WO2018080308A2 - Topographies de surface pour stimuler l'endothélialisation - Google Patents

Topographies de surface pour stimuler l'endothélialisation Download PDF

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Publication number
WO2018080308A2
WO2018080308A2 PCT/NL2017/050701 NL2017050701W WO2018080308A2 WO 2018080308 A2 WO2018080308 A2 WO 2018080308A2 NL 2017050701 W NL2017050701 W NL 2017050701W WO 2018080308 A2 WO2018080308 A2 WO 2018080308A2
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WO
WIPO (PCT)
Prior art keywords
protrusion
valley
protrusions
surface area
top surface
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PCT/NL2017/050701
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English (en)
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WO2018080308A8 (fr
WO2018080308A3 (fr
Inventor
Berendien Jacoba Papenburg
Jan De Boer
Godefridus Francicus Bernardus HULSHOF
Original Assignee
Materiomics B.V.
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Priority claimed from PCT/NL2017/050092 external-priority patent/WO2017142405A1/fr
Application filed by Materiomics B.V. filed Critical Materiomics B.V.
Publication of WO2018080308A2 publication Critical patent/WO2018080308A2/fr
Publication of WO2018080308A8 publication Critical patent/WO2018080308A8/fr
Publication of WO2018080308A3 publication Critical patent/WO2018080308A3/fr

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61F—FILTERS IMPLANTABLE INTO BLOOD VESSELS; PROSTHESES; DEVICES PROVIDING PATENCY TO, OR PREVENTING COLLAPSING OF, TUBULAR STRUCTURES OF THE BODY, e.g. STENTS; ORTHOPAEDIC, NURSING OR CONTRACEPTIVE DEVICES; FOMENTATION; TREATMENT OR PROTECTION OF EYES OR EARS; BANDAGES, DRESSINGS OR ABSORBENT PADS; FIRST-AID KITS
    • A61F2/00—Filters implantable into blood vessels; Prostheses, i.e. artificial substitutes or replacements for parts of the body; Appliances for connecting them with the body; Devices providing patency to, or preventing collapsing of, tubular structures of the body, e.g. stents
    • A61F2/02—Prostheses implantable into the body
    • A61F2/28—Bones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61C—DENTISTRY; APPARATUS OR METHODS FOR ORAL OR DENTAL HYGIENE
    • A61C8/00—Means to be fixed to the jaw-bone for consolidating natural teeth or for fixing dental prostheses thereon; Dental implants; Implanting tools
    • A61C8/0012—Means to be fixed to the jaw-bone for consolidating natural teeth or for fixing dental prostheses thereon; Dental implants; Implanting tools characterised by the material or composition, e.g. ceramics, surface layer, metal alloy
    • A61C8/0013—Means to be fixed to the jaw-bone for consolidating natural teeth or for fixing dental prostheses thereon; Dental implants; Implanting tools characterised by the material or composition, e.g. ceramics, surface layer, metal alloy with a surface layer, coating
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61F—FILTERS IMPLANTABLE INTO BLOOD VESSELS; PROSTHESES; DEVICES PROVIDING PATENCY TO, OR PREVENTING COLLAPSING OF, TUBULAR STRUCTURES OF THE BODY, e.g. STENTS; ORTHOPAEDIC, NURSING OR CONTRACEPTIVE DEVICES; FOMENTATION; TREATMENT OR PROTECTION OF EYES OR EARS; BANDAGES, DRESSINGS OR ABSORBENT PADS; FIRST-AID KITS
    • A61F2/00—Filters implantable into blood vessels; Prostheses, i.e. artificial substitutes or replacements for parts of the body; Appliances for connecting them with the body; Devices providing patency to, or preventing collapsing of, tubular structures of the body, e.g. stents
    • A61F2/02—Prostheses implantable into the body
    • A61F2/30—Joints
    • A61F2/30767—Special external or bone-contacting surface, e.g. coating for improving bone ingrowth
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61F—FILTERS IMPLANTABLE INTO BLOOD VESSELS; PROSTHESES; DEVICES PROVIDING PATENCY TO, OR PREVENTING COLLAPSING OF, TUBULAR STRUCTURES OF THE BODY, e.g. STENTS; ORTHOPAEDIC, NURSING OR CONTRACEPTIVE DEVICES; FOMENTATION; TREATMENT OR PROTECTION OF EYES OR EARS; BANDAGES, DRESSINGS OR ABSORBENT PADS; FIRST-AID KITS
    • A61F2/00—Filters implantable into blood vessels; Prostheses, i.e. artificial substitutes or replacements for parts of the body; Appliances for connecting them with the body; Devices providing patency to, or preventing collapsing of, tubular structures of the body, e.g. stents
    • A61F2/02—Prostheses implantable into the body
    • A61F2/30—Joints
    • A61F2/30767—Special external or bone-contacting surface, e.g. coating for improving bone ingrowth
    • A61F2/30771—Special external or bone-contacting surface, e.g. coating for improving bone ingrowth applied in original prostheses, e.g. holes or grooves
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61C—DENTISTRY; APPARATUS OR METHODS FOR ORAL OR DENTAL HYGIENE
    • A61C8/00—Means to be fixed to the jaw-bone for consolidating natural teeth or for fixing dental prostheses thereon; Dental implants; Implanting tools
    • A61C8/0018—Means to be fixed to the jaw-bone for consolidating natural teeth or for fixing dental prostheses thereon; Dental implants; Implanting tools characterised by the shape
    • A61C8/0037—Details of the shape
    • A61C2008/0046—Textured surface, e.g. roughness, microstructure
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61F—FILTERS IMPLANTABLE INTO BLOOD VESSELS; PROSTHESES; DEVICES PROVIDING PATENCY TO, OR PREVENTING COLLAPSING OF, TUBULAR STRUCTURES OF THE BODY, e.g. STENTS; ORTHOPAEDIC, NURSING OR CONTRACEPTIVE DEVICES; FOMENTATION; TREATMENT OR PROTECTION OF EYES OR EARS; BANDAGES, DRESSINGS OR ABSORBENT PADS; FIRST-AID KITS
    • A61F2/00—Filters implantable into blood vessels; Prostheses, i.e. artificial substitutes or replacements for parts of the body; Appliances for connecting them with the body; Devices providing patency to, or preventing collapsing of, tubular structures of the body, e.g. stents
    • A61F2/02—Prostheses implantable into the body
    • A61F2/30—Joints
    • A61F2/30767—Special external or bone-contacting surface, e.g. coating for improving bone ingrowth
    • A61F2/30771—Special external or bone-contacting surface, e.g. coating for improving bone ingrowth applied in original prostheses, e.g. holes or grooves
    • A61F2002/30838—Microstructures
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61F—FILTERS IMPLANTABLE INTO BLOOD VESSELS; PROSTHESES; DEVICES PROVIDING PATENCY TO, OR PREVENTING COLLAPSING OF, TUBULAR STRUCTURES OF THE BODY, e.g. STENTS; ORTHOPAEDIC, NURSING OR CONTRACEPTIVE DEVICES; FOMENTATION; TREATMENT OR PROTECTION OF EYES OR EARS; BANDAGES, DRESSINGS OR ABSORBENT PADS; FIRST-AID KITS
    • A61F2/00—Filters implantable into blood vessels; Prostheses, i.e. artificial substitutes or replacements for parts of the body; Appliances for connecting them with the body; Devices providing patency to, or preventing collapsing of, tubular structures of the body, e.g. stents
    • A61F2/02—Prostheses implantable into the body
    • A61F2/30—Joints
    • A61F2/30767—Special external or bone-contacting surface, e.g. coating for improving bone ingrowth
    • A61F2/30771—Special external or bone-contacting surface, e.g. coating for improving bone ingrowth applied in original prostheses, e.g. holes or grooves
    • A61F2002/30878—Special external or bone-contacting surface, e.g. coating for improving bone ingrowth applied in original prostheses, e.g. holes or grooves with non-sharp protrusions, for instance contacting the bone for anchoring, e.g. keels, pegs, pins, posts, shanks, stems, struts
    • A61F2002/30891—Plurality of protrusions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61F—FILTERS IMPLANTABLE INTO BLOOD VESSELS; PROSTHESES; DEVICES PROVIDING PATENCY TO, OR PREVENTING COLLAPSING OF, TUBULAR STRUCTURES OF THE BODY, e.g. STENTS; ORTHOPAEDIC, NURSING OR CONTRACEPTIVE DEVICES; FOMENTATION; TREATMENT OR PROTECTION OF EYES OR EARS; BANDAGES, DRESSINGS OR ABSORBENT PADS; FIRST-AID KITS
    • A61F2/00—Filters implantable into blood vessels; Prostheses, i.e. artificial substitutes or replacements for parts of the body; Appliances for connecting them with the body; Devices providing patency to, or preventing collapsing of, tubular structures of the body, e.g. stents
    • A61F2/02—Prostheses implantable into the body
    • A61F2/30—Joints
    • A61F2/30767—Special external or bone-contacting surface, e.g. coating for improving bone ingrowth
    • A61F2002/3093—Special external or bone-contacting surface, e.g. coating for improving bone ingrowth for promoting ingrowth of bone tissue

Definitions

  • cells can be placed on a surface while in a suitable culture medium, the goal being to have these cells to proliferate as natural as possible, and/or express specific biological properties such as differentiation, and/or responding to a specific chemical, physical or electrical stimulus.
  • a conventional strategy to change cell behavior is to add a variety of hormones, chemical compounds, growth factors, enzymes, salts and the like, which may have the effect of an altered cell response, such as a change in proliferation or an induced differentiation.
  • altered cell behavior in these conventional cultures is generally chemically induced, through interaction of the hormones, chemical compounds, growth factors, enzymes, salts and the like with the cell surface and/or cell interior.
  • potential micropatterns present on the surface generally are not taken into account.
  • topographical cues at cell level has been observed in different cell types. For example, it is shown that hepatocyte cell attachment, morphology and function were remarkably improved when cultured on surfaces featuring nano-topographies as compared to smooth substrates [You J et al. ACS Appl. Mater. Interfaces 2015; 5: 12299- 12308.].
  • Endothelial cells make up the inner lining of blood vessels and lumen forming the interface between the circulating blood or lymph and the rest of the vessel wall. Endothelial cells are involved in many vascular functions, including the prevention of blood clotting at the vessel wall. Proper endothelialization, i.e. formation of a uniform endothelial layer, is highly important in neovascularization (regeneration of blood vessels) and on the surface of implanted medical devices that are in contact with circulating blood or lymph. For example, accelerated formation of the endothelial layer on vascular medical devices such as stents, grafts and stent grafts is highly beneficial for proper functionality and performance of these medical device. Cell migration as well as cell attachment and proliferation are the major processes that drive endothelialization of a surface, either on their own or in combination.
  • Figure 1 a, b Schematic top view of two adjacent protrusions comprising (a) one and (b) two protrusion elements. (Imaginary) sample lines indicate the method used for determining the average distance between the two adjacent protrusions.
  • Figure 1 c, d 3D schematic top view of 2x2 pattern of protrusions, containing (c) one and (d) two protrusion elements. Protrusions, valley walls, valley wall sections and valley wall punctures are indicated.
  • Figure 2 Topographies El - E20 stimulate overall endothelialization. Images show visualization of endothelial cell migration through nano-bead uptake by the cells cultured on substrates featuring selected surface topographies, nonpatterned (NP) substrates and control substrates with basic topographies (B3, B4). Accumulation of the nano-beads in the cells, which is a measure for migration, are indicated by the white arrows.
  • FIG. 3 Topographies El - E20 stimulate overall endothelialization.
  • Graph shows topographies El-20 steering endothelial cell migration, attachment and proliferation. Analyzed for (a) nano-bead uptake, (b) the number of attached cells, (c) the number of EdU-positive cells and (d) the percentage of EdU-positive cells over all cells on the surface. NP control levels are indicated by the dashed line, basic topographies (B3, B4) are included for comparison.
  • Modulation is defined as altering the physiological state of the cells by altering morphology, proliferation, biochemical functioning, differentiation, attachment, migration, signaling, and/or cell death of an endothelial cell population by physical stimulation through the surface topography.
  • modulation is defined as modifying morphology, proliferation, biochemical functioning, differentiation, attachment, and/or migration of endothelial cells, in vivo or in vitro.
  • modulation means enhancing attachment, enhancing proliferation and increasing migration, in particular enhancing migration and/or enhancing proliferation, in vivo or in vitro.
  • modulation means enhancing migration.
  • modulation means enhancing proliferation.
  • modulation means enhancing attachment.
  • Modulation of endothelial cells thus means the modulation of morphology, proliferation, biochemical functioning, differentiation, attachment, migration, signaling, and/or cell death of an endothelial cell population. Modulation of an endothelial cell population results in stimulated endothelialization. This also results in enhanced growth of blood and/or lymph vessels, preferably at the site of the surface topography.
  • the invention furthermore provides methods to modulate endothelial cells, as well as an object provided with a surface topography as defined below for use in modulating endothelial cells.
  • the invention provides a surface topography as defined below for the preparation of an object for use in the treatment of various (blood or lymphatic) vessel conditions. Furthermore, the invention provides for a surface topography as defined herein, for the preparation of a medicament for the treatment of various vessel conditions.
  • Endothelial cells are defined as the cells found in the inner layer of blood vessels or lymphatic vessels, and are well-known in the art.
  • the inner layer of blood vessels or lymphatic vessels comprises endothelial cells, which in the case of blood vessels are known as vascular endothelial cells, and in the case of lymphatic vessels are known as lymphatic endothelial cells. Both types are comprised in the term "endothelial cells" as used herein.
  • the inner layer of blood vessels and lymphatic vessels comprising endothelial cells are also known as the endothelium (or endothelium layer).
  • the endothelium in the blood vessels and lymphatic vessels is involved in several functions.
  • the endothelium forms an interface between the circulating blood or lymph and the vessel wall, and acts as a selective barrier between the blood or lymph in the vessels and vessel wall.
  • the endothelium affects among others the quantity and type of fluid passing to and from the vessel fluid to and from the other side of the vessel wall.
  • the endothelium is also involved in hormone trafficking and background muscle activity, as well as in formation of new (blood or lymphatic) vessels (angiogenesis).
  • angiogenesis new (blood or lymphatic) vessels
  • a major function of endothelial cells in blood vessels is to provide a non-thrombogenic surface to avoid blood clotting.
  • Endothelial cells in the present context, refers to primary endothelial cells as present in the blood or lymphatic vessels, as well as to progenitor endothelial cells, endothelial-like cells, and progenitor endothelial-like cells.
  • Endothelial-like cells are cells which are not, and will not become, primary endothelial cells, but which have largely similar biophysical and biochemical function.
  • Endothelial-like cells include for example immortalized endothelial cell lines such as HMEC-1, as well as endothelial cells derived from any type of stem cell, such as from induced pluripotent stem cells (iPSCs).
  • iPSCs induced pluripotent stem cells
  • endothelial cells are primary endothelial cells or endothelial-like cells, and most preferably, endothelial cells are primary endothelial cells (herein also referred to as "primary cells").
  • Endothelial cells as used herein may be from human or animal origin, and are preferably mammalian cells, in particular cells from human, monkey, bovine, pig, rat or mouse. Most preferably, the cells are human endothelial cells.
  • Endothelialization is the formation of an endothelium on the surface of implanted medical devices. This is crucial for maintaining tissue functioning when it comes to the interactions with blood or lymph. Stimulating one or more of the parameters involved in endothelialization, including attachment, proliferation and migration of endothelial cells to or over the surface of the implant or tissue allows for (accelerated) formation of an endothelium and thus stimulates
  • a group of surface topographies has been identified that is able to modulate endothelial cells, in particular by promoting attachment, proliferation and/or migration of endothelial cells, in vivo or in vitro.
  • cell population is to be understood as referring to an endothelial cell population.
  • cell refers to an endothelial cell as defined above.
  • topographies allow for higher cell motility and migration capacity compared to surfaces without such topography (nonpatterned surface) or with basic (square, circular, triangular and the like) topographies. Higher motility results in increased presence of endothelial cells at the surface with topography.
  • the identified topographies stimulate cell attachment compared to nonpatterned surfaces or surfaces with basic topographies. Increased attachment results in anchoring of migrating endothelial cells to the surface topography, thus also increasing the presence of endothelial cells at the location of the surface topography.
  • the identified surface topographies do not negatively affect endothelial cell proliferation.
  • Surface topographies of the invention either increase the cell proliferation capacity or maintain a natural proliferation capacity, in comparison to a nonpatterned surface or a surface with a basic topography.
  • the invention pertains to a surface topography, as well as to an object comprising a surface part provided with one or more of such topographies.
  • the surface topography is formed by the presence of regularly spaced protrusions on the surface part, and can be defined by two alternative definitions.
  • the surface topography can be defined by the average distance between adjacent protrusions, the top surface area of the protrusions, and the coverage of the surface part, and furthermore by the length and width of the protrusions (the "protrusion '-definition).
  • the topography can be defined by the valleys which are located between the protrusions (the "valley" -definition).
  • the present invention provides an object, comprising a surface part provided with one or more topographies capable of modulating the morphology, proliferation, biochemical functioning, differentiation, attachment, migration, signaling, and/or cell death of a cell population by physical stimulation, and wherein the topography comprises a surface provided with a regular pattern of protrusions, which protrusions comprise one or more protrusion elements, which protrusion elements are defined as surface portions elevated above the surface having a top surface area and a circumferential side face connecting the top surface area with the surface, wherein each protrusion element has a maximum height of between 0.5 and 50 ⁇ m above the surface, and wherein
  • the average distance between adjacent protrusions is between 0 and 50 ⁇ m; b) the top surface area of the protrusion is between 1 and 6000 ⁇ m 2 ; and c) the protrusions cover between 3 and 90 % of the surface.
  • the object of the invention has a length and width
  • the length of the protrusion defined as the length of the longest straight- line fitting within the circumference of the top surface area parallel to the surface, is 0.01 - 100 ⁇ m;
  • the width of the protrusion defined as the length of the longest straight- line fitting within the circumference of the top surface area parallel to the surface, perpendicular to the length, is 0.01 - ⁇ m;
  • the length of each protrusion element defined as the length of the longest straight-line fitting within the circumference of the top surface area parallel to the surface, is 0.01 - 100 ⁇ m;
  • each protrusion element defined as the length of the longest straight-line fitting within the circumference of the top surface area parallel to the surface perpendicular to the length, is 0.01 - 100 ⁇ m;
  • the average distance between two circumferences of adjacent protrusion elements of the same protrusion is 0 - 50 ⁇ m.
  • the topography comprises a surface provided with a regular pattern of protrusions.
  • Protrusions are comprised of one or more protrusion elements, which elements are surface portions elevated above the surface on which the protrusions are formed, i.e. a surface surrounding the relevant protrusion or protrusion element.
  • Each protrusion element is defined by an elevated surface portion and by a top surface area, as well as by a circumferential side face which connects the top surface area with the surface.
  • the elevated surface portion is the total of the top surface area and the circumferential side face.
  • One protrusion may be defined as a single protrusion element, but one protrusion may also comprise multiple protrusion elements.
  • a protrusion comprises a single protrusion element.
  • a protrusion comprises at least two protrusion elements.
  • protrusions are small bumps or groups of bumps on the surface.
  • the protrusions define between them a three-dimensional network of valleys in the surface of the object. It has been found that a regular pattern of protrusions with a specific size, shape and configuration results in a topography that is capable of modulating the cell response, most notably morphology, proliferation, biochemical functioning, differentiation, attachment, migration, signaling, and/or cell death of a cell population by physical stimulation.
  • the regular pattern can be defined by an imaginary grid of intersecting gridlines which can be laid over the surface, which gridlines define a pattern of unit cells, such that each unit cell comprises a maximum of one protrusion.
  • the unit cells may have any shape.
  • a unit cell has a rectangular, especially square, trapezoid, triangular or hexagonal shape.
  • a regular pattern in this context means that any section of the topography which consists of m unit cells, for instance in a n X n arrangement, is repetitively present in sections adjacent to that section.
  • m and n are 1, but m may be 4 - 16 and n may be 2 - 4, in particular in case of unit cells comprising differently shaped or differently oriented protrusions, or in case of triangular, hexagonal or trapezoid unit cells.
  • a unit cell may for instance have a surface area of between 1 and 10000 ⁇ m 2 , preferably between 1 and 2500 ⁇ m 2 , more preferably between 25 and 2000 ⁇ m 2 , more preferably between 100 and 1000 ⁇ m 2 .
  • the unit cell may have length X width dimensions of (1-100) ⁇ m X (1- 100) ⁇ m, preferably (1-50) ⁇ m X (1-50) ⁇ m.
  • unit cells are square and have length X width dimensions of 5 ⁇ m X 5 ⁇ m to 45 X 45 ⁇ m, preferably 5 X 5 to 30 ⁇ m X 30 ⁇ m such as for example 5 ⁇ m X 5 ⁇ m, 10 ⁇ m X 10 ⁇ m, 15 ⁇ m X 15 ⁇ m, 20 ⁇ m X 20 ⁇ m, 25 ⁇ m X 25 ⁇ m or 28 ⁇ m X 28 ⁇ m.
  • each unit cell comprises a protrusion of equal dimensions, i.e., each unit cell comprises protrusions identical to one another.
  • the dimensions of a protrusion are defined as the total
  • characterizing features including for instance the height of the protrusion, the number and relative position of protrusion elements inside the unit cell, the length and width of each protrusion element, the angle of the circumferential side face relative to the surface, and the shape of the top surface area of each protrusion element.
  • the height of a protrusion, or at least of a protrusion element is between 0.5 and 50 ⁇ m above the surface. That is, the height of each protrusion element may be between 0.5 and 50 ⁇ m above the surface, even though in case a protrusion comprises multiple protrusion elements, the height of various protrusion elements in the protrusion may be different within the mentioned boundaries. Preferably, however, the height of each protrusion element is the same. The height is measured as a maximum height of the top surface area of a protrusion or protrusion element above the surface, especially above a surrounding surface area within a unit cell.
  • the height of a protrusion may be between 1 and 45, preferably between 2 and 40 ⁇ m, more preferably between 3 and 35 more preferably between 4 and 30 ⁇ m, even more preferably between 4 and 28 ⁇ m.
  • the number and relative position of protrusion elements inside a unit cell may vary from one unit cell to another as long as a regular pattern of protrusions is obtained. That is, two or more different unit cells comprising the same or different protrusions may be defined, which unit cells are placed in a regular pattern, for example alternate regularly on the surface. In case of triangular or trapezoid unit cells with protrusions of equal dimensions, this may result in a regular pattern of the same protrusions which have different orientations, for example alternatingly opposite orientation on the surface.
  • an alternating pattern of said unit cells results in a regular pattern of rows of said protrusions, which rows are either oriented parallel to the edges of the unit cells, or parallel to the diagonal of the unit cell.
  • square unit cells all having the same protrusion may be oriented differently on the surface, so as to obtain a surface in which the
  • protrusions are regularly distributed with varying orientation.
  • unit cells may be devised, and can be used in the present invention.
  • square, rectangular and hexagonal unit cells are all oriented in the same direction, while triangular unit cells are alternatingly oriented.
  • all unit cells contain an identical protrusion.
  • the length of each protrusion (or in case of protrusions comprising multiple protrusion elements, the length of each protrusion element), is defined as the length of the longest straight-line fitting within a circumference of the top surface area of the protrusion or protrusion element, parallel to the surface.
  • the length of a protrusion is 0.01 - 100 ⁇ m.
  • the length is 0.5 - 50 ⁇ m, more preferably 1 - 45, more preferably 2 - 40 ⁇ m.
  • the length of a protrusion element is 0.01 - 100 ⁇ m.
  • the length is 0.1 - 45 ⁇ m, more preferably 0.5 - 40 ⁇ m, even more preferably 1 - 35 ⁇ m.
  • each protrusion (or in case of protrusions comprising multiple protrusion elements, the width of each protrusion element), is defined as the length of the longest straight-line fitting within the circumference of the top surface area of the protrusion or protrusion element parallel to the surface, perpendicular to the length.
  • the width of a protrusion is 0.01 - 100 ⁇ m.
  • the width is 0.5 - 50 ⁇ m, more preferably 1 - 45 ⁇ m, more preferably 2 - 40 ⁇ m.
  • the width of a protrusion element is 0.01 - 100 ⁇ m.
  • the width is 0.1 - 45 ⁇ m, more preferably 0.5 - 40 ⁇ m, even more preferably 1 - 35 ⁇ m.
  • the number and relative position of protrusion elements inside the unit cell is an important feature of the dimensions of the protrusion.
  • the average distance between adjacent protrusion elements of the same protrusion is determined by dividing the sides of the protrusion elements which face each other into equal line segments of about 0.4 ⁇ m, and determining the distance of each line segment of the first protrusion element to the facing line segment of the adjacent protrusion element (see figure).
  • the average of all distances is the average distance between the protrusion elements.
  • the average distance of one protrusion element to a second protrusion element of the same protrusion may be different from the average distance of the same protrusion element to a third protrusion element of the same protrusion.
  • the average distance between adjacent protrusions is determined at half the height of the protrusion.
  • the same type of calculation is applied for determining the average distance between two facing protrusions, as has been exemplified in figure la and lb.
  • "Facing" in this context means that the distance between adjacent protrusions is determined on the basis of line segments stretching between those protrusions, over the length of the protrusion which is the shortest.
  • the average distance between two protrusions is 0 - 50 ⁇ m, preferably 0.5 - 40 ⁇ m, even more preferably 1 - 30 ⁇ m, even more preferably 2 - 20 ⁇ m.
  • the average distance between two protrusion elements in the same protrusion is similarly defined.
  • the average distance between two adjacent protrusions in one direction is 0, then the average distance between adjacent protrusions in the direction perpendicular to that distance should be larger than 0.
  • the average distance between adjacent protrusions in any direction is larger than 0.
  • the surface topography can be defined by determining the shortest and the longest distance between facing protrusions.
  • the shortest distance is defined by the shortest straight line as defined above for the calculation of average distance which can be drawn between two adjacent protrusions or protrusion elements.
  • the shortest distance is preferably 0 - 50 ⁇ m, more preferably 0 - 40 ⁇ m, more preferably 0 - 20 ⁇ m, and more preferably 0 - 15 ⁇ m.
  • the shortest distance is 1 - 50 ⁇ m, preferably 2 - 40 ⁇ m, more preferably 3 - 30 ⁇ m, even more preferably 4 -20 ⁇ m.
  • the longest distance is defined as the longest straight line as defined above for the calculation of average distance which can be drawn between two adjacent protrusions or protrusion elements.
  • the longest distance can be 0 - 50 ⁇ m, preferably 0.5 - 45 ⁇ m, more preferably 1 - 40 ⁇ m and more preferably 5 - 35 ⁇ m.
  • the circumferential side face of the protrusion elements may have any angle between 0 and 180° with the surface at the position where the side face intersects with the surface.
  • An angle of 90° is defined as normal to the surface, and an angle between 0° and 90° is defined as the situation where the top surface area is larger than the elevated portion of the surface, so that the top surface area at least partially covers the surface.
  • An angle between 90° and 180° is defined as the situation wherein the top surface area is smaller than the elevated portion of the surface.
  • the elevated portion of the surface is defined as the circumference of the protrusion at the point where it intersects the surface.
  • the circumferential side face of at least a portion of the protrusion or protrusion elements has an angle between 45° and 135° with the surface, more preferably between 60° and 120°, even more preferably between 75° and 115°, and even more preferably between 80° and 100°.
  • the circumferential side face of at least a portion of the protrusion or protrusion elements extends substantially normal to the surface at the position where the side face intersects with the surface, such as at an angle of 88 - 92°.
  • all protrusion elements have about the same angle with the surface at the position where the side face intersects with the surface.
  • each protrusion element is also an important feature of the dimensions of the protrusion, and may also be referred to as the shape of the protrusion (or protrusion element). This is because this shape defines the shape of the valley walls where cells of adjacent tissue may grow to provide fixation.
  • the shape of the circumference of the top surface area is determined parallel to the surface. As such, it is a top-view of the protrusion element, which may have any geometrical form.
  • the shape may comprise a circular, oval, triangular, square, rectangular, trapezoid, pentagonal, hexagonal, heptagonal or octagonal shape.
  • Such generally known shapes will be referred to as basic shapes herein.
  • a basic shape is symmetric, i.e. has at least one symmetry axis. In much preferred embodiments, the shape may comprise a combination of basic shapes.
  • the shape of the top surface area of each protrusion /protrusion element may not be a single geometric shape, such as a square, triangle, circle, octagonal, 5-pointed star, hexagonal, 3-pointed star, half moon, or a circle with a corner taken out ("pacman").
  • the shape may not be circular, oval, or a shape such as a polygon, triangle, rectangle, square, hexagon, star, parallelogram.
  • protrusions may have a shape as shown in table 1.
  • the topography does not comprise a protrusion shaped as shown in Table 1.
  • a topography does not have shape 1 from table 1.
  • a topography does not have shape 2 from table 1.
  • a topography does not have shape 3 from table 1.
  • a topography does not have, independently, shape 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, or 32 from table 1.
  • the topography does not comprise a selection of the topographies in table 1, and in further embodiments, the
  • topography does not comprise all of the shapes in table 1.
  • the top surface area is a surface positioned substantially parallel to the surface, although the top surface area may be slightly concave or convex, such as for instance slightly dome-shaped.
  • protrusions comprise overlapping or adjacent combinations of basic shapes, preferably interconnected by one or more bridging or overlapping portions, so as to obtain a complex shape. It has been found that the effect of modulating the cell response, most notably morphology, proliferation, biochemical functioning, differentiation, attachment, migration, signaling, and/or cell death of a cell population by physical stimulation by protrusions with specifically defined complex shapes is influenced not only by the dimensions of the protrusion, but also by the shape of the protrusion (or protrusion elements) alone. For protrusions of similar height, weight, length and width, but of different shape, there is a distinct effect on the cell response of the physically stimulating surface part. The individual effect of the shape of
  • protrusions on modulating morphology, proliferation, biochemical functioning, differentiation, attachment, and/or migration of endothelial cells of a cell population by physical stimulation will be further elaborated upon in the examples.
  • a complex shape can be obtained.
  • Complex shapes may comprise overlapping or adjacent circular, oval, triangular, square, rectangular, trapezoid, pentagonal, hexagonal, heptagonal or octagonal shapes.
  • adjacent basic shapes the adjacent basic shapes are preferably connected by an overlapping (bridging) portion, so that the top-view of the basic shape has a larger surface area than the surface area of the basic shapes alone.
  • the increased surface area relative to the separate shapes is preferably located between the basic shapes, effectively increasing the width of any point of contact between the basic shapes.
  • the circumferential side face of a protrusion is a continuous side face, which may be a connected series of straight line portions, connected by angles.
  • the straight line segments and/or the angles can be smoothly curved, so as to obtain a smoothly curved circumferential side face.
  • a smoothly curved circumferential side face in this context means that the circumferential side face only has rounded corners. Corners may stem from their presence in a shape, such as the corners in squares, triangles and hexagons, but such corners may also stem from the contact point between two adjacent shapes in a protrusion element, or from the presence of an overlapping (or bridging) portion between two basic shapes in a protrusion element. That is, corners in this sense refers to corners when the protrusion is seen with a top -view. When all corners in the circumferential side face of a protrusion element are rounded, a smoothly curved circumferential side face is obtained.
  • a protrusion has a complex shape.
  • a complex shape is a combination of basic shapes, in which the basic shapes are present as single protrusion elements, or as overlapping or adjacent basic shapes.
  • a complex shape is generally non-symmetric, i.e. has no symmetry axis.
  • a complex shape is further defined by the amount of corners in the circumferential side face.
  • a corner in this respect, may be a straight or rounded corner, and can be defined as any change in direction in the circumferential side face (top-view). Corners which take the shape of a line with a length of 5 % of the length of the circumferential side face will be dubbed a "bent". Straight lines in the circumferential side face will be called "straights".
  • a corner can be narrow or wide.
  • Narrow corners are corners with an angle of less than 90 °. That is, narrow corners result in a "spike" in the shape, which spike is either directed inward or outward, relative to the protrusion.
  • Wide corners are corners in which protrusion's circumferential side face displays an angle of more than 90 °.
  • Straight corners are corners with an angle of 90 °.
  • Complex shapes are defined as shapes having at least one wide corner, wherein preferably, the quantity of narrow corners is not the same as the quantity of wide corners.
  • complex shapes comprise at least one bent
  • complex shapes comprise both at least one bent and at least one straight.
  • a complex shape is a shape comprising more than two straights, preferably more than three, more preferably more than four, in which at least 75 % (preferably at least 85 %, more preferably at least 95 %) of all straights are of different length.
  • complex shapes comprise at least 3 bents.
  • An adjacent (or facing) protrusion to a protrusion in a particular unit cell is a protrusion which is present in a unit cell which shares a side with that particular unit cell. Adjacent unit cells are similarly defined. Unit cells which only share a corner with the particular unit cell are not adjacent. Given a regular pattern of protrusions, one protrusion is for example adjacent to three protrusions in case of triangular unit cells, to four protrusions in case of trapezoid, square or rectangular unit cells, and to six protrusions in case of hexagonal unit cells.
  • the top surface area of the protrusion is defined as the surface area of the protrusion at its most elevated circumference. It follows from the above that if a protrusion comprises multiple protrusion elements, that the top surface area of the protrusion is the sum of all surface areas of all protrusion elements of the protrusion, each determined at the most elevated circumference of the protrusion element.
  • the top surface area of a protrusion may be determined by for instance counting the number of pixels (as 0.4 ⁇ m X 0.4 ⁇ m elements) which make up the top surface area of the protrusion (or protrusion element).
  • the top surface area of the protrusion is between 1 and 6000 ⁇ m 2 , preferably 10 and 3000 ⁇ m 2 , more preferably between 20 and 1500 ⁇ m 2 , more preferably between 30 and 1000 ⁇ m 2 , even more preferably between 35 and
  • the coverage of the protrusions is defined as the percentage of top surface area relative to the full area which is covered by protrusions. Thus, the coverage is the sum of all top surface area, divided by the total area of the surface topography, times 100 %. This is the same as calculating which percentage of a unit cell comprises the top surface area of the protrusion.
  • the protrusions cover between 3 and 90 % of the surface part, preferably between 5 and 85 %, more preferably between 10 and 80 % of the surface part, more preferably between 20 and 78 %, more preferably between 25 and 75 %.
  • a protrusion covers between 3 and 90 % of a unit cell, preferably between 5 and 85 % of a unit cell, more preferably between 10 and 80 % of a unit cell, more preferably between 20 and 78 % of a unit cell, more preferably between 25 and 75 % of a unit cell.
  • the regular pattern of protrusions at the same time defines a pattern of valleys.
  • the surface topography of the invention may alternatively be defined by a surface part for modulating morphology, proliferation, biochemical functioning, differentiation, attachment, migration, signaling, and/or cell death of a cell population by physical stimulation, comprising a top surface area provided with a regular pattern of valleys defined by a regular pattern of protrusions, which valleys comprise a valley bottom, a first valley wall and a second valley wall, which first valley wall comprises first valley wall sections and optionally first valley wall punctures, and which second valley wall comprises second valley wall sections and optionally second valley wall punctures, wherein said first and second valley wall sections are defined by a side of a protrusion adjacent to the valley, and wherein said first and second valley wall punctures are defined as portions of the valley wall where a line perpendicular to the valley and parallel to the valley bottom does not contact the protrusion adjacent to the valley, and wherein
  • the valley wall has a height, defined as the distance normal to the valley bottom from the valley bottom to the top surface area, of 0.5 - 50 ⁇ m;
  • the valley wall profile of the first and second valley wall is independently between 0 and 40 ⁇ m.
  • the average width of the valley is between 0 and 50 ⁇ m.
  • the top surface area has been defined above, and is the area defined by all top surface areas of the protrusions. Between the protrusions, a series of valleys is defined.
  • the valleys are defined by two valley walls, which valley walls comprise those sections of the circumferential side face of the protrusions which are adjacent to the valley.
  • the valley wall is the hypothetical straight-line average of those parts of the circumferential side face of the protrusions which are adjacent to the valley, in the direction of the valley. See figure lc and Id.
  • a valley wall comprises valley wall sections, wherein a valley wall section is defined by a side of the protrusion (i.e. one side of a protrusion's circumferential side face) adjacent to the valley.
  • Valley wall sections are thus hypothetical straight lines on the valley wall, of a length defined by the presence of a protrusion.
  • the valley wall in that direction comprises only valley wall sections, and no valley wall punctures.
  • a valley wall further comprises valley wall punctures.
  • Valley wall punctures are defined as portions of the valley wall where a line perpendicular to the valley and parallel to the valley bottom does not contact a protrusion adjacent to the valley. As such, it is a portion of the valley wall where there is no valley wall section, i.e. the point in a valley wall where no protrusion is adjacent to the valley. See figure lc and Id.
  • the first and second valley wall defining a valley may be the same or different, and have the same or different valley wall profile.
  • the valley bottom is defined as the surface which is located between the protrusions, at the farthest possible distance from the top surface area.
  • the valley bottom may have some slight curvature due to processing, in which case the valley bottom extends from the point where the first valley wall rises to the point where the second valley wall rises. As such, it can also be defined by the average distance between the protrusions which form the valley.
  • a valley wall In line with the height of the protrusions, a valley wall has a height, defined as the distance normal to the valley bottom from the valley bottom to the top surface area, of 0.5 - 50 ⁇ m.
  • the height of the valley wall is between 1 and 45 ⁇ m, more preferably between 2 and 40 ⁇ m, more preferably between 3 and 35 ⁇ m, more preferably between 4 and 30 ⁇ m, even more preferably between 4 and 28 ⁇ m.
  • a valley wall is defined by the shape of the protrusions adjacent to the valley. As these shapes may be irregular, a valley wall is also defined by the valley wall profile.
  • the valley wall profile in this respect, is defined as the deviation in the direction normal to the valley wall, and is calculated on the basis of the valley wall sections only. Any valley wall puncture in the valley wall is excluded from the calculation.
  • the valley wall profile is expressed as the arithmetic mean of the distance of each point of a valley wall section from the valley wall. It is
  • R a is the valley wall profile
  • n is the number of data points and wherein
  • yi I is a positive number which indicates the distance to the valley wall segment, normal to the valley wall, i.e. normal to the average surface of the valley wall section.
  • the size of n is normally dependent on the resolution of the device used to measure R a , which can for instance be 0.4 ⁇ m, in which case n is the length of the valley wall section in ⁇ m.
  • R a can suitably be determined by
  • the valley wall profile is between 0 and 40 ⁇ m, preferably 0.1 and 30 ⁇ m, more preferably between 0.5 and 25 ⁇ m, even more preferably between 0.8 and 20.
  • a valley wall profile of 0 indicates that all protrusions which define the valley wall have a straight line parallel to the valley, such as may for instance occur in the case of square or rectangular protrusions.
  • the valley wall profile of the first and second valley wall may be different.
  • valley wall profile can be equated with the term “roughness”, although the term “roughness” is usually applied for the surface roughness of a surface, which is determined by the deviations of the average surface in vertical direction (for a horizontal surface), normal to the surface.
  • the valley wall profile is the roughness of the valley wall, i.e. the roughness of a vertical surface comprised on a horizontally oriented topography, which is determined by the deviation of the average valley wall in a direction parallel to the surface topography, and normal to the valley wall.
  • the length of valley wall sections can be the same as the length of a protrusion in the case when the longest straight-line fitting within the
  • a valley wall section is shorter than the length of the protrusion (or protrusion element).
  • the length of valley wall section is 0.01 - 100 ⁇ m, preferably, the length of a valley wall section is 0.05 - 50 ⁇ m, more preferably 0.1 - 40 ⁇ m, even more preferably 1 - 35 ⁇ m.
  • the length of valley wall sections in the two valley walls which define a valley may be the same or different.
  • the length of the valley wall punctures is generally between 0 and 50 ⁇ m, preferably 0.5 - 40 ⁇ m, more preferably 1 - 30 ⁇ m, even more preferably between 2 and 20 ⁇ m.
  • Valley wall punctures themselves define a valley, which is oriented perpendicular to the valley of interest.
  • the length of valley wall punctures in the first and second valley wall may be the same or different.
  • the average width of the valley is between 0 and 50 ⁇ m.
  • the average width of a valley is defined in line with the average distance between protrusions, or protrusion elements, and is calculated by dividing valley wall segments on opposing sides of the valley in line segments of about 0.4 ⁇ m, and determining the distance between each line segment of a valley wall section on one side of the valley and the facing line segment of the valley wall section of the adjacent protrusion on the other side of the (same) valley, and then averaging those distances.
  • the average valley width is the average distance between two adjacent protrusions.
  • the average width of the valley is between 0-50, preferably between 0.5 and 40 ⁇ m, more preferably between 1 and 30 ⁇ m, more preferably 2 and 20 ⁇ m.
  • the valley width may be defined by the shortest and longest distance between facing protrusions on opposite sides of the same valley, in line with the shortest and longest distance between facing protrusions defined above.
  • the surface topography comprises individually spaced protrusions, which do not touch adjacent protrusions.
  • the distance between protrusions i.e. the valley width
  • each valley wall comprises valley wall punctures.
  • a regular pattern of crossing valleys is obtained.
  • each valley wall comprises valley wall sections and valley wall punctures.
  • the valley wall is substantially normal relative to the valley bottom.
  • the angle that a valley wall can have relative to the valley bottom is defined in line with the angle that the circumferential side face may have with the surface.
  • a single protrusion defines multiple valley wall sections which may have a different profile, and multiple valley wall punctures, which may be of non-equal length.
  • the valley wall sections alternate with valley wall punctures in a regular order.
  • the topography as defined above is preferably present on an object having a surface part comprising a metal, polymeric, composite or ceramic material.
  • the surface part may be an exterior or interior surface part of the object.
  • An exterior surface part is a surface part which can be freely contacted by cells which are located at the outside of the object, when the cells are brought into contact with the object.
  • An exterior or interior surface part may include holes, pores or wells.
  • Suitable materials are known in the art, and include any material which is suitable for the application at hand. That is, for application of a topography of the invention on for instance a culture plate, a material suitable to construct a culture plate should be used. Similarly, for application of a topography of the invention on a vascular implant, a material suitable to construct a vascular implant should be used. The skilled person knows what type of material is suitable for what type of application, and is capable of choosing an appropriate material accordingly.
  • the object is made of a ceramic, glass, metal, polymeric material or composite materials.
  • the object comprises a surface part comprising carbon such as graphite, graphene, or LTI carbon.
  • a metal alloy can be used, such as an alloy comprising one or more elements selected from the group of silver, cobalt, iron, gold, platinum, zinc and titanium, as well as one or more elements selected from the group of aluminum, carbon, chromium, copper, iridium, manganese, magnesium, mercury, molybdenum, nickel, niobium, neodymium, palladium, tantalum, vanadium, tungsten, and zirconium.
  • Preferred alloys are Nitinol, a cobalt-chromium alloy, a Co/Cr/Mo alloy, as well as micro- galvanic alloys such as a Zn/Ag, Mg/Zn, Mg/Fe, Mg/Ni, or Mg/Cu, ferromagnetic alloys such as Mg/Al/Nd.
  • conductive polymers preferably polypyrrole, polyaniline and poly(3,4-ethylenedioxythiophene), polyurethane, polytetrafluoroethylene (Teflon), and polyethylene terephthalate, and biodegradable polymers like polyesters such as polylactic acid, polyethylene glycol (PEG or PEO) and polyglycolic acid, polyether ether ketone (PEEK), and composites of these materials.
  • An object comprising one (or more) topographies according to the invention can be made by any technique known for creating specifically shaped microstructures onto specific materials.
  • suitable techniques are 3D printing, laser printing, writing, layer-by-layer coating, electrospinning, deposition techniques, spraying and sputtering, stamping, (hot) pressing, (hot) embossing, (nano) imprinting, (injection) molding, casting, supercritical fluid-based processes, etching, laser machining, laser cutting and ablation, (precision) electrochemical machining, (precision) electrochemical gridding and (precision) electrical discharge machining, etc.
  • the object of the invention is preferably made by laser-machining, precision-electro(chemical) technologies, precision-electro assembly, engraving, printing, coating, stamping, (injection) molding, (hot) embossing, or etching the topography into or onto the object.
  • the object may be formed with the topography in a single process, such as by injection molding, pressing and hot- embossing. Suitable techniques to obtain an object provided with a topography as described are well-known in the art.
  • Printing may be achieved by employing 3D printing, laser printing and writing surface protrusions on a surface part of a metallic, polymeric, ceramic, composite or other substrate, as is known in the art.
  • Coating may be achieved by producing the protrusion on a substrate and then coat the surface part using spraying, sputtering, layer by layer coating, electrospinning, or deposition from solutions.
  • Stamping may be achieved by first producing a mold containing the negative form of the protrusions and then later stamp that mold onto a surface part by using techniques such as pressing, hot press, embossing, hot embossing, imprinting and nanoimprinting. Other techniques including injection molding, processing from a melt, or casting is also possible.
  • Etching may be achieved by using different solvents in the form of liquid and/or vapor, such as acids and/or organic solvents, as well as a suitable mold, such as to remove selective portions of the surface part defined by the mold design to obtain the surface topography.
  • solvents in the form of liquid and/or vapor such as acids and/or organic solvents
  • a suitable mold such as to remove selective portions of the surface part defined by the mold design to obtain the surface topography.
  • Precision-electro(chemical) technologies could include (precision) electrochemical machining, (precision) electrochemical gridding, (precision) electrical discharge machining.
  • the topography comprising a regular pattern of protrusions is a non-separable part of the object. This may be achieved by stamping, printing, etching, coating the topography onto the object or by using other methods mentioned above, for example by forming the object in a single process such as by injection molding.
  • the surface between the protrusions (valley bottom), the circumferential side face (the valley wall sections) and/or the top surface area of the protrusions may be smooth or substantially smooth, i.e. having a roughness of about 0 (i.e. 0 ⁇ 0.01 ⁇ m).
  • the surface between the protrusions and/or the top surface area and/or the circumferential side face may have a roughness, commonly abbreviated R a , of 0.01 ⁇ m - 10 ⁇ m, preferably 0.05 - 8 ⁇ m, even more preferably 0.1 - 5 ⁇ m.
  • the roughness may be 0.2 - 20 or 0.15 - 3 ⁇ m.
  • Roughness in this respect, is the surface roughness obtained by techniques such as etching, blasting or brushing the surface part prior to or after forming of the topography.
  • the valley wall profile abbreviated and calculated by the same formula, can thus be seen as the roughness of the valley wall normal to that surface.
  • the roughness can be determined using atomic force microscopy analysis.
  • a randomly roughened surface part may be obtained by conventional etching, brushing, blasting or the like, of the surface topography comprising a regular pattern of protrusions.
  • a flat surface part such as a surface part of an implant, may be roughened first, where after the surface topography of the invention is formed.
  • any randomly roughened surface part does still comprise the protrusions of the invention, so that a randomly roughened surface part may not have a roughness which is larger than the height and/or width of the protrusions, so that protrusions with the sizes indicated elsewhere remain present.
  • the topography of the invention is a surface topography consisting of a multitude of regularly spaced protrusions.
  • unit cells comprising a protrusion are distributed over the surface part so as to provide a single topography, as defined above.
  • the quantity of unit cells in a single topography is preferably at least 50, more preferably at least 100, even more preferably at least 200, even more preferably at least 500, even more preferably at least 1000.
  • Topographies which extend over a considerable surface part can be of high potential influence on the functioning of large cell collections, such as specific tissue. This is deemed appropriate for in vitro use, such as the growth of cell cultures or tissue, or in vivo use, such as the creation of implants.
  • a topography of the invention is capable of modulating the cell response, most notably modulating morphology, proliferation, biochemical functioning, differentiation, attachment, migration, signaling, and/or cell death of an
  • the topography of the invention is capable of altering the behavior of one or more living endothelial cells by inducing changes in morphology, proliferation, biochemical functioning, differentiation, attachment, migration, signaling, and/or cell death.
  • the behavior is altered by physical stimulation.
  • Physical stimulation in this context means that the signals which induce the change in behavior are transferred by among others shape, hardness and size of the surroundings where the cells are located, i.e. the topography.
  • “surroundings” of the cell(s) directly modulate the cell(s).
  • Physical stimulation means that the surface topography itself alters the cell response on various aspects, among which cell morphology, proliferation, biochemical functioning, differentiation, attachment, migration, signaling, and/or cell death, and that this response is different for different topographies.
  • cell morphology, proliferation, biochemical functioning, differentiation, attachment, migration, signaling, and/or cell death By changing a surface topography in contact with a cell or cell population, the cell's morphology, proliferation, biochemical functioning, differentiation, attachment, migration, signaling, and/or cell death can be influenced, all other parameters kept equal.
  • the physical stimulation which is responsible for the altered behavior is a separate effect from chemical stimulation to alter behavior.
  • chemical stimulation the onset of the signals received by the cell to alter behavior is chemically induced through an added compound or through surface chemistry (e.g. choice of bulk material, coating, or surface-functionalization) and is subsequently transferred by signaling molecules which interact with receptors on the cell surface or which enter the cell to interact with receptor molecules within the cell.
  • the mechanism behind the presently observed physical stimulation to alter behavior is still not fully understood, but the examples show that shape of the surface topography, rather than chemical stimulation, are causing the onset of the observed modulation of cell morphology, proliferation, biochemical functioning, differentiation, attachment, migration, signaling, and/or cell death of a cell population. So, the physical stimulation of cells is independent of the chemistry of the surface and/or environment.
  • the altered cell physiological behavior initiated by the physical stimulus of a surface topography might affect the cell's response to and interaction with signaling molecules and receptor molecules.
  • Modulation of endothelial cells includes stimulating endothelial-like behavior, which can be derived from stimulated morphology, stimulated
  • Stimulated morphology can be ascertained by imaging the cells through different techniques including immunofluorescent labeling and scanning electron microscopy. Stimulated differentiation and biochemical functioning can be
  • VE-cadherin Vascular endothelial cadherin
  • VEGF vascular endothelial growth factor
  • cell markers can be evaluated at gene- and protein- level using various techniques such as qPCR, ELISA, immunofluorescent staining, imaging, western blot and fluorescent- activated cell sorting (FACS), etc.
  • Stimulated attachment and proliferation can be ascertained by (1) Measuring the number of cells on a surface, through DNA-assay levels, imaging, counting, or otherwise, shortly after seeding (normally within 24 hrs) which indicates cell attachment to that surface and (2) by counting the number of viable cells over time, quantifying the DNA content or quantifying the number of cells over time or label cells that have or are replicating, e.g. by a EdU-positive cell assay.
  • EdU or 5-ethynyl-2'-deoxyuridine is a biomolecule that accumulates in the DNA of the cells during DNA replication and can be visualized by
  • Stimulated migration can be ascertained by migration assays such as live or time laps imaging or quantifying the migration over a surface through labeling migrating cells through e.g. a nano-bead uptake assay where the surface is, prior to adding the cells, coated with nano-beads that are taken up by cells when they adhere to that specific part of the surface.
  • migration assays such as live or time laps imaging or quantifying the migration over a surface through labeling migrating cells through e.g. a nano-bead uptake assay where the surface is, prior to adding the cells, coated with nano-beads that are taken up by cells when they adhere to that specific part of the surface.
  • the more a cell migrates the more surface that cell will have covered, the more nano-beads that will be taken up by that cell, which is subsequently measured.
  • an object of the invention can be a vascular or lymphatic implant, such as a cardiovascular implant, part of a cardiovascular implant, a lymphatic vessel implant or part of a lymphatic vessel implant and includes but is not limited to implants such as an artificial vessel, vascular graft, stent, AAA stent, stent graft, cardiac valve, cardiac pump, cardiac rhythm prevention product, total heart implant (artificial heart), pacemaker including (leadless) stimulator, blood pump, aneurism implants (coil), transcatheter pacing system, (intravascular) catheter, 2D or 3D scaffold for tissue regeneration, sensor.
  • endothelial cells are modulated in vitro.
  • an object of the invention can be a culture dish such as culture flask, plate, dish, slide, bottle, chamber or bag, microfluidic device, 2D or 3D scaffold, chips, (micro)fluidic devices, biore actors, membranes, etc. comprising a surface for culturing of endothelial cells.
  • a culture dish such as culture flask, plate, dish, slide, bottle, chamber or bag, microfluidic device, 2D or 3D scaffold, chips, (micro)fluidic devices, biore actors, membranes, etc. comprising a surface for culturing of endothelial cells.
  • Suitable culture media for this embodiment include any medium suitable for culturing endothelial cells, as known in the art.
  • Such media generally comprise water, and furthermore may comprise glucose, proteins, amino acids, vitamins, penicillin, antibiotics, and inorganic salts and whole blood or derivatives thereof.
  • the cells may be cultured in other liquids such as phosphate buffered saline (PBS), water, or on a gel such as Matrigel or agarose gel, on a coating such as collagen, laminin, fibronectin, fibrinogen, or polyamine or be embedded into a biomaterial such as a hydrogel or polymer carrier.
  • PBS phosphate buffered saline
  • Matrigel or agarose gel on a coating such as collagen, laminin, fibronectin, fibrinogen, or polyamine or be embedded into a biomaterial such as a hydrogel or polymer carrier.
  • Cells may be fixed using fixation solution and analyzed under wet or dry conditions.
  • Endothelial migration was determined using a fluorescent nano-bead uptake assay in which, prior to adding the cells, the substrate was coated with a homogeneous layer of fluorescently-labeled nano-beads. Cells migrating over the surface will take up these nano-beads so the more a cell migrates, the more nano- beads it will take up which is being measured. Endothelial proliferation was assessed using an EdU incorporation assay.
  • EdU (5-ethynyl-2 ' -deoxyuridine) is an alkyne -containing thymidine analog that incorporates in DNA during DNA replication and can be detected by a chemical reaction using a fluorescent dye.
  • the number of cells positive for EdU indicates cells that are or have been replicating, and when given in a percentage over all cells present, is a good indication for overall proliferation. Especially considering the early time point (18 hrs) only those surfaces that allow cells to immediately start proliferating, important for rapid endothelialization, will have higher levels of EdU-positive cells. The overall number of cells by itself is a good measure for cell attachment, especially in such early time point (18 hrs.) and taking into account proliferation. Culturing human coronary endothelial cells
  • the cultures were harvested after 18 hours of culture, washed with PBS, fixed with 3.7% formaldehyde, permeabilized with 0.1% Triton X-100 and treated with 2% BSA for blocking of non-specific binding sites.
  • the cell body was labeled with CellMaskTM Deep Red (L f set of substrates) or Orange (2 nd set of substrates, ThermoFisher).
  • the 2 nd set of substrates were then labeled with EdU labeling solution and DAPI (Sigma Aldrich).
  • the stained topographies were mounted onto glass slides.
  • Stimulated endothelialization is defined based on: high levels of nano- beads accumulated inside the cells reflecting strong motility of the cells over the surface, and total number of cells attached and total number of EdU-positive cells reflecting stimulation of cellular proliferation in a short time.
  • endothelialization had either higher bead uptake compared to the nonpatterned control (NP), up to 60% higher (for El- 15) or bead uptake level similar to NP (E16- E20) ( Figure 2, Figure 3a).
  • topographies of the invention had higher bead uptake, from 30% higher to even up to 130% higher (Table 2, Figure 3a).
  • the bead uptake on surface El was 230%) of that on basic surface B3 and B4 ( Figure 3a).
  • Cell attachment to the topographies of the invention was generally higher than to the non-patterned control ( Figure 3b).
  • topographies of the invention El- 15 had similar levels of EdU-positive cells (54-67%) compared to the nonpatterned control (68%) and slightly higher compared to basic topographies (50%) while topographies E16-E20 showed significantly increased number of EdU-positive cells (up to 80%>) (Figure 3c- d).
  • the surface topographies of the invention stimulate endothelialization by triggering migration and/or proliferation processes.
  • Surface topographies E1-E15 have a more dominant effect through the migration process in endothelial cells while surface topographies E16-E20 affect the proliferation process more dominantly. In strong contrast, the basic shapes do not stimulate
  • basic shape B4 has similar levels of EdU-positive cells compared to the nonpatterned control, but reduces migration by 30%.
  • basic shape B3 reduces both migration and the number of EdU-positive cells, both with 30% compared to the nonpatterned control.
  • endothelial cells need to quickly migrate from the out linings of the surrounding blood vessels or lumen over the surface and subsequently attach and proliferate to cover the surface. Simulating one of those three parameters (migration, attachment, and proliferation), while keeping the other parameters stable or stimulate those too, allows more rapid endothelialization of that surface.
  • the surface topographies that perform the best in improving endothelialization, through either stimulating endothelial cell attachment, - proliferation or - migration, or a combination of those, while maintaining the levels of the other parameters, have protrusions with a top surface area of 1-6000 and preferably 30- 1000 ⁇ m 2 , surface coverage of 3-90% and preferably 10-80 % and average distance between protrusions of 0-50 ⁇ m and preferably 1-30 ⁇ m.
  • the ranges are as defined in the general text above.

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  • Materials For Medical Uses (AREA)
  • Prostheses (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

L'invention concerne des topographies de surface qui peuvent être utilisées de manière à moduler la morphologie, la prolifération, le fonctionnement biochimique, la différenciation, la fixation, la migration, la signalisation et/ou la mort cellulaire d'une population de cellules endothéliales par stimulation physique. Ces topographies peuvent être appliquées in vitro et in vivo de manière à moduler le comportement cellulaire. Des exemples spécifiques comprennent des implants vasculaires ou lymphatiques dotés d'une topographie de l'invention qui augmentent l'endothélialisation.
PCT/NL2017/050701 2016-10-28 2017-10-30 Topographies de surface pour stimuler l'endothélialisation WO2018080308A2 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
NL2017684 2016-10-28
NL2017684 2016-10-28
PCT/NL2017/050092 WO2017142405A1 (fr) 2016-02-16 2017-02-16 Topographies de surface permettant de modifier la physiologie de cellules vivantes
NLPCT/NL2017/050092 2017-02-16

Publications (3)

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WO2018080308A2 true WO2018080308A2 (fr) 2018-05-03
WO2018080308A8 WO2018080308A8 (fr) 2018-05-31
WO2018080308A3 WO2018080308A3 (fr) 2018-07-05

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WO2018080308A3 (fr) 2018-07-05

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