An antibacterial bone-promoting metal implant and its preparation method and application

Abstract

This invention discloses an antibacterial osteogenic metal implant, its preparation method, and its application. The antibacterial osteogenic metal implant is a metal implant surface loaded with an exosome carrier containing antibacterial components and a core-shell structure. The exosome carrier with the core-shell structure consists of a core portion and a shell portion; the core portion is composed of exosomes loaded with a drug; the shell portion is an inorganic salt containing calcium and phosphorus; the loading amount of the exosome carrier containing antibacterial components on the metal implant surface is 90-100%. The preparation method includes the following steps: first, preparing the exosome carrier containing antibacterial components and a core-shell structure; oxidizing the metal implant, then mixing it with the exosome carrier containing antibacterial components and a core-shell structure, followed by vacuum drying or incubation to obtain the antibacterial osteogenic metal implant. This invention is applied in the preparation of orthopedic and / or dental metal implants, and has the effects of enhancing the fusion of bone tissue with the implant surface and inhibiting and killing bacteria.

Description

Technical Field

[0001] The invention relates to an antibacterial bone-promoting metal implant and a preparation method and application thereof, and belongs to the field of biomedicine and biomaterials. Background Art

[0002] Effective reconstruction of bone defects is a challenging problem in orthopedic surgery. Autologous bone transplantation and allogeneic bone transplantation are currently the most widely used methods for repairing bone defects. However, for autologous grafts, they are limited by the source of donors and do not match the defect site. The additional damage to the donor site and the frequent need for secondary surgery inhibit autologous bone transplantation; allogeneic and xenografts have the risk of disease transmission and immune rejection, and also face problems such as high failure rate and poor mechanical stability. At the same time, the implantation of stem cells, such as bone marrow mesenchymal stem cells and adipose-derived stem cells, is also considered as an alternative strategy. However, the implantation of stem cells faces major challenges, including immune rejection, teratoma formation, and undirected cell differentiation. Metal bone implants such as titanium alloys have been used in clinical practice. However, metal implants are inert and occupy the space for bone growth. In addition, fibrous tissue is easily formed around the implant after implantation, making the implant and bone not firmly integrated, affecting the stability of implant use. In addition, biomedical device-related infections caused by bacteria have led to a large number of implant treatment failures. Severe infections can lead to complications such as pain, loss of function, systemic diseases, and even death, and the original metal implants have no antibacterial properties. After the infection occurs, it is often necessary to remove the implant and perform surgical debridement. In secondary revision surgeries caused by bacterial infections, after the old prosthesis is removed, the infectious bacteria may still remain in the host. Once the new implant is re-implanted, these residual bacteria will quickly adhere to the implant surface, proliferate rapidly, and eventually form a dense bacterial film, hindering subsequent osteogenesis.

[0003] Surface modification of metal implants has been widely studied in order to effectively improve the osteogenic and antibacterial properties of implants. Previous studies have successfully loaded many antibiotics or antimicrobial peptides onto the surface of implants for antibacterial applications. However, bacterial resistance and rapid drug release limit their application. In addition, the drug retention rate is low, requiring high doses of drugs, which affects the safe application of drugs. Summary of the invention

[0004] The purpose of the present invention is to provide an antibacterial bone-promoting metal implant and a preparation method and application thereof.

[0005] The present invention loads an exosome carrier with a core-shell structure containing antibacterial components on the surface of a metal implant. It is the first time that exosomes are used as a sustained-release carrier of antibacterial components and for antibacterial purposes on the surface of a metal implant, which can greatly promote surface bone integration and prevent and treat implant infection.

[0006] The present invention provides an antibacterial bone-promoting metal implant, which is a metal implant surface loaded with an exosome carrier having a core-shell structure and containing antibacterial components;

[0007] The exosome carrier containing antibacterial components and having a core-shell structure is composed of a core part and a shell part; the core part is composed of exosome-loaded drugs; and the shell part is an inorganic salt containing calcium and phosphorus;

[0008] The loading amount of the exosome carrier with a core-shell structure and containing antibacterial components on the surface of the metal implant can be 90-100%.

[0009] In the present invention, the metal implant can be any conventional metal implant in the art.

[0010] In the above-mentioned antibacterial bone-promoting metal implant, the thickness of the shell portion may be 100 nm to 200 nm, specifically 110 nm to 200 nm;

[0011] The drug loading amount of the exosome-loaded drug may be 10% to 50%.

[0012] In the above-mentioned antibacterial bone-promoting metal implant, the exosomes are derived from cells and / or body fluids;

[0013] The cells include at least one of human, rat or mouse stem cells, macrophages, endothelial cells and epithelial cells; the stem cells include at least one of adipose mesenchymal stem cells, bone marrow mesenchymal stem cells, induced pluripotent stem cells and human umbilical cord mesenchymal stem cells;

[0014] The drug includes at least one of antibiotics, nucleic acids, proteins and polypeptides;

[0015] The inorganic salt containing calcium and phosphorus includes calcium phosphate.

[0016] The method for preparing the antibacterial bone-promoting metal implant provided by the present invention comprises the following steps: 1) co-incubating or ultrasonicating the exosomes and the drug in a cell culture medium, and then centrifuging; taking the precipitate after centrifugation and adding an inorganic salt solution containing calcium and phosphorus to continue incubation, so as to obtain the exosome carrier containing antibacterial components and having a core-shell structure;

[0017] 2) The metal implant is anodized, and then mixed with the exosome carrier with a core-shell structure and containing antibacterial components, and vacuum dried or incubated to obtain the antibacterial bone-promoting metal implant.

[0018] In the above-mentioned preparation method, the exosomes are prepared as follows: cells and / or body fluids are subjected to differential centrifugation to collect the exosomes;

[0019] The incubation time of the exosomes and the drug may be 1 to 5 hours, specifically 2 hours, 1 to 2 hours, 2 to 5 hours or 1.5 to 4.5 hours, and the reaction temperature may be 37°C;

[0020] The ultrasound is performed according to a method comprising the following steps: the exosomes are mixed with the drug, and an ultrasound probe is immersed in the mixed solution for ultrasound under ice bath conditions; the ultrasound is performed for 10 cycles, and the setting conditions are 500v, 2kHz;

[0021] The centrifugal force of the centrifugation may be 5000 g to 20000 g, and the centrifugal time may be 5 to 20 min.

[0022] In the above preparation method, the incubation time in the calcium-phosphorus-containing inorganic salt solution can be 1 to 5 hours, and the reaction temperature can be 37° C.;

[0023] The concentration of the exosomes may be 0.1 mg / ml to 100 mg / ml;

[0024] The concentration of the drug may be 0.1 mg / ml to 100 mg / ml;

[0025] The concentration of the calcium-phosphorus-containing inorganic salt solution may be 1 mmol / ml to 100 mol / ml;

[0026] The mass ratio of the exosomes, the drug, and the calcium-phosphorus-containing inorganic salt solution may be 1-3:1:0.001-3, specifically 1:1:1.11, 1-1.5:1:0.001-1.5, 1-2:1:0.001-2, or 1-2.5:1:0.01-1.5.

[0027] In the above preparation method, in step 1), after incubation in the inorganic salt solution containing calcium and phosphorus, the following post-treatment steps are also included: centrifugation and / or washing the precipitate with a buffer solution;

[0028] The centrifugal speed is specifically 5000g-20000g, and the centrifugal time can be 5-20min;

[0029] The buffer specifically includes physiological saline and / or PBS buffer;

[0030] The washing times are 1 to 6 times.

[0031] In the above-mentioned preparation method, in step 2), the metal implant is anodized by electrolysis;

[0032] The electrolysis method is specifically a constant potential electrolysis, and the conditions are as follows: the voltage can be 10-80V, the electrolyte is an ethylene glycol electrolyte of NH4F, and the concentration thereof can be 0.1wt%-1wt%.

[0033] In the above preparation method, in step 2), the incubation time is 0.5 to 5 hours, specifically 1 hour, 0.5 to 1 hour, 1 to 5 hours, 0.5 to 3 hours or 1 to 4 hours, and the temperature is 24 to 30°C, specifically 25°C.

[0034] The antibacterial bone-promoting metal implant of the present invention is used in preparing orthopedic and / or dental metal implants.

[0035] The antibacterial bone-promoting metal implant of the present invention can be directly used as an orthopedic and / or dental metal implant.

[0036] The present invention has the following advantages:

[0037] 1. Compared with ordinary titanium stent implants, the core-shell structure carrier-coated stent containing a calcium phosphate shell and a drug-loaded exosome core has the effect of enhancing the fusion between bone tissue and the implant surface and inhibiting bacteria and killing bacteria;

[0038] 2. Anodized nanotubes have the dual functions of loading exosome carriers and promoting osteogenesis;

[0039] 3. The core-shell structure carrier coating has the characteristic of pH-responsive release of antibacterial components, which is slowly released under normal physiological conditions and rapidly released under infection conditions; the calcium phosphate shell can promote bone repair and reconstruction.

[0040] 4. The proteins, RNA and DNA carried by exosomes themselves promote osteogenesis. At the same time, not using stem cells cultured in vitro for bone regeneration can avoid the immune rejection reaction caused by the implantation of foreign cells into the body. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 The drug release of the core-shell exosome-coated metal implant of the present invention under different pH PBS conditions.

[0042] Figure 2 This is the in vitro antibacterial experiment of the core-shell exosome-coated metal implant of the present invention in Escherichia coli. Figure 2 A represents titanium, B represents anodized titanium nanotubes, and C represents anodized titanium with a core-shell structure.

[0043] Figure 3 This is an in vitro antibacterial experiment of the core-shell structure exosome-coated metal implant of the present invention on Staphylococcus aureus. Figure 3 A represents titanium, B represents anodized titanium nanotubes, and C represents anodized titanium with a core-shell structure.

[0044] Figure 4 This is a cytotoxicity experiment of the core-shell structure exosome-coated metal implant of the present invention.

[0045] Figure 5 This is an in vitro osteogenic differentiation experiment of the shell-core structure exosome-coated metal implant of the present invention. DETAILED DESCRIPTION

[0046] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0047] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0048] Example

[0049] 1. Extraction of exosomes from bone marrow mesenchymal stem cells

[0050] Bone marrow mesenchymal stem cells were cultured with α-MEM medium (Gibco, catalog number: 12571063) containing 10% fetal bovine serum and 1% antibiotics. The cells were placed in a 37°C, 5% carbon dioxide incubator. After the cells grew to 70%, the medium was discarded, washed three times with 1×PBS, and replaced with exosome-free serum medium. The cells were cultured for 48 hours. The cell supernatant was collected and extracted by differential ultracentrifugation. The specific steps are as follows: 300g, 4°C centrifugation for 15min to remove dead cells; 3000g, 4°C centrifugation for 15min to remove cell debris; 10000g, 4°C centrifugation for 70min to remove other large extracellular vesicles; 120000g, 4°C centrifugation for 120min, the supernatant was removed, and the precipitate was exosomes. The exosomes were resuspended in an appropriate amount of PBS, filtered with a 0.22μm filter, and stored in a -80°C refrigerator for later use. The exosome concentration was detected by the BCA protein concentration kit.

[0051] 2. Preparation of drug-loaded core-shell exosome carriers

[0052] Exosomes (1 mg / ml) and gentamicin (1 mg / ml) at a mass ratio of 1:1 were mixed in 1 ml DMEM medium (Gibco), incubated at 37°C for 2 hours, ultrafiltration centrifuged to remove unbound drugs, and 10 μl CaCl2 solution (1 mol / L) was added and continued to be incubated at 37°C for 2 hours (calcium phosphate generated by the added calcium chloride and phosphorus in the α-MEM medium, the mass ratio of exosomes to calcium phosphate solution is 1:1.1). After the incubation, centrifuged at 14000g for 10 minutes and washed 3 times with ultrapure water. The mineralized exosomes loaded with gentamicin were resuspended with physiological saline or ultrapure water to obtain mineralized bone marrow mesenchymal stem cell-derived exosomes loaded with gentamicin, which is an exosome carrier with a core-shell structure for use in subsequent steps.

[0053] In this step, the mass ratio of exosomes to gentamicin can be 2:1, and exosome carriers with a core-shell structure can also be obtained.

[0054] 3. Construction of anodized titanium nanotubes and loading of core-shell exosomes

[0055] Titanium nanotubes were generated by electrochemical anodization. Titanium sheets were rubbed with sandpaper (400-2000 mesh), ultrasonically cleaned in acetone, ethanol (Beijing Chemical Plant, China) and deionized water, and dried in nitrogen. In a two-electrode cell with a DC power supply (graphite electrode IT6123, ITECH), 0.5wt% NH4F in ethylene glycol electrolyte was anodized for 3h at room temperature (25°C) and a constant potential of 50V. Dopamine solution was coated with titanium nanotubes (conventional adsorption was sufficient), and equal amounts of core-shell exosomes and titanium nanotubes were placed in a vacuum drying oven, vacuumed at 37°C for 5 minutes, and dried. The loading amount of core-shell exosomes on the surface of titanium nanotubes was 100%, and an antibacterial bone-promoting metal implant (also known as core-shell exosome coating implant) was obtained. It was placed in a vacuum package and refrigerated at 4°C for later use.

[0056] 4. Drug release from core-shell exosome-coated implants

[0057] 5 ml of pH 7.4 phosphate buffer (PBS) or pH 5.5 PBS was added to the core-shell structure exosome-coated implant in a 15 ml centrifuge tube, and the sample was completely immersed in PBS and placed in a 37 °C shaker at 40 rpm. 200 μl of the extract was taken at 0, 1, 2, 4, 8, 16, 24, and 48 h, and the same volume was added to the centrifuge tube. The gentamicin content was determined by HPLC, as shown in Figure 1 As shown. Figure 1 The results showed that pH changes had a significant effect on drug release from core-shell exosome-coated implants, with rapid release at low pH.

[0058] 5. In vitro antibacterial experiment of core-shell exosome-coated metal implants

[0059] Escherichia coli (E. coli) Gram-negative bacteria and Staphylococcus aureus (S. aureus) Gram-positive bacteria were used as experimental bacteria. The bacteria were 1×10 6 CFU mL -1 The concentration was inoculated in a 24-well plate and cultured for 24 hours. A titanium (denoted as A), anodized titanium nanotubes (denoted as B), and anodized titanium with a core-shell structure (denoted as C) were added to each well. Then the sample was taken out, gently rinsed, and placed in a new well with sterilized nutrient broth. After culturing at 37°C for 24 hours, the bacteria in the solution were quantified using the colony counting method. The number of colonies after 24 hours was counted, and the results were as follows: Figure 2 and Figure 3 As shown. Figure 2 and Figure 3The results show that the number of colonies on the core-shell structure anodized titanium (C) is significantly less than that on titanium (A) and anodized titanium nanotubes (B).

[0060] 6. Cytotoxicity Experiment of Core-shell Exosome-coated Metal Implants

[0061] Mouse embryonic osteoblast precursor (MC3T3-E1) cells in logarithmic growth phase were centrifuged and resuspended in sterile fresh α-MEM medium at a concentration of 1×10 4 cells / ml, seeded in a 24-well culture plate and cultured overnight in a CO2 incubator. After adding titanium or anodized titanium nanotubes with a core-shell structure, the samples were divided into three groups: titanium (A), anodized titanium nanotubes (B), and anodized titanium with a core-shell structure (C). Titanium was used as a basic control. Cell proliferation was detected by the CCK-8 method. After treatment for 1, 3, 5, and 7 days, the culture medium was replaced with fresh culture medium containing 10% CCK-8 solution, and the samples were incubated for 4 hours. The absorbance value (OD value) was detected at 450nm using a multifunctional microplate reader.

[0062] The results are as follows Figure 4 As shown, titanium (A), anodized titanium nanotubes (B) and anodized titanium nanotubes with shell-core structure (C) are non-toxic to cells. Nanotubes (B) formed by surface anodization and anodized titanium nanotubes with shell-core structure (C) both promote cell proliferation, and the OD value of anodized titanium nanotubes with shell-core structure (C) is larger, indicating that the effect of promoting cell proliferation is better. This result proves that anodized titanium nanotubes with shell-core structure are harmless to MC3T3-E1 cells, that is, the shell-core structure exosome-coated metal implants of the present invention are harmless to MC3T3-E1 cells.

[0063] 7. In vitro osteogenic differentiation experiment of shell-core structure exosome-coated metal implants

[0064] Cells were collected at 5×10 4 ml -1 After culturing for 24 hours, the original culture medium was removed and the culture was continued with osteogenic induction medium containing 10% fetal bovine serum, 100nM dexamethasone, 50M ascorbic acid, and 10mM sodium β-glycerophosphate. The culture medium was changed every 3 days. qRT-PCR was used to detect the expression of osteogenic markers ALP, osteopontin (OPN) and run-related transcription factor 2 (Runx2). Figure 5 As shown, in the cells treated with the core-shell exosome metal implant of the present invention (B), the expressions of ALP, OPN and Runx2 were significantly increased, while there was no such effect in the control group (A).

Claims

1. An antibacterial bone-promoting metal implant, characterized in that: The antibacterial bone-promoting metal implant is a metal implant surface loaded with an exosome carrier having a core-shell structure and containing antibacterial components; The exosome carrier containing antibacterial components and having a core-shell structure is composed of a core part and a shell part; the core part is composed of exosome-loaded drugs; and the shell part is an inorganic salt containing calcium and phosphorus; The loading amount of the exosome carrier with a core-shell structure and containing antibacterial components on the surface of the metal implant is 90-100%.

2. The antibacterial bone-promoting metal implant according to claim 1, characterized in that: The thickness of the shell portion is 100nm~200nm; The drug loading amount of the exosome-loaded drug is 10% to 50%.

3. The antibacterial bone-promoting metal implant according to claim 1 or 2, characterized in that: The exosome sources include cells and / or body fluids; The cells include at least one of human, rat or mouse stem cells, macrophages, endothelial cells and epithelial cells; The drug includes at least one of antibiotics, nucleic acids, proteins and polypeptides; The inorganic salt containing calcium and phosphorus includes calcium phosphate.

4. The method for preparing the antibacterial bone-promoting metal implant according to any one of claims 1 to 3, comprising the following steps: 1) co-incubating or ultrasonicating the exosomes and the drug in a cell culture medium, and then centrifuging; taking the precipitate after centrifugation and adding an inorganic salt solution containing calcium and phosphorus to continue incubation, so as to obtain the exosome carrier containing the antibacterial component and having a core-shell structure; 2) The metal implant is anodized, and then mixed with the exosome carrier with a core-shell structure and containing antibacterial components, and vacuum dried or incubated to obtain the antibacterial bone-promoting metal implant.

5. The preparation method according to claim 4, characterized in that: The exosomes are prepared as follows: cells and / or body fluids are subjected to differential centrifugation to collect the exosomes; The exosomes and the drug are incubated for 1 to 5 hours at a reaction temperature of 37°C; The ultrasound is performed according to a method comprising the following steps: the exosomes are mixed with the drug, and an ultrasound probe is immersed in the mixed solution for ultrasound under ice bath conditions; the ultrasound is performed for 10 cycles, and the setting conditions are 500 v, 2 kHz; The centrifugal force of the centrifugation is 5000g~20000g, and the centrifugation time is 5~20min.

6. The preparation method according to claim 4 or 5, characterized in that: The incubation time in the calcium-phosphorus-containing inorganic salt solution is 1 to 5 hours, and the reaction temperature is 37° C.; The concentration of the exosomes is 0.1 mg / ml to 100 mg / ml; The concentration of the drug is 0.1 mg / ml to 100 mg / ml; The concentration of the calcium-phosphorus-containing inorganic salt solution is 1mmol / ml to 100mol / ml; The mass ratio of the exosomes, the drug, and the calcium-phosphorus-containing inorganic salt solution is 1-3:1:0.001-3.

7. The preparation method according to claim 4 or 5, characterized in that: In step 1), after incubation in the inorganic salt solution containing calcium and phosphorus, the following post-treatment steps are also included: centrifugation and / or washing the precipitate with a buffer solution; The centrifugal speed is 5000g~20000g, and the centrifugal time is 5~20min; The buffer comprises physiological saline and / or PBS buffer; The washing times are 1 to 6 times.

8. The preparation method according to claim 4 or 5, characterized in that: In step 2), the metal implant is anodized by electrolysis; The electrolysis method is constant potential electrolysis, and the conditions are as follows: the voltage is 10-80 V, and the electrolyte is ethylene glycol electrolyte of NH4F, and the concentration thereof is 0.1wt%-1wt%.

9. The preparation method according to claim 4 or 5, characterized in that: In step 2), the incubation time is 0.5 to 5 hours and the temperature is 24 to 30°C.

10. Use of the antibacterial bone-promoting metal implant according to any one of claims 1 to 3 in the preparation of orthopedic and / or dental metal implants.

Images