A product with a topological microstructure capable of accelerating endothelial cell migration, and its preparation method and application

Abstract

The present invention discloses a product having a topological microstructure that can accelerate endothelial cell migration, including a substrate and a topological microstructure on the substrate surface. The topological microstructure is a continuous groove array composed of a number of continuous grooves arranged in a regular pattern. A single continuous groove is a continuous teardrop-shaped structure, and the cross-section of the teardrop-shaped structure is composed of a semicircle and a trapezoid. The radius r of the semicircle is selected from 2 to 50 μm, the lower base of the trapezoid is 2r, the height l of the trapezoid is selected from 10 to 100 μm, and the upper base a of the trapezoid is selected from 1 to 80 μm. The topological microstructure disclosed in the present invention is a continuous groove array in a continuous teardrop-shaped structure. Its special structure can improve the orientation of endothelial cells and accelerate the efficiency of endothelial cell migration. The structure is simple to prepare, has a controllable process, and has stable properties. It can be applied to medical devices such as occluders, arterial stent grafts, and balloons.

Description

Technical Field

[0001] The present invention relates to the technical field of biomaterials, and in particular to a product with a topological microstructure capable of accelerating endothelial cell migration, a preparation method thereof, and application in medical devices. Background Art

[0002] After implantation, the surface of an implantable medical device is completely covered with endothelial cells, achieving endothelialization, which is crucial for the device's proper function and the suppression of surface coagulation. During the endothelialization process, endothelial cells in contact with the device migrate to the device surface, providing a crucial cell source for endothelialization. Therefore, designing surfaces that promote rapid endothelial cell migration is crucial for improving the biosafety of implantable medical devices.

[0003] The existing methods of accelerating endothelialization mainly include chemical / biomolecule modification of medical device surfaces and physical construction of surface microstructures to guide cell migration. However, the chemical or biological modification of medical device surfaces is complex, has poor stability, high requirements for the substrate, and the bioactive substances may be inactivated during processing. Compared with chemical / biological modification methods, directly constructing microstructures on the surface by physical means is safer, more stable, and has a controllable process, which is conducive to industrial production applications. However, the existing surface microstructures are single in design (mainly irregular and rough surfaces or surface grooves), and the efficiency of guiding cell migration is low. Therefore, it is very important to design a topological microstructure surface that accelerates endothelial cell migration. Summary of the Invention

[0004] In response to the above-mentioned problems existing in the prior art, the present invention discloses a topological microstructure that can accelerate the migration of endothelial cells. This special topological microstructure can improve the orientation of endothelial cells and accelerate the efficiency of endothelial cell migration. In addition, the preparation of this structure is simple, the process is controllable, and the properties are stable.

[0005] The specific technical solutions are as follows:

[0006] A product having a topographical microstructure that can accelerate endothelial cell migration, including a substrate and a topographical microstructure on the substrate surface;

[0007] The topological microstructure is a continuous groove array composed of a number of continuous grooves arranged regularly;

[0008] A single continuous groove presents a continuous teardrop-shaped structure, the cross section of which is composed of a semicircle and a trapezoid, the radius r of the semicircle is selected from 2 to 50 μm, the lower base of the trapezoid is 2r, the height l of the trapezoid is selected from 10 to 100 μm, and the upper base a of the trapezoid is selected from 1 to 80 μm.

[0009] The present invention forms a continuous groove array of a specific shape on the substrate surface, inducing directional cell migration while restricting the direction of cell migration, significantly improving cell migration efficiency and accelerating endothelialization. Because of the unique teardrop-like structure, when cells migrate from the wide to the narrow portion of the droplet, the cell structure in the narrow portion guides the cell polarization, with the wide portion being wider and the narrow portion being narrower. Therefore, the resistance to cell migration toward the narrow portion is low, while the resistance to migration toward the wide portion is high, allowing cells to migrate along the droplet from the wide portion to the narrow portion. If the droplet is aligned with the target endothelialization area, rapid endothelialization of the target area can be achieved.

[0010] Compared with traditional parallel microgrooves or other common topological microstructures (micropit topological structures), the special topological microstructure disclosed in the present invention can significantly improve the migration efficiency of cells.

[0011] The topological microstructures disclosed in the present invention can be present on different substrates, such as thermoplastics, thermoplastic elastomers, thermosetting polymers, metals, etc.

[0012] Specifically selected from one or more of nylon, acrylonitrile-butadiene-styrene, polycarbonate, polyetheretherketone, polydimethylsiloxane elastomer, polylactic acid-glycolic acid, polycaprolactone, epoxy polymer, unsaturated polyester polymer, polyimide polymer, nickel titanium alloy, and stainless steel.

[0013] The regular arrangement includes parallel arrangement, arrangement in an arc shape, arrangement in a triangle, arrangement in a parallelogram, etc., and preferably a parallel arrangement is adopted.

[0014] In the topological microstructure disclosed in the present invention:

[0015] The arrangement spacing s between adjacent continuous grooves is 1 to 100 μm, preferably 30 to 80 μm, and more preferably 50 μm.

[0016] Experiments have shown that the depth of the grooves in the topological microstructures disclosed herein affects the degree of endothelial cell orientation, and thus the efficiency of directional cell migration. When the depth d of the continuous grooves is as low as 1 μm, the degree of cell orientation is low. Preferably, the depth d of the continuous grooves is 2 to 50 μm; more preferably, the depth d of the continuous grooves is 3 to 10 μm; and even more preferably, it is 3 to 7 μm.

[0017] Experiments have shown that, in the topological microstructure disclosed in the present invention, the value of the upper base a of the trapezoid and the ratio of a to r have a critical impact on the efficiency of directional cell migration.

[0018] Preferred:

[0019] The upper base a of the trapezoid is selected from 5 to 10 μm, the radius r of the semicircle is selected from 5 to 20 μm, and the ratio of a to r is 1:(1 to 4);

[0020] More preferably, the upper base a of the trapezoid is selected from 5 μm, and the ratio of a to r is 1:1.

[0021] With the continuous optimization of the above parameters, the efficiency of this topological microstructure for directional cell migration increases accordingly.

[0022] Preferably, the height l of the trapezoid is selected from 40 to 100 μm, and more preferably is 80 μm.

[0023] The present invention also discloses a method for preparing the above-mentioned product having a topological microstructure capable of accelerating endothelial cell migration, comprising the following steps:

[0024] When thermoplastic plastic and / or thermoplastic elastomer is used as the substrate, the substrate material is heated and then solidified in a mold with the continuous groove array and then demoulded to obtain the topological microstructure that can accelerate endothelial cell migration;

[0025] When a thermosetting polymer or metal is used as the substrate, the continuous groove array is formed on the surface of the substrate by hot pressing to obtain the topological microstructure that can accelerate the migration of endothelial cells.

[0026] The present invention also discloses the application of the above-mentioned product with a topological microstructure that can accelerate endothelial cell migration in medical devices. When the substrate is replaced with an occluder, an arterial covered stent, a balloon, etc., it can be specifically applied to the fields of occluders, arterial covered stents, balloons, etc.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] The present invention discloses a product with a topological microstructure that can accelerate the migration of endothelial cells. The topological microstructure is a continuous groove array in a continuous teardrop-shaped structure. Its special structure can improve the orientation of endothelial cells and accelerate the efficiency of endothelial cell migration. In addition, the structure is simple to prepare, the process is controllable, and the properties are stable. It can be applied to medical devices such as occluders, arterial covered stents, and balloons. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 Schematic diagram of the topological microstructure of the present invention that can accelerate endothelial cell migration, in which 1 is a continuous groove, 2 is a substrate;

[0030] Figure 2 Schematic cross-sectional view of the topological microstructure of the present invention that can accelerate endothelial cell migration;

[0031] Figure 3 A partial schematic diagram of a teardrop-shaped groove in a topological microstructure capable of accelerating endothelial cell migration in the present invention;

[0032] Figure 4 This is a scanning electron microscope image of the topological microstructure surface that can accelerate endothelial cell migration prepared in Example 1;

[0033] Figure 5 This is a scanning electron microscope image of the topological microstructure surface that can accelerate endothelial cell migration prepared in Example 2;

[0034] Figure 6 This is a scanning electron microscope image of the substrate surface without a groove structure prepared in Comparative Example 1;

[0035] Figure 7 This is a scanning electron microscope image of the substrate surface with a parallel groove structure prepared in Comparative Example 2;

[0036] Figure 8 Comparative diagram of the endothelial cell states on the substrate surfaces prepared in Examples 1 and 2 and Comparative Examples 1 and 2, respectively;

[0037] Figure 9 Statistical graphs of endothelial cell orientation angles on the substrate surface prepared in Examples 1, 2, 4 and Comparative Examples 1 to 4, respectively;

[0038] Figure 10 The figure is a bar graph showing the migration speed of endothelial cells on the substrate surfaces prepared in Examples 1 to 7 and Comparative Examples 1 to 4, respectively. DETAILED DESCRIPTION

[0039] The present invention will be further described in detail below with reference to examples and comparative examples, but the embodiments of the present invention are not limited thereto.

[0040] Figure 1 The schematic diagram of the topological microstructure disclosed in the present invention that can accelerate endothelial cell migration includes a substrate 2 and a topological microstructure on the substrate surface. The topological microstructure is a continuous groove array composed of a plurality of continuous grooves 1 arranged in parallel. The depth of a single continuous groove is denoted as d, and the spacing between adjacent continuous grooves is denoted as s (the cross-sectional schematic diagram is shown in FIG. Figure 2 As shown); a single continuous groove presents a continuous water drop-shaped structure, and the local schematic diagram is shown Figure 3 As shown, the cross-section of the water drop-shaped structure is composed of a semicircle and a trapezoid, the radius of the semicircle (i.e., the groove corner radius) is recorded as r, the lower base of the trapezoid (i.e., the wide part of the groove) is recorded as 2r, the upper base of the trapezoid (i.e., the narrow part of the groove) is recorded as a, and the height of the trapezoid (i.e., the groove unit length) is recorded as l.

[0041] Example 1

[0042] Using polydimethylsiloxane elastomer (PDMS) as the material, a topological microstructure that can accelerate endothelial cell migration was prepared by hot pressing.

[0043] Step 1: Draw a topological microstructure diagram using the computer drawing software AutoCAD, and process it to obtain the corresponding photomask.

[0044] In this embodiment, a single continuous groove is a continuous teardrop-shaped structure. Several continuous grooves are arranged in parallel to form a continuous groove array. The depth d of a single continuous groove is 3 μm, and the spacing s between adjacent continuous grooves is 50 μm. The specific dimensions of a single continuous groove are:

[0045] A single continuous groove has a continuous teardrop-shaped structure, and its cross-section is composed of a semicircle and a trapezoid. The radius r of the semicircle (i.e., the groove corner radius) is selected from 5μm, the lower base 2r of the trapezoid (i.e., the wide part of the groove) is 10μm, the upper base a of the trapezoid (i.e., the narrow part of the groove) is 5μm, and the height l of the trapezoid (i.e., the groove unit length) is 80μm.

[0046] Step 2: Preparation of a negative silicon template with a topological microstructure. The silicon wafer is cleaned and the design is transferred to the wafer using a standard photolithography process, resulting in a silicon wafer with a topological microstructure. The specific process involves using a spreader to evenly distribute the photoresist on the wafer, pre-baking the photoresist on a hot plate, and then placing the patterned mask into the photolithography machine for exposure and development. The resulting silicon template features continuous, narrow, water-droplet-like protrusions.

[0047] Step 3: Fabrication of PDMS Surface Microtopology. PDMS and its crosslinker (Dow Corning SYLGARD 184) were mixed (mass ratio 10:1) and allowed to stand at room temperature for 1 hour to remove bubbles. The PDMS mixture, prepared in Step 2, was added to a two-inch silicon template and hot-pressed at 80°C for 1 hour at a pressure of 5 MPa. This resulted in a PDMS surface with continuous, narrow grooves.

[0048] Figure 4 This is an SEM image of the surface of the PDMS with continuous narrow water droplet grooves prepared in this example.

[0049] Example 2

[0050] The preparation process is basically the same as that in Example 1, except that the specific size of the single continuous groove designed in step 1 is changed, specifically:

[0051] The radius r of the semicircle (ie, the groove corner radius) is adjusted to 20 μm. At this time, the lower base 2r of the trapezoid (ie, the groove width) is 40 μm.

[0052] PDMS with continuous wide water droplet grooves on the surface was prepared.

[0053] Figure 5 This is an SEM image of the surface of the PDMS with continuous wide water droplet grooves prepared in this example.

[0054] Example 3

[0055] The preparation process is basically the same as that in Example 2, except that the specific size of the single continuous groove designed in step 1 is changed, specifically:

[0056] The upper base a of the trapezoid (ie, the narrow portion of the groove) was adjusted to 10 μm.

[0057] Examples 4 to 7

[0058] The preparation process is basically the same as that in Example 1, except that the specific size of the single continuous groove designed in step 1 is changed, specifically:

[0059] The depth d of a single continuous groove was adjusted to 2 μm, 5 μm, 7 μm, and 10 μm in sequence.

[0060] Comparative Example 1

[0061] In this comparative example, a silicon wafer with a smooth surface was hot-pressed. The hot-pressing conditions were the same as those in step 3 of Example 1. A planar PDMS without a groove structure was prepared in this comparative example.

[0062] Figure 6 This is an SEM image of the surface of the PDMS without groove structure prepared in this comparative example.

[0063] Comparative Example 2

[0064] The preparation process was essentially the same as that in Example 1, differing only in the topological microstructure design. This comparative example employed parallel grooves with a groove width a of 10 μm, a groove spacing s of 50 μm, and no asymmetric water droplet design. A PDMS surface with parallel grooves was prepared.

[0065] Figure 7 This is an SEM image of the surface of the PDMS with parallel grooves prepared in this comparative example.

[0066] Comparative Example 3

[0067] The preparation process is basically the same as that in Example 1, the only difference is that the topological microstructure of the design is different. This comparative example adopts a micro-pit structure with a diameter of 40 μm, a spacing of 50 μm, and a depth of 3 μm. The specific design refers to the Chinese patent document with application publication number CN115089772A.

[0068] Comparative Example 4

[0069] The preparation process is basically the same as that in Example 1, except that the specific size of the single continuous groove designed in step 1 is changed, specifically, the depth d of the single continuous groove is adjusted to 1 μm. Performance test:

[0070] 1. Observation of endothelial cell status:

[0071] Human umbilical vein endothelial cells were harvested and labeled with red fluorescent live cell dye. 2 The cells were seeded at a density of 100 nm on the PDMS surface treated in different embodiments or comparative examples, and the cells were taken out after 48 h to observe the cell morphology under a fluorescence microscope.

[0072] Figure 8 Comparison of endothelial cell patterns on substrates prepared in Examples 1 and 2 and Comparative Examples 1 and 2, respectively. Observations show that the narrow waterdrop groove (Example 1), wide waterdrop groove (Example 2), and parallel groove PDMS (Comparative Example 2) surfaces all clearly guide cell orientation, with the orientation aligned with the direction of the microstructured grooves. In contrast, the flat PDMS surface (Comparative Example 1) exhibits a chaotic cell orientation with no apparent directionality.

[0073] 2. Cell Orientation Angle Statistics

[0074] Human umbilical vein endothelial cells were harvested and labeled with red fluorescent live cell dye. 2 The cells were seeded at a density of 100 nm on the PDMS surface treated in different embodiments or comparative examples. After 48 h, the cells were taken out and the cell morphology was photographed under a fluorescence microscope. The angle between the long axis of each cell and the horizontal direction, i.e., the cell orientation angle, was counted using image analysis software (ImageJ).

[0075] Figure 9 This is a statistical graph of the orientation angle data of endothelial cells on the PDMS surfaces prepared in Examples 1, 2, 4 and Comparative Examples 1 to 4, respectively. In the graph, the more concentrated the orientation angle, the higher the degree of orientation. By observing the graph, it can be found that the cell orientation angles in Example 1, Example 2 and Example 4 with the special topological microstructure disclosed in the present invention are more concentrated at 90° (microstructure grooves), indicating that the microstructure we designed can guide the cells to be oriented along the direction of the microstructure. The parallel groove structure in Comparative Example 2 can also guide the cells to be oriented along the direction of the microstructure, but the micropit structure disclosed in Comparative Example 3 has no effect on the cell orientation. Further comparison of Example 1, Example 4 and Comparative Example 4 shows that as the depth decreases from 3μm (Example 1) to 2μm (Example 4) and 1μm (Comparative Example 4), the cell orientation angles gradually become dispersed, indicating that the cell orientation is affected by the microstructure.

[0076] 3. Migration Speed ​​Statistics:

[0077] Endothelial cell movement on the PDMS surfaces treated in different examples or comparative examples was observed microscopically, and the distance the cells collectively moved in a given direction over 24 hours was calculated. Speed ​​= 24-hour migration distance / 24 hours. A faster speed indicates more unidirectional cell movement and higher migration efficiency.

[0078] Figure 10 The histograms of the migration speed of endothelial cells on the PDMS surfaces prepared in Examples 1 to 7 and Comparative Examples 1 to 4 respectively show that the endothelial cells migrated the fastest on the PDMS surfaces treated in Examples 1, 5, 6 and 7, and had the highest migration efficiency. The migration speed of endothelial cells on the PDMS surfaces treated in Examples 1 to 4 was faster than that of the parallel microgrooves without a structural design (Comparative Example 2), Comparative Example 1 without a groove structure, and Comparative Example 3 with a topological microstructure of a micropit structure, indicating that the water drop-shaped grooves in the present invention can promote unidirectional migration of cells along the groove direction, thereby improving the migration efficiency of endothelial cells. Further comparison of Example 1, Example 4 and Comparative Example 4 revealed that as the depth decreased from 3 μm (Example 1) to 2 μm (Example 4) and 1 μm (Comparative Example 4), the migration speed of endothelial cells decreased significantly.

[0079] The applicant declares that the present invention illustrates the detailed method of the present invention through the above embodiments, but the present invention is not limited to the above detailed method.

Claims

1. A product having a topological microstructure that can accelerate endothelial cell migration, comprising a substrate and a topological microstructure on the substrate surface, characterized in that: The topological microstructure is a continuous groove array composed of a number of continuous grooves arranged regularly; A single continuous groove presents a continuous teardrop-shaped structure, wherein the cross section of the teardrop-shaped structure is composed of a semicircle and a trapezoid, wherein the radius r of the semicircle is selected from 2 to 50 μm, the lower base of the trapezoid is 2r, the height l of the trapezoid is selected from 10 to 100 μm, and the upper base a of the trapezoid is selected from 1 to 80 μm; The depth d of the continuous groove is 2-50 μm.

2. The product having a topological microstructure capable of accelerating endothelial cell migration according to claim 1, characterized in that: The substrate is selected from one or more of thermoplastics, thermoplastic elastomers, thermosetting polymers, and metals.

3. The product having a topological microstructure capable of accelerating endothelial cell migration according to claim 2, characterized in that: The substrate is selected from one or more of nylon, acrylonitrile-butadiene-styrene, polycarbonate, polyetheretherketone, polydimethylsiloxane elastomer, polylactic acid-glycolic acid, polycaprolactone, epoxy polymer, unsaturated polyester polymer, polyimide polymer, nickel titanium alloy, and stainless steel.

4. The product having a topological microstructure capable of accelerating endothelial cell migration according to claim 1, characterized in that: The regular arrangement includes parallel arrangement, arrangement in an arc shape, arrangement in a triangle or arrangement in a parallelogram.

5. The product having a topological microstructure capable of accelerating endothelial cell migration according to claim 4, characterized in that: The arrangement spacing s between adjacent continuous grooves is 1~100μm.

6. The product having a topological microstructure capable of accelerating endothelial cell migration according to claim 1, characterized in that: The radius r of the semicircle is selected from 5 to 20 μm, the height l of the trapezoid is selected from 40 to 100 μm, the upper base a of the trapezoid is selected from 5 to 10 μm, and the ratio of a to r is 1:(1 to 4).

7. The product having a topological microstructure capable of accelerating endothelial cell migration according to claim 6, characterized in that: The continuous groove array is composed of a plurality of continuous grooves arranged in parallel; The spacing s between adjacent continuous grooves is 30~80μm; The depth d of the continuous groove is 3-10 μm.

8. The product having a topological microstructure capable of accelerating endothelial cell migration according to claim 7, characterized in that: In the continuous groove, the upper base a of the trapezoid is selected from 5 μm, and the ratio of a to r is 1:

1.

9. A method for preparing a product having a topological microstructure capable of accelerating endothelial cell migration according to any one of claims 1 to 8, characterized in that: The steps include: When thermoplastic plastic and / or thermoplastic elastomer is used as the substrate, the substrate material is heated and then solidified in a mold with the continuous groove array and then demoulded to obtain the topological microstructure that can accelerate endothelial cell migration; When a thermosetting polymer or metal is used as the substrate, the continuous groove array is formed on the surface of the substrate by hot pressing to obtain the topological microstructure that can accelerate the migration of endothelial cells.

10. Use of the product having a topological microstructure capable of accelerating endothelial cell migration according to any one of claims 1 to 8 in medical devices.

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