JPH0342294B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a biocompatible material molded article used as a biocompatible material, such as a culture bed for culturing biological cells. Recently, research has been actively conducted on the reactions and other properties of biological cells under various conditions, and on the products obtained from specific cell activities. In particular, the production of substances that cannot be synthesized purely artificially or that are extremely difficult to synthesize by utilizing the activities of specific cells has been studied in many fields. Specific examples of such substances include interferons, hormones, lymphokines, and others. Cell culture is generally carried out by implanting or inoculating cells on a culture bed, placing them in a medium, and incubating them under environmental conditions suitable for the cells. There are various restrictions. One of the major limitations is the problem of culture beds. For example, cells with adhesion dependence for proliferation, such as normal diploid cells, proliferate in a monolayer on the surface of the culture bed after adhering to the culture bed.
The culture yield is greatly influenced by the conditions of the culture bed used, especially the quality of cell adhesion, that is, the biocompatibility. Conventionally, polysaccharide polymers, certain synthetic polymers, and the like have been used as biocompatible materials suitable for culture beds, but good results cannot always be obtained. In addition, constructing a culture bed using only a specific biocompatible material is not only economically disadvantageous because the material is expensive, but also has restrictions on molding and processing due to its physical properties, such as mechanical strength. For example, it is often difficult to mold biocompatible materials into the shape of a Petri dish or microtiter plate. In addition, in the case of biocompatible materials such as culture beds, it is sufficient that the specific surface on which cells etc. adhere and operate is biocompatible, so using the casting method, A biocompatible material having a surface layer made of a biocompatible substance is obtained by applying a solution of a biocompatible substance in a volatile solvent onto the surface of a substrate having a specific shape, and then allowing the solvent to evaporate and evaporate. However, in this case, the adhesion or integrity between the surface layer made of a biocompatible material and the substrate would be low, making it difficult to obtain great durability, and furthermore, casting would require a long time. ,
Furthermore, when the surface shape of the substrate is complex, a surface layer made of a biocompatible material cannot be uniformly formed on the surface of the substrate. In addition, as a particulate culture bed for culturing cells, low-temperature plasma is brought into contact with the resin particle surface, thereby oxidizing the resin particle surface, increasing its hydrophilicity, and improving the adhesion of cells to the resin surface. Some are known that have increased proliferative properties. (Tokukai Akira
57-22691). Specifically, this culture bed is made by oxidizing the polystyrene surface in oxygen plasma or air plasma to make the surface hydrophilic. However, oxygen plasma has the following drawbacks. That is, oxygen atoms and oxygen molecules have a large electron affinity and easily become negative ions in plasma, resulting in an auto-ionization phenomenon. Therefore, the plasma state inside the reaction vessel is not uniquely determined by energy applied from the outside, such as high-frequency power or microwave power, but spatially there are areas where ions are sparse and areas where they are dense, and It becomes unstable because it vibrates. For these reasons, it is not easy to achieve a uniform surface treatment using oxygen plasma. Considering that one of the requirements for a culture bed is that the surface properties be uniform, manufacturing a culture bed using oxygen plasma is difficult. Not necessarily an advantageous method. The present invention was completed as a result of intensive research based on the above circumstances, and its purpose is to
The present invention provides a molded article for a biocompatible material that has great durability and can be easily manufactured into any shape in a short period of time and is used as a biocompatible material. That is, the present invention provides a plasma polymer film having at least one kind of hydrophilic group selected from a sulfur-containing hydrophilic group and a nitrogen-containing hydrophilic group on the surface of a substrate by plasma treatment in the presence of a plasma polymerizable substance gas. The object of the present invention is to provide a molded article for a biocompatible material, which is characterized in that it is formed by molding. The present invention will be specifically explained below. The molded article for biocompatible materials of the present invention is produced by plasma treatment in the presence of a plasma polymerizable substance gas to form at least one kind of hydrophilic group selected from sulfur-containing hydrophilic groups and nitrogen-containing hydrophilic groups on the surface of the substrate. For example, the following molded products for biocompatible materials can be exemplified. 1. A plasma polymer film having sulfur-containing hydrophilic groups on the surface is formed by plasma treatment in the presence of a plasma polymerizable substance gas having sulfur-containing hydrophilic groups, or a mixed gas of the above gas and oxygen or oxygen compound gas. A molded article for biocompatible material, characterized in that: 2. Plasma treatment in the presence of a plasma polymerizable substance gas having a nitrogen-containing hydrophilic group, a mixed gas of nitrogen and/or ammonia and a hydrocarbon compound, or a mixed gas of the above gas and oxygen or an oxygen compound gas. A molded article for biocompatible material, characterized in that a nitrogen-containing plasma polymer film is formed on the surface of the molded article. In the first example, a plasma polymer film of a plasma polymerizable substance having a sulfur-containing hydrophilic group is formed on the outer surface of a substrate made of a suitable solid material to construct a molded article for a biocompatible material. . For example, as shown in FIG. 1, a coil 2 is provided at the small diameter portion of one end of a reaction vessel 1 connected to a vacuum pump (not shown), a high frequency power source 3 is connected to this, and a support base 4 inside the reaction vessel 1 is provided. A substrate S to be a molded article for a biocompatible material is held on top, and plasma polymerization having a sulfur-containing hydrophilic group is carried out inside the reaction vessel 1 through a gas inlet 5 while the inside of the reaction vessel 1 is evacuated. A gas containing only a chemical substance or a mixture thereof with oxygen or an oxygen compound is introduced, and the coil 2 is connected to a power source 3.
A higher frequency voltage is applied to generate plasma in the reaction vessel 1, and this plasma is applied to the outer surface of the substrate S to form a plasma polymer film of the plasma polymerizable substance thereon. Alternatively, as shown in FIG. 2, a pair of electrodes 11, 11 are provided in a reaction vessel 10 constituted by a bell jar so as to face each other, and a substrate S to be a molded article for biocompatible material is held therebetween. , electrode 1
For example, a power supply 12 with a frequency of 10 KHz is connected between electrodes 1 and 11 to generate plasma between the electrodes 11 and 11, and this plasma is applied to the outer surface of the substrate S to cause plasma generated by the plasma polymerizable material to be applied to the outer surface of the substrate S. A polymer film is formed. 13 is an exhaust pipe, and 14, 14 is a reaction gas introduction pipe. In the above, the substrate is a solid material that does not emit a large amount of gas in a vacuum, for example, the amount of gas emitted in a plasma reaction system is 0.1 Pa・m ³・sec ^-1・
Any solid substance with a particle size of m ^-1 or less can be used without particular limitation, such as polystyrene, polyethylene, polyvinyl chloride,
Resin-like or rubber-like polymers such as polyester, organopolysiloxane, styrene-butadiene copolymer rubber, fluorocarbon rubber, and polyisoprene rubber, and inorganic substances such as metals, semiconductor materials, and ceramics can be used. Further, the shape of the solid substance is not particularly limited, and any shape can be used, such as a particle shape with a diameter of 100 Å to 10 mm, a Petri dish shape, a microtiter plate shape, etc. Examples of the above-mentioned sulfur-containing hydrophilic groups include sulfonic groups and mercapto groups. Examples of plasma-polymerizable substances having sulfonic groups that can be gasified in a plasma reaction system include methanesulfonic acid and ethanesulfonic acid. , sulfonated hydrocarbons such as propanesulfonic acid, aromatic sulfonated hydrocarbons such as benzenesulfonic acid and styrenesulfonic acid, and plasma polymerizable substances having a mercapto group that can be gasified in a plasma reaction system. By way of example, mention may be made of mercaptans such as methyl mercaptan, ethyl mercaptan, propyl mercaptan and the like. Examples of the above-mentioned oxygen compounds include oxygen compounds that can be gasified in a plasma reaction system, such as carbon monoxide, carbon dioxide, nitrogen monoxide, nitrogen dioxide, and nitrous oxide. When used, the amount used is preferably 0.2 mol or less per 1 mol of the plasma polymerizable substance having a sulfur-containing hydrophilic group. In addition, methane, ethane,
propane, ethylene, propylene, acetylene,
Hydrocarbon plasma polymerizable substances that can be gasified, such as benzene and styrene, may also be present. Apparatuses for plasma processing are not limited to those shown in FIGS. 1 and 2; for example, the energy source for plasma generation may be direct current,
It may be any alternating current power source, and in the case of alternating current, it may be of any frequency, low frequency, high frequency, or microwave. In the case of microwaves, the coupling method between the amplifier and the plasma system may be either a ladder type or a cavity type. Further, the type of plasma generating electrode, ie, inductive type, capacitive type, etc., is not limited. Furthermore, the conditions for plasma treatment, such as the degree of vacuum inside the reaction vessel, the flow rate of plasma polymerizable material gas, and the discharge power, are the same as those for ordinary plasma polymerization reactions, but the degree of vacuum inside the reaction vessel is 1. The appropriate flow rate of the plasma polymerizable material gas is from several cc to several hundred cc per minute under standard conditions when the capacity of the reaction vessel is 50 mTorr to 10 Torr. Further, the discharge power is preferably set so that the electron temperature of the plasma is 80,000 degrees or less, particularly 5,000 to 60,000 degrees, in order to reduce variations in the biocompatibility of the molded product obtained. The plasma treatment time varies depending on the thickness of the plasma polymer film to be formed on the substrate. The thickness of this plasma polymer film is not particularly limited, but it usually only needs to be about 100 to 5000 Å, so the plasma treatment time can be shortened.
For example, it is several tens of minutes or less. Further, the ratio of sulfur atoms to carbon atoms (S/C) on the surface of the plasma polymer film having sulfur-containing hydrophilic groups is not particularly limited, but is generally preferably 0.05 to 0.5, particularly 0.05 to
0.2 is preferred. According to this first example, the outer surface of the substrate is covered with the plasma polymer film, and therefore a molded article for biocompatible material is obtained whose surface is formed by the plasma polymer film. That is, the plasma polymer membrane is made of an organic material having a sulfur-containing hydrophilic group, and has suitable biocompatibility. For example, when this is used as a cell culture bed, cells have high adhesion and proliferation progresses smoothly. Therefore, it can be used for initial cell culture, especially adhesion-dependent cells that grow in a monolayer on the culture bed. can be cultured with high efficiency. The cell type or cells are not particularly limited, but examples include hamster lung cells, human fetal lung cells, chimpanzee lung fibroblasts, human foreskin cells, chicken fetal fibroblasts, and the like. In the second example, a plasma polymer film of a plasma polymerizable material having nitrogen-containing hydrophilic groups is formed on the outer surface of a substrate made of a suitable solid material to construct a molded article for biocompatible material. In the above, the plasma generation device and plasma generation conditions are substantially the same as in the first example. Examples of nitrogen-containing hydrophilic groups include cyano groups, amino groups, and imino groups, and examples of plasma-polymerizable substances having nitrogen-containing hydrophilic groups include methylamine, dimethylamine, trimethylamine, aniline, ethylenediamine, urea, and allylamine. Examples include compounds having a nitrogen-containing hydrophilic group that can be gasified in a plasma reaction system, such as , pyridine, and pyrimidine. Further, in the second example, a plasma polymer film is formed from ammonia and a hydrocarbon compound. Hydrocarbon compounds include methane, ethane, propane, ethylene, propylene,
Examples include hydrocarbon compounds that can be gasified in a plasma reaction system and can be plasma polymerized, such as acetylene, benzene, and styrene. The mixing ratio of these hydrocarbon compounds to 1 mole of nitrogen and/or ammonia is preferably 1 mole or less, and in this case, the resulting plasma polymer membrane having nitrogen-containing hydrophilic groups has particularly excellent biocompatibility. become. Further, as the oxygen compound in the second example, the same compounds as the oxygen compound in the first example can be exemplified. In the second example, when using oxygen or an oxygen compound, the amount used is:
Plasma polymerizable substance having nitrogen-containing hydrophilic group or nitrogen and/or ammonia and hydrocarbon compound 1
It is preferably 0.2 mol or less per mole. According to this second example, a plasma polymer film having a nitrogen-containing hydrophilic group is formed on the surface of the base by subjecting the surface of the base to the action of plasma in the presence of the above gas, and this plasma polymer film has the same biocompatibility as the plasma polymer membrane of the first example, and therefore a molded article for biocompatible material that can be suitably used as a biocompatible material can be obtained. In this case, the ratio of nitrogen atoms to carbon atoms (N/C) on the surface of the plasma polymer film having nitrogen-containing hydrophilic groups is:
It is preferably 0.1 or more, and particularly excellent biocompatibility is exhibited in this case as well. N/C can be adjusted as appropriate depending on the type and amount of gas, plasma conditions, etc., but is generally 1 or less. Note that the above-mentioned hydrocarbon compounds and the like can also be used in combination with the plasma polymerization material gas having a nitrogen-containing hydrophilic group, thereby making it possible to easily adjust N/C. Further, in the first and second examples described above, the gas used in each example may be mixed with the gas used in other examples. According to the first and second examples above, the plasma polymer film formed on the outer surface of the substrate has high adhesion or integrity with the substrate, and therefore has high durability, and the plasma polymerization is performed in the gas phase. As the process proceeds, a uniform plasma polymer film is formed on the surface of the substrate, no matter what the surface shape of the substrate is.
Therefore, it is possible to obtain a biocompatible material molded product having any shape, such as a flat surface, a surface with deep grooves, an uneven surface, and the like. Furthermore, it is easy to manufacture, takes a short time to manufacture, and has almost no restrictions on the material of the base, making it extremely advantageous. As described above, according to the present invention, a biocompatible material is used as an excellent biocompatible material that has great durability and can be easily manufactured into any shape in a short time. Molded products can be provided. Furthermore, the molded article for biocompatible materials of the present invention can be used not only as a cell culture bed but also as a material for forming artificial human bodies such as artificial blood vessels, artificial femoral heads, artificial joints, and artificial teeth by appropriately selecting the base material. In addition, it can be suitably used as a suture thread for the human body, a hemostatic material, and a carrier for immunoreactive substances such as antigens and antibodies. Examples of the present invention will be described below, but the present invention is not limited thereto. Example 1 A reaction apparatus having the configuration shown in Fig. 1 was used, a polystyrene shear was held as a base on a support stand, and styrene sulfonic acid gas was supplied as a plasma polymerizable substance at a rate of 4 c.c. (STP)/min. While introducing the material into the reaction vessel at a flow rate, the inside of the vessel was maintained at a vacuum level of 20 millitorr, and a high frequency voltage was applied to the coil to generate plasma with an electron temperature of 2.5 ± 0.3 million degrees, and the reaction continued for 10 minutes. A plasma polymer film was formed on the surface of the substrate. Here, the plasma electron temperature is
This is a value measured using a heated probe (not shown). The thickness of the formed plasma polymer film can be determined by changing the density of the polymer film by 1 from the change in oscillation frequency detected by a sensor of a quartz crystal film thickness meter (not shown) placed near the substrate. It was recognized that it corresponds to 400±100 Å in the case of assuming that. Ten chalets were made using the above method. The presence of sulfonic groups on these Schare surfaces indicates that FT
-IR-ATR (reflection type Fourier transform infrared spectrometer)
This was confirmed by measuring the characteristic absorption of the sulfone group appearing at 1225 to 1195 cm ^-1 using . moreover
ESCA (Electron Spectroscopy for Chemical
C _1S (284eV) on the Schare surface by Analysis)
The S/C value obtained by measuring the peaks of and S _2S 1/2 (229 eV) and considering the ionization cross section from each peak area was 0.12 to 0.14. Cell line V derived from Chinese hamster lungs was stored in the air for 24 hours, subjected to high temperature sterilization treatment for 24 hours, and cultured separately.
-79 was implanted as free cells in a 0.25 wt% trypsin solution into each cell, and using Eagle MEM medium (manufactured by Nissui Pharmaceutical Co., Ltd.) containing 10 wt% fetal bovine serum, 5% carbon dioxide by volume and 95% air were used. In an incubator with an atmosphere of vol%, the temperature is 37℃.
Cell culture was performed using When the culture time reaches 120 minutes, each shell is removed from the incubator, the medium is removed, and after washing with phosphate buffered saline, 1 ml of pronase EDTA solution is added to each shell to ensure adhesion to the culture bed surface. The cells were released and the number of cells was measured using a hemocytometer. Then, the ratio of the number of adhered cells to the total number of cells (adhesion rate) was determined. The results are shown in Table 1. Further, Table 1 shows the results of a comparative test conducted in the same manner as in this example except that the plasma polymer film forming treatment was not performed.

[Table] Example 2 An aluminum shear plate was used as the substrate, ethanesulfonic acid gas was used as the plasma polymerizable substance, and the flow rate of the ethanesulfonic acid gas was 20c.c.
(STP)/min, plasma with an electron temperature of 4.5±0.3 million degrees was generated, and the treatment time was 30 minutes. Make 10 pieces of chare,
Along with 10 comparative test plates without plasma treatment, the cells were cultured in the same manner as in Example 1.
The adhesion rate for each was determined. The results are shown in Table 2. In addition, the presence of sulfone groups on the surface of Shearle in this example was confirmed by FT-IR-ATR. Also
The S/C value of the Sheare surface obtained from ESCA is
It was 0.06-0.07.

[Table] Example 3 Except for using a mixed gas of methanesulfonic acid gas 20 c.c. (STP)/min and oxygen gas 2 c.c. (STP)/min instead of ethanesulfonic acid gas in Example 2. Ten shears coated with a plasma polymer film having a thickness of 400±100 Å were prepared in the same manner as in Example 2. This shear dish was subjected to the same cell culture as in Example 1, and the adhesion rate in each case was determined. The results are shown in Table 3. The presence of sulfone groups on the surface of Shearle in this example was confirmed by FT-IR-ATR. Also
The S/C value of the Sheare surface obtained from ESCA is
It was 0.08-0.10.

[Table] Example 4 To make the electron temperature of the plasma 7 ± 0,500 degrees,
Example 1 except that the high frequency power was increased
Ten sheets coated with a plasma polymer film with a thickness of 300 ± 100 Å were made in exactly the same manner as above, and these were subjected to no plasma treatment for comparison test (1).
The 10 shears and the polystyrene shears for comparison test (2) were subjected to the same cell culture as in Example 1 together with the 10 sulfonated polystyrene shears that had been sulfonated by immersing them in sulfuric acid, and the adhesion rates of each were determined. I asked for The results are shown in Table 4. The presence of sulfone groups on the surface of the sulfonated polystyrene shear in this example and in the comparative test (2) was confirmed by FT-IR-ATR. In addition, the S/C value of the Shear surface in this example determined from ESCA is 0.02 to 0.05,
The S/C value of the sulfonated polystyrene shear surface for comparative test (2) was 0.02 to 0.04.

[Table] Example 5 Using a reaction apparatus having the configuration shown in Fig. 2, a polystyrene sheared substrate was held, and 50 c.c. of monoethylamine gas was used as a plasma polymerizable substance.
(STP)/min, vacuum level of 50 millitorr, electron temperature of 20,000 degrees, approximately 500 Å on the Schare surface.
A plasma polymer film with a thickness of . When the obtained surface of the surface of the surface was thoroughly dried and the absorption of the surface of the surface of the surface of the surface was measured using FT-IR-ATR in a dry atmosphere, two characteristic absorptions of amino groups were observed near 3450 cm ^-1 . A characteristic absorption of imino groups was observed near 3350 cm ^-1 . Also, C _1S (284eV) on the Schare surface by ESCA
N/ obtained from the peak area of and N _1S 1/2 (399eV)
The value of C was 0.1-0.2. This shear dish was subjected to the same cell culture as in Example 1, and the adhesion rate in each case was determined. The results are shown in Table 5. Example 6 Same as Example 5 except that a mixed gas of 50 c.c. (STP)/min of monoethylamine gas and 5 c.c. (STP)/min of oxygen gas was used instead of the monoethylamine gas in Example 5. Ten shears coated with plasma polymer films with a thickness of 300±100 Å were fabricated. This shear dish was subjected to the same cell culture as in Example 1, and the adhesion rate in each case was determined. 5th result
Shown in the table. In this example, the presence of amino groups on the surface of Sheare was confirmed by FT-IR-ATR. Also ESCA
The N/C value of the Sheare surface calculated from 0.1 to 0.2
It was hot. Example 7 Using a reaction apparatus with the configuration shown in Figure 2, a polystyrene shear is held as a substrate, and 100% of methyl mercaptan gas is used as a plasma polymerizable substance.
cc (STP)/min, vacuum level of 100 millitorr, electron temperature of 45,000 degrees, the surface of the sheared surface is 250±
A 100 Å plasma polymer film was formed. Ten pieces were made using the above method. The presence of mercapto groups on these Schare surfaces is ET-IR-ATR.
This was confirmed by the characteristic absorption near 2550 cm ^-1 . Further, the S/C value of the Shear surface determined by ESCA was 0.13 to 0.17. This shear dish was subjected to the same cell culture as in Example 1, and the adhesion rate in each case was determined. The results are shown in Table 5. Example 8 Ten shears coated with a plasma polymer film having a thickness of 150±100 Å were prepared in the same manner as in Example 7, except that the electron temperature was changed to 75,000 degrees. This shear dish was subjected to the same cell culture as in Example 1, and the adhesion rate in each case was determined. The results are shown in Table 5. In this example, the presence of mercapto groups on the surface of Shearle was confirmed by ET-IR-ATR. Also
The S/C value determined from ESCA was 0.08 to 0.10.

【table】 [Brief explanation of drawings]

FIG. 1 and FIG. 2 are explanatory diagrams each showing the configuration of a reaction apparatus that can be used for manufacturing a molded article according to the present invention. 1, 10... Reaction container, S... Substrate, 2... Coil,
11...electrode, 4...support stand, 13...exhaust pipe.

Claims (1)

[Claims] 1 A plasma polymer film having at least one type of hydrophilic group selected from sulfur-containing hydrophilic groups and nitrogen-containing hydrophilic groups is formed on the surface of the substrate by plasma treatment in the presence of plasma polymerizable substance gas. A molded article for biocompatible materials characterized by: