KR102051983B1 - Implant for inhibiting fibrosis and method for preparing thereof - Google Patents

Abstract

The implant according to the present invention relates to a prosthesis having a multilayer thin film coated on the surface where pores are formed. When the implant is implanted, the prosthesis can suppress the fibrosis reaction primarily by the multilayer thin film, and from the roughness caused by the pores formed on the surface of the prosthesis. Secondly, the fibrosis reaction can be prevented or suppressed, so that the fibrosis reaction can be more effectively suppressed and spherical construction can be prevented.

Description

Implants for inhibiting fibrotic reaction and preparation method thereof

The present invention relates to a prosthesis coated with a multi-layered thin film, and more particularly, by coating a multi-layered thin film on the surface of the prosthesis formed by the particles prepared by the film emulsification method, the pores formed on the surface of the multi-layered thin film and the implant It relates to an implant that can prevent fibrosis through.

Silicone implants are used for breast augmentation for cosmetic or patients who require breast reconstruction due to the treatment of breast cancer. Breast cancer has been steadily increasing in modern times, and Korea has the highest incidence of breast cancer among OECD countries. Breast plasticity is also increasing in demand. However, when a silicone implant is inserted for breast reconstruction or plastic surgery, the body recognizes it as an external substance and causes an immune response. This results in a fibrosis reaction in which the silicone implant is surrounded by fibers in the human body, which has been reported as the most frequent side effect of the silicone implant.

To prevent the fibrosis reaction caused by silicone implants, it is currently recommended to take oral anti-fibrosis drug for 2 weeks and 1 year after implant implantation. Have. Another method is to insert a silicone implant with pores on the surface. It is to reduce the fibrosis by allowing the living tissue to grow in the pores present on the surface of the implant. However, these pore-textured silicone implants do not completely prevent the fibrosis reaction, so studies are being conducted to mix drug delivery systems or biocompatible materials.

Accordingly, techniques for modifying the surface of the silicon implant to be more biocompatible using natural polymers such as collagen, hyaluronic acid, and chitosan have been studied.

Korea Patent Registration No. 10-1288115 (2013.07.08.)

An object of the present invention is to provide a prosthesis and a method for producing the prosthesis for preventing or inhibiting spherical contraction and fibrosis reaction induced after implantation of the prosthesis.

The inventors of the present invention while studying the surface modification method of the implant to suppress the excessive fibrosis reaction that causes spherical building, when the implant is coated with a multilayer thin film on the surface of the pores is formed, the multilayer thin film is primarily suppressed by the multilayer thin film The present invention has been completed by confirming that the fiberization reaction can be prevented or suppressed from the roughness due to the pores formed on the surface of the implant.

Thus, the present invention,

A prosthesis in which pores are formed on a surface by particles prepared by a film emulsification method; And

Provided is a fibrous reaction inhibiting prosthesis coated with a multi-layered thin film comprising a cationic material layer laminated with a cationic material on the surface of the implant and an anionic material layer stacked with anionic material on the cationic material layer.

Below. The configuration of the present invention will be described in detail.

In the present invention, the term "capsular contracture" refers to a phenomenon in which the coating around the implanted implant is thick and hard due to excessive fibrosis, and is one of side effects that occur during implant implantation. In the present invention, the prosthesis can prevent spherical contraction by inhibiting excessive fibrosis reaction after transplantation.

In the present invention, the term "prosthesis" is available to the site of the depression, such as skin depressions or wrinkles, and means all implants available for improving the volume of cosmetic purposes. In the present invention, the implant may be a medical implant for the purpose of preserving the shape or size of the breast during breast reconstructive surgery or breast plastic surgery for cosmetic purposes, but is not limited thereto.

In the present invention, the prosthesis may be a prosthesis formed of silicone or polyurethane. The implant may include, but is not limited to, conventional silicone back implants, silicone gel implants, cohesive silicone gel implants or polytech implants. In one embodiment, the implant may be a silicone implant.

In the present invention, particles prepared by the film emulsification method can be used to form pores on the surface of the implant. Specifically, pores may be formed on the surface of the implant by attaching the particles to the surface of the implant and then applying heat to melt the particles. At this time, the particles are not particularly limited in kind and may be applied as long as they are easily soluble in water or an organic solvent. In addition, the particles may include particles having a diameter of 10 to 150 μm, for example, 15 to 140 μm, 20 to 130 μm, 25 to 120 μm, 30 to 110 μm, and 35 to 100 μm. When implanting the prosthesis formed with pores on the surface through the particles of the diameter as described above, it is possible to suppress the fibrosis reaction from the small and constant size pores, thereby preventing the spherical building.

In the present invention, the particles produced by the membrane emulsification method may be obtained by applying a conventional membrane emulsification method. As the emulsion film used in the film emulsification method, for example, a membrane having a pore size of 10 to 30 µm may be used, and a shirasu porous glass (SPG) membrane may be used, but is not limited thereto. In one embodiment, when the particles are prepared by the film emulsification method using the SPG film, since particles of a certain size may be manufactured, the implant to which the particles are attached may have uniform and fine pores, thereby inhibiting the fibrosis reaction from the pores. Can be maximized.

Therefore, the pores formed on the surface of the prosthesis from the particles may have a diameter of 10 to 150㎛, such as 15 to 140㎛, 20 to 130㎛, 25 to 120㎛, 30 to 110㎛, 35 to 100㎛, particles Depending on the size of the pore size can be adjusted.

In addition, the present invention includes a prosthesis having a multilayer thin film coated on the surface of the prosthesis having pores formed on the surface thereof. The multilayer thin film includes a cationic material layer stacked with a cationic material and an anionic material layer stacked with an anionic material on the cationic material layer.

In the present invention, the cationic material and the anionic material are natural polymers that can reduce the fibrosis reaction, and the cationic material is poly L-lysine (PLL), collagen, chitosan, polyethylene Imine (polyethyleneimine (PEI), polyamidoamine (PAA), polyaminoester (PAE), poly [2- (N, N-dimethylamino) ethyl methacrylate] (poly [2- (N, N-dimethylamino) ethylmetacrylate]; PDMAEMA), cationic cellulose, cationic dextrin and gelatin may be selected from the group consisting of, for example, poly L- lysine. The anionic material is hyaluronic acid, alginate, albumin, albumin, dextran sulfate, heparin, sodium alginate, poly gamma-glutamic acid, and poly gamma-glutamic acid. acid, poly aspartic acid, poly styrene sulfonate, poly phthalic ethylene glycol ester, poly sulfonated aniline and poly allyl It may be one selected from the group consisting of amine hydrogen chloride (poly allyamine hydrogen chloride), for example hyaluronic acid.

The implant according to the present invention may be to coat the cationic material layer and the anionic material layer through a layered self-assembly method, and after the plasma treatment with oxygen on the surface of the implant to give a negative charge, the cation through the electromagnetic attraction The material layer and the anion material layer may be coated by bonding themselves.

In the present invention, the implant for inhibiting the fibrosis reaction may be to further coat the drug layer on the multilayer thin film. The drug of the drug layer may include a drug that can inhibit the fibrosis reaction in implantation of the implant, for example, tranilast, pirfenidone, mitomycin, acetylsalicylic acid ( one or more fibrosis inhibitors selected from the group consisting of acetylsalicylic acid, genistein, selenocystine, triamcinolone acetonide, suplatast tosilate, and montelukast However, it is not limited thereto. The coating of the drug layer may be coated by electromagnetic attraction, such as a layered self-assembly method, and may be coated on the surface of the implant by supporting the implant in a solution in which the drug is dissolved, but using a conventional coating method It may include all to be coated on the surface of the implant.

In the following examples, it was confirmed that the silicone implant (polydimethylsiloxane) as described above was excellent in biocompatibility, exhibited low cytotoxicity, and was excellent in inhibiting fibrosis. In addition, as a result of implanting the implant in a mouse model, the inflammatory response was low due to the fine pores on the surface of the implant, and the levels of TGF-β, fibroblast, and myofibroblast involved in the fibrosis reaction were also observed. It was found to decrease significantly.

In addition, the present invention,

Forming pores on the surface of the implant from the particles prepared by the film emulsification; And

Provided is a method for producing a fibrous reaction inhibiting implant comprising the step of coating a multi-layered thin film comprising a cationic material layer laminated with a cationic material and an anionic material layer laminated with an anionic material on the prosthesis formed with pores on the surface.

In the above production method, all of the above descriptions may be applied or mutatis mutandis to the particles, implants, and multilayer thin films produced by the film emulsification method.

Specifically, the manufacturing method includes forming pores on the surface of the prosthesis from the particles prepared by the film emulsification method. In one embodiment, the method may further comprise polymerizing the polystyrene solution through a membrane to produce particles. The polystyrene solution may be obtained by dissolving polystyrene in a solvent, and the solvent may be a solvent capable of dissolving polystyrene, for example, an organic solvent such as methylene chloride (DCM), acetone, methanol, or the like. It is not limited to this.

At this time, the membrane may be an emulsion film having a pore size of 20 to 50㎛, particles can be prepared by using a conventional membrane emulsification method. Specifically, the polystyrene solution was made into a discontinuous phase, passed through an emulsion film, and dispersed in a spherical droplet in the continuous phase. At this time, the polystyrene solution can be passed through the emulsion film under the conditions of a pressure of 0.5kPa to 3.0kPa and a stirring speed of 100 to 300rpm, thereby controlling the size of the particles. The solution in which the droplets are dispersed may be prepared in a powder form by drying the solvent for 12 to 36 hours after removing the solvent.

In addition, in the manufacturing method according to the invention, the step of forming pores on the surface of the prosthesis from the particles, uniformly spraying the particles on the surface of the prosthesis and pressure is applied before completely curing the prosthesis formed of silicone or polyurethane Adding; And completely curing the implant at 30 ° C. to 50 ° C.

In the above manufacturing method, the prosthesis formed of silicone or polyurethane can adjust the curing temperature according to the material or material of the prosthesis, for example, may be 70 ℃ to 90 ℃.

In this step, if the prosthesis is completely cured before attaching the particles, the particles are difficult to attach, and if the drug particles are attached before the prosthesis is cured, the particles may be buried in the silicone or polyurethane, thereby completely curing the silicone or polyurethane. It may be desirable to perform the step of spraying the particles before.

In addition, the drug particles may further include the step of applying a pressure after the injection, wherein the pressure applied to the implant is not limited as long as the particles can be embedded in the surface of the implant formed of silicone or polyurethane, This can be properly adjusted by a person skilled in the art according to the material of the implant, the stress that the implant can withstand, the particles and the like.

By going through the above process, the particles are embedded in the surface of the implant and can form pores on the surface through melting process, it can give a roughness to the implant.

In addition, the manufacturing method includes the step of coating a multi-layered thin film on the prosthesis formed with pores on the surface. Specifically, forming a cationic material layer by layering a cationic material on the implant by layered self-assembly, and forming an anionic material layer by laminating anionic material on the cationic material layer by layered self-assembly. In this case, the cation material layer and the anion material layer may be alternately repeated one or more times to form a multilayer thin film. The implant includes plasma treatment with oxygen to treat the surface to have a negative charge. Next, the method may include inducing a positively charged material (cationic material) solution to bind the cationic material to the surface of the implant. Specifically, the step is a step of immersing the implant in the cationic material solution for 5 to 20 minutes, and then immersed in the washing solution for 5 to 20 minutes. Thereafter, a solution having a negative charge on the implant coated with the cationic material (anion material) may be induced to bind the anionic material to the implant, thereby coating the multilayer thin film on the surface of the implant. The step can be carried out in the same manner as inducing a cationic material to be bonded, except that an anionic material solution is used. By repeating the above process, it is possible to obtain a prosthesis coated with a multilayer thin film of a cationic material layer and an anionic material layer on the surface. As described above, by coating the surface of the implant with a natural polymer that reduces the fiberization reaction, the fiberization reaction can be primarily suppressed.

Therefore, the implant according to the present invention can primarily inhibit the fibrosis reaction through the multilayer thin film coated on the surface when implanted in the body, and pores are formed on the surface of the implant from particles prepared by the film emulsification method to impart roughness. It is possible to suppress the fibrosis reaction secondary, so that it is possible to more effectively suppress the fibrosis reaction and to prevent spherical building.

The implant according to the present invention relates to a prosthesis having a multilayer thin film coated on the surface where pores are formed. When the implant is implanted, the prosthesis can suppress the fibrosis reaction primarily by the multilayer thin film, and from the roughness caused by the pores formed on the surface of the prosthesis. Secondly, the fibrosis reaction can be prevented or suppressed, so that the fibrosis reaction can be more effectively suppressed and spherical construction can be prevented.

1 to 3 are photographs of the polystyrene particles according to the size of the SPG film (20 μm, 30 μm, 50 μm) observed through SEM.
Figure 4 is a photograph observing the pore size of the surface of polydimethylsiloxane (PDMS) formed by polystyrene particles.
5 is a graph measuring the thickness of a multilayer thin film on the surface of polydimethylsiloxane using an ellipsometer.
FIG. 6 illustrates a process of forming pores on the surface of a prosthesis from particles prepared by a film emulsification method and coating a multilayer thin film.
7 shows the results of CKK-8 analysis of polydimethylsiloxane having pores on the surface and polydimethylsiloxane treated with a multilayer thin film.
8 is a result of observing cell morphology through immunostaining in polydimethylsiloxane having pores on the surface and polydimethylsiloxane treated with a multilayer thin film.
9 shows the degree of TGF-β release from polydimethylsiloxane having pores on the surface and polydimethylsiloxane treated with a multilayer thin film.
10 is a sample of silicone implant (polydimethylsiloxane) in vivo of the present invention to be inserted into a mouse model.
11 is a result of analyzing the inflammatory response of the mouse model in which the silicone implant sample of the present invention is inserted.
12 is a result of analyzing the capsule-related factors (TGF-β, the number of fibroblasts and myofibroblasts) of the mouse model in which the silicone implant sample of the present invention was inserted.
13 is a result of measuring the collagen density of the mouse model in which the silicone implant sample of the present invention was inserted.
14 is a result of measuring the capsule thickness of the mouse model in which the silicone implant sample of the present invention is inserted.

Advantages and features of the present invention and methods for achieving them will be apparent with reference to the embodiments described below in detail. However, the present invention is not limited to the embodiments disclosed below, but will be implemented in various forms, and only the embodiments are intended to complete the disclosure of the present invention, and the general knowledge in the technical field to which the present invention pertains. It is provided to fully convey the scope of the invention to those skilled in the art, and the present invention is defined only by the scope of the claims.

[ Example  1] Preparation of particle and particle size verification using membrane emulsification

1) Preparation of Polystyrene Particles

About 38 μm, 72 μm, and 100 μm particles were prepared using SPG film emulsification under the same conditions as in Table 1 below. Prepare a solution in which polystyrene is dissolved in solvent methylene chloride in a discontinuous phase, and polystyrene particles are prepared by SPG film emulsification using 20 ml of water containing sodium lauryl sulfate in a continuous phase. Prepared. In order to remove the solvent remaining in the particles, the continuous phase solution in which the particles were dispersed was centrifuged at 2,000 rpm for 1 minute to separate the particles from the solution, and the supernatant was discarded and the particles were dispersed again using DW. The particles dispersed solution was centrifuged twice for 1 minute at 2,000 rpm to wash the particles. Next, the particles were dried overnight at 80 ° C. to obtain particles in powder form.

Particle diameter
(Μm) Membrane Pore Size (㎛) Critical pressure (kPa) Discontinuity
(w / v%) Continuous phase
(w / v%) Stirring Speed (rpm) 38 20 1.6 22.5 3 250 70 40 1.2 15 3 250 100 50 1.3 30 3 250

2) Check the size of the particles

In order to determine the size of the particles prepared in 1), the particles were observed with a scanning electron microscope (SEM) and the size was measured. 1 to 3 is a result of observing the shape and size of the particles by SEM, the diameter of the particles according to the size of each SPG film is shown in Table 2.

SPG  membrane 20 ㎛ 30 μm 50 μm Particle diameter 38.97 μm 72.25 μm 100.05㎛

As shown in Table 2, the size of the particles were confirmed to represent the size of 38.97㎛, 72.25㎛, 100.05㎛ corresponding to about 1.5 to 2.5 times of the hydrophilic membrane of 20㎛, 30㎛, 50㎛, respectively It was confirmed that the particles having a very uniform size distribution could be prepared by the SPG membrane emulsification method. In addition, when the particles were prepared using the SPG membrane, it was confirmed that the particle size had the greatest influence on the viscosity due to the oil phase concentration.

[ Example  2] manufacture of implants

1) Preparation of the prosthesis with pores

A solution of sylgard 184 A: sylgard 184 B = 10: 1, a precursor of polydimethylsiloxane (PDMS), was applied on a glass plate in a predetermined amount, and then partially cured in an oven at 80 ° C. for 17 minutes 30 seconds to 18 minutes, The particles prepared in 1) were uniformly sprayed onto the polydimethylsiloxane so that the particles were pressurized by hand so that the particles could be embedded in the polydimethylsiloxane surface. Thereafter, overnight in an oven at 80 ℃ to completely cure. The cured polydimethylsiloxane melted the polystyrene particles embedded in the surface by using chloroform to form pores in the polydimethylsiloxane due to the imprinted marks as the particles melted. The photo of observing the polydimethylsiloxane with pores formed on the surface through SEM is shown in FIG. 4, and the size of the pores formed by the particles is shown in Table 3 below.

SPG  membrane 20 ㎛ 30 μm 50 μm Groundbreaking diameter 37.16 ± 1.38 μm 70.22 ± 3.26 μm 97.64 ± 5.21 μm

2) Preparation of prosthesis coated with multilayer thin film

The polydimethylsiloxane prepared in 1) was plasma treated with oxygen for 10 minutes to induce a negative charge on the surface. After dissolving DW in NaCl 0.15mM and dissolving 100 mg of poly L-lysine in 100 ml of solution adjusted to pH 6.0-6.5 using NaOH and HCl, the polydimethylsiloxane was immersed for 10 minutes. The surface of the poly L- lysine was to be coated. Then, NaCl 0.15mM was dissolved in DW and polydimethylsiloxane was immersed for 10 minutes again in a washing solution adjusted to pH 6.0-6.5 using NaOH and HCl. The prosthesis coated with poly L-lysine was immersed in a solution of 100 mg of hyaluronic acid in 100 ml of a solvent (the washing solution) for 10 minutes to coat hyaluronic acid on the surface of the prosthesis through electromagnetic attraction, and this process was repeated three times. By doing so, a polydimethylsiloxane coated with a multilayer thin film was prepared. The multilayer thin film was confirmed by measuring the thickness of the thin film of polydimethylsiloxane using an ellipsometer (Wooram, KR) (Fig. 5).

The process of forming pores on the surface of the prosthesis from the particles prepared by the film emulsification method and coating the multilayer thin film is illustrated in FIG. 6.

As can be seen in FIG. 5, as the number of multilayer thin films increased, the thickness of the thin films stacked on the polydimethylsiloxane surface was increased by 1 nm.

Experimental Example 1 Biocompatibility Evaluation (CCK-8 assay)

As a control, polydimethylsiloxane (bare PDMS) without any treatment on the surface, polydimethylsiloxane having pores on the surface of about 38 μm, 70 μm, and 100 μm, respectively, and PLL / HA multilayer thin films were treated on each surface by the experimental group. One polydimethylsiloxane was prepared. Each sample was cut to a size of 1 mm thick, 1 cm ⅹ 1 cm, put into a 24-well plate and soaked in ethanol for 1 hour and washed twice with Dulbecco's phosphate buffered saline (DPBS). NIH 3T3 of passages 8 to 11 passaged using DMEM-high (Fetal bovine serum 10%, Penicillin streptomycin 1%) medium was seeded onto the sample at a cell density of 20,000 cells / well, 1 ml medium. And then cultured. After that, the CCK-8 assay was performed on the 1st and 3rd day. CCK-8 assay first remove the medium and wash twice with DPBS, mix 1ml medium and 100μl CCK-8 solution, incubate for 1 hour and extract 100μl into 96-well plate The absorbance (Absorbance) was measured at 450nm. The results are shown in FIG.

As a result, based on the absorbance of untreated polydimethylsiloxane (Bare PDMS) with proven biocompatibility, the degree of similarity is obtained even when pores are formed or pores are formed and PLL / HA multilayer thin films are laminated. It was confirmed that the absorbance was shown. That is, it was confirmed that the implant according to the present invention is also biocompatible.

Experimental Example 2 Cell Morphology Evaluation (Immunostaining)

As a control, polydimethylsiloxane (bare PDMS) without any treatment on the surface, polydimethylsiloxane having pores on the surface of about 38 μm, 70 μm, and 100 μm, respectively, and PLL / HA multilayer thin films were treated on each surface by the experimental group. One polydimethylsiloxane was prepared. Each sample was cut to a size of 1 mm thick, 1 cm ⅹ 1 cm, put into a 24-well plate and soaked in ethanol for 1 hour and washed twice with Dulbecco's phosphate buffered saline (DPBS). NIH 3T3 of passages 8 to 11 passaged using DMEM-high (Fetal bovine serum 10%, Penicillin streptomycin 1%) medium was seeded onto the sample at a cell density of 20,000 cells / well, 1 ml medium. And then cultured. Immunostaining is then conducted on the third day. Immunostaining was performed by first removing the medium, soaking in 4% paraformaldehyde (v / v) to fix the cells for 20 minutes, removing paraformaldehyde, followed by 0.2% Triton-X 100 (v / v) and 1% BSA (w / v) was immersed in a buffer solution dissolved in DPBS for 1 hour for permeabilization and blocking. The solution is then removed and diluted with anti-vimentin (1: 100 dilution) and anti-alpha smooth muscle actin (1: 100 dilution) in the previously used buffer solution. The primary antibody was allowed to adhere to the cells for a period of time. The solution was then removed and diluted with DPBS diluted goat anti-rabbit IgG H & L (Alexa Fluor® 488) (1: 200 dilution) and goat anti-mouse IgG H & L (Alexa Fluor® 594) (1: 200 dilution). The phosphor was left at room temperature for 1 hour. Finally, the solution was removed for nuclear staining and soaked for 3 minutes in a solution of DAPI solution (1: 1000 dilution) diluted in DPBS. Samples were washed three times with DPBS between each step. Fluorescence images were taken using a confocal laser scanning microscope (LSM 700, Carl Zeiss, Oberkochen, Germany).

As a result, looking at Figure 8 it was observed that the fibroblasts grow in groups in bare PDMS untreated. In addition, it was observed that cells expressing α-SMA, a marker of myofibroblast, grew around cells expressing vimentin, a marker of fibroblast, and confirmed that the differentiation of fibroblast into myofibroblast proceeds from the inside when fibroblasts flock. Fibroblasts still grew in clusters when they grew on pore surfaces but were affected by the pore size. On the surface with pores of 38 mu m, the pores were covered and grown in clusters. As the pores grew in size, they were observed to grow completely trapped in the pores on surfaces with 100 µm pores. On the surface coated with the multilayer thin film, fibroblasts were distributed evenly on the sample surface. As a result, it was confirmed that the intensity of expression of α-SMA, which appears strongly when the fibroblasts grew in groups, was weakened and differentiation into myofibroblast was suppressed. The synergistic effect was confirmed through the best suppression of differentiation into myofibroblast in the samples coated with pores and multilayer thin films of 70 to 100 μm.

Experimental Example 3 Evaluation of TGF-β Release

As a control, polydimethylsiloxane (bare PDMS) without any treatment on the surface, polydimethylsiloxane having pores of about 38 μm, 70 μm and 100 μm on the surface, respectively, and PLL / HA multilayer thin film on each surface as an experimental group Treated polydimethylsiloxane was prepared. Each sample was cut to a size of 1 mm thick, 1 cm 크기 1 cm, put into a 24-well plate and soaked in ethanol for 1 hour and washed twice with DPBS. NIH 3T3 of passages 8 to 11 passaged using DMEM-high (Fetal bovine serum 10%, Penicillin streptomycin 1%) medium was seeded onto the sample at a cell density of 20,000 cells / well, 1 ml medium. After culturing for 3 days without changing the medium. After 3 days, the supernatant of the culture medium was obtained and diluted 0.5-fold with fresh medium. The TGF-β ELISA assay of the diluted solution as a sample was performed using a ^{TGF-β Quantikine ® ELISA Kit (} R & D systems, Minneapolis, MN, USA). Since the amount of TGF-β was corrected by 2 times.

As a result, as shown in FIG. 9, the release of TGF-β, a representative immunity promoting fibrosis, was statistically significant when compared to bare PDMS without any treatment at 100 μm pore and multilayer thin film coated sample surface.

Example 3 Mouse Model Construction

Experiments were performed using 250 to 300 g of 9-week-old Sprague-Dawley rats with water and food in a specific antigen-free environment. The experiment used an in vivo protocol approved by the Institutional Animal Care and Use Committee of Seoul national University Bundang Hospital (Approved Number: BA 1608-206 / 045-02).

The experimental group was divided into Bare, S, M, L, Bare_LbL, S_LbL, M_LbL and L_LbL, Bare is a PDMS group without any treatment on the surface, S is a PDMS having a pore of 40 ㎛, M is a 70 ㎛ pores PDMS having, L indicates a PDMS group having a pore of 100㎛, LbL represents a PDMS group treated with a PLL / HA multilayer (LbL (Layer-by-Layer) thin film).

Rats had their hair removed in the middle of their back and then cut in 2 cm to create a subcutaneous pocket. Then, the in vivo sample (2cm diameter, both sides) as shown in FIG. 10 was inserted and sutured using Nylon 4/0 (Ethicon, Somerville, USA) and then sterilized with 70% alcohol and betadine. Samples were inserted into 18 rats in each group, and 6 animals were randomly sacrificed every 2 weeks, 4 weeks, and 8 weeks with carbon dioxide. Epidermis and dermis tissues were biopsied with silicone samples inserted for analysis.

Experimental Example 4 Inflammation Response Analysis

Paraffin tissue block obtained through the biopsy in Example 3 was cut to a thickness of 4 ㎛ to remove paraffin with xylene (xylene) and ethanol. 25 slides were stained using Haematoxylin and eosin (H & E) staining, and images obtained by 200-fold magnification were semi-quantitative using ImageJ software (ver. 1.47, National Institutes Health, Rockville, USA). Measured by. Finally, nuclei were stained and slides were mounted using VECTASHIELD Mounting Medium with DAPI (H-1200, Vector Laboratories, Burlingame, USA). The results are shown in FIG.

As a result, as shown in Figure 11, in all groups showed an initial high inflammatory response, but statistically significantly lower inflammatory response was confirmed in M and L groups. The LbL group also showed lower inflammation than the group not coated by the LbL coating. This trend persisted for up to 8 weeks, indicating a decrease in the levels of TGF-β, fibroblasts, and myofibroblasts involved in the fibrotic response by controlling the initial inflammatory response (butterfly effect).

Experimental Example 5 Measurement of Capsule Related Factors (TGF-β, Fibroblasts and Myofibroblasts)

Paraffin tissue block obtained through the biopsy in Example 3 was cut to a thickness of 4 ㎛ to remove paraffin with xylene (xylene) and ethanol. 25 slides were stained using immunofluorescence and 200-fold magnified images were semi-quantitative using ImageJ software (ver. 1.47, National Institutes Health, Rockville, USA). Measured.

The primary antibody used at this time is as follows:

TGF-β: anti-TGF rabbit antibody diluted 1: 300,

Fibroblast: anti-vimentin rabbit antibody diluted 1: 250,

Myofibroblast: anti-α-SMA mouse antibody diluted 1:50.

Secondary antibody was used as anti-rabbit and anti-mouse antibody diluted 1: 2000. Dilution was performed with 1X PBS containing 1% (w / v) BSA and 0.1% (v / v) sodium dodecyl sulfate (SDS). Finally, nuclei were stained and slides were mounted using VECTASHIELD Mounting Medium with DAPI (H-1200, Vector Laboratories, Burlingame, USA). The results are shown in FIG.

As a result, statistically significant decrease in TGF-β expression was observed at week 8 in the M and L groups compared to the Bare and S groups. In particular, the expression of TGF-β was further reduced by LbL.

In addition, the number of fibroblasts was significantly decreased at week 8 in the M and L groups compared to the Bare and S groups. The number of fibroblasts was further reduced by LbL. The effect of microtexture was greater than that of LbL in reducing the number of fibroblasts, but the synergistic effect of LbL significantly decreased the number of fibroblasts at 100 μm (L_LbL group).

In addition, the number of myofibroblasts was significantly decreased at 8 weeks in the M and L groups compared to the Bare and S groups. LbL further decreased the number of myofibroblasts. In the L_LbL group, significantly lower fibroblast counts were observed from 2 weeks to 8 weeks compared to Bare.

It was judged that the immune response was reduced because the micro movement of the silicon sample was reduced in the texture of 70 μm or more (groups M and L). This is because the M and L groups are more tightly coupled to the tissues than the Bare or S groups. Capsule-related factors also decreased with early (2 weeks) suppression of immune responses.

Experimental Example 6 Check Collagen Density and Capsule Thickness

1) collagen density measurement

Paraffin tissue block obtained through the biopsy in Example 3 was cut to a thickness of 4 ㎛ to remove paraffin with xylene (xylene) and ethanol. Twenty-five slides were stained using Masson's trichrome (MT) method and 200-fold magnified images were semi-quantitative using ImageJ software (ver. 1.47, National Institutes Health, Rockville, USA). Measured. The blue stained collagen was measured and expressed as percentile. Finally, nuclei were stained and slides were mounted using VECTASHIELD Mounting Medium with DAPI (H-1200, Vector Laboratories, Burlingame, USA). The results are shown in FIG.

As a result, a statistically significant decrease in collagen density was observed on the LbL treated microtextured surface at week 4. In particular, collagen density due to LbL in the S and L groups was significantly decreased. At week 8, collagen density was significantly decreased in M and L groups, and synergistic with LbL. In particular, L_LbL showed the lowest collagen density.

2) Capsule thickness measurement

Paraffin tissue block obtained through the biopsy in Example 3 was cut to a thickness of 4 ㎛ to remove paraffin with xylene (xylene) and ethanol. 25 slides were stained using Haematoxylin and eosin (H & E) staining, and the image obtained by 50 times magnification was semi-quantitative using ImageJ software (ver. 1.47, National Institutes Health, Rockville, USA). Measured by. Finally, nuclei were stained and slides were mounted using VECTASHIELD Mounting Medium with DAPI (H-1200, Vector Laboratories, Burlingame, USA). The results are shown in FIG.

As a result, the thickness of capsules in Bare and S groups increased steadily from 2 to 8 weeks, with or without LbL, whereas in M and L groups, capsule thickness remained similar to week 2 until week 8. At week 8, the capsule thickness was significantly reduced in the M and L groups compared to the Bare group.

According to the results, it was shown that the effect of LbL is smaller than in vitro experiments, which are relatively short-term reactions. This may be due to the degradation of LbL in an in vivo environment. The micro-texture and LbL reduced the capsule-relateed factor according to the inhibitory effect of the initial immune (two-week) response, and thus collagen at 8 weeks. It was found that the density and capsule thickness decreased.

Claims (17)

The surface of the implant having pores formed on the surface by the particles prepared by the film emulsification method; And
As a prosthesis for inhibiting a fibrous reaction coated with a multilayer thin film comprising a cationic material layer stacked with a cationic material on the surface of the implant and an anionic material layer stacked with an anionic material on the cationic material layer,
The pores formed on the surface of the implant by the particles prepared by the membrane emulsification method has a diameter of 35 to 100㎛, the pores are formed by adhering the particles to the surface of the implant before the implant is completely cured Implants. The method of claim 1,
Cationic materials include poly L-lysine (PLL), collagen, collagen, chitosan, polyethyleneimine (PEI), polyamidoamine (PAA), polyaminoester (PAE) ), Poly [2- (N, N-dimethylamino) ethyl methacrylate] (poly [2- (N, N-dimethylamino) ethylmetacrylate]; PDMAEMA), cationic cellulose, cationic dextrin dextrin) and gelatin (gelatin) one or more selected from the group consisting of fibrosis reaction inhibitory. The method of claim 1,
Anionic materials include hyaluronic acid, alginate, albumin, dextran sulfate, heparin, sodium alginate, poly gamma-glutamic acid ), Poly aspartic acid, poly styrene sulfonate, poly phthalic ethylene glycol ester, poly sulfonated aniline and poly allylamine Implants for inhibiting fibrosis reaction, which is at least one selected from the group consisting of poly allyamine hydrogen chloride. The method according to claim 2 or 3,
The cationic material is poly L-lysine, and the anionic material is hyaluronic acid. The method of claim 1,
The multilayer thin film is a implant for inhibiting a fibrosis reaction is coated on the surface of the implant by a layered self-assembly. delete The method of claim 1,
Prosthesis is the implant for inhibiting the fibrosis reaction is further coated with a drug layer on the multilayer thin film. The method of claim 7, wherein
The drug layer is tranilast, pirfenidone, mitomycin, acetylsalicylic acid, genistein, selenocystine, triamcinolone acetonide Prosthesis for inhibiting fibrosis, comprising at least one drug selected from the group consisting of, tolatic acid spratast (Suplatast tosilate) and montelukast (Montelukast). The method of claim 1,
Prosthesis is a prosthesis for inhibiting the fibrosis reaction is formed of silicone or polyurethane. Forming pores on the surface of the implant from the particles prepared by the film emulsification; And
A method of manufacturing a fibrous reaction inhibiting implant comprising: coating a multilayer thin film including a cation material layer stacked with a cation material and an anion material layer stacked with an anion material on a shape having pores formed on a surface thereof,
The pores formed on the surface of the implant by the particles prepared by the membrane emulsification method is 35 to 100㎛ diameter, the pores are formed by adhering the particles to the surface of the implant before the implant is completely cured Method of manufacturing the implant. delete The method of claim 10,
Cationic materials include poly L-lysine (PLL), collagen, collagen, chitosan, polyethyleneimine (PEI), polyamidoamine (PAA), polyaminoester (PAE) ), Poly [2- (N, N-dimethylamino) ethyl methacrylate] (poly [2- (N, N-dimethylamino) ethylmetacrylate]; PDMAEMA), cationic cellulose, cationic dextrin dextrin) and gelatin (gelatin) of at least one selected from the group consisting of fibrosis reaction inhibiting prosthesis. The method of claim 10,
Anionic materials include hyaluronic acid, alginate, albumin, dextran sulfate, heparin, sodium alginate, poly gamma-glutamic acid ), Poly aspartic acid, poly styrene sulfonate, poly phthalic ethylene glycol ester, poly sulfonated aniline and poly allylamine Method for producing a fibrosis reaction inhibiting prosthesis which is at least one selected from the group consisting of hydrogen chloride (poly allyamine hydrogen chloride). The method according to claim 12 or 13,
The cationic material is poly L-lysine, and the anionic material is hyaluronic acid. The method of claim 10,
The multi-layered thin film is coated on the surface of the implant by the layered self-assembly method for producing a fibrous reaction inhibiting implant. The method of claim 10,
A method of manufacturing a prosthesis further comprising coating a drug layer on the multilayer thin film. The method of claim 16,
The drug layer is tranilast, pirfenidone, mitomycin, acetylsalicylic acid, genistein, selenocystine, triamcinolone acetonide , Tosyl acid Spratast (Suplatast tosilate) and montelukast (Montelukast) A method for producing a fibrosis reaction suppression comprising one or more drugs selected from the group consisting of.

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