KR101905072B1 - Orthopedic or dental metal complex for promoting angiogenesis by production of reactive oxygen species - Google Patents

Abstract

The present invention relates to an orthopedic and dental metal complex which accelerates angiogenesis by the production of active oxygen species, and a method of using the same. According to one aspect of the present invention, there is provided an orthopedic and dental metal complex for promoting angiogenesis, It is possible to effectively regenerate the periosteum and bones of the surrounding tissues in which the orthopedic and dental metal complexes are used and to shorten the treatment period by promoting angiogenesis caused by active oxygen species. The metal composite can be used directly in existing medical implants.

Description

TECHNICAL FIELD [0001] The present invention relates to an orthopedic or dental metal complex for promoting angiogenesis by the production of reactive oxygen species

To an orthopedic and dental metal complex which promotes angiogenesis by the production of active oxygen species, and a method using the same.

If a metal plate is used orthopedically for the treatment of fractures, perforation damage and loss will inevitably occur. These periosteal injuries are the cause of nonunion of the fracture, metal fracture, and osteomyelitis. Therefore, regeneration and preservation of periosteum during bone union is one of the key factors of fracture treatment. Various surgical techniques have been developed to maximize preservation of the periosteum.

On the other hand, various physiological mechanisms of reactive oxygen species (ROS) have been studied, and it has been found that the effect of promoting angiogenesis of ROS is promoted. However, existing techniques for generating ROS require external energy supply such as light or electric energy for production of ROS, and excessive production of ROS results in cell death rather than application of ROS production system in vivo There was a difficulty.

Therefore, the inventors of the present invention invented a metal complex which naturally generates ROS without external energy supply, and proved that the metal complex is effective for regeneration of periosteum and bone by promoting angiogenesis of peripheral tissues. Thus, It can be used as a metal complex for orthopedic surgery. In addition, the metal complex of the present invention can be used with the same mechanism in a dental implant that restores a tooth lost in a dentistry.

One aspect provides orthopedic and dental metal complexes that promote angiogenesis.

Another aspect provides a method of generating active oxygen species using a metal complex.

Another aspect provides a method of making orthopedic and dental metal complexes that promote angiogenesis.

One aspect provides a metal complex that promotes angiogenesis.

In the metal composite,

An anode comprising a first metal; And

And a cathode electrically connected to the anode including the second metal layer.

The metal complex may be inserted into the body for orthopedic surgery and dentistry.

The metal complex may be inserted into the body and brought into contact with body fluid, blood, lymph fluid, tissue fluid, body fluid, intracellular fluid, or extracellular fluid.

The term " angiogenesis " may mean a mechanism by which new blood vessels are generated from existing blood vessels to supply oxygen and nutrients to the cells.

The term " anode " may refer to an electrode having a higher potential than a cathode and generating electrons in an oxidation reaction to deprive electrons from the cathode.

The term " cathode " may refer to an electrode having a lower potential than the anode and absorbing electrons from the anode to cause a reduction reaction. The negative electrode is -0.2 V vs.. It may be possible to generate ROS or to promote production by an oxygen reduction reaction (ORR) under a voltage of Ag / AgCl or less and a current density of 1 μA / cm ² or more. The place where the ROS is generated may be a body fluid, blood, lymph fluid, tissue fluid, body fluid, intracellular fluid, extracellular fluid, or electrolytic solution.

The " electrically connected " means that the electrodes are connected so that electrons can move between the electrodes, and includes both direct electrode contact and indirect connection through a conductor or the like.

The first metal may be at least one selected from the group consisting of Mg, Al, Co, K, Na, Ca, Fe, Si, Ti, V, Sn, Zn, Li, Sr, Ba, Nb, Ta, W, Co, , Se, Ge, Ga, B, Be, As, Zr, alloys thereof, or metals based thereon.

The second metal is selected from the group consisting of titanium and titanium alloys, steel alloys including stainless steel, cobalt alloys including cobalt and cobalt-chromium alloys, nickel-titanium alloys, noble metals (Au, Ag, Pt, Pd) And may include metals similar to these.

The second metal may have a lower ionization tendency than the first metal.

The cathode may further comprise a second metal oxide layer. The cathode may further comprise a nitride or other catalyst coating layer in addition to the oxidized second metal layer.

The method of forming the metal oxide layer may include anodizing, thermal processing, oxygen-plasma treatment, and arc oxidation / plasma electrolytic oxidation. .

The " anodizing " means that when the metal is placed on the anode in the electrolyte solution and electricity is passed through it, the oxygen anion is transferred to the anode of the metal by an electrical force and they oxidize the metal surface to produce a metal oxide layer on the surface . As a result, the metal may have effects such as corrosion resistance, abrasion resistance, color development, dyeing, and electrical insulation. The metal oxide layer generated by the anodic treatment is a catalyst of the ORR, but when it is thicker than necessary, the movement of the electrons is disturbed, so that the ORR reaction in the cathode of the metal complex can be controlled according to the thickness thereof.

The first metal and the second metal may further include a coating layer on the surface. The coating layer may comprise an oxide, a hydroxide of a coating element, an oxyhydroxide of a coating element, an oxycarbonate of a coating element, or a coating element compound of a hydroxycarbonate of a coating element. The compound forming the coating layer may be amorphous or crystalline. As the coating element included in the coating layer, Mg, Al, Co, K, Na, Ca, Fe, Si, Ti, V, Sn, Zn, Li, Sr, Ba, Nb, Ta, , Ag, Bi, Se, Ge, Ga, B, Be, As, Zr or alloys thereof. The first metal and the second metal are expressed by the above-described chemical formulas, and the alloy of the first metal and the second metal having no coating layer and the first metal and the second metal represented by the above formulas, .

The metal complex may be a metal complex that generates active oxygen species without external energy supply.

The " external energy supply " may mean the supply of energy that can induce the emission of electrons such as light, voltage, and current from the outside.

&Quot; Reactive oxygen species (ROS) " means a molecule that is in a chemically active state, including oxygen atoms, and may include O2-, H2O2, O, OH, and O2H.

The generation rate of active oxygen species generated from the cathode of the metal complex per unit time (1 hour) may be 1, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 μM / , 100, 90, 80, 70, 60, 50, 40, 45, 40, 35, 30, 25, 24, 23, 22, 21, 20 μM / h.

The number of molecules generated per unit time (1 hour) of reactive oxygen species generated from the negative electrode of the metal complex may be 2, 5, 10, 20, 30 nmol or more, and 200, 180, 160, 140, 120, 60, 40, 30 nmol or less.

The number of molecules generated per unit time (1 hour) of reactive oxygen species generated from the negative electrode of the metal complex may be 5, 10, 20, 30, 40, 50 pmol or more per unit area (1 mm 2) , 400, 200, 100, 80, 60 pmol or less.

The active oxygen species generated from the negative electrode of the metal complex can be maintained at a concentration of 1, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, Or at a rate that can be maintained at or below 100, 90, 80, 70, 60, 50, 40, 45, 40, 35, 30, 25, 24, 23, 22, Lt; / RTI >

In one embodiment, certain concentrations of active oxygen species have been shown to promote angiogenesis in peripheral cells and are effective in regenerating periosteum and bone surrounding tissue. The concentration of the active oxygen species may be varied depending on the generation rate of the active oxygen species, the state of the cells around the metal complex, the type of disease, the progress of disease, the sex, age, body weight and race of the patient.

The thickness of the second metal oxide layer may be 1, 5, 10, 15, 20, 30, 40, 50, 100, 200, 300, 400, 500, 600, 700, 800 nm, 70, 600, 500, 400, 300, 200, 180, 160, 140, 120, 100, 90, 80, 70, 60, 50 nm or 100, 90, 80, 70, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 μm or less.

The second metal oxide layer may be formed on two or more different portions of the cathode. The second metal oxide layers formed on the different portions may have different thicknesses. Since the oxidized second metal layer having different thicknesses generates active oxygen species at different concentrations, the amount of active oxygen species generated depending on the region of the negative electrode can be controlled.

The cathode may further comprise a catalytic metal layer formed on the oxidized second metal layer.

Wherein the catalyst metal is selected from the group consisting of Au, Mg, Al, Co, K, Na, Ca, Fe, Si, Ti, V, Sn, Zn, Li, Sr, Ba, Nb, Ta, W, Co, Bi, Se, Ge, Ga, B, Be, As, Zr or alloys thereof.

The method of forming the catalytic metal layer may include deposition by sputtering, adsorption through ion reduction, spin coating of nanoparticles, and drop-casting.

The " sputtering " is a kind of vacuum deposition method in which a plasma is generated at a low degree of vacuum so that a gas such as ionized argon collides with a metal such as Au to repel the metal particles, and the repelled metal particles are re- Collision to create a cluster, particle, or film. The film thus formed is referred to as a thin film. The cluster, particle or film formed as described above may absorb electrons emitted from the anode to inhibit the reduction reaction at the cathode, so that the ORR reaction at the cathode can be controlled by controlling the thickness of the thin film layer.

The thickness of the catalytic metal layer may be 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, , 100, 90, 80, 70, 60, 50, 45, 40, 35, 30, 25, 24, 23, 22, 21, 20 nm.

At least two catalyst metal layers may be formed on different portions of the cathode. The Au layers formed on the different portions may have different thicknesses. Since the catalytic metal layers having different thicknesses produce different concentrations of active oxygen species, the amount of reactive oxygen species generated according to the sites of the negative electrodes can be controlled.

Another aspect provides a method of generating active oxygen species using a metal complex.

The method comprises:

And electrically connecting the anode including the first metal and the cathode including the second metal having a lower ionization tendency than the first metal.

When metals having different ionization tendencies are connected to each other, electrons generated by the oxidation reaction in a metal having a high ionization tendency can be transferred to a metal having a low ionization tendency and used for a reduction reaction. Therefore, oxygen can be reduced by the reduction reaction to produce active oxygen species.

In the above method, considering the initial Mg volume and corrosion rate, the active oxygen species may be added for 1, 5, 10, 15, 20, 25, 30 minutes or 1 hour, 2 hours, 3 hours, 4 hours Hours, 5 hours, 7 hours, 9 hours, 12 hours, 24 hours, 36 hours, 48 hours, 60 hours, 72 hours, 100 hours, 200 hours, 300 hours, 400 hours, 500 hours, 600 hours, 700 hours, 800 hours, 900 hours, 1000 hours or more, or 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 14 days or more, or 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks or more, or 1 month, 2 months, 3 Months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months or more. This is sufficient time to induce angiogenesis and tissue regeneration therethrough, taking into account the characteristics of the present invention acting in the initial regeneration phase of the lesion.

The concentration of active oxygen around the cathode can be controlled by controlling the generation time of the active oxygen species.

It is to be understood that what has already been mentioned in the description of the metal complex among the terms or elements mentioned in the promoting method.

Another aspect provides a method of making orthopedic and dental metal complexes that promote angiogenesis.

The method comprises:

And then producing a positive electrode comprising the first metal.

And a negative electrode comprising a second metal.

And oxidizing the second metal to form a second metal oxide layer.

And forming a catalytic metal layer on the oxidized second metal layer.

And electrically connecting the anode and the cathode to each other

It is to be understood that what has already been mentioned in the description of the metal complex or the method of promoting among the terms or elements mentioned in the above-mentioned method of manufacturing.

According to one aspect of the present invention, there is provided an orthopedic and dental metal composite which promotes angiogenesis, and a method using the same, which promotes angiogenesis caused by reactive oxygen species to effectively enhance the periosteum and bone of peripheral tissues using orthopedic and dental metal complexes Regenerated, and the treatment time can be shortened.

1A is a TEM image of a Ti / TiO ₂ / Au plate fabricated with a focused ion beam.
Figure 1b is an HR-TEM image, and a fast Fourier transform image of the individual layers of Ti / TiO ₂ / Au plate.
Fig. 1C shows a point EDX spectrum in the spherical aberration correction STEM mode of the Au, TiO ₂ , and Ti regions.
FIG. 1D shows the EDX analysis result of magnifying the spherical aberration correction STEM image of the cross section of the Ti / TiO ₂ / Au layer and a part thereof.
FIG. 1E shows the XPS, and FIG. 1F shows the results measured by atomic force microscopy.
1F shows an AFM image of a Ti / TiO ₂ / Au plate surface.
FIG. 1G is a graph showing the results of XPS depth analysis of oxygen (O), titanium (Ti), and gold (Au) atomic ratios of a Ti / TiO ₂ / Au plate.
FIG. 2A is a graph showing the changes in the Mg vs. Mg concentration in O ₂ -saturated PBS buffer. Ag / AgCl, and Ti / TiO ₂ / Au vs. Ag / AgCl as a function of discharge current density.
Figure 2b is N ₂ - Mg in the saturation vs. PBS buffer Ag / AgCl, and Ti / TiO ₂ / Au vs. Ag / AgCl as a function of discharge current density.
2c is _{O 2 - Ti / TiO 2 /} Au vs. saturation in PBS buffer Mg voltage as a function of various discharge current densities.
3A is a graph showing the results of CV measurement of a Ti / TiO ₂ / Au plate in an O ₂ and N ₂ saturated PBS solution.
FIG. Graphs showing the concentration and the Faraday effect of H ₂ O ₂ produced from Ti / TiO ₂ / Au plates during bulk-electrolysis with Ag / AgCl voltage.
3C is a graph showing the linear sweep voltage curves of Ti / TiO ₂ / Au plates at O ₂ -saturated PBS buffer at circulation rates of 400, 625, 900, 1225, and 1600 rpm.
Figure 3d shows a Koutecky-Levich graph as a function of the voltage applied to the Ti / TiO ₂ / Au plate.
4A is a graph showing the concentration of H ₂ O ₂ produced when Mg and Ti / TiO ₂ / Au are connected by a metal conduit (black) in the EBM-2 medium and when not connected as a control (red) to be.
FIG. 4B is a graph showing the concentration of H ₂ O ₂ produced in the PBS buffer when Mg and Ti / TiO ₂ / Au are connected by a metal conduit (black) and when not connected as a control (red).
5A is a schematic diagram showing an experiment for measuring the effect on the angiogenesis of the Mg-Ti / TiO ₂ / Au system.
FIG. 5B is a fluorescence image showing HUVECs cultured in metal complexes in which Mg and Ti / TiO ₂ / Au are connected by metal conduits or in separated metal, and angiogenesis of positive and negative control cells.
5C is a graph showing data on the number of HUVECs cultured in metal complexes in which Mg and Ti / TiO ₂ / Au are connected by metal conduits or in separated metal complexes, and the number of vascular branches generated per unit area of positive control and negative control cells .
FIG. 5d shows quantitative DNA content of HUVECs cultured in metal complexes in which Mg and Ti / TiO ₂ / Au are connected by metal conduits or in separate metal complexes, and HUVECs after 8 and 24 hours of culture of positive and negative controls, The results of the analysis are shown.
FIG. 6 is an image (scale bar = 500 μm) showing the degree of tube formation after incubation of positive control, negative control, and HUVEC exposed to 100 μM H ₂ O ₂ for 30 min at a relatively high concentration.
FIG. 7A shows the result of quantitatively measuring the concentration of H ₂ O ₂ with DPD / POD according to the discharge time at various thicknesses of various TiO ₂ layers, which are variously generated according to the anodizing potential. FIG. Represents the concentration of H ₂ O ₂ with anodizing potential.
FIG. 8A shows the results of quantitatively measuring the concentration of H ₂ O ₂ with DPD / POD depending on the discharge time at various thicknesses of Au layers formed by Au sputtering time, FIG. 8B shows a result of sputtering time of 20 seconds, And the H ₂ O ₂ concentration according to the gold plasma current when the discharge time is 60 minutes.
9A is a schematic diagram of a Mg-Ti / TiO ₂ / Au metal composite having TiO ₂ layers of different thicknesses.
FIG. 9B is an application example of a Mg-Ti / TiO2 / Au metal composite implant having TiO ₂ layers of different thicknesses at different sites.
FIG. 10A is a schematic view of the manufacture of an implant using a Mg-Ti / TiO ₂ / Au metal composite.
10B is an image of an implant manufactured using a Mg-Ti / TiO ₂ / Au metal composite.
FIG. 10C is an image showing that the implants prepared using the Mg-Ti / TiO ₂ / Au metal composite are cultured together in EBM-2 medium containing HUVEC cells.
FIG. 10D is a graph showing the number of blood vessel branching per unit area according to the contact time of the HUVEC cells with the implant prototype manufactured using the Mg-Ti / TiO ₂ / Au metal complex.
FIG. 10E is an image obtained by observing the angiogenesis of HUVEC cells by fluorescence microscope according to the contact time with the implant prototype manufactured using the Mg-Ti / TiO ₂ / Au metal complex.
FIG. 11 shows the result of immersion test for 5 days (120 hours) in a Hanks solution after inserting a pure zinc bar into a Ti-6Al-4V coin.
12 shows the results of evaluating the amount of ROS generated in a PBS buffer after inserting a pure zinc bar into a Ti-6Al-4V screw.

Hereinafter, the present invention will be described in more detail with reference to examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

Manufacturing example  One. Ti / TiO ₂ / Au Au cathode manufacturing

A Ti / TiO ₂ / Au metal complex with improved catalytic performance of an oxygen reduction reaction (ORR) using magnesium as an anode and an oxidized Ti alloy as a cathode was prepared.

Specifically, an anodic oxidation treatment was performed by applying a 10 V voltage for 30 seconds to a Ti plate (2.0 mm thickness, 99.7% purity, Sigma Aldrich) in 1.7 M acetic acid. Thereafter, a current of 2 mA was applied for 20 seconds at a chamber pressure of 2 × 10 ^-6 Torr using a magnetron sputtering deposition machine, and the anodized Ti was subjected to Au sputtering. Commercially purchased Mg (99.9%, Yinguang) was used as an anode material without further purification.

Example  One. Ti / TiO ₂ / Au metal composite

The local structure of the cathode of the formed Ti / TiO ₂ / Au metal device manufactured in Production Example 1 was observed using a transmission electron microscope (TEM).

Specifically, a high-resolution transmission electron microscopy (HR-TEM) and a Cs-corrected scanning transmission electron microscopy (STEM) analysis were carried out using an FEI Titan 80-300 microscope keV. The cross-sections of Ti / TiO ₂ / Au for TEM analysis were made using a focused ion beam (Helios NanoLab 600, FEI). An energy dispersive X-ray (EDX) analysis was performed in a Cs-corrected STEM mode and spectra were obtained from three different layer regions. SEM images of the corroded Mg after discharge were obtained with Quantata 3D FEG (FEI, Netherland). AFM images of the Ti / TiO ₂ / Au plate surface were obtained in the non-contact mode of the XE-100 (Park systems, korea). It was set to the operating environment of 0.4 Hz scan rate. X-ray photoelectron spectroscopy (XPS) spectra were obtained by adjusting the pass energy to 30 eV for each plate and 0.1 eV for each step. All bond energies were calculated based on C 1s (284.5 eV). XPS depth analysis at a depth of 80 nm from the plate surface was performed by stepwise surface etching in 1 nm increments.

FIG. 1A shows a TEM image of a Ti / TiO ₂ / Au plate fabricated with a focused ion beam. Three distinct regions of Au, TiO ₂ , and Ti were clearly observed. Au clusters of about 3 nm in thickness were deposited on the amorphous 20 nm thick TiO ₂ layer on the uppermost surface of the cathode.

Figure IB shows HR-TEM images of the three regions and their fast Fourier transforms (FFT). The grid patterns of the Ti and Au regions are indicated by white and yellow arrows. The lattice spacing of Au clusters is 0.230 nm and 205 nm, respectively.

1C shows the point EDX spectra of the Au, TiO ₂ , and Ti regions in the spherical aberration correction STEM mode. The EDX peaks of Au were found near the Au clusters of about 3 nm. The EDX peaks of Ti and O were observed in the amorphous 20 nm thick TiO2 layer and the EDX peaks of Ti were observed in the orthotopic Ti plate. The small Cu peak emerging from the EDX spectrum is due to the Cu TEM-grid.

FIG. 1D shows the EDX analysis result of magnifying the spherical aberration correction STEM image of the cross section of the Ti / TiO ₂ / Au layer and a part thereof. Three well-ordered structures can be identified.

FIG. 1E shows the XPS, and FIG. 1F shows the results measured by atomic force microscopy. Surface XPS results showed Au 4d and 4f peaks of the Au clusters (Fig. 1e), and clusters with a height of about 3 nm from the Atomic Microscopy results are attributed to the deposited Au clusters (Fig. 1f). Also, it can be confirmed from the XPS results obtained by etching in the depth direction that Au clusters having a size of about 3 nm are present on the surface, and atypical TiO2 and Ti are sequentially present in the depth direction (FIG. 1G). Au clusters of about 3 nm in height deposited on the Ti phase with distinct Au 4d and 4f peaks can be identified.

Example  2. Mg- Ti / TiO ₂ / Au Metal Complexes

The electrochemical properties of Mg and the Ti / TiO ₂ / Au metal composite system prepared in Preparation Example 1 were evaluated in presence or absence of O ₂ in PBS of pH 7.4.

Specifically, the electrochemical characteristics of the Mg-Ti / TiO ₂ / Au metal system were analyzed using a 3-electrode electrochemical cell system. An Ag / AgCl reference electrode (BASi Ag / AgCl / 3M NaCl) was used. Electrochemical properties were analyzed using a potentiostat at 37 ° C in O ₂ and N ₂ saturated PBS buffer (1 ×, pH = 7.4, Gibco, Life technology). Each buffer was saturated with each gas by bubbling high purity O ₂ (99.999%) or N ₂ (99.999%) for at least 2 hours before beginning each measurement. During the course of the measurement, the O ₂ feed flow was maintained to maintain the O ₂ saturation. The electrode potential was converted to a RHE scale using the following equation: E (NHE) = E (Ag / AgCl) + 0.059 X pH + 0.197 V Mg vs. Et in O ₂ and N ₂ saturated PBS buffer. Ag / AgCl, Ti / TiO ₂ / Au, etc. Ag / AgCl, and Mg < / RTI > The Ti / TiO ₂ / Au voltage was recorded at a scan rate of 0.005 mA / cm ² s as a function of discharge current density. In addition, Mg. Discharge performance of Ti / TiO ₂ / Au was measured up to 0.5 mAh discharge capacity under a constant discharge current density (0.05 mA / cm2, 0.10 mA / cm2, and 0.20 mA / cm2) using a two-electrode configuration.

FIG. 2A is a graph showing the changes in the Mg vs. Mg concentration in O ₂ -saturated PBS buffer. Ag / AgCl, and Ti / TiO ₂ / Au vs. Ag / AgCl voltage as a function of current density discharged. Mg. The voltage of Ag / AgCl was maintained at about -1.5 V and the current of about 1.0 mA / cm ² was discharged. Such a constant voltage indicates that the dominant anodic reaction is that Mg is oxidized to Mg ^{2 +} ions. In contrast to the voltage of the Mg anode, Ti / TiO ₂ / Au vs.. Voltage of the Ag / AgCl was observed that a discharge current density is gradually falling from -0.2V while increased from 0 to 1.0 mA / cm ² to -1.2V, especially in the current density between about 0.6 mA / cm ² abrupt voltage Reduction. The change of the voltage according to the discharge current density as described above indicates that the negative electrode reaction has changed with the change of the current density. From these results, it can be considered that a reduction reaction from -0.2 V to -1.2 V (vs. Ag / AgCl) in PBS buffer at pH 7.4 is as follows: oxygen reduction with H ₂ O ₂ or H ₂ O (0.59 V and 0.065 V (vs. Ag / AgCl)), or hydrogen evolution (-0.64 V vs. Ag / AgCl). Based on the theoretical reduction potential of the three reactions, ORR is the main reaction at low discharge currents, and the hydrogen generation reaction is the main reaction at high discharge currents. It is reduced according to the discharge current Ti / TiO ₂ / Au vs. Due to the Ag / AgCl voltage, the overall Ti / TiO ₂ / Au vs.. The Mg voltage is Ti / TiO ₂ / Au vs.. It decreases with increasing discharge current, similar to Ag / AgCl voltage.

Figure 2b is N ₂ - Mg in the saturation vs. PBS buffer Ag / AgCl, and Ti / TiO ₂ / Au vs. Ag / AgCl voltage as a function of current density discharged. Ti / TiO ₂ / Au vs. O ₂ saturation condition. In contrast to the Ag / AgCl voltage abruptly dropping from the current density of about 0.6 mA / cm ² to -1.0 V, the voltage in the N ₂ -saturated PBS solution was reduced from a relatively low current density of 0.1 mA / cm ² to -1.0 V . ≪ / RTI > The result of varying the voltage value in the low current density region depending on the presence or absence of O _{2 in} the buffer clearly indicates that the ORR occurs at the cathode during current discharge. However, the discharge current is 0.6 mA / cm ² than it indicates the presence of the O ₂ in the buffer regardless of Ti / TiO ₂ / Au vs. The Ag / AgCl voltages showed similar values. As a result, it was confirmed that hydrogen generation reaction was the main reaction at high discharge current.

FIG. 2C shows a comparison of Ti / TiO ₂ / Au vs. O ₂ in an O ₂ -saturated PBS buffer. Mg voltage as a function of various current densities. The voltage profile at constant current dissipation indicates that the overall output voltage in an O ₂ -saturated PBS buffer can be maintained at 0.05 mAh capacity. As the current density increases from 0.05 mA / cm ² to 0.20 mA / cm ² , the overall output voltage decreases from 1.35 V to 0.8 V.

These results indicate that it is possible to control the Mg-Ti / TiO ₂ / Au metal complex so that ORR occurs naturally in the cathode according to the control of the discharge current density, that is, the time.

Example  3. Ti / TiO ₂ From Au ORR  Identification of Cathodic Reduction Reaction

In order to confirm exactly what type of reduction reaction the reaction in Ti / TiO ₂ / Au is, the following experiment was carried out.

Ti / TiO ₂ / Au plates, Mg plates and Ag / AgCl electrodes were used as working electrode, counter electrode and reference electrode, respectively, to obtain a cyclic voltammetry (CV) curve of the Ti / TiO ₂ / . CV curves were recorded in O ₂ and N ₂ -saturated PBS at a scan rate of 5 mV / sec, from 0.5 V to -0.5 V (vs. Ag / AgCl). Prior to all electrochemical testing, solution resistance was measured and all data was iR-compensated accordingly.

FIG. 3A shows the CV measurement result of Ti / TiO ₂ / Au plate in O ₂ and N ₂ saturated PBS solution. Under O ₂ -saturated conditions. The apparent reduction and oxidation peaks of Ti / TiO ₂ / Au were observed at about -0.3 V and 0.1 V (vs. Ag / AgCl), respectively. On the other hand, a weak current was observed at similar voltages under N ₂ -saturated conditions. The difference in electrochemical behavior as described above is dependent on the presence of O _{2 in} the buffer, and it can be more clearly confirmed that ORR occurs in Ti / TiO ₂ / Au.

Example  4. Mg- Ti / TiO ₂ / Au metal complex without the need for external voltage H ₂ O ₂ end  Determining if it Occurs

It was confirmed whether or not H ₂ O ₂ was produced in the Mg-Ti / TiO ₂ / Au metal composite without any external voltage or current. Specifically, the Mg-Ti / TiO ₂ / Au metal complex having a surface area of about 230 mm ² was placed in a 2 ml volume of EBM-2 medium or PBS buffer, and the amount of H ₂ O ₂ produced was measured.

Figures 4a and 4b show the concentration of H ₂ O ₂ produced when Mg and Ti / TiO ₂ / Au are connected by a metal conduit (black) and not connected as a control (red) EBM-2 medium (endothelial growth basal medium), and Figure 4b shows the results of experiments in PBS buffer. The experiment was carried out without an external voltage source, and the amount of H ₂ O ₂ produced was determined by the DPD-POD method after the anode and cathode were connected to the metal conduit.

As can be seen in Figure 4a, the amount of H ₂ O ₂ produced from the metal complexes connected by metal conduits increased over time and reached 17 μM after 30 min. On the other hand, the amount of H ₂ O ₂ produced in the control group after 30 minutes was almost negligible (<5 μM). The formation of H ₂ O ₂ in the control group is presumed to be due to the formation of H ₂ O ₂ on the Mg surface due to the natural Mg corrosion.

As shown in FIG. 4B, although a relatively high level of H ₂ O ₂ production was observed in the PBS buffer as compared with the case of EBM-2, overall results were similar to those of EBM-2. Concentration difference in the H ₂ O ₂ is assumed to be due to different chemical composition that affect the formation of H ₂ O ₂ PBS with EBM-2.

Example  5. Mg- Ti / TiO ₂ / Au Metal Complexes to Promote Angiogenesis Promoting Effect

The angiogenic effect of Mg-Ti / TiO ₂ / Au metal complex was confirmed based on the effect of H ₂ O ₂ production.

First, HUVEC (C2517; Lonza) cells were cultured in an endothelial growth medium-2 (EGM-2) (CC-3162; Lonza) at 37 ° C in a humidified atmosphere of 5% CO ₂ . Four to six passages of HUVEC were used in the experiments. For HUVEC angiogenesis analysis, EBM-2 medium containing an appropriate number of HUVECs was pre-introduced into a system in which Mg and Ti / TiO ₂ / Au metal complexes were electrically connected or separated. The amount of H ₂ O ₂ produced can be controlled by the incubation time of the HUVEC in the Mg-Ti / TiO ₂ / Au system. After removal of the Mg-Ti / TiO ₂ / Au system, the HUVECs were centrifuged to remove the EBM-medium supernatant and then resuspended in fresh medium. Thereafter, H ₂ O ₂ -stimulated HUVECs were grown in 96-well plates (CC-3156, Lonza) coated with growth factor-reduced Matrigel ^® (354230, Corning) It was busy. The Matrigel ^® coated wells were prepared according to the following protocol. Matrigel ^® was thawed on ice overnight at 4 ° C and diluted 1: 1 in serum free-media. 75 μl of diluted Matrigel ^® was added to each of the 96-well plates and incubated for 30 min at 37 ° C before cell division. H ₂ O ₂ -stimulated HUVECs were dispensed onto coated 96-well plates at a density of 1.5 × 10 ⁴ cells / well in 100 μl EBM-2 medium. HUVEC cultured on EBM-2 medium supplemented with growth factors without H ₂ O ₂ stimulation was used as a negative control, and HUVEC cultured on EBM-2 medium without growth factor was used as a negative control. The proliferative morphology of HUVEC was observed by phase contrast microscopy at several hours (3, 8 and 24 hours). Various cell morphologies were observed at 8 h, after which the samples were stained with Calcein-AM (C3 100 MP, Invitrogen). For calcine-AM staining, 2 [mu] M calcein-AM was prepared in PBS and HUVEC was incubated with the saline reagent for 30 minutes. Live cells are visualized in green under a fluorescence microscope (Zeiss Axio Vert. A1). To extract quantitative data during CLS formation, five non-overlapping regions 50x of samples (n = 5, each group) were obtained. The number of blood vessels per area was manually counted.

2 x 10 ⁴ cells / well HUVEC cells were plated in 24-well plates to determine the effect of H ₂ O ₂ on the proliferation and survival rate of HUVEC cells at different incubation times. The divided cell groups were cultured in EBM-2 medium for 5, 15, 30, and 60 minutes. At the same time, the group cultured in EGM-2 or EBM-2 medium without H ₂ O ₂ stimulation was used as a positive or negative control. After 8 hours and 24 hours of the above-mentioned division, HUVEC was dissolved using a cell lysis buffer. The DNA content of each group was quantitatively analyzed using the Quant-iT ™ PicoGreen® Assay Kit (P11496; Invitrogen). The samples were subjected to fluorescence measurement using a fluorescent microplate reader (Infinite 200 pro, Tecan, Switzerland) at a fluorescent wavelength (excitation wavelength: 485 nm, emission wavelength: 525 nm). Statistical analysis of the current data was performed through ANOVA and Tukey's post-mortem analysis using the Graph prism 5 program. Statistical significance was determined as * (p <0.05), ** (p <0.01), and *** (p <0.001), respectively.

5A is a schematic diagram showing a manner of performing the above experiment. FIG. 5B is a fluorescence image showing HUVECs cultured in metal complexes in which Mg and Ti / TiO ₂ / Au are connected by metal conduits or in isolated metal, and tube formation of positive and negative control cells. As in FIG. 5b, HUVEC cells exposed to a metal complex connected by a metal conduit for 30 minutes, stimulated with H ₂ O ₂ at a concentration of about 16 μM, were then cultured on a Matrigel ^® plate for 8 hours, The most prominent angiogenesis was observed compared to the control group. On the other hand, HUVEC, which was cultured under Mg and Ti / TiO ₂ / Au metal complexes not connected with metal conduits, showed similar degree of tubular formation as the negative control group. It was confirmed that the Mg-Ti / TiO ₂ / Au metal complex connected to the metal conduit can promote angiogenesis without growth factors.

Figure 5c shows data from a number of vascular branches generated per unit area from five independent, repeated iterations of the experiment. T1 and T2 represent the case where HUVEC was incubated for 30 minutes in metal complexes and metal complexes in which Mg and Ti / TiO ₂ / Au are connected, respectively. On average, 23 vessels were produced in T1, and less than 5 vessels were observed in T2 and negative control groups.

FIG. 5D shows the results of quantitative analysis of DNA content of HUVECs after 8 and 24 hours culture of T1, T2, positive control and negative control by PicoGreen analysis. In the case of T1, the amount of HUVEC cells was significantly increased compared to T2 and negative control.

Example  6. H ₂ O ₂ of  Determination of effect on cell by concentration

H ₂ O ₂ is generally known to exhibit cytotoxicity at high concentrations. Therefore, it was confirmed how the effect of H ₂ O ₂ was changed when the concentration of H ₂ O ₂ was changed in the same manner as in Example 5.

Figure 6 is an image showing angiogenesis after incubation on Matrigel ( ^R) of HUVECs exposed for 30 min to 100 uM H ₂ O ₂ at a relatively high concentration of positive control, negative control plus growth factor. As can be seen in FIG. 6, HUVEC exposed to H ₂ O ₂ at a high concentration of 100 μM did not stimulate angiogenesis, but rather decreased cell numbers. This was in contrast to the fact that the angiogenesis was promoted at an H ₂ O ₂ concentration of about 16 μM as confirmed in Example 5 above.

Example  7. Mg- Ti / TiO ₂ / Au metal complex H ₂ O ₂  Production control

(One) TiO ₂ Layer  Thickness H ₂ O ₂ Occurrence amount  control

In order to control the concentration of H ₂ O ₂ generated in the Mg-Ti / TiO ₂ / Au metal complex, the amount of H ₂ O ₂ generated according to the thickness of TiO ₂ was measured.

FIG. 7A shows the result of quantitatively measuring the concentration of H ₂ O ₂ with DPD / POD according to the discharge time at various thicknesses of various TiO ₂ layers, which are variously generated according to the anodizing potential. FIG. Represents the concentration of H ₂ O ₂ with anodizing potential. That is, it was confirmed that different concentrations of H ₂ O ₂ were produced at the same discharge time depending on the thickness of the TiO ₂ layer formed differently depending on the anode treatment potential.

Therefore, it was confirmed that the concentration of H ₂ O ₂ can be controlled by adjusting the thickness of the TiO ₂ layer. When an amorphous TiO ₂ layer of about 90 nm was formed by anodic oxidation treatment at 50 V potential for 30 seconds, it was confirmed that 16 μM of H ₂ O ₂ was spontaneously generated after 5 minutes of binding to the Mg anode.

(2) Au layer  Thickness H ₂ O ₂ Occurrence amount  control

In order to control the concentration of H ₂ O ₂ generated in the Mg-Ti / TiO ₂ / Au metal complex, the amount of H ₂ O ₂ generated according to the thickness of the Au layer was measured.

FIG. 8A shows the results of quantitatively measuring the concentration of H ₂ O ₂ with DPD / POD depending on the discharge time at various thicknesses of Au layers formed by Au sputtering time, FIG. 8B shows a result of sputtering time of 20 seconds, And the H ₂ O ₂ concentration according to the gold plasma current when the discharge time is 60 minutes. As shown in FIG. 8, it was confirmed that different concentrations of H ₂ O ₂ were produced at the same discharge time depending on the thickness of the Au layer formed differently according to the conditions of the sputtering. When Au was deposited for 20 seconds at a current density of 10 mA on a 20 nm thick TiO2 layer, it was confirmed that H ₂ O ₂ was generated at a cumulative concentration of about 16 μM after 5 minutes of bonding with the Mg anode.

(3) a separate &lt; RTI ID = 0.0 &gt; TiO ₂ layer  Due to formation H ₂ O ₂ Occurrence amount  control

A metal complex as shown in FIG. 9A was designed to control the amount of H ₂ O ₂ generation depending on the position of the Mg-Ti / TiO ₂ / Au metal complex.

As was confirmed in Example 6, by using H ₂ O ₂ generated in the 90 nm TiO ₂ layer higher than H ₂ O ₂ generated in the 200 nm TiO ₂ layer, 90 nm and 200 Mg / Ti / TiO ₂ / Au metal complexes having different TiO ₂ layers were prepared.

As a result, H ₂ O ₂ was confirmed to have more than 1.5 times the H ₂ O ₂ selectively generated compared to the area of 200 nm in 90 nm TiO ₂ layer region. This can be applied to the development of a selective tissue regeneration inducing type implant that generates H ₂ O ₂ at different concentrations aiming at a specific tissue damaged as shown in FIG. 9B.

Example  8. Mg- Ti / TiO ₂ / Au Metal Composite and Its Vascular Neovascularization Effect

Based on the angiogenic effects of the Mg-Ti / TiO ₂ / Au metal complex, novel Ti-based implant prototypes were prepared as shown in FIGS. 10a and 10b. Anodic oxidation was carried out at a voltage of 10 V for 30 seconds to form a TiO2 layer with a thickness of about 20 nm and thereafter an Au cluster with a size of about 3 nm was deposited thereon. In EBM-2 medium, the implant prototype was found to generate about 15 μM H ₂ O ₂ after 15 min. In particular, the Mg cylinder was physically integrated within the screw-shaped Ti-6-4 alloy. The Ti-6-4 alloy is a common material used in orthopedic and dental implants. The surface of the Ti alloy was oxidized in the same manner as in Production Example 1, and was deposited with Au. Electrons generated in Mg can be easily transferred to the surface of Ti without the connection of Mg and Ti by a separate metal conduit or the like. The prepared implant prototype was tested for its angiogenic effect in EMB-2 medium containing HUVEC.

Figure 10C shows EBM-2 medium in which the implant prototype is incubated. FIG. 10D is a graph showing the number of blood vessel branching per unit area according to the time of contact of the HUVEC cells with the implant prototype. FIG. FIG. 10E is an image (HUVEC) of blood vessel formation observed with a fluorescent microscope (scale bar = 500 μm) according to the time of contact with the implant prototype. High level of angiogenesis of HUVEC stimulated by the implant prototype was observed compared to the negative control, and angiogenesis was most frequently observed in the case of incubation for 15 minutes in contact with the above implanted prototype.

Manufacturing example  2. Preparation of metal complex using zinc

A metal complex was prepared in the same manner as described in Preparation Example 1, except that zinc was used as a positive electrode instead of magnesium.

Example  9. Using zinc Immersion  Test result

To determine if zinc could be held for more than 2 weeks after insertion into the body, a pure zinc bar was inserted into the Ti-6Al-4V coin and immersed in Hanks solution for 5 days (120 hours) Respectively.

As a result, as shown in Fig. 11, even though the immersion test was conducted for 5 days, it was found that at least 80% of zinc remained. The metal in the middle portion of FIG. 11 is zinc and the portion surrounding it is a Ti-6Al-4V alloy, and the dotted line at the top represents the initial surface of zinc.

Example  10. Measurement of active oxygen production using metal complex containing zinc

Similar to the method described in Example 4, the amount of active oxygen generated in the PBS buffer solution (2 ml) was measured three times by inserting a zinc bar into a Ti-6Al-4V disk (? 10 x 2T).

As a result, it was observed that the amount of H ₂ O ₂ produced in the PBS buffer increased in all three tests as shown in FIG. Especially, the concentration of H ₂ O ₂ after 30 minutes was measured to be about 33 μM on the average, and the effect was more excellent than that of Mg.

Claims (21)

delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete Producing a positive electrode comprising a first metal;
Producing a negative electrode comprising a second metal;
Oxidizing the second metal to form a second metal oxide layer;
Forming an Au layer on the oxidized second metal layer; And
And electrically connecting the positive electrode and the negative electrode, wherein the connecting step connects the positive electrode into the negative electrode in an integrated manner, the method comprising:
The second metal oxide layer has a thickness of 20 nm to 10 mu m,
Wherein the Au layer has a thickness of from 1.5 nm to 25 nm. 21. The method of claim 20, wherein forming the second metal oxide layer comprises forming separate second metal oxide layers having different thicknesses on different portions.

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