Porous TiO2 coating capable of resuscitating mitochondrial function of macrophages and further promoting M2 polarization of macrophages and preparation method of porous TiO2 coating
By preparing porous TiO2 coating, microarc oxidation and magnesium thermal reduction reactions, the problems of mitochondrial function resuscitation and low M2 polarization efficiency of macrophages were solved, rapid resuscitation of mitochondrial function and promotion of M2 polarization were achieved, and osteointegration and anti-inflammatory ability were improved.
Patent Information
- Application Number
- CN202510319662.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-03-18
- Publication Date
- 2025-06-13
AI Technical Summary
The prior art is difficult to quickly resuscitate the mitochondrial function of macrophages, resulting in inefficient polarization of macrophages M2.
Porous TiO2 coating was used to prepare a porous TiO2 coating rich in oxygen vacancies and Mg2+ doped porous TiO2 coating through microarc oxidation treatment and magnesium thermal reduction reaction, which promoted the increase of mitochondrial respiration in macrophages and increased membrane potential, thereby achieving rapid M2 polarization of macrophages.
The rapid resuscitation of mitochondrial function of macrophages has been achieved, the M2 polarization of macrophages has been promoted, the osteointegration and anti-inflammatory ability have been improved, and the new osteogenesis and bone mass have been enhanced.
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Figure CN120138753A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of biomaterials, and particularly relates to a porous TiO coating that can revive the mitochondrial function of macrophages and thus promote the M2 polarization of macrophages 2 and a preparation method thereof. Background Art
[0002] Mitochondria are key organelles that regulate macrophage polarization. M1 macrophages and M2 macrophages are the two main subtypes of macrophages. Among them, M1 macrophages mainly obtain energy through the glycolytic pathway. By decomposing glucose, pyruvate is converted into lactate under the action of pyruvate dehydrogenase kinase 1 (PDK1), inhibiting the tricarboxylic acid cycle, while weakening the cellular oxidative respiratory chain and increasing reactive oxygen species (ROS) and nitric oxide (NO). On the contrary, M2 macrophages mainly obtain energy through the oxidative phosphorylation pathway, promoting pyruvate to enter the mitochondria, and finely coordinating the synthesis of the electron transport chain (ETC) and adenosine triphosphate (ATP) on the inner mitochondrial membrane (IMM). This process is accompanied by the transfer of electrons, the delivery of H, and the formation of membrane potential. It can be seen that maintaining or even improving the stability of mitochondrial metabolism, that is, maintaining mitochondrial status, respiratory function, and membrane potential, is a prerequisite for ensuring the normal function of M2 macrophages.
[0003] The existing regulation of mitochondrial metabolism is mainly based on aerobic exercise, nutritional support therapy, and the use of supplements. For example, powerful antioxidants such as coenzyme Q10 and resveratrol can protect mitochondria and enhance their function. NAD+ supplements such as nicotinamide adenine dinucleotide (NAD+) help to enhance mitochondrial energy metabolism, and mitochondrial-targeted drugs can also be used to improve the efficiency of intracellular energy production. In addition, molecular hydrogen is also considered to be applicable to the regulation of mitochondrial metabolism due to its antioxidant and anti-inflammatory properties.
[0004] Mitochondria are considered to be key organelles that regulate macrophage polarization, and they can play a role in regulating macrophage polarization by affecting the cell metabolism mode. However, the existing methods for regulating mitochondria have problems such as a long period and many uncontrollable factors.
[0005] Therefore, there is an urgent need for a method that can quickly revive mitochondrial function to achieve M2 polarization of macrophages to solve the above problems. Summary of the Invention
[0006] To solve the above technical problems, the present invention proposes a porous TiO that can revive the mitochondrial function of macrophages and thus promote the M2 polarization of macrophages 2Coating and its preparation method.
[0007] To achieve the above object, the present invention provides the following technical solutions:
[0008] The present invention provides a preparation method of a porous TiO coating that can revive the mitochondrial function of macrophages and thereby promote the M2 polarization of macrophages, including the following steps: 2 (1) Using a titanium substrate as the anode, performing micro-arc oxidation treatment in an electrolyte containing calcium acetate and β-glycerophosphate sodium hydrate to obtain a TiO coating;
[0009] (1) Using a titanium substrate as the anode, performing micro-arc oxidation treatment in an electrolyte containing calcium acetate and β-glycerophosphate sodium hydrate to obtain a TiO coating; 2 coating;
[0010] (2) Annealing the TiO coating obtained in step (1) and Mg powder under a protective atmosphere to obtain the porous TiO coating that can revive the mitochondrial function of macrophages and thereby promote the M2 polarization of macrophages; the holding time of the annealing treatment is 55 - 65 min. 2 (2) Annealing the TiO coating obtained in step (1) and Mg powder under a protective atmosphere to obtain the porous TiO coating that can revive the mitochondrial function of macrophages and thereby promote the M2 polarization of macrophages; the holding time of the annealing treatment is 55 - 65 min. 2 coating; the holding time of the annealing treatment is 55 - 65 min.
[0011] Technical principle: The present invention first prepares a TiO coating with a microporous structure by performing micro-arc oxidation treatment on a titanium substrate using an electrolyte containing calcium acetate and β-glycerophosphate sodium hydrate, and then uses a magnesiothermic reduction reaction to control the holding time of the reduction reaction simultaneously, obtaining a porous TiO coating rich in oxygen vacancy defects and doped with Mg. The obtained porous TiO coating can achieve the release of Mg and electron transfer, thereby increasing the mitochondrial respiration volume of macrophages, raising the membrane potential, promoting ATP synthesis, and thus realizing the rapid M2 polarization of macrophages. 2 Technical principle: The present invention first prepares a TiO coating with a microporous structure by performing micro-arc oxidation treatment on a titanium substrate using an electrolyte containing calcium acetate and β-glycerophosphate sodium hydrate, and then uses a magnesiothermic reduction reaction to control the holding time of the reduction reaction simultaneously, obtaining a porous TiO coating rich in oxygen vacancy defects and doped with Mg. The obtained porous TiO coating can achieve the release of Mg and electron transfer, thereby increasing the mitochondrial respiration volume of macrophages, raising the membrane potential, promoting ATP synthesis, and thus realizing the rapid M2 polarization of macrophages. 2+ coating, 2 the obtained porous TiO coating 2 can achieve the release of Mg 2+ and electron transfer, thereby increasing the mitochondrial respiration volume of macrophages, raising the membrane potential, promoting ATP synthesis, and thus realizing the rapid M2 polarization of macrophages.
[0012] Further, in step (1), the arc frequency of the micro-arc oxidation treatment is 100 Hz, the positive voltage is 400 V, and the negative voltage is 50 V.
[0013] Further, in step (2), the mass ratio of the Mg powder to the surface area of the TiO coating is 0.2 g∶153.94 mm 2 coating is 0.2 g∶153.94 mm 2 .
[0014] Further, in step (2), the heating process of the annealing treatment is: heating from room temperature to 650 °C in 90 min, then holding for a period of time, and then cooling to room temperature with the furnace.
[0015] Further, in step (2), the holding time of the annealing treatment is 60 min.
[0016] The present invention provides a porous TiO coating prepared by the preparation method described in the above technical solution, which can revive the mitochondrial function of macrophages and thereby promote the M2 polarization of macrophages. 2 coating.
[0017] Compared with the prior art, the present invention has the following advantages and technical effects:
[0018] When macrophages are inoculated on the surface of the porous TiO coating provided by the present invention and ultrasonic treatment is applied for 2 minutes, it can cause changes in the mitochondrial function of macrophages and thereby polarize them towards M2, achieving the purpose of rapidly reviving mitochondrial function. 2
[0019] The porous TiO coating provided by the present invention has the dual effects of promoting the M2 polarization of macrophages and improving bone integration, and has strong immune regulation ability under ultrasonic mediation; after implantation, it can significantly reduce the expression of inflammatory factors through ultrasound, improve the anti-inflammatory ability, and at the same time enhance mitochondrial respiration and membrane potential MMP, promoting new bone formation and increasing bone mass. 2 BRIEF DESCRIPTION OF THE DRAWINGS
[0020] The drawings constituting a part of the present invention are used to provide a further understanding of the present invention. The schematic embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation to the present invention. In the drawings:
[0021] Figure 1 is the TEM image of the coatings prepared in Example 1 and Comparative Examples 1-2;
[0022] Figure 2 is the expression of Arg-1 of macrophages inoculated on the coating surface;
[0023] Figure 3 is the expression of iNOS of macrophages inoculated on the coating surface;
[0024] Figure 4 is the fluorescence imaging diagram of MAO+U and BT-60+U after JC-1 staining;
[0025] Figure 5 is the Micro-CT imaging examination (A), hard tissue section VG staining (B) of the femurs of rats in the MAO group, BT-20 group, BT-60 group and BT-120 group, and the double fluorescence labeling diagram of the bone around the implant under a laser confocal microscope (C);
[0026] Figure 6 Micro-CT imaging examinations (A), VG staining of hard tissue sections (B), and double fluorescence labeling maps of the bone around the implant under a laser confocal microscope (C) for the MAO+U group, BT-20+U group, BT-60+U group, and BT-120+U group. Detailed implementation manners
[0027] The following will clearly and completely describe the technical solutions in the embodiments of the present invention with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.
[0028] To make the above objects, features, and advantages of the present invention more obvious and understandable, the present invention will be further described in detail below with reference to the accompanying drawings and specific implementation manners.
[0029] The embodiments of the present invention provide a preparation method for a porous TiO coating that can resuscitate the mitochondrial function of macrophages and thus promote the M2 polarization of macrophages, including the following steps: 2 (1) Using a titanium substrate as the anode, performing micro-arc oxidation treatment in an electrolyte containing calcium acetate and β-glycerophosphate sodium hydrate to obtain a TiO coating;
[0030] (2) Annealing the TiO coating obtained in step (1) and Mg powder under a protective atmosphere to obtain the porous TiO coating that can resuscitate the mitochondrial function of macrophages and thus promote the M2 polarization of macrophages. 2 coating;
[0031] (2) Annealing the TiO coating obtained in step (1) and Mg powder under a protective atmosphere to obtain the porous TiO coating that can resuscitate the mitochondrial function of macrophages and thus promote the M2 polarization of macrophages. 2 coating; 2 coating.
[0032] In a preferred embodiment, in step (1), the titanium substrate is a pure titanium disc with a size of Φ14×2 mm; before the micro-arc oxidation treatment, the titanium substrate further includes being polished with 400-mesh and 1500-mesh sandpapers respectively, then ultrasonically cleaned in absolute ethanol and deionized water for 10 min each, and then dried.
[0033] In a preferred embodiment, in step (1), the concentration of calcium acetate in the electrolyte is 0.22 mol / L.
[0034] In a preferred embodiment, in step (1), the concentration of β-glycerophosphate sodium hydrate in the electrolyte is 0.02 mol / L; the β-glycerophosphate sodium hydrate is β-glycerophosphate pentahydrate (β-GP).
[0035] In a preferred embodiment, in step (1), the electrolyte is composed of calcium acetate, sodium β-glycerophosphate hydrate, and sodium hydroxide; the concentration of sodium hydroxide in the electrolyte is 0.005 mol / L.
[0036] In a preferred embodiment, in step (1), the arc frequency of the micro-arc oxidation treatment is 100 Hz, the positive voltage is 400 V, the negative voltage is 50 V, and the duty cycle is 7.5%; the micro-arc oxidation treatment uses a DC pulse power supply, and the stainless steel pot is used as the cathode. In the present invention, the process parameters of the micro-arc oxidation treatment are controlled within the above range, which is beneficial to obtaining a TiO 2 coating with a microporous structure.
[0037] In a preferred embodiment, in step (1), during the micro-arc oxidation treatment, a cooling circulating water system is used to control the temperature of the electrolyte at 20-30 °C.
[0038] In a preferred embodiment, in step (2), the mass ratio of the Mg powder to the surface area of the TiO 2 coating is 0.2 g∶153.94 mm 2 .
[0039] In a preferred embodiment, in step (2), the flow rate of the protective atmosphere is 300 sccm; the heating process of the annealing treatment is as follows: from room temperature, it is heated to 650 °C in 90 min, then held for a period of time, and then cooled to room temperature with the furnace.
[0040] In a preferred embodiment, in step (2), the holding time of the annealing treatment is 55-65 min, and more preferably 60 min. During the annealing treatment, a magnesium thermal reduction reaction occurs, realizing the doping of Mg 2+ , and at the same time, oxygen vacancy defects are formed. The holding time of the annealing treatment is a key factor affecting the effect of promoting macrophage M2 polarization of the porous TiO 2 coating. Too short or too long holding time of the annealing treatment is not conducive to achieving a better M2 polarization effect.
[0041] The present invention provides a porous TiO 2 coating prepared by the preparation method described in the above technical solution, which can recover the mitochondrial function of macrophages and thus promote macrophage M2 polarization.
[0042] In the embodiments of the present invention, room temperature refers to "25±2 °C".
[0043] Unless otherwise specified, the raw materials in the embodiments of the present invention are all obtained through commercial channels.
[0044] Example 1
[0045] A preparation method of a porous TiO coating that can resuscitate macrophage mitochondrial function and thus promote macrophage M2 polarization, the specific steps are as follows: 2 The coating preparation method is as follows:
[0046] (1) First, use 400-mesh and 1500-mesh sandpapers to polish a pure titanium disc with a size of Φ14×2 mm. Subsequently, ultrasonically clean it in absolute ethanol and deionized water for 10 min each, and then put it into an oven to dry. Using a DC pulse power supply, with the treated pure titanium disc as the anode and a stainless steel pot as the cathode, perform micro-arc oxidation treatment in an electrolyte containing 0.22 mol / L calcium acetate (Ca(CH 3 COO) 2 )), 0.02 mol / L β-glycerophosphate pentahydrate (β-GP), and 0.005 mol / L sodium hydroxide. Set the micro-arc oxidation treatment parameters as follows: arc frequency 100 Hz, positive voltage 400 V, negative voltage 50 V, duty cycle 7.5%. During the micro-arc oxidation treatment, use a cooling circulating water system to control the temperature of the micro-arc oxidation electrolyte at 20 °C. After the micro-arc oxidation treatment, obtain a TiO 2 coating, denoted as MAO.
[0047] (2) Put the TiO 2 coating obtained in step (1) into a crucible, add 0.2 g of Mg powder to the crucible, put the crucible into a tube furnace, and then perform annealing treatment under an Ar atmosphere with a flow rate of 300 sccm. Set the heating curve of the tube furnace as follows: heat from room temperature to 650 °C in 90 min, hold for 60 min, and cool to room temperature with the furnace. Obtain a porous TiO 2 coating that can resuscitate macrophage mitochondrial function and thus promote macrophage M2 polarization, denoted as BT-60.
[0048] Comparative Example 1
[0049] The difference from Example 1 is only that in step (2), the holding time is 20 min, and the others are the same as in Example 1, denoted as BT-20.
[0050] Comparative Example 2
[0051] The difference from Example 1 is only that in step (2), the holding time is 120 min, and the others are the same as in Example 1, denoted as BT-120.
[0052] Figure 1 FIG. is the TEM images of the coatings prepared in Example 1 and Comparative Examples 1-2. Among them, a is MAO in step (1) of Example 1, b is BT-60 prepared in Example 1, c is BT-20 prepared in Comparative Example 1, and d is BT-120 prepared in Comparative Example 2. From Figure 1It can be seen that the surface morphologies of the four groups of coatings, namely MAO, BT-20, BT-60, and BT-120, are basically the same, all being porous structures. The periphery of the large pores is crater-shaped, and the pore size ranges from 1 to 4 μm. The results show that a rough and porous TiO 2 coating is prepared on the titanium substrate surface after micro-arc oxidation, with pores of micron size evenly distributed, and the magnesiothermic reduction reaction has little effect on the surface morphology of the coating.
[0053] Macrophages RAW264.7 were respectively inoculated on the surfaces of the coatings prepared in Example 1 and Comparative Examples 1-2, and then ultrasonic treatment (2.5 W·cm -2 , 1 MHz, 2 min) was carried out. The polarization of macrophages was evaluated by immunofluorescence staining, and the results are shown in Figure 2 and Figure 3 .
[0054] Figure 2 shows the Arg-1 expression of macrophages inoculated on the coating surface, Figure 3 shows the iNOS expression of macrophages inoculated on the coating surface. In Figure 2 and Figure 3 , a is MAO in step (1) of Example 1, b is BT-20 prepared in Comparative Example 1, c is BT-60 prepared in Example 1, d is BT-120 prepared in Comparative Example 2, e is MAO after ultrasound (MAO+U), f is BT-20 after ultrasound (BT-20+U), g is BT-60 after ultrasound (BT-60+U), h is BT-120 after ultrasound (BT-120+U). Figure 2 and Figure 3 , red represents Arg-1 and iNOS respectively. From Figure 2 and Figure 3 , it can be seen that after ultrasound, the iNOS expression of macrophages in the BT-60 coating group (BT-60+U) is greatly reduced compared with the MAO+U group and the BT-20+U group, and the Arg-1 expression is increased compared with the other three groups, indicating that under the combined action of electron transition and magnesium ion release on the titanium dioxide surface, BT-60+U greatly reduces the expression of inflammation, improves the anti-inflammatory ability, and macrophages tend to M2 polarization, indicating that BT-60+U has the best mitochondrial resuscitation function.
[0055] JC-1 was used to evaluate the mitochondrial membrane potential of MAO+U and BT-60+U. JC-1 is a dual-emission fluorescent dye, existing in the form of monomers and aggregates. At low mitochondrial membrane potential, JC-1 exists in the monomer form and emits green fluorescence, while at high membrane potential, JC-1 forms aggregates and emits red fluorescence. The results of the mitochondrial membrane potential are shown in Figure 4 .
[0056] Figure 4 Fluorescence imaging diagrams of MAO+U and BT-60+U after JC-1 staining. Among them, A is the fluorescence imaging diagram of MAO+U with JC-1 aggregates, JC-1 monomers, and co-staining of JC-1 aggregates and JC-1 monomers, and B is the fluorescence imaging diagram of BT-60+U with JC-1 aggregates, JC-1 monomers, and co-staining of JC-1 aggregates and JC-1 monomers. From Figure 4 it can be seen that the BT-60+U group showed a higher red / green fluorescence intensity ratio, indicating that BT-60+U maximally maintained the mitochondrial membrane potential, indicating that BT-60 can play a role in resuscitating mitochondrial function and exhibited excellent regenerative performance at the cellular level.
[0057] The coating materials prepared in Example 1 and Comparative Examples 1-2 were implanted into the femurs of rats and grouped as (n = 3): MAO group, BT-20 group, BT-60 group, BT-120 group, MAO+U group, BT-20+U group, BT-60+U group, BT-120+U group. The MAO group, BT-20 group, BT-60 group, and BT-120 group were the groups without ultrasonic treatment, and the MAO+U group, BT-20+U group, BT-60+U group, and BT-120+U group were the groups with ultrasonic treatment. The ultrasonic treatment was performed on the 2nd and 4th days after surgery (2.5 W·cm -2 , 1 MHz, 15 min). After 4 weeks of surgery, samples were taken for Micro-CT imaging examination, hard tissue sectioning, and double fluorescence staining. Calcein and tetracycline were used as staining agents for double fluorescence staining. The results are shown in Figure 5 and Figure 6 .
[0058] Figure 5 Micro-CT imaging examination (A), VG staining of hard tissue sections (B), and double fluorescence labeling diagrams of the bone around the implant under a laser confocal microscope (C) of the femurs of rats in the MAO group, BT-20 group, BT-60 group, and BT-120 group. From Figure 5 it can be seen that without ultrasonic action, compared with other coatings, the yellow area of the BT-60 coating was the largest, that is, the most bone mass was generated; from the VG staining of the hard tissue sections, it can be seen that the tissues around the BT-60 coating and the BT-120 coating had a larger red area, indicating that these two coatings had a higher bone bonding rate. The red fluorescence band labeled with tetracycline and the green fluorescence band labeled with calcein represent bone mineralization 13 days and 3 days before sacrifice, respectively. The distance between them represents the new bone formation rate of the implant 10 days after being implanted into the body. The larger the distance, the greater the new bone formation rate, corresponding to the best bone regeneration ability. The results showed that the BT-60 coating had the largest new bone formation rate.
[0059] Figure 6Micro-CT imaging examinations (A), VG staining of hard tissue sections (B), and double fluorescence labeling maps of the bone around the implant under a laser confocal microscope (C) for the MAO+U group, BT-20+U group, BT-60+U group, and BT-120+U group. From Figure 6 It can be seen that under ultrasonic conditions, the yellow area of the BT-20+U group and the BT-60+U group is larger, that is, the most bone mass is generated; the distance between the double fluorescences of the BT-60+U coating is significantly greater than that of other coatings, that is, the new bone formation rate of BT-60+U is the largest, indicating the best bone regeneration ability.
[0060] Based on the above results, it can be known that the porous TiO 2 coating can transform macrophages from a pro-inflammatory state to an anti-inflammatory state, and then has the best bone regeneration ability, playing a key role in promoting macrophage M2 polarization and then regulating the bone integration process.
[0061] The above is only a preferred specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention should be covered by the protection scope of the present invention. Therefore, the protection scope of the present invention should be subject to the protection scope of the claims.
Claims
1. A method for preparing a porous TiO2 coating that can revive macrophage mitochondrial function and promote macrophage M2 polarization, characterized in that: The following steps are involved: (1) using a titanium substrate as an anode, performing micro-arc oxidation treatment in an electrolyte containing calcium acetate and sodium β-glycerophosphate hydrate to obtain a TiO2 coating; (2) Annealing the TiO2 coating and Mg powder obtained in step (1) under a protective atmosphere to obtain the porous TiO2 coating that can revive the mitochondrial function of macrophages and promote the polarization of macrophage M2; the annealing treatment has a holding time of 55 to 65 minutes.
2. The preparation method according to claim 1, characterized in that: In step (1), the arc frequency of the micro-arc oxidation treatment is 100 Hz, the positive voltage is 400 V, and the negative voltage is 50 V.
3. The preparation method according to claim 1, characterized in that: In step (2), the ratio of the mass of the Mg powder to the surface area of the TiO2 coating is 0.2 g: 153.94 mm 2 .
4. The preparation method according to claim 1, characterized in that: In step (2), the heating process of the annealing treatment is: heating from room temperature to 650° C. over 90 minutes, then keeping the temperature for a period of time, and then cooling to room temperature in the furnace.
5. The preparation method according to claim 1, characterized in that: In step (2), the holding time of the annealing treatment is 60 minutes.
6. A porous TiO2 coating prepared by the preparation method according to any one of claims 1 to 5, which can restore the mitochondrial function of macrophages and promote the polarization of macrophage M2.