MEDICAL IMPLANT SURFACE-MODIFIED WITH FUNCTIONAL POLYPEPTIDE

Abstract

Provided is a medical implant including: an implant base having a surface made of a silicon material; a linker having one end attached onto the surface of the implant base; and a cytokine bound to another end of the linker. By inducing the secretion of anti-inflammatory cytokines, a capsular contracture, which is one of the complications that may occur after transplantation of the patient's breast implant, may occur less.

Claims

1. A medical implant comprising: an implant base having a surface made of a silicon material; a linker having one end attached onto the surface of the implant base; and a functional polypeptide bound to another end of the linker.

2. The medical implant of claim 1, wherein the surface of the implant base includes a shell of a silicon material.

3. The medical implant of claim 1, wherein the functional polypeptide is at least one selected from a cytokine and a chemokine.

4. The medical implant of claim 3, wherein the cytokine is at least one selected from interleukin-4 (IL-4), interleukin-10 (IL-10), and interleukin-13 (IL-13).

5. The medical implant of claim 1, wherein the linker is represented by Formula 1: ##STR00002## wherein, in Formula 1, A is an implant with a silicon surface, B is the end of the functional polypeptide, and n is an integer from 5 to 15.

6. The medical implant of claim 1, wherein the medical implant induces secretion of an anti-inflammatory cytokine.

7. The medical implant of claim 1, wherein the medical implant is a breast implant.

8. The medical implant of claim 7, wherein the breast implant suppresses the formation of breast capsular contracture.

9. The medical implant of claim 6, wherein the breast implant has a roughness value (Rq) of about 4 nm to about 10 nm.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0032] FIG. 1 shows a diagram showing the process of attaching the cytokine to the surface of the implant.

[0033] FIG. 2 shows an image of the medical implant manufactured through the process illustrated in FIG. 1.

[0034] FIG. 3 is a graph of the nitrogen concentration of the silicon surface according to the treatment time when APTMS is bonded to the silicon surface.

[0035] FIG. 4A is a graph showing the roughness value (Rq) measured in untreated silicon and the surface thereof in three dimensions.

[0036] FIG. 4B is a graph showing the roughness value (Rq) measured after treating silicon with O.sub.2 plasma and the surface thereof in three dimensions.

[0037] FIG. 4C is a graph showing Rq measured after treating silicon with O.sub.2 plasma and APTMS and the surface thereof in three dimensions.

[0038] FIG. 4D is a graph showing Rq measured after treating silicon with O.sub.2 plasma, APTMS, and Bis diPEG®.sub.5 and the surface thereof in three dimensions.

[0039] FIG. 4E is a graph showing Rq measured after treating silicon with O.sub.2 plasma, APTMS, Bis diPEG®.sub.5, and IL-4 and the surface thereof in three dimensions.

[0040] FIG. 4F is a graph showing Rq measured after treating silicon with O.sub.2 plasma, APTMS, Bis diPEG®.sub.5, and IL-13 and the surface thereof in three dimensions.

[0041] FIG. 5 is a graph showing the viability (toxicity) of cells measured in a group having a smooth surface and a group having a smooth surface with IL-4 attached thereonto (smooth+IL-4 group).

[0042] FIG. 6A shows an image showing the results of Western blot performed to measure the degree of differentiation of M2 macrophages.

[0043] FIG. 6B shows an image showing the results of Western blot performed to measure the degree of differentiation of M1 macrophages.

[0044] FIG. 6C shows an image showing the results of immunofluorescence performed to measure the degree of differentiation of M2 macrophages.

[0045] FIG. 7 is a graph showing the results of an enzyme-linked immunosorbent assay (ELISA) performed to measure proinflammatory cytokines.

[0046] FIG. 8 is a graph showing the results of ELISA performed to measure anti-inflammatory cytokines.

[0047] FIG. 9 is an image showing the result of Western blot performed to determine whether the IL-4-modified implant alone can activate the STAT6 pathway.

[0048] FIGS. 10A and 10B are graphs and microscopic images showing the changed capsular thickness and collagen density after transplantation of the implant into a mouse as an animal experimental model for a medical implant according to an aspect.

[0049] FIG. 11A shows comparison results of the production amount of TNF-α through ELISA in a group having a smooth surface and a group having a smooth surface with IL-10 attached thereonto (smooth+IL-10 group).

[0050] FIG. 11B shows comparison results of the production amount of IL-6 through ELISA in the smooth group and the smooth+IL-10 group.

[0051] FIG. 11C shows comparison results of the production amount of IL-1β through ELISA in the smooth group and the smooth+IL-10 group.

[0052] FIG. 11D shows comparison results of the production amount of IL-10 through ELISA in the smooth group and the smooth+IL-10 group.

[0053] FIG. 11E shows comparison results of the production amount of IL-4 through ELISA in the smooth group and the smooth+IL-10 group.

[0054] FIG. 12 is a graph showing the viability of cells measured in the smooth group and smooth+IL-10 group.

[0055] FIG. 13 is an image showing the results of immunofluorescence measurement performed to measure the degree of differentiation of M2 macrophages in the smooth group and the smooth+IL-10 group.

[0056] FIG. 14 shows comparison results of Arg-1, IL-10, IL-1β, and TNF-α in a group having a smooth surface and a group having a smooth surface with IL-13 attached thereonto (smooth+IL-13 group).

[0057] FIG. 15 is a graph showing the viability of cells measured in the smooth group and the smooth+IL-13 group.

MODE OF DISCLOSURE

[0058] Hereinafter, the present disclosure will be described in more detail through examples. However, these examples are for illustrative purposes of the present disclosure, and the scope of the present disclosure is not limited to these examples.

REFERENCE EXAMPLE

Reference Example 1. Cell Culture

[0059] RAW 264.7 cells were purchased from the American Type Culture Collection (Rockville, Md., USA) and incubated in a medium-supplemented with 10% heat-inactivated fetal bovine serum (FBS), 100 U/ml of penicillin, and 100 μg/ml of streptomycin (Gibco, Carlsbad, Calif.) under a humidified condition containing 5% CO.sub.2 at a temperature of 37° C.

Reference Example 2. Western Blot Analysis

[0060] After 24 hours, RAW 264.7 cells were dissociated with cold PBS and homogenized on ice using cell lysis buffer (Cell Signaling Technology, Danvers, Mass., USA). After heating the sample at a temperature of 95° C. for 5 minutes and cooling briefly on ice, 30 μg of protein was loaded onto a 10% SDS-PAGE polyacrylamide gel. After gel electrophoresis, the gel was transferred to a nitrocellulose membrane (GE Healthcare, Piscataway, N.J., USA). The membrane was blocked with 5% BSA in PBS for 2 hours at room temperature, and primary antibodies against iNOS (Abcam, Cambridge, UK), Arg-I (Santa Cruz, Calif., USA) and control GAPDH were incubated overnight at a temperature of 4° C. After washing 4 times with PBS-T (pH 7.4), the cell membrane was diluted 1:2,000 with HRP-conjugated an anti-mouse or anti-rabbit IgG secondary antibody (Santa Cruz Biotechnology, Santa Cruz, Calif., USA) and stored at room temperature for 2 hours. Next, the membrane was washed 4 times with PBS-T. A Western blot detection kit (EZ-Western Lumi pico, Dogen, Korea) was used for protein detection. Finally, protein was quantified in the blot, and analysis for densitometry of the blot was performed in Image J (Image J, National Institutes of Health, USA). Relative quantitation was calculated after being converted to GAPDH levels. The above analysis method was repeated twice.

Reference Example 3. Immunofluorescence Assay

[0061] Cells were washed 3 times with PBS (pH 7.4) for 5 min each. Then, the slides were treated with a blocking solution (0.2% Triton X-100, 1% BSA in PBS) for 1 hour to block non-specific antigen bindings. The slides were then incubated overnight with diluted primary antibody. The next day, after washing 3 times with PBS, the plate was incubated at room temperature for 1 hour with a secondary antibody diluted 1:2000. Then, the slides were thoroughly washed with PBS and then staining was performed thereon using DAPI (DAPI, VECTASHIELD, Vector Laboratories, USA) to stain cell nuclei. Images were then taken using a z-stack with a confocal microscope.

Reference Example 4. Reverse Transcription Polymerase Chain Reaction (RT-PCR) and Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR)

[0062] RNA of RAW 264.7 cells was extracted according to the instructions of the RNA extraction kit (easy-BLUE RNA extraction kit, iNtRON Biotechnology, Gyeonggi-do, Korea). RNA was quantified with a spectrometer (Nanodrop 1000, Wilmington, Del.). From 2 μg of RNA, 20 μl of cDNA was synthesized using reverse transcriptase (AccuPower® RT PreMix, Bioneeer Corporation, Daejeon, Korea) according to the manufacturer's instructions. The reaction was performed using an ABI 7500 Real-Time PCR System (Applied Biosystems). The expression level of the gene was normalized using GAPDH mRNA. Expression levels presented were the mean values of each sample. In the case of RT-PCR, the annealing temperature for IL-6 and GAPDH was 62° C. The resultant product was electrophoresed on a 2% agarose gel and stained with ethidium bromide.

Reference Example 5. Statistical Analysis Method

[0063] Each data was presented as mean±standard error (SEM). One-way ANOVA was used for multi-group comparisons after Tukey's test. Power analysis was applied to determine the difference between the control group and the treatment group. P<0.05 was considered as being significant.

EXAMPLE

Example 1. Preparation of IL-4 Surface-Modified Silicon

[0064] This embodiment was performed to manufacture a medical implant according to an aspect. FIGS. 1 and 2 schematically show the surface modification of the silicon and the production process of the cytokine, which is one of the functional polypeptides, attached thereonto. Specifically, the surface of the silicon was surface-treated with oxygen plasma (O.sub.2 plasma) at 100 W for 5 minutes, and then, 3-aminopropyltrimethoxysilane (APTMS) was attached thereonto. APTMS attachment was performed for 1 or 2 hours. The concentration of nitrogen according to the coating time of APTMS is shown in FIG. 3.

[0065] As shown in FIG. 3, the concentration of nitrogen was increased as the coating time of APTMS was increased, and this result indicates that the modification of the silicon surface through APTMS proceeded normally.

[0066] After coating APTMS, bis-dPEG®5 NHS ester was added to modify the surface. As shown in FIG. 2, IL-4, one of the functional polypeptides, was finally introduced onto the modified surface in a weakly basic state of pH 8.2.

Example 2. Contact Angle (WCA) Analysis

[0067] In this example, the contact angle was measured to measure the degree of modification of the silicon surface prepared by the method of Example 1. Specifically, as the contact angle, a water contact angle (WCA) was measured using a program (First Ten Angstroms FTA 1000 C Class) in which Sessile drop technology was combined with drop shape analysis software. To measure static advancing contact angles, 2.0 μL of water droplet was added to the droplet every 2 seconds to grow the droplet, and then the droplet was added and within 5 seconds, images thereof were captured. This procedure was repeated 20 times. For the concrete reliability of WCA, the contact angle of a non-ideal surface such as APTMS SAM was calculated using the tangent-leaning method. The WCA of each of: a sample including Si/O.sub.2 plasma/APTMS/bis-dPEG®.sub.5 NHS ester/IL-4, which was a surface-modified silicon prepared by the method of Preparation Example 1; a control sample including Si, Si/O.sub.2 plasma, Si/02 plasma/APTMS, Si/O.sub.2 plasma/APTMS/bis-dPEG®5 NHS ester, or Si/02 plasma/APTMS/bis-dPEG®.sub.5 NHS ester/(IL-4 or IL-13), was measured. The WCA value is the average of at least three measurements. The measurement results are shown in Table 1.

TABLE-US-00001 TABLE 1 Sample WCA (°) (a) Bare silicone prosthetic material (Si) 93.90 (b) Si/O.sub.2 plasma 0.16 (c) Si/O.sub.2 plasma/APTMS 97.80 (d) Si/O.sub.2 plasma/APTMS/Bis diPEG.sub.@5 NHS ester 100.60 (e) (d)/IL4 (Cytokine immobilization) 78.1 (f) (d)/IL13 (Cytokine immobilization) 76.6

[0068] As shown in Table 1, the untreated silicon surface had strong hydrophobicity, and thus, a large WCA value was measured therefor. When the silicon surface was formed with oxygen plasma, due to the enhanced hydrophilicity caused by the presence of OH— functional groups, the WCA value was decreased rapidly to 0.16°. In addition, as APTMS was introduced to the surface, the WCA value was 97.80°, that is, the hydrophobicity became stronger. After introduction of bis-diPEG®5 NHS ester, the WCA value was 100.60°, that is, the hydrophobicity was further enhanced. Additionally, the WCA values corresponding to the case in which functional polypeptides, IL-4 or IL-13 were introduced, were 78.1° C. and 76.6° C., respectively. That is, hydrophobicity was slightly attenuated, which is considered to be due to the hydrophilicity of cytokines. These WCA values and the changes thereof indicate that the silicon surface was modified step by step.

Example 3. AFM Analysis

[0069] In this example, atomic force microscopy (AFM) analysis was performed in order to obtain an image of the surface layer of each sample used in Example 2. XE-100 AFM (Park Systems) was used for biofilm imaging, the resonant frequency was 200 kHz to 400 kHz, and the nominal force constant was set to be 42 N/m. Surface imaging was obtained in non-contact mode by using a silicone tip of a 125 μm-long nitride lever coated cantilever (PPP-NCHR 10M; Park Systems). The scan frequency was typically 1 Hz per line. Roughness was calculated with 3 μm×3 μm images. The results are shown in FIGS. 4A to 4F.

[0070] Regarding the results of FIGS. 4A to 4F, the surface mean square roughness (Rq) value of Si/O.sub.2 Plasma/APTMS was slightly increased from 2.06 nm, which is the Rq value of the control group, to 3.72 nm, which is the Rq value of the APTMS treatment group, and after treatment with bis-dPEG®.sub.5 NHS ester, the Rq value was increased to 10.4 nm. Additionally, as shown in FIGS. 4E and 4F, when IL-4 or IL-13 was added, the roughness values were decreased again to 6.14 nm and 6.02 nm, respectively. These results indicate that each of the additive materials successfully modified the silicon surface.

EXPERIMENTAL EXAMPLE

Experimental Example 1. Cell Viability Assay (MTT Assay)

[0071] This experimental example was performed to measure the cytotoxicity of a medical implant according to an aspect. In order to measure cell viability as an indicator of cytotoxicity, RAW 264.7 cells were prepared by the method described in Reference Example 1. Cells were divided into two groups, which were then brought into contact with a smooth silicon surface which was not unmodified (smooth), or a silicon surface which was modified with IL-4 prepared according to Example 1 (smooth+IL-4). After detaching cells from each group at time points of 24, 48, and 72 hours, the cells were washed once with PBS. In DMEM medium, 0.5 mg/mL of MTT was added to each well, then the cells were incubated at a temperature of 37° C. for 4 hours, and then the MTT solution was removed. Finally, formazan crystals were dissolved in DMSO and the absorbance thereof at 560 nm was read in a microplate reader (EPOCH2, BioTek). The results are shown in FIG. 5.

[0072] According to the results shown in FIG. 5, the cell viability tended to increase from 24 hours to 48 hours, and at the time point of 72 hours, the cell viability was slightly decreased. This was the same in both groups. As shown in FIG. 4, the cells of the smooth+IL-4 group showed significantly better viability than the cells of the smooth group at all time points of 24, 48 and 72 hours. These results indicate that the cytotoxicity was significantly reduced by IL-4 introduced to the silicon surface.

Experimental Example 2. Measurement of M1 and M2 Macrophage Differentiation

[0073] This experimental example was performed to identify whether IL-4 introduced to the silicon surface affects the healing of M2 or M1 wounds and tissue recovery of macrophages and affects the inflammatory immune response. After preparing RAW 264.7 cells by the method described in Reference Example 1, the cells were divided into two groups, which were respectively brought into contact with a non-modified smooth silicon surface, or a silicon surface modified with IL-4 prepared according to Example 1 (smooth+IL-4). Then, the immunofluorescence staining method of Reference Example 3 and Western blotting of Reference Example 2 were performed to confirm the expression levels of genes and proteins. The results are shown in FIGS. 6A to 6C.

[0074] As shown in FIGS. 6A and 6B, it was confirmed that Arg-1 and IL-10, which are markers of M2 macrophages, were more highly expressed in the smooth+IL-4 group. In addition, Western blot results showed that the M1 marker iNOS was expressed at a high level in the smooth group, and the M2 marker Arg-1 was significantly highly expressed in the smooth+IL-4 group. In addition, referring to FIG. 6C, the results of immunofluorescence staining showed that, in the smooth group, CD206 was hardly observed and, in the smooth+IL-4 group, CD206 was remarkably highly expressed. These results indicate that RAW 264.7 cells are more actively differentiated into M2 macrophages on the surface of IL-4 introduced silicon than in the smooth group.

Experimental Example 3. Cytokine Production Measurement

[0075] In this experimental example, the production of proinflammatory cytokines IL-6 and TNF-α was measured. Enzyme-linked immunosorbent assay (ELISA) was performed. Captured antibodies were diluted with PBS and coated on a 96-well plate at room temperature for 24 hours. Then, the plate was washed twice with PBS, and blocked with PBS with 10% FBS for 2 hours. After adding the sample extracted from the cell culture supernatant of each of the smooth group and the smooth+IL-4 group thereto, the reaction was performed at room temperature for 2 hours. After treatment with secondary antibodies, substrate reagents were reacted and reading was carried out at a 405 nm wavelength in an ELISA reader (EPOCH2, BioTek). The results are shown in FIG. 7.

[0076] As shown in FIG. 7, IL-6 tended to decrease over time, and from the first 24.sup.th date, in the case of the smooth+IL-4 group, the detected concentration thereof was statistically significantly low. Regarding TNF-α, both groups showed the decreasing tendency over time, but at all time points, in the smooth+IL-4 group, the concentration thereof was significant low. These results indicate that IL-4-modified silicon can reduce the expression of pro-inflammatory cytokines, which could cause the generation of inflammation.

Experimental Example 4. Measurement of Th2 Cell Cytokine Production

[0077] This experimental example was performed to measure the production of IL-4 and IL-10, which are anti-inflammatory cytokines. The measurement method was performed by the methods of RT-PCR and qRT-PCR described in Reference Example 4. The results are shown in the graph of FIG. 8.

[0078] As shown in FIG. 8, IL-4 was secreted significantly high in the Smooth+IL-4 group. In the case of IL-10, the two groups showed no difference after 24 and 48 hours, but after 72 hours, significantly high secretion occurred in the Smooth+IL-4 group. These results indicate that the Smooth+IL-4 group induced the expression of anti-inflammatory cytokines, and thus, the anti-inflammatory response and wound healing effects thereof were greater than those of the smooth group.

Experimental Example 5. Measurement of STAT6 Pathway Activity

[0079] Activation of the STAT6 pathway is an important factor in differentiating macrophages to the M2 type. Accordingly, in this Experimental Example, Western blotting according to Reference Example 2. was performed on STAT6 and pSTAT6 to determine whether IL-4-modified silicon alone could activate the STAT6 pathway. The results are shown in FIG. 9.

[0080] As shown in FIG. 9, there was no significant difference in STAT6 between the Smooth+IL-4 group and the Smooth group. On the other hand, pSTAT6, which is the active form, was detected more in the Smooth+IL-4 group. These results indicate that in the smooth+IL-4 group, M2-type macrophages were generated more.

Experimental Example 6. In Vivo Experiments

[0081] In this experimental example, an animal experiment was performed to measure the in vivo effect of the medical implant. For animal experiments, 10 Sprague-Dawley mice weighing 250 g to 300 g at 9 weeks of age were used. Five animals in each group were randomly distributed into each of two groups. Animals were exposed in a 12/12 h light/dark cycle in specific-pathogen-free (SPF) conditions with free access to food and water. Approval for this protocol was approved by the Bundang Seoul National University Hospital Animal Experiment and Use Committee (approval number: BA1801-240/011-01), and all procedures were in accordance with the guidelines of the NIH. There were an animal group in which an intact silicon was inserted as an implant and an animal group in which silicon modified with IL-4 was inserted as an implant. The former group was used as a control group.

[0082] The process of inserting the implant is specifically as follows. The subject mice were anesthetized by inhalation of isoflurane (Hana Pharm, Korea), the hair on the back was shaved, and the surgical site was disinfected with 70% alcohol and betadine. Then, a 2-3 cm incision was made in the back with a #15 scalpel blade, and the implant was inserted into the cortical pouch. The incision site was closed with surgical sutures (Nylon 4/0, Ethicon, USA). The surgical site was disinfected again with 70% alcohol and betadine and a light dressing was applied thereon.

[0083] Animals were monitored for 12 weeks after transplantation, confirming the development of cascade inflammation. Therefore, at predetermined time points of 1, 2, 4, 8 and 12 weeks, all animals in each group were tissue biopsied. For biopsies, selected animals were euthanized with carbon dioxide, and tissues and implants in the dorsal region with epidermis, dermis, posterior and anterior capsules were removed.

[0084] 6.1. Evaluation of Capsular Thickness and Collagen Density In Vivo

[0085] The thickness of the capsule tends to be increased over time due to the accumulation of collagen. Accordingly, the in vivo capsular thickness and collagen density were investigated. Capsular thickness was determined by analyzing tissue slides which were H&E-stained using a microscope (LSM 700, Carl Zeiss, Oberkochen, Germany) at 40× magnification. The capsular range was defined from the top of the silicone insertion area to the bottom of the dorsal subcutaneous muscle. To evaluate the overall capsular thickness from the tissue slides, three different parts of the capsule were randomly photographed, and the capsular thickness was measured with ZEN software. The results thereof are shown in FIG. 10A.

[0086] Collagen density was analyzed using image analysis of 5 randomly selected regions on slides stained with MT staining at 40× magnification. Collagen bundles were stained as being blue to analyze the density of collagen over the entire microscopic area with Image J software. The results thereof are shown in FIG. 10B.

[0087] Regarding the results of FIG. 10A, the capsular thickness of the control group was 688.5 μm±177.9 μm, whereas the capsular thickness of the mice implanted with IL-4 modified silicon was measured to be 317.8 μm±31.5 μm. It was confirmed that the group implanted with IL-4 modified silicon had a statistically significant inhibition of capsular formation on the 7.sup.th day.

[0088] FIG. 10B shows that, regarding the density of capsule-constructing collagen, the collagen density of the group implanted with IL-4 modified silicon used as an implant was 56.5±10.8%, which was significantly reduced than the collagen density of the control group, which was 78.4±2.3%.

Experimental Example 7. Evaluation of Biological Activity for IL-10 Surface-Modified Silicon Implants

[0089] The present experimental example was based on the IL-10 surface-modified silicon implant prepared in the same manner as in Example 1, and the levels of pro-inflammatory cytokines TNF-α, IL-6, and IL-13 produced in RAW 264.7 cells and the levels of IL-10 and IL-4 were measured. In addition, cytotoxicity evaluation with respect to IL-10 introduced into the silicon surface was performed, and the differentiation level of M2 or M1 macrophages was evaluated, and the effect on wound healing and tissue recovery of macrophages was confirmed. This experiment was performed in the same manner as in Experimental Examples 1 to 3, and the control group was a group (smooth) in which the subject was in contact with a smooth silicon surface, which was not modified.

[0090] As a result, as shown in FIGS. 11A to 11E, the concentrations of TNF-α, IL-6, and IL-13 were significantly lower in the smooth+IL-10 group, whereas the concentrations of IL-10 and IL-4 were significantly higher in the smooth+IL-10 group. In addition, as shown in FIGS. 12 and 13, cytotoxicity did not occur in the IL-10-modified silicone, but rather showed higher viability compared to the smooth group, CD206, which is a marker indicating differentiation into M2 macrophages, and was not present in the smooth group, whereas was significantly highly expressed in the smooth+IL-10 group. These results indicate that IL-10 introduced into the silicon surface may reduce the side effects of in vivo transplantation by inhibiting the production of proinflammatory cytokines around the implanted site and promoting differentiation into M2 macrophages.

Experimental Example 8. Biological Activity Evaluation of IL-13 Surface Modified Silicon Implants

[0091] In the present experimental example, regarding the IL-13 surface-modified silicon implant prepared in the same manner as in Example 1, the level of proinflammatory cytokines TNF-α, and IL-1β produced in RAW 264.7 cells and the level of IL-10 were measured, and the level of Arg-1, a marker of M2 macrophages, was measured. In addition, cytotoxicity evaluation with respect to IL-13 introduced to the silicon surface was performed. This experiment was performed in the same manner as in Experimental Examples 1 to 3, and the control group was a group (smooth) in which the subject was in contact with a smooth silicon surface, which was not modified.

[0092] As a result, as shown in FIG. 14, the level of TNF-α in the smooth+IL-13 group (IL-13) was decreased, while the levels of Arg-1 and IL-10 were increased. In addition, as shown in FIG. 15, the smooth+IL-13 group showed better viability than the cells of the smooth group at the time points of 24 and 48 hours. These experimental results show that IL-13 can also contribute to reducing the side effects caused by implantation in vivo, although the degree of the reducing was smaller than that of IL-4.

[0093] The description of the present disclosure described above is for illustration, and those of ordinary skill in the art to which the present disclosure pertains will understand that it can be easily modified into other specific forms without changing the technical spirit or essential features of the present disclosure. Therefore, it should be understood that the embodiments described above are illustrative in all respects and not restrictive.