Patent Application
20200268937
The disclosure relates to a composition for surface modification of a medical implant and a medical implant surface-modified thereby.
Medical implants such as implants, dental implants, stents, screws and bone replacements are inserted for the purpose of treatment or plastic surgery. However, being inserted into the body, the medical implants are recognized as foreign substances in the body, and a clotting mechanism is activated, or foreign body reaction is generated to prevent bleeding and blood loss. In addition, since medical implants make permanent contact with biological tissues, the surface biocompatibility and in vivo affinity thereof are required.
Accordingly, technique for modifying the surface of medical implants and effective methods for fixing bioactive materials are required,
Meanwhile, recently, it has been reported that itaconic acid inhibits the decomposition enzyme of isocitric acid, which is an important enzyme of the energy metabolic pathway of microorganisms, thereby contributing to the antibacterial activity of macrophages.
Accordingly, the inventors of the disclosure confirmed the fibrosis inhibition and inflammatory response inhibition at the implantation site of medical implants by using itaconic acid and completed the inventive concept.
An aspect is to provide a composition including itaconic acid for surface modification of a medical implant.
Another aspect is to provide a medical implant surface-modified with the itaconic acid.
Another aspect is to provide a method of manufacturing the medical implant.
An aspect provides a composition including itaconic acid for surface modification of a medical implant.
In the disclosure, the itaconic acid may be represented by Formula 1 below.
The composition may further include gelatin.
In the disclosure, the term “gelatin” may mean a protein obtained by partial hydrolysis of collagen which is the main protein component of connective tissues such as bones, cartilages, skins, etc., of animals. The gelatin may include gelatin derivatives in addition to pure gelatin. For example, the gelatin derivative may include at least one of a phthalated gelatin, an esterified gelatin, an amidated gelatin, or a formylated gelatin. Relating to gelatin, the kind (source) thereof is not specifically limited, and various kinds of gelatin derived from, for example, mammals and fishes, for example, cow bones, cow skins, swine bones, swine skins, etc., may be used. In addition, the gelatin may have a molecular weight from about 10,000 to about 30,000.
The gelatin may be crosslinked. In addition, the gelatin may be chemically binded with itaconic acid.
In an embodiment, the composition may be provided in a powder type.
Another aspect provides a medical implant surface-modified with itaconic acid.
Particularly, a surface-modified medical implant of which at least a portion of the surface of the implant is modified through the binding with itaconic acid may be provided as a medical implant.
In the disclosure, the term “surface modification” may mean the change of the chemical structure and physical structure of the surface of particles.
The medical implant may be additionally binded with gelatin. The gelatin may be crosslinked and binded with the surface of the implant through itaconic acid.
The itaconic acid and gelatin may have a nanoparticle type or a polymer type.
At least a portion of the surface of the medical implant may introduce an amino group. Particularly, the surface of the medical implant may be silanized for the binding with itaconic acid. An aminosilane compound for silanization may be 3-aminopropyltriethoxysilane (APTES), (3-aminopropyl)-tetraethylorthosilicate, [3-(2-aminoethylarnino)propyl]trimethoxysilane, 3-(2-(2-aminoethylamino)ethylamino)propyltrimethoxysilane, or combinations thereof.
In addition, the itaconic acid may be directly binded with the surface of the implant through the fixing compound at the surface of the implant. The fixing compound may include a compound having biotin, avidin, streptavidin, carbohydrate, poly L-lycine, a thiol group, an amine group, an alcohol group, a carboxyl group, an amino group, a sulfur group, an aldehyde group, a carbonyl group, a succinimide group, a maleimide group, an epoxy group, or an isothiocyanate group, or combinations thereof. Examples of the compound having an amino group may include 3-aminopropyltrimethoxysilane, N-(2-aminoethyl)-3-aminopropyltrimethoxysilane (EDA), trimethoxysilylpropyldiethylenetriarnine (DETA), 3-(2-aminoethylaminopropyl) trimethoxysilane, and 3-aminopropyltriethoxysilane, and the compound having an aldehyde group may include glutaraldehyde. Examples of the compound having a thiol group may include 4-mercaptopropyltrimethoxysilane (MPTS). In addition, examples of the compound having an epoxy group may include 3-glycidoxypropyltrimethoxysilane, examples of the compound having an isothiocyanate group may include 4-phenylenediisothiocyanate (PDITC), and examples of the compound having succinimide and maleimide may include disuccinimidyl carbonate (DSC) or succinimidyl 4-(maleimidephenyl)butyrate (SMPB).
The medical implant may be a biocompatible material. Examples of the biocompatible material may include one or more materials selected from the group consisting of gold (Au), silver (Ag), platinum (Pt), palladium (Pd), copper (Cu), steel, tantalum (Ta), magnesium (Mg), nickel (Ni), chromium (Cr), iron (Fe), a nitinol alloy (NiTi), a cobalt-chromium alloy (CoCr) gallium arsenic (GaAs), titanium (Ti), polylactic acid, poly(glycolic acid) (PGA), poly(lactic-co-glycolic acid) (PLGA), poly-ε-(caprolactone), polyanhydride, polyorthoester, polyvinyl alcohol, polyethylene glycol, polyurethane, polyacrylic acid, poly-N-isopropylacrylamide, poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide), polytetrafluoroethylene, polycarbonate, polyurethane, nitrocellulose, polystyrene, polyethylene, polyethylene terephthalate (PET), polydimethylsiloxane (PDMS), polyether ether ketone (PEEK), silicon oxide (SiO2), titanium oxide (TiO2), aluminum oxide (Al2O3), niobium oxide (Nb2O5), silicon, silicone rubber, and glass.
In addition, the medical implant may be selected from the group consisting of breast implants, spinal implants, dental implants, cardiovascular implants, cardiac implants, stents, artificial joints, artificial head bones, and cell culture mediums.
In a particular embodiment, the surface modified medical implant may have a water contact angle from about 20° to about 90°, from about 25° to about 85°, from about 25° to about 80°, from about 25° to about 70°, from about 30° to about 60°, or from about 30° to about 40°.
In another particular embodiment, the surface modified medical implant may have improved fibrosis inhibition or inflammatory response inhibition at an implantation site when compared with that before surface modification.
Another aspect provides a method of surface modifying a medical implant, including a step of functionalizing at least a portion of a surface of a medical implant; and a step of binding itaconic acid with the surface of the functionalized medical implant.
The functionalization step may be a process for binding itaconic acid and/or gelatin with the surface of the implant. The functionalization may include a step of introducing an amino group, or a step of functionalizing using a fixing compound as described above.
In addition, the step of binding itaconic acid may be a step of binding gelatin additionally. This step may include a step of preparing a composite of itaconic acid and gelatin and then, binding, binding itaconic acid and then, binding gelatin, or binding gelatin and then, combining itaconic acid.
According to the composition for surface modification of a medical implant according to an aspect and a medical implant surface-modified thereby, itaconic acid may be binded with the surface of the medical implant in high binding stability, and effects showing activity on fibrosis inhibition and inflammatory response inhibition at an implantation site may be achieved.
Hereinafter, the present disclosure will be explained in more detail referring to embodiments. However, the embodiments are illustrated for explaining the present disclosure, and the scope of the present disclosure is not limited thereto.
A medical implant surface-modified with itaconic acid was manufactured as shown in
Particularly, a polydimethylsiloxane (PDMS) substrate (Sylgard® 184 silicone elastomer kit, Dow Corning, USA) was treated with oxygen plasma (CUTE-1B, Femto Science, USA) for 1 minute so as to attach hydroxyl groups (—OH) on the surface of PDMS. After that, the surface of the substrate was silanized using 3-aminopropyl triethoxysilane (APTES) at 60° C. for 2 hours. Then, 50 mmol, or 150 mmol of itaconic acid (IA, analytical grade, assay ≥99%, MW: 130.10 g/mol, density: 1.573 g/mL at 25° C. (lit.), Sigma-Aldrich, South Korea) was reacted at 60° C. for 2 hours in the presence of 1-ethyl-3-(3-dimethylamino)propyl carbodiimide (EDC) and N-hydroxy succinimide (NHS) (EDC/NHS) to bind the itaconic acid with the surface of the PDMS substrate. Then, EDC/NHS residues were removed, and drying was performed for 12 hours to manufacture a medical implant surface-modified with itaconic acid (hereinafter, will be referred to as “IA-PDMS”).
A medical implant surface-modified with itaconic acid and gelatin (IA-GT) was manufactured as shown in
Particularly, in order to prepare a polymer of itaconic acid and gelatin, a gelatin type B (derived from cow skin, Bloon 75) powder was mixed with DPBS and dissolved at 135° C. to 140° C. for 16 hours. Then, with the dissolved gelatin type B, 50 mmol of EDC and 50 mmol of NHS solutions were mixed, and 100 mmol of itaconic acid was put and reacted for 2 hours. Additionally, DPBS was added to the mixture, and the reaction with gelatin and itaconic acid was performed at 60° C. for 30 minutes and then finished. The final mixture was dialyzed through a cellulose membrane (MWCO 6-8 kD), and unreacted itaconic acid, EDC/NHS, and salts were removed. After that, freeze drying was performed at −80° C. for 48 hours to obtain an IA-GT powder.
The IA-GT powder thus obtained was binded with the surface of a PDMS substrate as in Example 1 to manufacture a medical implant surface-modified with itaconic acid and gelatin (hereinafter, will be referred to as “IA-GTpoly-PDMS”). Briefly, a surface modified PDMS was manufactured by the same method as in Example 1 except for using DW+EDC 10 mmol+NHS 10 mmol to manufacture the surface modified PDMS of IA-GTpoly-0.25 wt %, and using DW+EDC 20 mmol+NHS 20 mmol to manufacture the surface modified PDMS of IA-GTpoly-0.50 wt %.
1.1. Hydrophilicity Analysis
On the surface of the implants manufactured in Examples 1 and 2, 4 μl of distilled water was dropped, and contact angles were measured at room temperature using a laboratory-made contact angle goniometer with a charge-coupled device camera (IMT 3, IMT Solutions), and the results are shown in
As shown in shown
1.2. Surface Morphology Analysis
The surfaces of the implants manufactured in Examples 1 and 2 were observed by a scanning electron microscope (SEM) (SEM, S-3400N, Hitachi, Tokyo, Japan), and the results are shown in
As shown in
1.3. Surface Chemical Bond Analysis
In order to confirm the chemical bond at the surface of the implant, attenuated total reflection (ATR)-FTIR analysis was performed,
Particularly, with respect to the implants manufactured in Examples 1 and 2, each spectrum at room temperature was recorded from 400 cm−1 to 4000 cm−1 with a resolution of 4 cm−1 using a FT-IR Spectroscopy (Frontier spectrophotometer equipped with and attenuated total reflection (ATR-FTIR) module (Nicolet 6700, Thermo Scientific, USA), the scanning was performed 200 times and averaged, and the results are shown in
As shown in
1.4. Protein Absorbing Capacity Analysis
The protein absorbing capacity of the surfaces of the implants manufactured in Examples 1 and 2 was analyzed.
Particularly, bovine serum albumin (BSA) (4.5 mg/mL) was dissolved in DPBS. Separately, the surfaces of IA-PDMS and IA-GTpoly-PDMS (size: 1 cm2) were cultured on a hot stirring plate in a protein solution while stirring constantly for 1 hour at 37° C. in 200 rpm conditions. After washing with clean DPBS twice, BSA protein-cultured bare, IA-conjugated and IA-GTpoly conjugated PDMS surfaces (triple) were transported to a 24-well plate containing DW and an operation reagent mixture. After culturing the well plate at 37° C. for 30 minutes, the absorbance value of each sample was analyzed. The amount of absorbed protein was quantified using Micro BCA™ protein analysis kit according to the instruction of a manufacturer. The absorbance value was measured using a microplate spectrophotometer with a fixed wavelength of 570 nm. All values were represented by average±standard deviation, and the results are shown in
As shown in
1.5. Binding Stability Analysis
In order to analyze the binding stability of itaconic acid and/or gelatin, heat treatment was performed with respect to the surface of the implants manufactured in Examples 1 and 2.
Particularly, IA-GTpoly-PDMS (IA-GTpoly-0.50 wt %) having a size of 2 cm2 was used as a specimen for heat treatment, and the heat treatment was performed three times in total at 120° C. for 1 hour. After that, contact angles were measured by the same method as in Experimental Example 1.1, and the results are shown in
As shown in
2.1. In Vitro Analysis of Cell Culture Ability
2.1.1. Analysis of Cell Proliferation Rate and Viability
In order to analyze the cell proliferation rate and viability in the implants manufactured in Examples 1 and 2, NIH3T3 mouse fibroblast lines were used.
Particularly, the fibroblast lines were cultured at the surface of PDMS having a size of 1 cm×1 cm with cells of 20,000 cell/ml at 37° C. After initiating the culture, the cell proliferation rates and viability at 12 hour, 24 hour and 48 hour were analyzed. The cell proliferation rate was observed by an optical microscope (Euromex IF-Series, Netherlands), by treating cells with trypsin (GE Healthcare Life Sciences HyClone Laboratories, USA.) and counting the number of cells, or by counting the number of cells through an MTT assay (Cell Biolabs, INC, USA). The observation results by the optical microscope are shown in
In addition, the cell viability was observed through a Live/Dead analysis method (L-3224, Invitrogen, LIVE/DEAD® Viability/Cytotoxicity Kit, for mammalian cells (Molecular Probes®)). Particularly, in order to prepare a Live/Dead analysis solution, 10 ml of PBS was added to a 15 ml tube, and 2 mM EthD-1 and 4 mM calcein AM were added in 20 μl and 5 μl, respectively. Then, the tube was wrapped with an aluminum foil, well mixed through pipetting, and stored under refrigeration. In addition, the cultured cells were put in PBS and washed for 20 minutes, PBS was removed, and the Live/Dead analysis solution thus prepared was added to a cell specimen. After that, culturing was performed in an incubator for 30 minutes, photographs were taken using a fluorescence and confocal microscope, and the results are shown in
As shown in
In addition, as shown in
In addition, as shown in
2.1.2. Analysis of Cell Attachment Pattern
In order to analyze cell attachment pattern, the cells were stained with a Rhodamin/DAPI staining (Invitrogen, ThermoFisher Scientific, USA). This was observed with a fluorescence microscope (CKX-41, OLYMPUS, Japan), and the results are shown in
As shown in
2.2. In Vivo Activity Analysis
2.2.1. Analysis of Fibrosis at Implantation Site
The implants manufactured in Examples 1 and 2 were transplanted to animal models, and the fibrosis states at implantation sites were observed.
First, four 8-week Sprague-Daley rats (Young Bio, KOREA) were used for each group as the animal models. Animal experiment was conducted after final approval from Institutional Animal Care and Use Committee (IACAU) by Seoul National University Hospital in Bundang (approval number: BA1608-206/045-01). Experiment was conducted by dividing into five groups as follows. In order to prevent inflammation and infection at a wounded area during transplanting an implant, disinfection was conducted using betadine. The disinfection was performed twice before and after surgical procedure. For transplantation, the back was incised by 2 cm, a subcutaneous pocket was formed, and implants of each group were transplanted in the pocket. After the transplantation, the wounded area was sutured using Nylon 4/0 (Ethicon), the wounded area was finally disinfected using betadine, and a gauze dressing was applied.
Five groups of implants below were all transplanted in one rat, the rat was euthanized after 2, 4 and 8 weeks, and tissues at an implantation site were obtained.
Group 1 (control group): untreated PDMS
Group 2 (IA 50 mmol (low) PDMS): PDMS surface treated with IA having a low concentration
Group 3 (IA 150 mmol (high) PDMS): PDMS surface treated with IA having a high concentration
Group 4 (IA-GT 0.25% (low) PDMS): PDMS surface treated with IA chemically bonded to gelatin, having a low concentration
Group 5 (IA-GT 0.50% (high) PDMS): PDMS surface treated with IA chemically bonded to gelatin, having a high concentration
In order to evaluate a fibrosis degree, i) capsule thickness, ii) number of fibroblast and iii) number of myofibroblast, were quantified.
Particularly, capsule thickness was analyzed through staining with Hematoxylin & Eosin. The capsule thickness was measured with respect to collagen and tissue layers accumulated on the bottom part of subcutaneous muscles, the analysis was performed by measuring a portion where the thinnest image phase capsule was formed, and the results are shown in Table 1.
As shown in Table 1, IA-PDMS and IA-GT-PDMS groups were confirmed to show decreased capsule thicknesses in all cases after 2, 4 and 8 weeks when compared with the control group, and accordingly, it could be found that the fibrosis was reduced by IA. Particularly, the thinnest capsule thickness was observed in IA-GT-PDMS high, and this means that the capsule thickness of IA-GT high PDMS was reduced to about 63% when compared with the control group (IA-GT high: 528 μm (37%) against control group: 1426 μm (100%)).
In order to evaluate the number of fibroblast in tissues, immunostaining was performed, only fibroblast was specifically stained using an antibody (ab92546, Abcam, CA, USA) binded with Vimentin which is a specific factor of fibroblast, and only cells expressing fluorescence were selectively evaluated to confirm the number of cells. Particularly, for the progress of the experiment, the biopsy of tissues was performed after a certain time period, and the biopsied tissues were manufactured into slides through a paraffin embedded technique. After the pre-treatment of the slide (Deparaffine, Rehydration), an immune factor bonding to the fibroblast specific factor was used, color expressed by the bonding of the specific factor and DAPI where staining of a cell nucleus occurs were selectively counted and analyzed per week, and the results are shown in Table 2.
32 ± 4.73
39 ± 11.83
As shown in Table 2, somewhat lower number of fibroblasts was confirmed in all groups until 2 weeks and 4 weeks from transplantation when compared with the control group, but the difference was insignificant. However, in groups after 8 weeks from the transplantation, smaller fibroblasts were observed in IA high and IA-GT high groups when compared with the control group (p<0.05). This corresponds to an effect of reducing the collagen density of IA-GT high PDMS to about 50% when compared with the control group (IA high: 21 (53.8%)/IA-GT high: 20.75 (53.2%) against control group: 39 (100%)).
In order to evaluate the number of myofibroblasts in tissues, immunostaining was performed, only myofibroblasts were specifically stained using an antibody binded with alpha-SMA which is a specific factor of myofibroblast, and only cells expressing fluorescence were selectively evaluated to confirm the number of cells. Particularly, for the progress of the experiment, the biopsy of tissues was performed after a certain time period, and the biopsied tissues were manufactured into slides through a paraffin embedded technique. After the pre-treatment of the slide (Deparaffine, Rehydration), an immune factor bonding to a myofibroblast specific factor was used, color expressed by the bonding of the specific factor and DAPI where staining of a cell nucleus occurs were selectively counted and analyzed per week, and the results are shown in Table 3.
33 ± 6.72
As shown in Table 3, the significant decrease of myofibroblasts was observed in IA high group after 2 weeks from transplantation. In addition, after 4 weeks, the significant decrease of myofibroblasts was confirmed in all experimental groups (IA high, IA-GT low, IA-GT high) excluding the IA low group when compared with the control group (p<0.05). In the groups after 8 weeks, the decrease of the myofibroblast number was observed in IA high and IA-GT high groups when compared with the control group. This corresponds to an effect of decreasing the collagen density of IA-GT high PDMS to about 50% when compared with the control group (IA high: 21 (53.8%)/IA-GT high: 20.75 (53.2%) against control group: 39 (100%)).
2.2.2. Inflammatory Response Analysis
In order to analyze inflammatory response at an implantation site, biopsied tissues were stained using Hematoxylin & Eosin. Then, only polynuclear cells (inflammatory mediated cells) in the capsule formation area of stained tissues were selected and quantified using an image software. Particularly, the staining with Hematoxylin & Eosin was performed for biopsied tissues, and only cells considered to be polynuclear cells after the staining of the tissues were specifically selected and counted. Scores were configured based on the counted specific polynuclear cells, inflammatory response score in each group was analyzed according to the range of the configuration, and the results are shown in Table 4.
1 ± 0.3
As shown in Table 4, it could be found that the inflammatory response was significantly decreased in all experimental groups when compared with the control group.
From the results above, it could be found that an implant surface-modified with itaconic acid and/or gelatin according to an embodiment reduces fibrosis or inflammatory response at an implantation site when compared with a control group and accordingly, the implant could be useful as a medical implant.
| Number | Date | Country | Kind |
|---|---|---|---|
| 10-2017-0135862 | Oct 2017 | KR | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/KR2018/008100 | 7/18/2018 | WO | 00 |
