JP2010004755A - Endothelial cell-proliferative material - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To achieve an endothelial cell-proliferative material having selectively improved proliferative properties of a vascular endothelial cell. <P>SOLUTION: The endothelial cell-proliferative material has a membrane body 32 formed on the surface of a base material 31, and formed by bonding carbons to each other, and a plasma treated layer 32A formed on the surface of the membrane body 32, and having nitrogen and oxygen bonded to the carbon forming the membrane body 32. The ratio of the nitrogen to the oxygen in the plasma treated layer 32A is ≥0.3. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to an endothelial cell proliferating material, and more particularly to an endothelial cell proliferating material having a selective proliferation property of endothelial cells.

  With the development of medical treatment, the use of medical instruments used in vivo such as stents placed in blood vessels and implantable artificial hearts is increasing. However, when a medical instrument is embedded in a living body, the living body recognizes it as a foreign substance, which causes a problem that a thrombus is formed.

  As a method of reducing thrombus generated in a medical device, a method of coating the medical device with an antithrombotic drug such as heparin or phosphorylcholine is known. However, there is a problem that the drug coating is easily peeled off and the effect is not sustained. In addition, the problem of side effects caused by drugs is also known.

  In view of this, a method of forming the medical device itself with an antithrombotic material or forming a strong film with excellent antithrombogenicity on the surface of the medical device is being studied instead of depending on a drug. Among them, a method of covering a medical device with a carbonaceous film such as a diamond-like carbon (DLC) film is particularly attracting attention because of its excellent durability.

Furthermore, in order to improve the antithrombogenicity of the carbonaceous film, introduction of a hydrophilic functional group on the surface of the carbonaceous film or fixing an antithrombotic drug has been studied (for example, (See Patent Document 1).
JP 2007-195883 A

  However, when examining the antithrombogenicity of the carbonaceous membrane, the interaction with blood cells such as platelets has been studied, but the interaction with vascular endothelial cells has hardly been studied.

  It is known that vascular endothelial cells undergo external stimulation and finely adjust the fibrinolytic dynamics of blood. When dysfunction occurs in vascular endothelial cells, the fibrinolytic regulation function is lost and a thrombus is formed. For this reason, in order to improve the antithrombogenicity of a medical device, it is important not to cause abnormality in vascular endothelial cells.

  In addition, when a blood vessel is expanded by a stent, there is a possibility that a laceration may occur in the endothelial layer during the blood vessel expansion. In order to prevent blood clots, it is important to quickly heal lacerations generated in the endothelial layer. Accordingly, it is preferable that the endothelial cells proliferate rapidly on the stent surface. However, generally a carbonaceous film has a mirror-like smooth surface. For this reason, vascular endothelial cells hardly proliferate on the surface of the carbonaceous film. Further, when an antithrombotic drug or the like is carried on the surface, the proliferation of vascular endothelial cells is further suppressed.

  On the other hand, it is known that when vascular smooth muscle cells proliferate abnormally, vascular restenosis occurs. For this reason, it is necessary not only to simply improve the proliferation of cells but also to selectively improve the proliferation of vascular endothelial cells.

  The present invention solves the above-mentioned conventional problems and makes it possible to realize an endothelial cell proliferating material that selectively improves the proliferation of vascular endothelial cells.

  In order to achieve the above object, the present invention is configured such that the endothelial cell proliferating material has a plasma treatment layer on the surface of the carbonaceous film.

  Specifically, the first endothelial cell proliferating material according to the present invention is formed on the surface of the base material, and is formed on the surface of the membrane body formed by bonding carbon to each other. And a plasma treatment layer having nitrogen and oxygen bonded to carbon, and a ratio of nitrogen to oxygen in the plasma treatment layer is 0.3 or more.

  The first endothelial cell proliferating material includes a plasma treatment layer in which the ratio of nitrogen to oxygen is 0.3 or more. Thereby, it is considered that a functional group having nitrogen such as an amino group and a functional group having oxygen such as a carboxyl group are formed on the surface of the material. Therefore, the surface has excellent compatibility with endothelial cells, and the endothelial cells are more likely to proliferate than smooth muscle cells. As a result, an inner cell proliferating material excellent in selective proliferation of endothelial cells can be realized.

  The first endothelial cell proliferating material may have a zeta potential of −20 mV or more on the surface of the membrane body.

  The second endothelial cell proliferating material according to the present invention includes a membrane body formed on the surface of the base material and formed by bonding carbon to each other, and carbon formed on the surface of the membrane body and constituting the membrane body. And a plasma treatment layer having combined oxygen, wherein the ratio of oxygen to carbon in the plasma treatment layer is 0.1 or more.

  The second endothelial cell proliferating material includes a plasma treatment layer in which the ratio of oxygen to carbon is 0.1 or more. Thereby, it is considered that a functional group having oxygen such as a carboxyl group is formed on the surface of the material. Therefore, the surface has excellent compatibility with endothelial cells, and the endothelial cells are more likely to proliferate than smooth muscle cells. As a result, an inner cell proliferating material excellent in selective proliferation of endothelial cells can be realized.

  The second endothelial cell proliferating material may have a zeta potential of −30 mV or less on the surface of the membrane body.

  In the first and second endothelial cell proliferating materials, the proliferation rate of the endothelial cells on the surface of the plasma treatment layer is preferably higher than the proliferation rate of the smooth muscle cells.

  In this case, the proliferation rate of the endothelial cells is preferably 1.1 times or more that of the smooth muscle cells.

  According to the endothelial cell proliferating material according to the present invention, an endothelial cell proliferating material having a selective proliferative property of vascular endothelial cells can be realized.

(First embodiment)
A first embodiment of the present invention will be described with reference to the drawings. FIG. 1 shows a cross-sectional configuration of the endothelial cell proliferating material according to the first embodiment. An endothelial cell proliferating material is a material excellent in selective proliferation of endothelial cells. As shown in FIG. 1, the endothelial cell proliferating material of the present embodiment is a carbonaceous film 32 that covers a base material 31, and a plasma treatment layer 32 </ b> A is formed on the surface of the carbonaceous film 32.

  The base material 31 may be any material. Specifically, although not particularly limited, for example, a metal such as iron, nickel, chromium, copper, titanium, platinum, tungsten, or tantalum can be used as the base material. Further, these alloys are stainless steel such as SUS316L, shape memory alloy such as Ti—Ni alloy or Cu—Al—Mn alloy, Cu—Zn alloy, Ni—Al alloy, titanium alloy, tantalum alloy, platinum alloy or An alloy such as a tungsten alloy can also be used. Moreover, bioactive ceramics, such as oxides, such as aluminum, a silicon | silicone, or a zircon, nitride, or carbide | carbonized_material, or ceramics which have bioactivity, such as apatite or a biological glass, may be sufficient. Further, polyester such as polymethyl methacrylate (PMMA), high density polyethylene, polyacetal, polyethylene terephthalate (PET), polymer resin such as polycarbonate resin or polysulfone, silicon polymer such as polydimethylsiloxane, or fluorine such as polytetrafluoroethylene A polymer may be used.

  Further, the shape may be any shape, and it may be formed in a state of a medical instrument or the like, or may be in a state of a material before forming, such as a plate material, a rod material, or a wire material.

  The carbonaceous film 32 is a film formed by bonding carbon atoms such as a diamond-like carbon (DLC) film. Moreover, silicon (Si) or fluorine (F) may be contained.

  Although the film thickness of the carbonaceous film 32 is not specifically limited, The range of 0.005 micrometer-3 micrometers is preferable, More preferably, it is the range of 0.01 micrometer-1 micrometer.

  The carbonaceous film 32 may be formed directly on the substrate 31 or may be formed with an intermediate layer interposed. Various intermediate layers can be used depending on the type of substrate, but silicon (Si) and carbon (C), titanium (Ti) and carbon (C), or chromium (Cr) and carbon (C). A known film such as an amorphous film can be used. Although the thickness is not specifically limited, the range of 0.005 micrometer-0.3 micrometer is preferable, More preferably, it is the range of 0.01 micrometer-0.1 micrometer.

  The plasma treatment layer 32A is formed by irradiating the surface of the carbonaceous film 32 with plasma, and contains nitrogen (N) and oxygen (O) bonded to carbon atoms.

  It is not clear what state the nitrogen introduced into the plasma treatment layer 32A is in. However, it is thought that functional groups containing nitrogen (nitrogen functional groups) such as amino groups, amide groups, and imide groups are formed. Although detailed analysis of nitrogenous functional groups is difficult, the N1s peak appeared at 398.9 eV in X-ray photoelectron spectroscopy (XPS) measurement. Since this is the binding energy (400 ± 1 eV) of N1s of an amino group and an amide group, it is considered that a functional group containing an amino group is generated on the surface of the carbonaceous film.

  Similarly, from the result of XPS measurement, oxygen is considered to form a functional group containing oxygen (oxygen functional group) such as C—O, C═O, and C (═O) —O.

  FIG. 2 shows the relationship between the nitrogen-to-oxygen ratio (N / O) of the plasma treatment layer 32A obtained by XPS measurement and the proliferation rate of vascular endothelial cells (ECs) and vascular smooth muscle cells (SMCs). .

  For the XPS measurement, a photoelectron spectrometer JPS-9010MC manufactured by JEOL Ltd. was used. Al was used as the X-ray source, and X-rays were generated under the conditions of an acceleration voltage of 12.5 kV and an emission current of 17.5 mA. Measurement was performed on an area of 5 mm in diameter arbitrarily selected from the sample. Further, the composition up to a depth of about 5 nm is measured by making X-rays incident on the sample perpendicularly and setting the detection angle to 90 degrees with respect to the sample surface.

  The binding energy measurement region was 274 eV to 294 eV, 389 eV to 409 eV, and 522 eV to 542 eV, and peaks of C1s, N1s, and O1s were obtained, respectively. By comparing the area ratios of the obtained peaks, the abundance ratio O / C of oxygen based on carbon and the abundance ratio N / C of nitrogen based on carbon were determined.

The cell growth rate was determined as follows. A 15 mm diameter cover glass coated with various carbonaceous films with different N / O values was sterilized with 70% ethanol, and placed on the bottom of a 24-well cell culture multiplate (Costar 3516, Corning). Washed 3 times with ultra pure water. Human coronary artery-derived endothelial cells (Cell Applications: HCAEC) and smooth muscle cells (Cell Applications: HCASMC) were seeded on the surface of the cover glass at a density of 1 × 10 ⁴ cells / well (500 μL). After sowing, the temperature was 37 ° C., and the culture was continued for 4 days in a 5% carbon dioxide atmosphere. As the medium, a medium dedicated to each cell sold by Cell Applications was used, and the medium was changed every day. The cell growth rate after culturing for 4 days was determined using Cell-Counting-Kit8 (Dojindo). The measurement method was performed according to the manual attached to the kit. The comparison of the growth rate in each carbonaceous membrane was determined by using a microplate reader to determine the concentration (absorbance) of WST-8 frmazan produced by intracellular mitochondrial activity corresponding to the number of cells grown. The absorbance was set to 100%, and the growth rate was compared by calculating the% growth rate of each sample.

  As shown in FIG. 2, the growth rate of SMCs hardly changes in the carbonaceous film having the plasma treatment layer 32A and having nitrogen functional groups introduced on the surface thereof. On the other hand, the proliferation rate of ECs increased as the value of N / O increased. This is because the growth rate of ECs can be selectively increased without increasing the growth rate of SMCs by forming a plasma treatment layer in which nitrogen and oxygen are introduced by plasma treatment on the surface of the carbonaceous film. Show. That is, by forming the plasma treatment layer 32A, it is possible to realize an endothelial cell proliferating material with further improved ECs proliferative ability.

  In the case of a stent or the like, the higher the ECs growth rate, the lower the risk of thrombosis because the stent platform is covered with endothelial cells in a short time. However, in the case of a blood vessel filter or the like, clogging or the like may occur even if the proliferation rate of the endothelial cells is too high. For this reason, what is necessary is just to select the value of N / O suitably according to a use. However, it is preferable that N / O in which the growth rate of ECs is 1.1 times or more that of SMCs is in the range of 0.3 or more. Further, if N / O is in the range of about 1.5 or less, the production is easy.

  The reason why the growth rate of ECs is increased by forming the plasma treatment layer 32A containing nitrogen and oxygen is not clear. However, as described above, the nitrogen introduced into the plasma treatment layer 32A is introduced as a nitrogenous functional group, and is considered to be an amino group in particular. Moreover, it is thought that a part of oxygen is a carboxyl group. Thus, it is presumed that the growth rate of ECs may be increased by the plasma treatment layer 32A having an amino group and a carboxyl group.

  FIG. 3 shows the relationship between the zeta potential of the carbonaceous film 32 on which the plasma treatment layer 32A is formed and the cell growth rate. As shown in FIG. 3, when the zeta potential is higher than that of an untreated carbonaceous film having no plasma treatment layer, the growth rate of ECs increases, but the growth rate of SMCs hardly changes. Also from this, it is presumed that at least a part of nitrogen introduced into the plasma treatment layer 32A is an amino group, and that the proliferation rate of SMCs increases as the amount of amino group introduced increases.

  The higher the zeta potential value, the higher the ECs growth rate, which is preferable. However, at least −20 mV or more at which a difference between the ECs growth rate and the SMCs growth rate is recognized.

  For the measurement of the zeta potential, a zeta potential / particle size measurement system ELS-Z manufactured by Otsuka Electronics Co., Ltd. was used. The sample on which the carbonaceous film was formed was brought into close contact with a flat plate sample cell, and monitoring particles were injected into the cell. The monitoring particles used were those manufactured by Otsuka Electronics Co., Ltd. dispersed in a 10 mM sodium chloride (NaCl) solution. The monitor particles were electrophoresed at each level in the cell depth direction, and the apparent velocity distribution inside the cell was measured. Electrophoresis was performed under the conditions of an average electric field of 17.33 V / cm and an average current of 1.02 mA. By analyzing the apparent velocity distribution obtained based on the Mori-Okamoto equation, the surface potential of the carbonaceous film surface was obtained. The plate sample cell was used after being coated with polyacrylamide in order to suppress the influence of the charge on the cell surface.

  Below, an example of the manufacturing method of the endothelial cell proliferative material which concerns on this embodiment is shown. First, the carbonaceous film 32 is formed on the surface of the substrate 31. The carbonaceous film 32 is formed by sputtering, DC magnetron sputtering, RF magnetron sputtering, chemical vapor deposition (CVD), plasma CVD, plasma ion implantation, superimposed RF plasma ion implantation, ion plating. It may be formed by a known method such as an arc ion plating method, an ion beam vapor deposition method or a laser ablation method.

For example, when forming by sputtering, first, the base material 31 such as a metal plate is set in the chamber of an ionization vapor deposition apparatus, and bombard cleaning is performed for about 30 minutes. Bombardment cleaning, after introducing to argon gas (Ar) pressure becomes ^{10 -1 Pa~10 -3 Pa (10 -3} Torr~10 -5 Torr) in the chamber, Ar ions generated by performing discharge The generated Ar ions may be made to collide with the surface of the substrate.

Subsequently, tetramethylsilane (Si (CH ₃ ) ₄ ) is introduced into the chamber for 3 minutes, and an amorphous intermediate layer having a thickness of 20 nm is mainly formed of silicon (Si) and carbon (C).

After forming the intermediate layer, C ₆ H ₆ gas is introduced into the chamber, and the gas pressure is set to 10 ⁻¹ Pa. The C ₆ H ₆ ionized by performing continuously introduced while discharging the C ₆ H ₆ at 30ml / min, performing ionized evaporation for about 2 minutes. Thereby, the carbonaceous film 32 which is a DLC film having a thickness of 30 nm is formed on the surface of the base material 31.

  For example, the target voltage when forming the carbonaceous film 32 is 1.5 kV, the target current is 50 mA, the filament voltage is 14 V, the filament current is 30 A, the anode voltage is 50 V, the anode current is 0.6 A, the reflector voltage is 50 V, The reflector current may be 6 mA. Moreover, the temperature of the base material 31 at the time of formation may be about 160 ° C.

  In addition, although the example which provides an intermediate | middle layer in order to improve the adhesiveness of the base material 31 and the carbonaceous film 32 was shown, it abbreviate | omits when the adhesiveness of the base material 31 and the carbonaceous film 32 can fully be ensured. May be.

  Next, the carbonaceous film 32 is irradiated with plasma to form a plasma treatment layer 32A. For example, the plasma irradiation may be performed by a parallel plate type plasma irradiation apparatus as shown in FIG. After setting the base material 31 on which the carbonaceous film is formed in the chamber 10 of the plasma irradiation apparatus, the pressure in the chamber 10 is exhausted to a predetermined pressure. When the pressure in the chamber 10 is set to a high vacuum state, exhaust may be performed using a turbo molecular pump. Next, a gas is introduced into the chamber 10 at a predetermined flow rate, and plasma is generated by applying high frequency power between the parallel plate electrodes 12A and 12B. The high frequency power may be applied using a high frequency power supply 15 connected via the matching box 14. The gas flow rate may be adjusted by the mass flow controller 13.

The plasma treatment layer 32A containing nitrogen and oxygen may be formed by irradiation with ammonia plasma. Further, after irradiation with other plasma such as argon (Ar), oxygen (O ₂ ), acetylene (C ₂ H ₂ ), or a mixed gas thereof, ammonia plasma may be irradiated.

It is also possible to use other basic nitrogen-containing compounds instead of ammonia. Examples of basic nitrogen-containing compounds include organic amines represented by the general formula NR ₁ R ₂ R ₃ (wherein R ₁ , R ₂ and R ₃ are hydrogen, —CH ₃ , —C ₂ H ₅ , —C ₃ H ₇ or —C ₄ H ₈ , and R ₁ , R ₂ and R ₃ may be the same or different from each other.) Or benzylamine and its secondary and tertiary amines. However, ammonia is preferable because of cost and ease of handling.

A higher ultimate vacuum in the chamber during plasma irradiation is preferable because the zeta potential can be increased. This is thought to be because the generation of carboxyl groups by oxygen in the air can be suppressed by increasing the ultimate vacuum in the chamber. For this reason, the ultimate vacuum may be about 5 × 10 ⁻³ Pa. However, it is possible to obtain a plasma treatment layer having a sufficiently high zeta potential even when the ultimate vacuum is about 2 Pa.

  Note that a plasma irradiation apparatus having any structure may be used. Also, any type of discharge may be used. For example, a parallel plate method, an after glow discharge method, an electromagnetic induction type, a magnetic field type, or the like may be used. Plasma irradiation conditions are not particularly limited. For example, as the power source for generating plasma, various power source frequencies such as a commercial frequency (50 Hz or 60 Hz), a high frequency (radio frequency), or a microwave region can be used. Furthermore, the pressure control method and supply structure of the source gas are not particularly limited. However, if plasma irradiation conditions with a very high etching rate are used, the carbonaceous film may be damaged.

(Second Embodiment)
The second embodiment of the present invention will be described below with reference to the drawings. FIG. 5 shows a cross-sectional configuration of the endothelial cell proliferating material according to the second embodiment. In FIG. 5, the same components as those of FIG.

  As shown in FIG. 5, the endothelial cell proliferating material of the present embodiment is a carbonaceous film 32 that covers the base material 31, and a plasma treatment layer 32 </ b> B is formed on the surface of the carbonaceous film 32. The plasma processing layer 32B of this embodiment is a layer that contains almost no nitrogen (N) component bonded to carbon atoms formed by the plasma processing and contains an oxygen (O) component.

  FIG. 6 shows the relationship between the ratio of oxygen to carbon (O / C) in the plasma treatment layer 32B obtained by XPS measurement and the proliferation rate of vascular endothelial cells (ECs) and vascular smooth muscle cells (SMCs). . The amount of nitrogen obtained by XPS measurement was 1 at. %, Which was almost a trace amount.

  By forming a plasma treatment layer containing almost no nitrogen component and O / C containing oxygen of 0.1 or more, the growth rate of ECs should be higher than the growth rate of SMCs as shown in FIG. I was able to.

  The reason why the growth rate of ECs is higher than the growth rate of SMCs when the value of O / C is about 0.1 to 0.4 is unknown. However, in this region, it is considered that the growth rate of ECs is increased by a mechanism different from the case where both nitrogen and oxygen are included.

  FIG. 7 shows the relationship between the zeta potential of the plasma treatment layer and the growth rate of ECs and SMCs. As shown in FIG. 7, when the zeta potential is lower than that of an untreated carbonaceous film that does not have a plasma treatment layer, the growth rate of ECs is increased. On the other hand, the growth rate of SMCs hardly increases even when the zeta potential is lowered. From this, it is thought that the growth rate of ECs is higher when the oxygen functional group has more C (═O) —O components.

  As described above, the growth rate of ECs can be increased without increasing the growth rate of SMCs by forming a plasma treatment layer that hardly contains nitrogen and has an O / C value of 0.1 or more. And an endothelial cell proliferating material excellent in antithrombogenicity can be realized. Further, the zeta potential is preferably −30 mV or lower, which is lower than that of the untreated carbonaceous film.

  When such a plasma processing layer 32B having a large O / C value is formed, the carbonaceous film formed by the same method as in the first embodiment may be irradiated with oxygen plasma. The same plasma processing apparatus as that in the first embodiment can be used. In addition, after irradiation with an inert gas such as argon or a plasma of a hydrocarbon gas such as acetylene, oxygen plasma may be irradiated, or plasma of a mixed gas of these gases and oxygen may be irradiated. Even when the plasma of an inert gas such as argon is irradiated without irradiating oxygen plasma, oxygen can be introduced into the plasma treatment layer by oxygen in the air.

  The endothelial cell proliferating material shown in each embodiment can be used for stents, vascular filters, artificial valves, artificial blood vessels, stent grafts, medical devices such as pacemakers and long-term implantable vascular indwelling catheters, and artificial organs such as artificial hearts. .

  The endothelial cell proliferating material according to the present invention can selectively improve the proliferative property of vascular endothelial cells, and is useful as an endothelial cell proliferating material having selective proliferative properties of endothelial cells.

It is sectional drawing which shows the endothelial cell proliferative material which concerns on the 1st Embodiment of this invention. It is a graph which shows the relationship between the ratio with respect to the oxygen with respect to the oxygen in the endothelial cell proliferative material which concerns on the 1st Embodiment of this invention, and the growth rate of a cell. It is a graph which shows the relationship between the zeta potential of the endothelial cell proliferative material which concerns on the 1st Embodiment of this invention, and the proliferation rate of a cell. It is the schematic which shows an example of the plasma irradiation apparatus used for manufacture of the endothelial cell proliferative material which concerns on the 1st Embodiment of this invention. It is sectional drawing which shows the endothelial cell proliferative material which concerns on the 2nd Embodiment of this invention. It is a graph which shows the relationship between the ratio with respect to the carbon of oxygen in the endothelial cell proliferative material which concerns on the 2nd Embodiment of this invention, and the growth rate of a cell. It is a graph which shows the relationship between the zeta potential of the endothelial cell proliferative material which concerns on the 2nd Embodiment of this invention, and the proliferation rate of a cell.

Explanation of symbols

DESCRIPTION OF SYMBOLS 10 Chamber 12A Parallel plate electrode 13 Mass flow controller 14 Matching box 15 High frequency power supply 31 Base material 32 Carbonaceous film 32A Plasma processing layer 32B Plasma processing layer

Claims (6)

A membrane body formed on the surface of the substrate and formed by bonding carbon to each other;
A plasma treatment layer having nitrogen and oxygen formed on the surface of the film main body and combined with carbon constituting the film main body,
The ratio of nitrogen to oxygen in the plasma treatment layer is 0.3 or more, wherein the endothelial cell proliferating material.   2. The endothelial cell proliferating material according to claim 1, wherein the zeta potential on the surface of the membrane body is −20 mV or more. A membrane body formed on the surface of the substrate and formed by bonding carbon to each other;
A plasma treatment layer formed on the surface of the membrane body and having oxygen combined with carbon constituting the membrane body;
The ratio of oxygen to carbon in the plasma treatment layer is 0.1 or more.   4. The endothelial cell proliferating material according to claim 3, wherein the zeta potential on the surface of the membrane body is −30 mV or less.   The endothelial cell proliferating material according to any one of claims 1 to 4, wherein the proliferation rate of endothelial cells on the surface of the plasma treatment layer is higher than the proliferation rate of smooth muscle cells.   The endothelial cell proliferating material according to claim 5, wherein the proliferation rate of the endothelial cells is 1.1 times or more of the proliferation rate of the smooth muscle cells.