Titanium surface composite coating with osteogenesis promoting and antibacterial functions and preparation and application thereof

Abstract

The invention relates to the technical field of medical materials, and particularly discloses a titanium surface composite coating with osteogenesis promoting and antibacterial functions. The micro-arc magnesium oxide coating is arranged on the surface of the titanium substrate; the chitosan coating is arranged on the surface of the micro-arc magnesium oxide coating; the chlorogenic acid is loaded in the chitosan coating; a magnesium-containing porous microstructure is constructed on the surface of a titanium substrate through a micro-arc oxidation technology, so that the biological activity and corrosion resistance of the coating are improved; furthermore, chitosan is used as a biocompatible carrier to load a traditional Chinese medicine active ingredient chlorogenic acid, so that the surface of the implant has excellent osteogenic differentiation promoting capability and broad-spectrum antibacterial property at the same time. Physical modification, biopolymer drug loading and traditional Chinese medicine active ingredients are combined, and the problems that a traditional titanium implant is high in surface biological inertness, infection is likely to be caused and osseointegration is poor are solved through the synergistic effect on the three aspects of the structure, the material and the biological function.

Description

Technical Field

[0001] This invention belongs to the field of medical materials technology, specifically relating to a titanium surface composite coating with bone-promoting and antibacterial functions, its preparation and application. Background Technology

[0002] Repairing bone defects is a significant challenge in orthopedic clinics. Currently, mainstream repair methods include autologous bone grafting, allogeneic / xenogeneic bone grafting, and implantation of biomedical materials such as titanium and its alloys. Among these, autologous bone grafting is considered the primary choice, but it has inherent drawbacks such as limited availability and the risk of secondary trauma. Allogeneic / xenogeneic grafting, on the other hand, faces the risks of immune rejection and disease transmission. With the development of bone tissue engineering, titanium has become the preferred material for implants due to its excellent mechanical properties. However, its inherent bioinertness leads to insufficient bone integration and susceptibility to bacterial infection, affecting the success rate of implantation.

[0003] To improve the biological properties of titanium implant surfaces, a series of surface modification techniques have been developed and applied. For example, micro-arc oxidation (MAO) technology can construct porous coatings on titanium surfaces to improve osseointegration; and using biopolymer carriers such as chitosan (CTS) to load active molecules can endow the surface with certain biological functions. However, existing technologies often have limited functionality: either focusing on improving osseointegration while lacking antibacterial ability, or focusing on antibacterial activity while having limited bone activity. Single modification strategies are insufficient to synergistically address the multiple clinical needs of early implant stability, long-term integration, and anti-infection, thus limiting their application in the repair of complex bone defects. Summary of the Invention

[0004] The purpose of this invention is to provide a titanium surface composite coating with bone-promoting and antibacterial functions, its preparation and application, so as to solve the problems mentioned in the background art.

[0005] To achieve the above objectives, the present invention provides the following technical solution:

[0006] A titanium surface composite coating with bone-promoting and antibacterial functions, comprising:

[0007] Titanium substrate;

[0008] Micro-arc magnesium oxide coating disposed on the surface of the titanium substrate;

[0009] Chitosan coating disposed on the surface of the micro-arc magnesium oxide coating;

[0010] And chlorogenic acid loaded in the chitosan coating.

[0011] Preferably, the thickness of the micro-arc magnesium oxide coating is 10–50 μm, the surface has a porous micron structure, and the porosity is 20–40%.

[0012] Preferably, the thickness of the chitosan coating is 1–10 μm.

[0013] Preferably, the chlorogenic acid loading is 0.5–2 mg / cm².

[0014] A method for preparing a composite coating on a titanium surface includes the following steps:

[0015] S1. Perform surface pretreatment on the titanium substrate;

[0016] S2. A magnesium-containing micro-arc oxidation coating is prepared on the surface of a titanium substrate using a micro-arc oxidation process;

[0017] S3. A chitosan coating is formed on the surface of the micro-arc oxidation coating by electrochemical deposition.

[0018] S4. The chitosan coating is immersed in a chlorogenic acid solution to load the chlorogenic acid into the coating.

[0019] 6. The method for preparing a titanium surface composite coating with bone-promoting and antibacterial functions according to claim 1, characterized in that: the electrolyte of the micro-arc oxidation process in step S2 contains NaH2PO4 and Mg(OH)2, and the oxidation treatment is carried out in a two-way mode of constant current followed by constant voltage.

[0020] Preferably, the electrolyte for electrochemical deposition in step S3 comprises chitosan, deionized water and acetic acid, the deposition voltage is 30–50V and the deposition time is 5–15 minutes.

[0021] Preferably, the concentration of the chlorogenic acid solution in step S4 is 0.5–2 mg / ml, and the immersion time is 12–36 hours.

[0022] An orthopedic implant having a surface coated with a titanium surface composite coating as described above.

[0023] Preferably, the implant is one of an artificial joint, a bone plate, a bone screw, or a bone defect filling material.

[0024] Compared with the prior art, the beneficial effects of the present invention are:

[0025] (1) This composite coating constructs a magnesium-containing porous microstructure on the surface of a titanium substrate using micro-arc oxidation technology, thereby improving the coating's bioactivity and corrosion resistance. Furthermore, chitosan is used as a biocompatible carrier to load chlorogenic acid, an active ingredient in traditional Chinese medicine, giving the implant surface both excellent bone differentiation-promoting ability and broad-spectrum antibacterial properties. By combining physical modification, biopolymer drug delivery, and active ingredients in traditional Chinese medicine, the coating works synergistically from three levels: structure, materials, and biological function, overcoming the problems of strong bioinertness, easy infection, and poor osseointegration on the surface of traditional titanium implants.

[0026] (2) The prepared coating can not only significantly promote early bone healing and enhance the long-term stability of the implant, but also effectively reduce the risk of postoperative infection. In addition, chlorogenic acid, a natural product with wide availability and high safety, is used as a functional molecule. Attached Figure Description

[0027] Figure 1 The images show a comparison of the scanning electron microscope (SEM) morphology of the three groups of samples in Example 1: micro-arc oxidation (Ti-Mg group), chitosan coating (Ti-Mg-CTS group), and chlorogenic acid-loaded coating (Ti-Mg-CTS-CGA group).

[0028] Figure 2 This is a schematic diagram showing the results of the CCK-8 assay in Example 2, which detected the proliferation activity of rat bone marrow mesenchymal stem cells (BMSCs) on days 1, 3, and 5 of culture in each group of samples.

[0029] Figure 3 These are representative images of the morphology and activity of BMSCs on the surface of each group of samples observed by fluorescent staining of live / dead cells in Example 2.

[0030] Figure 4 This is a diagram showing the results of alkaline phosphatase (ALP) staining in Example 2, which detected the effect of each coating group on the early osteogenic differentiation of BMSCs.

[0031] Figure 5 This is a graph showing the results of alizarin red staining in Example 2 to detect the effect of each coating group on the formation of late-stage mineralized nodules in BMSCs;

[0032] Figure 6 This is a schematic diagram showing the effect of qRT-PCR detection on the expression levels of osteogenic-related genes (Runx2, OCN, OPN, BMP2) in BMSCs in Example 2;

[0033] Figure 7 These are representative images from Example 3 showing the new bone growth of each group of implants in a rat femoral defect model, as displayed by Micro-CT three-dimensional reconstruction.

[0034] Figure 8 This is a schematic diagram of the statistical results of the Micro-CT quantitative analysis of the bone volume fraction (BV / TV), trabecular thickness (Tb.Th), and trabecular separation (Tb.Sp) around the implant in each group in Example 3. Detailed Implementation

[0035] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0036] Example 1:

[0037] Please see Figure 1 As shown, a method for preparing a titanium surface composite coating with bone-promoting and antibacterial functions includes the following steps:

[0038] 1. Substrate pretreatment: Take a pure titanium disc with a diameter of approximately 5mm and a thickness of approximately 1mm, and successively polish it using 400#, 600#, 800#, 1000#, and 2000# sandpaper until the surface is smooth and free of obvious scratches. Place the polished titanium disc in anhydrous ethanol for ultrasonic cleaning for 15 minutes, then rinse it three times with distilled water, and dry it in a 50℃ oven for later use.

[0039] 2. Preparation of micro-arc magnesium oxide coating: Using a pretreated titanium sheet as the anode and a stainless steel sheet as the cathode, the electrodes are placed in an electrolyte containing 0.1 mol / L NaH2PO4 and 0.05 mol / L Mg(OH)2;

[0040] The process employs a bidirectional power supply mode: First, in constant current mode, the positive current is increased from 1A to 5A, and the negative current is increased from 1A to 4A. When the positive voltage reaches 600V, the process switches to constant voltage mode, setting the positive voltage to 600V, the negative voltage to -90V, the duty cycle to 30%, and the processing time to 20 minutes. During the process, continuous electric spark discharge occurs on the surface of the titanium sheet, resulting in the in-situ growth of a gray porous oxide layer.

[0041] After the reaction was completed, the titanium sheet was removed, rinsed thoroughly with deionized water, and dried in an oven at 50°C to obtain a magnesium-containing micro-arc oxidation coating with a micron-scale porous structure on the surface (denoted as Ti-Mg group).

[0042] 3. Alkali treatment activation: Prepare a 3 mol / L NaOH solution and heat it in a 60-65℃ water bath. Completely immerse the Ti-Mg group samples in the NaOH solution and treat in a sealed environment for 3 hours. After removal, rinse with deionized water and spin dry.

[0043] 4. Electrochemical deposition of chitosan coating: The alkali-treated sample was used as the cathode, and a stainless steel sheet was used as the anode. The sample was immersed in an electrolyte containing 3g chitosan, 2L deionized water, and 10ml acetic acid, and deposition was performed at a constant voltage of 40V for 10 minutes. After deposition, the sample was removed, rinsed with deionized water, dried, and sterilized with ethylene oxide to obtain a sample with a chitosan coating (denoted as the Ti-Mg-CTS group).

[0044] 5. Chlorogenic Acid Loading: Prepare a chlorogenic acid (CGA) phosphate buffer (PBS) solution with a concentration of 1 mg / ml. Mix thoroughly with a magnetic stirrer. Immerse the Ti-Mg-CTS group sample completely in the solution and let it stand at 4°C in the dark for 24 hours. After that, remove the sample, rinse it gently with sterile PBS solution, and air dry it in a clean bench to obtain the final composite coating sample loaded with chlorogenic acid (denoted as Ti-Mg-CTS-CGA group, i.e., MCC group).

[0045] like Figure 1 As shown, the surface morphology of the three groups of samples (Ti-Mg, Ti-Mg-CTS, and Ti-Mg-CTS-CGA) was observed using scanning electron microscopy (SEM). The results showed that the Ti-Mg group exhibited a typical "crater-like" porous structure with pore sizes ranging from 1 to 5 μm. The pores on the surfaces of the Ti-Mg-CTS and Ti-Mg-CTS-CGA groups were partially filled with chitosan, resulting in a slight decrease in pore size, but the porous morphology remained intact. Spot scan analysis of the Ti-Mg group surface using energy dispersive spectroscopy (EDS) confirmed the successful incorporation of Mg.

[0046] Example 2:

[0047] 1. Cell proliferation assay (CCK-8 assay): Rat bone marrow mesenchymal stem cells (BMSCs) were seeded onto the surfaces of the following six sample groups: 1) blank culture plate control group, 2) pure titanium sheet group (Ti), 3) micro-arc oxidation group (Ti-Mg, MAO group), 4) chitosan coated group (Ti-Mg-CTS, MC group), 5) chlorogenic acid solution treatment group (CGA), and 6) chlorogenic acid-loaded composite coating group (Ti-Mg-CTS-CGA, MCC group). Cell viability was detected using a CCK-8 assay kit on days 1, 3, and 5 of culture. Figure 2 The results showed that cells in all groups proliferated on days 1 and 3 of culture, but there was no significant statistical difference compared with the control group. On day 5 of culture, the absorbance value of the MCC group was significantly higher than that of the control group and other experimental groups (p<0.05), indicating that the composite coating can significantly promote the proliferation of BMSCs.

[0048] 2. Cell morphology observation (live / dead staining and cytoskeleton staining): After 3 days of culture, cells were stained with calcein-AM (live cells, green) and propidium iodide (dead cells, red). Figure 3 As shown, under a fluorescence microscope, the MCC group exhibited a large number of viable green cells with a large spreading area, plump cell morphology, fully extended pseudopodia, tight intercellular connections, and very few dead cells. Staining the cytoskeleton with phalloidin further confirmed that the MCC group cells exhibited the best adhesion and spreading properties.

[0049] 3. Osteogenic differentiation capacity test: BMSCs were cultured in osteogenic induction medium.

[0050] Alkaline phosphatase (ALP) staining: After 7 days of culture, ALP staining was performed using the BCIP / NBT kit. Figure 4 As shown, ALP activity (blue-purple precipitate) could be detected in all experimental groups, with the staining depth and area of ​​the MCC group being significantly stronger than those of the other groups.

[0051] Alizarin Red S staining (mineralized nodules): After 21 days of culture, alizarin red S staining was used to detect calcium nodules, such as... Figure 5 As shown, the MCC group exhibits a large number of dense orange-red mineralized nodules, with both their number and area significantly exceeding those of the Ti, MAO, and MC groups.

[0052] Osteogenesis-related gene expression (qRT-PCR): After 5 days of culture, cellular RNA was extracted, and the mRNA expression levels of key osteogenic genes Runx2, OCN, OPN, and BMP2 were detected. GAPDH was used as an internal control. Figure 6 As shown, compared with the control group, the expression levels of these four genes in the MCC group were significantly upregulated (p<0.01), indicating that the coating effectively promoted osteogenic differentiation of BMSCs at the gene level.

[0053] Example 3:

[0054] Thirty-six healthy 6-week-old SD rats (weighing approximately 200g) were randomly divided into six groups (n=6): sham surgery group (defect created but no implantation), chlorogenic acid group (defect created but no implantation, chlorogenic acid solution administered by gavage), pure titanium sheet implantation group (Ti group), micro-arc oxidation coating implantation group (MAO group), chitosan coating implantation group (MC group), and chlorogenic acid-loaded composite coating implantation group (MCC group). A cylindrical bone defect model with a diameter of 3mm and a depth of 2mm was created in the distal femoral condyles of both rats.

[0055] Except for the sham surgery group and the chlorogenic acid group, the other groups had their corresponding titanium implants (3 mm in diameter and 1 mm in thickness) tightly implanted into the bone defects. The incisions were sutured in layers after the operation. The rats were fed normally. Eight weeks after the operation, all rats were sacrificed and the distal femur samples containing the implants were completely removed.

[0056] Micro-CT analysis: Samples were scanned using micro-CT scanners, and 3D reconstruction and bone metrology analysis were performed using accompanying software. Evaluation parameters included bone volume fraction (BV / TV), trabecular bone thickness (Tb.Th), and trabecular bone separation (Tb.Sp), such as... Figure 7-8 As shown, the MCC group exhibited the highest amount of new bone formation around the implant, with the most complete repair of bone defects. The BV / TV and Tb.Th values ​​were significantly higher than other implant groups (p<0.05), while the Tb.Sp value was significantly lower, indicating a denser new bone structure. The implant-bone interface was tightly bonded with no obvious gaps.

[0057] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A titanium surface composite coating with osteogenic and antibacterial functions, characterized in that, include: Titanium substrate; Micro-arc magnesium oxide coating disposed on the surface of the titanium substrate; Chitosan coating disposed on the surface of the micro-arc magnesium oxide coating; And chlorogenic acid loaded in the chitosan coating.

2. The titanium surface composite coating with bone-promoting and antibacterial functions according to claim 1, characterized in that: The thickness of the micro-arc magnesium oxide coating is 10–50 μm, and the surface has a porous micron structure with a porosity of 20–40%.

3. The titanium surface composite coating with bone-promoting and antibacterial functions according to claim 1, characterized in that: The thickness of the chitosan coating is 1–10 μm.

4. The titanium surface composite coating with bone-promoting and antibacterial functions according to claim 1, characterized in that: The chlorogenic acid loading is 0.5–2 mg / cm².

5. A method for preparing a composite coating on a titanium surface according to any one of claims 1-4, characterized in that, Includes the following steps: S1. Surface pretreatment of titanium substrate; S2. A magnesium-containing micro-arc oxidation coating is prepared on the surface of a titanium substrate using a micro-arc oxidation process; S3. A chitosan coating is formed on the surface of the micro-arc oxidation coating by electrochemical deposition. S4. The chitosan coating is immersed in a chlorogenic acid solution to load the chlorogenic acid into the coating.

6. The method for preparing a titanium surface composite coating with bone-promoting and antibacterial functions according to claim 5, characterized in that: In step S2, the electrolyte for the micro-arc oxidation process contains NaH2PO4 and Mg(OH)2, and the oxidation process is carried out in a two-way mode of constant current followed by constant voltage.

7. The method for preparing a titanium surface composite coating with bone-promoting and antibacterial functions according to claim 5, characterized in that: In step S3, the electrolyte for electrochemical deposition includes chitosan, deionized water, and acetic acid. The deposition voltage is 30–50V, and the deposition time is 5–15 minutes.

8. The method for preparing a titanium surface composite coating with osteogenic and antibacterial functions according to claim 5, characterized in that: The concentration of the chlorogenic acid solution in step S4 is 0.5–2 mg / ml, and the immersion time is 12–36 hours.

9. An orthopedic implant, characterized in that, Its surface is provided with a titanium surface composite coating according to any one of claims 1–4.

10. An orthopedic implant according to claim 9, characterized in that: The implant is one of the following: artificial joint, bone plate, bone screw, or bone defect filling material.

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