral buffered formalin and maintained in 70% EtOH for further analysis / characterization.

[0231] The analysis was performed as follows. (1) Pull-out tensile test using a mechanical pull bench: A tensile force was applied to the sample at a rate of 1 mm / min, and the implant was separated from the bone while recording all the applied forces, and (2) Histology: The fixed samples were dehydrated in an ethanol series and embedded in methyl methacrylate (MMA). Histological sections were prepared and stained with a 1% methylene blue and 0.3% basic fuchsin solution. Three sections per sample were scanned and the bone-implant contact rate (%BIC) was measured (defined as all areas with direct bone contact to the implant without gaps or fibrous tissue).

[0232] Mouse subcutaneous model for confirming immunomodulation (Example 7) To evaluate the immune response in mice, polyurethane (PU) implants were transplanted into 64 female mice (Harlan, approximately 9 months old, 18 - 20 g, 2 implants per mouse (n = 8) per condition) together with controls. Non-patterned (NP) implants, sham wounds (sham group) and silicone elastomer controls (SE, bisected catheters 2.5 mm in diameter and 0.6 mm thick, Medtronic Medical) were included as controls. This study was conducted under a protocol appropriately and ethically approved by Dutch law.

[0233] The transplantation was performed under general anesthesia and aseptic conditions. After sedation with 2% isoflurane (Pharmachemie) in oxygen in a laminar flow cabinet, subcutaneous injection of buprenorphine (Temgesic, RB Pharmaceuticals Limited, 0.05 mg / kg) was performed 15 minutes before the surgery for pain control. The back of the mouse was shaved and disinfected with 70% ethanol. On both sides of the mouse's back, a 0.4-cm incision was made 1 cm lateral to the spine. Subsequently, a 1-cm-long implant was placed subcutaneously using sterile forceps with minimal tissue damage. The incision was closed. The sham group and SE control were transplanted in the same manner. After the surgery, the mice were housed individually until the wound healed. Subsequently, the mice were housed in pairs until the end of the test. Nine days or 60 days after the test, indicating early and late immune responses respectively, the mice were sacrificed according to an ethically approved protocol, and samples were collected together with the subcutaneous tissue and the attached skin. Half of each biopsy was fixed in 4% paraformaldehyde, followed by methyl methacrylate / butyl methacrylate (MMA / MBA, Merck), 3-μm sections were cut, de-plasmafied with acetone, and washed with deionized water. For each biopsy, three consecutive sections were stained with hematoxylin and eosin (H&E), picrosirius red (PSR), or F4 / 80.

[0234] For H&E staining (for nuclear and cytoplasmic staining), the slides need to be incubated in hematoxylin for 5 minutes, followed by washing with water for 5 minutes, counterstaining with eosin, and washing again. Next, the slides are incubated in ethanol and xylene for dehydration and mounted on a coverslip using Vectamount (vector labs).

[0235] For PSR (to visualize collagen and collagen-rich tissues), the slides need to be incubated in the PSR solution (Klinipath) for 20 minutes, washed with 0.1 N HCl, and dehydrated with ethanol and xylene. The slides were mounted as described above.

[0236] F4 / 80 (a protein marker of the macrophage population) was incubated in methanol containing 0.3% H2O2 in the dark for 20 minutes, washed with water, incubated in 50 mM Tris, 0.9% NaCl buffered saline (TBS), washed again, incubated with Superblock (Klinipath) for 10 minutes, then incubated overnight with rat anti-mouse monoclonal F4 / 80 antibody (Serotec, 1:1000 in TBS), incubated with rabbit anti-rat IgG antibody (SBA / ITK, 1:3000 in TBS and 20% normal mouse serum) for 30 minutes, washed with BrightVision anti-rabbit AP antibody (Immunologic) for 30 minutes, and finally washed with Vector Blue (Vector Labs) for 10 minutes. The slides were then washed with TBS and then tap water, stained with hematoxylin for 1 minute, and washed with tap water again. After drying, the slides were mounted, scanned, imaged, and analyzed (digital pathology software, IntelliSite, Philips).

[0237] For immune cell infiltration, FBGC formation, implant encapsulation, and formation of fibrous tissue around the implant, the slides were scored from 0 to 3 (0 = none, 1 = mild, 2 = moderate, and 3 = severe). The thickness of the fibronectin tissue around the implant was measured at six random sites around the implant. Scoring and measurement of the thickness of the fibrous layer were performed separately by two blinded individuals.

[0238] Example 1: Surface Topography for Maintaining Hepatocytes Derived from Primary Rhesus Monkeys during Culture Table 7 shows the topography that can maintain and increase the functionality of hepatocytes (H1-10) during culture, and shows the performance of the analytical parameters when made of geometric shapes and polystyrene. As controls, non-patterned (NP) substrates (including polystyrene) and topographies of a more "basic" design (triangles, B1-2) are included. Low cell numbers indicate low attachment and / or low cell survival, while high cell numbers (>60) are undesirable because these cells do not express an appropriate hepatocyte morphology and phenotype. Therefore, a moderate number of cell levels show the best performance (40 - 60) when combined with particularly appropriate surface coverage and cell morphology (judged visually when selecting the hit topography).

[0239] Topographies H1 - H10 have a combined moderate number of adherent cells (44 - 60) with cell coverage of 29 - 36% of the cell number. In contrast, the NP substrate has a moderate number of cells that adhere leaning towards the low end (44) in combination with a lower cell coverage rate (27%). However, what is most prominent on the NP substrate is the loss of poor cell morphology and phenotype, which correlates with poor results after a long culture time (see Figure 2c, image on day 31 of culture). The basic topography results in either a small number of cells with low cell coverage (an appropriate hepatocyte culture cannot be maintained) or a large number of cells and / or cell coverage (even non-functional and non-phenotypic hepatocytes). B1 results in a large number of cells (73) with an average cell coverage rate (35%) at a protrusion coverage rate of 36% (exceeding the range of the highest performance topographies of 8 - 35%), and these cells are small, do not present well-diffused and appropriate morphology, and thus do not have a phenotype and functionality. B2 results in a small number of cells (35) with a low cell coverage rate (22%) at a protrusion coverage rate of 7% (less than the range of the best performance topographies) and does not show an appropriate hepatocyte culture.

[0240] Verification of Topography Verification confirmed the effectiveness of topography in supporting in vitro culture of hepatocytes. Normal tissue culture polystyrene (TCPS) supports hepatocytes for less than 5 days, and collagen-coated TCPS supports them for up to 14 days, while the topographies with the highest performance (regardless of collagen coating) support hepatocytes for up to 31 days.

[0241] A total of 10 topographies strongly promoted in vitro culture of hepatocytes. Substrates featuring topographies had 2 - 6 times more cells attached to the surface 31 days after culture, were CD81 positive, and were thus confirmed to be hepatocytes, compared to non-patterned control surfaces (Figure 2a). Also, the cells covered 2 - 5 times the surface area of the topography-featured surfaces compared to non-patterned control surfaces (Figure 2b). Finally, hepatocytes cultured on substrates featuring topographies strongly controlled the morphology of hepatocytes seen on non-patterned control substrates (Figure 2c) and had a morphology very similar to that of in vivo (native) tissue and morphology of hepatocytes in the liver (H3, Figure 2d).

[0242] Secondary screening of topographies The topographies with the highest performance in Experiment 1 (H3, H4, H5, H6, H8) were further verified. In addition to the 31-day verification, the 8th day of culture was included to compare the culture on the 8th day with the current standard. Also, a part of the samples was infected with malaria parasites to demonstrate hepatocyte function (malaria parasites can only infect functional hepatocytes).

[0243] Again, screening and verification results showing a large number of cells, high cell coverage (Figure 3a, b, d and e) and appropriate expression of CD81 were confirmed.

[0244] The infection efficiency against the substrate characterized by topography increased to 0.5% on the 8th day and 0.15% (Fig. 3c, f) on the 31st day. These infection levels are consistent with the standard levels using the non-patterned collagen-coated surface (8th day). The level on the 31st day cannot be compared because hepatocytes cannot be cultured on the non-patterned (collagen-coated) surface for more than 10 - 14 days. However, the fact that sporozoites of Plasmodium are still detected on the 31st day is unprecedented and is the only advantage of topography that brings a great opportunity for the research and development of antimalarial drugs.

[0245] Conclusion The identified surface topography promotes collagen-independent hepatocyte attachment and supports their in vitro culture for 31 days while expressing the hepatocyte phenotype, even in the presence of Plasmodium. In stark contrast, the current standard method allows only 8 - 11 days of in vitro culture at most and requires collagen. The best surface topography for supporting hepatocytes during culture has protrusions with an area of the top surface region of 20 - 250 μm 2 , a surface coverage of 8 - 35% and an average distance between protrusions of 3 - 25 μm.

[0246] Example 2: Surface Topography for Maintaining Primary Human-Derived Hepatocytes during Culture With or without collagen coating, the topography was verified to support the extension of in vitro culture of primary human-derived hepatocytes (PHH). The gold standard "collagen sandwich" (CS) for culturing hepatocytes between two layers of collagen is included as a benchmark (up to 14 days) and a non-patterned (NP) surface as a control.

[0247] Results Substrates featuring all topographies were superior to the unpatterned control and at both time points, regardless of the presence or absence of collagen. At both day 14 (Figs. 4a, b) and day 30 (Figs. 4c, d), the total cell number and surface coverage were higher on the topography-featured substrates than on CS (only at day 14) and the unpatterned control, and were significantly greater at day 30. However, the most notable effect was seen in cell morphology. On certain topographies, the cells were clearly similar to natural (in vivo) liver tissue and morphology, while on other topographies and the unpatterned control, the cells did not exhibit such tissue and cell shapes. Coating the topography-featured surface with collagen had little effect on the results.

[0248] Conclusion The topography of the present invention demonstrated excellent performance for extending the proper in vitro culture of PHH by one month, while the current gold standard CS can only be used for a maximum of two weeks and in practice can only be used for eight days. The most excellent surface topography for supporting hepatocyte culture has protrusions with an area of the top surface region of 20 - 250 μm 2 and a surface coverage of 8 - 35% and an average distance between protrusions of 3 - 25 μm.

[0249] Example 3: Surface Topography for Functionally Maintaining Primary Human-Derived Hepatocytes The most promising topographies of Examples 1 - 2 (H3, H4, and H8, with or without collagen coating) were analyzed for the expression of a PHH hepatocyte-specific phenotype that exhibits functionality during two weeks of in vitro culture. The CS and NP surfaces were included as controls. The gene expression levels on the topography-featured surfaces at day 14 (all normalized to the level of CS at day 3) were compared to the CS levels at days 14 and 3.

[0250] Results HNF4α (including the transcription of hepatocyte-specific proteins and the regulation of hepatocyte function) is highly expressed even at the level of 3-day CS as compared to 14-day CS on substrates characterized by all topographies with a collagen coating.

[0251] Albumin (Figure 5b, an indicator of hepatocyte metabolism) reveals the efficiency of surface topography for maintaining and perhaps even promoting the metabolic-related phenotype in long-term in vitro culture, and was substantially more highly expressed (more than 4-fold) on substrates characterized by topography, regardless of the presence or absence of collagen coating, as compared to CS at both time points.

[0252] Cyp3A4 (Figure 5c, an important detoxification marker) was highly expressed (up to 4-fold) on all substrates characterized by topography as compared to CS at both time points, indicating the improvement of this function-related phenotype by applying these surface topographies regardless of the presence or absence of collagen.

[0253] The expression of other Cyp markers (Cyp2B6, Cyp1A2, Cyp2C9 involved in detoxification) on the surface characterized by topography was higher than (Cyp2B6 and Cyp1A2) or similar to CS (Cyp2C9) at 14 days.

[0254] For CD81 and E-cadherin (involved in cell adhesion, tight junction formation and intercellular communication), the expression levels on the surface characterized by topography were comparable to CS and higher as compared to NP at 14 days.

[0255] Conclusion Topography was comparable to the current gold standard at early time points and was able to maintain the hepatocyte phenotype over a long period. In some cases, functionality increased even within 14 days of in vitro culture. The best surface topography to support hepatocyte culture is 20 - 250 μm in vitro 2It has protrusions with an area of the top surface region of 8 - 35%, a surface coverage rate of 8 - 35%, and an average distance between protrusions of 3 - 25 μm.

[0256] Example 4: Surface topography for functionally maintaining primary human-derived hepatocytes in long-term culture Regardless of the presence or absence of collagen, the most promising topography (H3) of Examples 1 - 3 is analyzed for its effect on PHHs that maintain or recover their phenotype during a 24-day culture period. The genes analyzed were the same as in Example 3. CS (up to 14 days) and NP controls are included.

[0257] Results On the H3 surface, regardless of the presence or absence of collagen coating, all analyzed genes (Figure 6) were expressed substantially higher at all time points (days 8 - 24) compared to the levels on day 3, which is considered the time when cells need to recover to a normal hepatocyte state. In contrast, the expression of all biomarkers in CS was maintained at the same level as on day 3 or decreased to (very) low levels over time (up to 14 days). When directly comparing the H3 surface with CS on day 24, all genes were still expressed at similar or (very) higher levels on H3 than in CS, even on day 8 (gold standard) and even on day 3.

[0258] Conclusion The PS substrate characterized by the surface shape H3 was able to not only maintain the hepatocyte phenotype and functionality compared to the current gold standard, but also further promote these functions including metabolic activity, signal transduction, protein secretion, and detoxification, and enable the extension of in vitro culture to at least 24 days (compared to 8 days of the current gold standard). The surface topography showing the best performance in supporting hepatocyte culture has protrusions with an area of the top surface region of 20 - 250 μm 2 It has protrusions with an area of the top surface region of 8 - 35%, a surface coverage rate of 8 - 35%, and an average distance between protrusions of 3 - 25 μm.

[0259]

Table 7-1

[0260]

Table 7-2

[0261] Example 5: Surface Topography for Promoting Osseointegration and Fixation of Bone / Dental Implants Titanium-coated chips with various different topographies were used to study surface topographies that improve osteogenic differentiation of human mesenchymal stromal cells (hMSCs) to stimulate new bone formation for the purpose of improving the fixation of orthopedic and dental implants to bone. When hMSCs differentiate into the osteogenic lineage to form new bone, they express the osteogenic marker ALP and were thus used as a marker.

[0262] In vitro Verification of Topography Topographies that stimulated ALP expression according to the combinations of parameters listed in Table 5 were selected as hit topographies (Table 8, O1 - O10). ALP expression at topographies O1 - 10 was in the range where the integrated ALP intensity (iALP) exceeded approximately 600 - 1000 and the relative integrated ALP intensity (riALP) ranged from 36 - 58. In stark contrast, the non-patterned (NP) control substrate had values of 14 (iALP) and 212 (riALP), and the basic topographies (triangles and rectangles, B3 - 5) had values of 14 - 19 (iALP) and approximately 350 - 500 or more (riALP). These basic topographies had a protrusion surface coverage far below the range of the topographies with the best performance (4 - 25% vs 30 - 65%), and the protrusion top surface area was in a lower range than that of the topographies with the highest performance (25 - 29μm 2 vs 30 - 750μm 2 ). This data demonstrates the superiority of surface topographies O1 - O10 in stimulating ALP expression and bone formation.

[0263] Verification of the hit surface using well-known in vitro techniques including protein expression (FACS), gene expression (qPCR) and mineralization (tetracycline staining) supported the effectiveness of the hit topography and its superior performance compared to the NP surface.

[0264] In vivo verification of topography Topographies with the best performance in in vitro experiments were fabricated within full-body titanium and implanted in vivo into the rabbit femur model to evaluate osseointegration ability at 4 and 8 weeks. An NP control was included in this study for comparison.

[0265] Both mechanical and histological analyses (Figure 7) revealed that implants featuring the topography stimulated the formation of new bone tissue. Pull-out tests (Figure 8a) showed that the NP control had the lowest level of osseointegration after 4 and 8 weeks. At 4 weeks, all implants featuring one of the hit topographies required 2 - 4 times the pull-out force of the NP control, and implants featuring one hit topography required more than 5 times the pull-out force of the NP control. At 8 weeks, the NP control still required less force to dislodge compared to the best scoring topography at 4 weeks. Implants featuring the hit topography required approximately twice the force of the NP control, and one topography required more than 3 times the force.

[0266] Histological bone implant (BIC) contact analysis (Figure 8b) showed that the NP control had approximately 10% lower BIC at both 4 and 8 weeks. Implants featuring the topography showed much higher BIC at both time points, although there was high variability between animals. The results showed up to 80% BIC for the hit topography featuring 8-week implants.

[0267] These results provide evidence of the effectiveness of hit topographies in inducing osseointegration in relevant in vivo models.

[0268] Conclusion The identified topographies have highly enhanced osseointegration properties compared to non-patterned control implants. The results further show that the topographies of the present invention having regular pattern protrusions / valleys provide better osseointegration than clinically applied benchmarks having randomly roughened surfaces. Thus, the topographies of the present invention exhibit increased fixation and increased bone formation compared to known implants.

[0269] The best surface topographies for stimulating bone formation have protrusions with a top surface area of 30 - 750 μm 2 , preferably 150 - 450 μm 2 , a surface coverage of 30 - 65%, preferably 35 - 60%, and an average distance between protrusions of 2 - 25 μm, preferably 5 - 15 μm.

[0270]

Table 8-1

[0271]

Table 8-2

[0272] Example 6: Surface Topography for Modulating Immune Response In vitro Surface topographies that regulate the response of immune cells to biomaterials were identified using topographies made of polyurethane. When a biomaterial is implanted into the body, the body attempts to heal the damaged tissue surrounding the biomaterial and an immune response is induced to remove / separate the foreign biomaterial. This reaction is called the foreign body reaction (FBR). First, the acute reaction is important for biomaterial acceptance but can lead to a chronic inflammatory process involving excessive formation of a fibrous capsule around the implant. Therefore, we use the FBR, and in particular the associated formation of foreign body giant cells (FBGC, multinucleated cells), as markers.

[0273] Surface topographies with the ability to regulate FBR were selected, scaled up to a substrate ( 2 cm 2 surface area), implanted, and verified using 1 donor.

[0274] Secondary screening by 2 donors included a more complex mixed population of mononuclear cells (including monocytes) by excluding the Ficoll gradient during isolation to better mimic the in vivo environment for the short stage of the immune response. Unpatterned (NP) substrates were included as controls.

[0275] In vitro verification of topographies The topography that most strongly affects macrophage adhesion and fusion to FBGC, as evaluated by the combination of parameters listed in Table 5, was selected as the hit topography (Table 9, I1 - 13). Topography can stimulate and inhibit macrophage adhesion and fusion in vitro. Stimulatory topographies (I1 - 4) inhibited a large number of macrophages (12 - 15) with a large number of multinuclei (12 - 16), and inhibition of topographies (I5 - 13) led to low macrophage adhesion (3 - 6) and low multinucleation (4 - 5). In contrast, B6 and basic topographies (circles and triangles, B6 - 7) induced a moderate effect on both macrophage adhesion (NP: 6, B6 - 7: 8 - 10) and multinucleation (NP: 5, B6 - 7: B7 - 10) and could not control the immune response. B6 had an area of the protrusion top surface region below the range of all three groups of topographies with the best performance (19μm 2 vs 20μm 2 and above), while B7 had a surface coverage outside the range of all three groups of topographies with the best performance (13% vs 15 - 25% of H / H, 5 - 10% of H / L, and 26 - 50% of L / L).

[0276] Verification of these surface topographies supports the effectiveness of topographies for significantly modulating the immune response. For illustration, Figure 9 shows the early stages of macrophage adhesion and FBGC formation in vitro on surfaces characterized by topographies (I1 and I5) compared to the NP surface. The I5 surface had low macrophage adhesion and fusion, while surface I1 had strong macrophage adhesion and FBGC formation and showed a strong FBR. NP had fewer adherent cells compared to macrophages, but some multinucleated cells were formed.

[0277] Secondary verification has revealed that topography strongly affects macrophage attachment and FBGC formation at early stages in vitro (Figure 10). The difference in macrophage attachment between low-attachment and high-attachment topographies was up to five-fold, and up to 1.5-fold compared to the NP surface for low-attachment topographies. FBGC formation decreased even further for some topographies, with the decrease being even greater between low-attachment and high-attachment topographies, and up to two-fold compared to the NP surface for low-attachment topographies. This effect also means that the average number of nuclei per cell was significantly higher for high-attachment compared to low-attachment topographies. Comparing the NP surface to surfaces characterized by low-attachment topographies, it is notable that in addition to affecting macrophage attachment itself, FBGC formation and the number of cells fusing with one large cell were significantly reduced.

[0278] Conclusion The identified topographies have been highly successful in manipulating early-stage immune regulatory mechanisms through macrophage attachment and their fusion into FBGCs under in vitro conditions. Surface topographies that stimulate high attachment at early stages of macrophage and / or FBGC formation in vitro can be divided into two groups with respect to their effects at the final stage. I2 and I4 stimulate the final-stage immune response, and this group has protrusions with a top surface area of 20 - 70 μm 2 , a surface coverage of 15 - 25% and an average distance between protrusions of 5 - 12 μm. I1 and I3 reduce the final-stage immune response, and this group has protrusions with a top surface area of 25 - 65 μm 2 , a surface coverage of 5 - 10% and an average distance between protrusions of 12.1 - 20 μm. On the other hand, surface topographies that induce low attachment of macrophages and FBGC formation at early stages in vitro (I5 - I13) and reduce the final-stage immune response have a top surface area of 25 - 200 μm 2It has protrusions with an area of the top surface region, a surface coverage rate of 26 to 50%, and an average distance between protrusions of 2 to 12 μm. All three groups of topographies affect the immune control mechanism at both early and late stages and can be used according to the end use for which the immune response should be downregulated or stimulated.

[0279]

Table 9-1

[0280]

Table 9-2

[0281]

Table 9-3

[0282] Example 7: In Vivo Surface Topography for Modulating the Immune Response To evaluate the in vivo efficacy of surface topographies selected for modulating the immune response, implants featuring these topographies were placed in a subcutaneous mouse model.

[0283] Results The surface topography substantially affected the immune system response of the mice at both time points (Figure 11) compared to the control.

[0284] Suspected conditions (where no foreign object is implanted and the reaction is only to the treatment itself) generally have the mildest immune responses at both the early stage (9d) and the late stage (60d) compared to all other conditions, as expected. The SE reference substance shows a stronger immune reaction compared to other implanted samples. By day 9, most are already at the high end compared to other implants, but especially by day 60, there is more immune reaction activity and fibrosis, resulting in a much thicker layer of connective tissue forming around the implant. This result is expected considering the well-known "normal" inflammatory reaction of silicone elastomers. The immune response to unpatterned PU implants designed for chronic implantation and good biocompatibility starts out as milder to moderate, resulting in moderate immune activity and fibrous capsule formation (9d). Generally lower than the SE reference substance, the reaction to the NP implant over time is most similar to the reaction to the SE control group.

[0285] The immune response to PU implants characterized by surface topography I1 (H / L type) starts as a mild reaction and decreases slightly compared to NP, but the immune reaction is very mild at 60 days. The in vivo early-stage results are consistent with the relatively high macrophage attachment and FBGC formation in the in vitro model at the early stage (Example 6). This phenomenon of topography showing one of the lowest immune activity levels at the final stage is very interesting and not yet fully understood, but is hypothesized to be related to the initial acute reaction being appropriate and controlled. This topographical FBGC formation and fibrosis at the final stage are very natural (very small variations) and mild results, similar to the case of low immune activity and thin fibrous capsule formation.

[0286] Topography I2 (H / H type) induced immune responses ranging from moderate to severe at both time points at an early stage comparable to I1, but remained at this level at this time point. Topography I5 (L / L type) also had immune responses ranging from moderate to severe at an early stage and became milder at a later stage, but had a thicker capsule compared to other topographies.

[0287] Topography I6 (type L / L) showed the mildest FBR compared to biomaterials with surface topography, NP, and SE groups. This reaction starts as a mild to moderate reaction at a stage closest to the sham group but leading to the thinnest fibrous capsule, and continues this reaction. Topography I7 (L / L type) first showed a similar mild to moderate FBR on day 9, but the FBR was more moderate at later time points.

[0288] Comparing all implants based on the formed connective tissue, the initial (9d) topographies I6 and I7 induced the thinnest fibrous capsules consistent with the early stage of the in vitro results shown in Example 6. At the final stage of FBR, surface topographies I1 and I6 were even lower than the sham control compared to other surface topographies and NP controls, inducing minimal fibrous encapsulation.

[0289] Conclusion Surface topography can regulate the foreign body (immune) response at both early (acute phase) and late (chronic phase) stages. The FBR to topography-featured implants varies from very mild to severe and affects the inflammatory response and the formation and thickness of the fibrous capsule. The surface topography of the present invention enables a low immune response at an early stage and a low immune response at a later stage, reducing fibrous encapsulation at the FBR (2 months) at a later stage, and these surfaces are 25 - 200 μm 2It had protrusions having an area of the top surface region of 26 to 50%, a surface coverage rate of 26 to 50%, and an average distance between protrusions of 2 to 12 μm. Further, the surface topography of the present invention enables a high immune response at an early stage and a high immune response at a later stage, leading to a stimulation of controlled fibrous encapsulation at FBR (2 months) at the later stage, and these surfaces are 20 to 70 μm 2 It had protrusions having an area of the top surface region of 15 to 25%, a surface coverage rate of 15 to 25%, and an average distance between protrusions of 5 to 12 μm. Further, the surface topography of the present invention enables a high and a low immune response at an early stage and a later stage, respectively, reducing fibrous encapsulation at FBR (2 months) at the later stage, and these surfaces are 25 to 65 μm 2 It had protrusions having an area of the top surface region of 5 to 10%, a surface coverage rate of 5 to 10%, and an average distance between protrusions of 12.1 to 20 μm.

[0290] (Appendix) (Appendix 1) An article comprising a surface portion having one or more topographies capable of regulating the morphology, proliferation, biochemical function, differentiation, adhesion, migration, signal transduction, and / or cell death of a cell population by physical stimulation, wherein the topography comprises a surface having protrusions in a regular pattern, the protrusions include one or more protrusion elements, the protrusion elements are defined as a surface portion raised on the surface having a top surface region and a surrounding side surface connecting the top surface region to the surface, and each protrusion element has a maximum height of 0.5 to 50 μm on the surface, a) The average distance between adjacent protrusions is 0 to 50 μm, b) The area of the top surface region of the protrusion is 1 to 6000 μm 2 and, c) The protrusions cover 3 to 90% of the surface, article.

[0291] (Appendix 2) a) When the protrusion has one protrusion element, The length of the protrusion defined as the length of the longest straight line fitting within the periphery of the top surface region parallel to the surface is 0.01 to 100 μm, The width of the protrusion defined as the length of the longest straight line fitting within the periphery of the top surface region perpendicular to the length and parallel to the surface is 0.01 to 100 μm, b) When the protrusion has a plurality of protrusion elements, The length of each protrusion element defined as the length of the longest straight line fitting within the periphery of the top surface region parallel to the surface is 0.01 to 100 μm, The width of each protrusion element defined as the length of the longest straight line fitting within the periphery of the top surface region perpendicular to the length and parallel to the surface is 0.01 to 100 μm, c) The average distance between two peripheries of adjacent protrusion elements of the same protrusion is 0 to 50 μm, The thing described in Appendix 1.

[0292] (Appendix 3) The surface part includes a metal material, a polymer material, a composite material or a ceramic material, The thing described in Appendix 1 or 2.

[0293] (Appendix 4) The regular pattern of the protrusions is defined by a grid of intersecting grid lines that can be placed on the surface, and the grid lines define the pattern of unit cells such that each unit cell contains at most one protrusion, The thing described in any one of Appendices 1 to 3.

[0294] (Appendix 5) On the surface part, preferably one, but not exceeding 10, different topographies are included for regulating the morphology, proliferation, biochemical function, differentiation, adhesion, migration, signal transduction, and / or cell death of a cell population by physical stimuli, The thing described in any one of Appendices 1 to 4.

[0295] (Appendix 6) For use in regulating the immune response to foreign bodies, or for cell culture, or for stimulating bone formation The substance according to appendix 5

[0296] (Appendix 7) The substance according to appendix 5 or 6 for use as an implant

[0297] (Appendix 8) For stimulating osteogenic differentiation, maintaining the phenotype and function of hepatocytes, or stimulating and / or suppressing the immune response The substance for the use according to appendix 7

[0298] (Appendix 9) The implant is a dental implant or an orthopedic implant, and the topography is a) The average distance between adjacent protrusions is 2 - 25 μm, and b) The area of the top surface region of the protrusion is 30 - 750 μm 2 and, c) The protrusions cover 30 - 65% of the surface defined by The substance for the use according to appendix 7 or 8

[0299] (Appendix 10) The implant is a liver implant, and the topography is a) The average distance between adjacent protrusions is 3 - 25 μm, and b) The area of the top surface region of the protrusion is 20 - 250 μm 2 and, c) The protrusions cover 8 - 35% of the surface defined by The substance for the use according to appendix 7 or 8

[0300] (Appendix 11) The substance regulates the immune response to foreign bodies by suppressing the immune response, and the topography is a) The average distance between adjacent ones of the protrusions is 2 to 12 μm, and b) the area of the top surface region of the protrusion is 20 to 200 μm 2 and, c) the protrusions cover 26 to 50% of the surface, as defined by an article for use according to claim 7 or 8.

[0301] (Annex 12) The article modulates the immune response to a foreign body by stimulating an immune response, and the topography is a) The average distance between adjacent ones of the protrusions is 5 to 12 μm, and b) the area of the top surface region of the protrusion is 20 to 70 μm 2 and, c) the protrusions cover 15 to 25% of the surface, as defined by an article for use according to claim 7 or 8.

[0302] (Annex 13) The article modulates the immune response to a foreign body by stimulating the immune response at an early stage to a foreign body, resulting in a lower immune response at a final stage, and the topography is a) The average distance between adjacent ones of the protrusions is 12.1 to 20 μm, and, b) the area of the top surface region of the protrusion is 25 to 65 μm 2 and, c) the protrusions cover 5 to 10% of the surface, as defined by an article for use according to claim 7 or 8.

[0303] (Annex 14) An article according to claim 5 or 6 for modulating the morphology, proliferation, biochemical function, differentiation, attachment, migration, signal transduction, and / or cell death of a cell population by physical stimulation in vitro.

[0304] (Supplementary Note 15) Said regulation includes maintaining the phenotype and function of hepatocytes, and each of said topographies a) The average distance between adjacent said protrusions is 3 to 25 μm, b) The area of the top surface region of said protrusion is 20 to 250 μm 2 and, c) said protrusions cover 8 to 35% of said surface part, as defined by the thing described in Supplementary Note 14.

[0305] (Supplementary Note 16) Said topography is defined as or includes a top surface region having valleys of a regular pattern defined by protrusions of a regular pattern, said valleys including valley bottoms, first valley walls and second valley walls, said first valley walls including first valley wall sections and, optionally, punctures of the first valley walls, said second valley walls including second valley wall sections and, optionally, punctures of the second valley walls, said first and second valley wall sections being defined by the sides of the protrusions adjacent to said valleys, and said first and second valley wall punctures being defined as the parts of said valley walls where lines perpendicular to said valleys and parallel to said valley bottoms do not contact the protrusions adjacent to said valleys, a) Said valley walls have a height of 0.5 to 50 μm, defined as the distance perpendicular to said valley bottoms from said valley bottoms to said top surface regions, b) The profiles of said first and second valley walls are independently 0 to 40 μm, c) The lengths of said first and second valley wall sections are independently 0.01 to 100 μm, d) When there are punctures in said first and second valley walls, their lengths are independently 0 to 50 μm, e) The average width of said valleys is 0 to 50 μm, the thing defined in Supplementary Notes 1 to 15.

[0306] (Supplementary Note 17) A method for regulating the morphology, proliferation, biochemical function, differentiation, adhesion, migration, signal transduction, and / or cell death of a cell population by physical stimuli, comprising: 1) contacting one or more cells in a suitable medium with a surface portion having protrusions in a regular pattern, wherein the protrusions in the regular pattern are defined by a grid of intersecting grid lines that can be placed on the surface portion, the grid lines defining a pattern of unit cells such that each unit cell contains at most one protrusion, the protrusions comprising one or more protrusion elements, the protrusion elements being defined as a surface portion raised on a surface having a top surface region and a surrounding side surface connecting the top surface region to the surface, each protrusion element having a maximum height of 0.5 to 50 μm on the surface, a) the average distance between adjacent protrusions being 0 to 50 μm, b) the area of the top surface region of the protrusion being 1 to 6000 μm 2 and c) the protrusions covering 3 to 90% of the surface, and 2) enabling the cells to respond to the surface, and a method comprising the steps.

[0307] (Appendix 18) The living cells are hepatocytes, and the topography is defined by a) the average distance between adjacent protrusions being 3 to 25 μm, b) the area of the top surface region of the protrusion being 20 to 250 μm 2 and c) the protrusions covering 8 to 35% of the surface portion, and the method according to Appendix 17. The method according to Appendix 17.

Claims

Item 1. An article comprising a surface portion having one or more topographies capable of regulating the morphology, proliferation, biochemical function, differentiation, adhesion, migration, signal transduction, and / or cell death of a cell population by physical stimulation of osteoblasts, wherein the topography comprises a surface having a regular pattern of protrusions, the protrusions include one or more protrusion elements, the protrusion elements are defined as surface portions raised on the surface having a top surface region and a peripheral side surface connecting the top surface region to the surface, and each of the protrusion elements has a maximum height of 0.5 to 50 μm on the surface, the shape of the plan view of the protrusions is a complex shape, the complex shape is defined as a combination of a plurality of overlapping or adjacent basic shapes, and the basic shapes are selected from a circle, an ellipse, a triangle, a square, a rectangle, a trapezoid, a pentagon, a hexagon, a heptagon, or an octagon, the topography is a) the average distance between adjacent protrusions is 2 to 25 μm, b) the area of the top surface region of the protrusion is 30 to 750 µm ２ and c) the protrusions cover 30 to 65% of the surface, defined by, an article. Item 2. An article comprising a surface portion having one or more topographies capable of regulating the morphology, proliferation, biochemical function, differentiation, adhesion, migration, signal transduction, and / or cell death of a cell population by physical stimulation of hepatocytes, wherein the topography comprises a surface having a regular pattern of protrusions, the protrusions include one or more protrusion elements, the protrusion elements are defined as surface portions raised on the surface having a top surface region and a peripheral side surface connecting the top surface region to the surface, and each of the protrusion elements has a maximum height of 0.5 to 50 μm on the surface, the shape of the plan view of the protrusions is a complex shape, the complex shape is defined as a combination of a plurality of overlapping or adjacent basic shapes, and the basic shapes are selected from a circle, an ellipse, a triangle, a square, a rectangle, a trapezoid, a pentagon, a hexagon, a heptagon, or an octagon, the topography is a) the average distance between adjacent protrusions is 3 to 25 μm, b) the area of the top surface region of the protrusion is 20 to 250 µm ２ and c) the protrusions cover 8 to 35% of the surface, defined by, an article. Item 3. An article comprising a surface portion having one or more topographies for physically stimulating immune cells, wherein the topography comprises a surface having a regular pattern of protrusions, the protrusions comprising one or more protrusion elements, the protrusion elements being defined as surface portions raised on the surface having a top surface region and a surrounding side surface connecting the top surface region to the surface, each of the protrusion elements having a maximum height of 0.5 to 50 μm on the surface, wherein the shape of the protrusion in plan view is a complex shape, the complex shape being defined as a combination of a plurality of overlapping or adjacent basic shapes, the basic shapes being selected from a circle, an ellipse, a triangle, a square, a rectangle, a trapezoid, a pentagon, a hexagon, a heptagon or an octagon, the article regulating the immune response to a foreign substance by suppressing the immune response, and the topography being a) the average distance between adjacent protrusions being 2 to 12 μm, b) the area of the top surface region of the projection is 20 to 200 µm ２ and c) the protrusions covering 26 to 50% of the surface, defined by the above, an article. Item 4. An article comprising a surface portion having one or more topographies for physically stimulating immune cells, wherein the topography comprises a surface having a regular pattern of protrusions, the protrusions comprising one or more protrusion elements, the protrusion elements being defined as surface portions raised on the surface having a top surface region and a surrounding side surface connecting the top surface region to the surface, each of the protrusion elements having a maximum height of 0.5 to 50 μm on the surface, wherein the shape of the protrusion in plan view is a complex shape, the complex shape being defined as a combination of a plurality of overlapping or adjacent basic shapes, the basic shapes being selected from a circle, an ellipse, a triangle, a square, a rectangle, a trapezoid, a pentagon, a hexagon, a heptagon or an octagon, the article regulating the immune response to a foreign substance by stimulating the immune response, and the topography being a) the average distance between adjacent protrusions being 5 to 12 μm, b) The area of the top surface region of the protrusion is 20 to 70 μm ２ and c) the protrusions covering 15 to 25% of the surface, defined by the above, an article. Item 5. An article comprising a surface portion having one or more topographies for physically stimulating immune cells, wherein the topography comprises a surface having a regular pattern of protrusions, the protrusions comprising one or more protrusion elements, the protrusion elements being defined as surface portions raised on the surface having a top surface region and a surrounding side surface connecting the top surface region to the surface, each of the protrusion elements having a maximum height of 0.5 to 50 μm on the surface, wherein the shape of the protrusion in plan view is a complex shape, the complex shape being defined as a combination of a plurality of overlapping or adjacent basic shapes, the basic shapes being selected from a circle, an ellipse, a triangle, a square, a rectangle, a trapezoid, a pentagon, a hexagon, a heptagon or an octagon, and the article regulates the immune response to a foreign substance by stimulating the immune response at an early stage against the foreign substance, resulting in a lower immune response at a final stage, and the topography is a) the average distance between adjacent protrusions being 12.1 to 20 μm, b) the area of the top surface region of the protrusion is 25 to 65 μm ２ and c) the protrusions covering 5 to 10% of the surface, and being defined by the above, the article. Item 6. An article comprising a surface portion having one or more topographies capable of regulating the morphology, proliferation, biochemical function, differentiation, adhesion, migration, signal transduction, and / or cell death of a cell population by physical stimulation in vitro, wherein the topography comprises a surface having a regular pattern of protrusions, the protrusions comprising one or more protrusion elements, the protrusion elements being defined as surface portions raised on the surface having a top surface region and a surrounding side surface connecting the top surface region to the surface, each of the protrusion elements having a maximum height of 0.5 to 50 μm on the surface, wherein the shape of the protrusion in plan view is a complex shape, the complex shape being defined as a combination of a plurality of overlapping or adjacent basic shapes, the basic shapes being selected from a circle, an ellipse, a triangle, a square, a rectangle, a trapezoid, a pentagon, a hexagon, a heptagon or an octagon, the regulation including maintaining the phenotype and function of hepatocytes, and each topography is a) the average distance between adjacent protrusions being 3 to 25 μm, b) the area of the top surface region of the protrusion is 20 to 250 μm ２ and c) the protrusions covering 8 to 35% of the surface, and being defined by the above, the article. Item 7. a) when the protrusion has one protrusion element, The length of the protrusion defined as the length of the longest linear fitting within the perimeter of the top surface region parallel to the surface is 0.01 to 100 μm, The width of the protrusion defined as the length of the longest linear fitting within the perimeter of the top surface region perpendicular to the length and parallel to the surface is 0.01 to 100 μm, b) When the protrusion has a plurality of protrusion elements, The length of each protrusion element defined as the length of the longest linear fitting within the perimeter of the top surface region parallel to the surface is 0.01 to 100 μm, The width of each protrusion element defined as the length of the longest linear fitting within the perimeter of the top surface region perpendicular to the length and parallel to the surface is 0.01 to 100 μm, The average distance between two perimeters of adjacent protrusion elements of the same protrusion is 0 to 50 μm, The object according to any one of claims 1 to 6.

8. The surface part includes a metal material, a polymer material, a composite material or a ceramic material, The object according to any one of claims 1 to 7.

9. The regular pattern of the protrusions is defined by a grid of intersecting grid lines that can be placed on the surface, and the grid lines define a pattern of unit cells such that each unit cell contains at most one protrusion. The object according to any one of claims 1 to 8.

10. The surface part includes 1 to 10 different topographies, The object according to any one of claims 1 to 9.

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