Preparation method of bionic composite micro-nano structure metal surface for promoting bone fusion

Abstract

The invention relates to the technical field of orthopedic implants, in particular to a preparation method of a bionic composite micro-nano structure metal surface for promoting bone fusion, which comprises the following steps: S1, preparing a periodic groove structure on the metal surface by using double-beam laser equipment; s2, a blind hole structure is secondarily machined in the metal surface of the periodic groove structure through laser scanning equipment, and a bionic composite micron structure is formed; and S3, thermal oxidation treatment is conducted on the surface of the bionic composite micron structure, the bionic composite micron structure obtained after thermal oxidation treatment is cooled to the room temperature, then deionized water is used for cleaning, drying is conducted, and the metal bionic composite micro-nano structure is obtained. According to the metal surface prepared by the method, the surface structure is directly constructed on the surface of the metal implant, so that the problem that the coating is easy to fall off is avoided, and meanwhile, the problem that the surface structure and osteoblast reaction are unstable due to superposition of a plurality of preparation technologies is also avoided.

Description

Technical Field

[0001] The present invention relates to the technical field of orthopedic implants, and provides a method for preparing a bionic composite micro-nano structured metal surface that promotes bone fusion. Background Art

[0002] Bone replacement surgery has been widely used in the fields of orthopedics and dentistry with the intensification of aging. Although bone replacement surgery provides many conveniences for many patients, problems such as loosening, wear, and incompatibility of implants may occur, and the implants may be unstable due to the influence of the surrounding environment, thus bringing irreparable consequences to patients. It has become particularly important to find ways to reduce implant loosening and improve its stability. Currently, traditional techniques mainly include micro-machining, polishing, surface sandblasting, surface coating, acid etching, and laser texturing to produce micro-texture patterns on the implant metal surface. However, these methods have problems such as a high concentration of cytotoxic materials on the implant metal surface and complex equipment. Laser interference lithography technology was born under this background. Metal materials are commonly used in medical implants and industrial production, and improving cell proliferation and spreading has become a research hotspot at home and abroad.

[0003] In recent years, researchers have proposed various techniques to solve the surface topography of metal implants, including laser processing, chemical etching, and electrochemical techniques, etc. However, the cumbersome manufacturing process and high manufacturing cost limit the application of metal implants in actual medical treatment. At the same time, the complex natural environment also poses higher requirements for the stability and compatibility of the surface with cells.

[0004] Therefore, there is an urgent need to develop an efficient manufacturing method that can regulate cell behavior while improving the fusion of cells with metal implants to overcome these challenges and achieve wider applications. Summary of the Invention

[0005] To solve the above problems, the present invention provides a method for preparing a bionic composite micro-nano structured metal surface that promotes bone fusion. The metal surface prepared by this method directly constructs a surface structure on the metal implant surface, avoiding the problem that the coating is prone to falling off, and at the same time avoiding the problem of unstable reaction between the surface structure and osteoblasts caused by the superposition of multiple preparation techniques.

[0006] To achieve the above object, the technical solution adopted by the present invention is:

[0007] In the first technical solution, a method for preparing a bionic composite micro-nano structured metal surface that promotes bone fusion is characterized in that it includes the following steps:

[0008] S1. Use a double-beam laser device to prepare a periodic groove structure on the metal surface;

[0009] S2. Use a laser scanning device to perform secondary processing on the metal surface with a periodic groove structure to form a bionic composite micro-structure;

[0010] S3. Perform thermal oxidation treatment on the surface of the bionic composite micro-structure. After the thermal oxidation treatment, the bionic composite micro-structure is cooled to room temperature, and then it is washed with deionized water and dried to obtain the metal bionic composite micro-nano structure.

[0011] In the first technical solution, preferably, in S3, during the thermal oxidation treatment of the obtained bionic composite micro-structure surface, the temperature is set at 700 °C, and after 4 hours of thermal oxidation treatment, it is cooled to room temperature.

[0012] In the first technical solution, preferably, the dual-beam laser device is a nanosecond laser dual-beam interference direct writing system; the secondary processing device is a laser marking machine; the thermal oxidation treatment of the bionic composite micro-structure surface is carried out in a quartz tube furnace.

[0013] In the first technical solution, preferably, in S1, the spatial angle of the interference beams of the dual-beam laser interference direct writing system is centrosymmetric, the polarization state is TE-TE, and the light intensity energy density ratio of the interference beams is 1:1; the laser wavelength is 1064 nm, the repetition frequency is 10 Hz, the pulse duration is 7 - 9 ns, and the period is 200 μm.

[0014] In the first technical solution, preferably, in S2, when using the laser marking machine for scanning, the laser power is 4 W, the scanning speed is 2000 mm / s, the structure height is 8.7 μm, and the spacing is about 49 μm, the laser output power is 20 W, the laser rated power is 0.5 KW, and the laser pulse width is 20 - 100 ns.

[0015] In the second technical solution, a metal surface prepared by the method as described in the first technical solution.

[0016] In the third technical solution, for use in attaching cells to the metal surface described in the second technical solution, the preparation method for the reaction of human osteosarcoma-like osteoblast cells to the implanted metal surface structure includes:

[0017] Prepare a minimum essential medium for culturing osteoblasts. The osteoblasts are attached to the metal surface and maintained under incubation conditions of an environmental temperature of 37 °C and an environmental air CO 2 content of 5%.

[0018] The beneficial effects of using the present invention are:

[0019] Compared with the prior art, the present invention uses two techniques, namely nanosecond laser double-beam interference direct writing and laser scanning, to prepare a bionic composite micro-structure, combines a high-temperature annealing technique to construct a cross-scale micro-nano structure, and generates a bionic composite micro-nano structure on the metal surface. By changing the surface topography, the surface hydrophilicity and roughness are changed, the cell behavior is regulated, the development process of osteoblasts in bone tissue is simulated, the loosening factors of metal implants are reduced, and the fusion of metal implants is improved by at least 30%. Description of the Drawings

[0020] Figure 1 The double-beam interference optical path system used in the present invention.

[0021] Figure 2 The bionic structure obtained by laser scanning and secondary processing of the metal surface in the present invention.

[0022] Figure 3 The bionic micro-nano structure obtained by high-temperature annealing in the present invention

[0023] Figure 4 The roughness and depth measurement results of the US sample, SGM sample, BMN, BMN2, BMN4, and BMN6 samples in the present invention.

[0024] Figure 5 The hydrophilicity measurement results of the US sample, SGM sample, and BMN2 sample in the present invention.

[0025] Figure 6 The quantitative analysis of the cell deflection angle on the SGM sample and BMN2 sample in the present invention.

[0026] Figure 7 The cell length and width after culturing cells for 24 h on the US sample, SGM sample, and BMN2 sample in the present invention.

[0027] Figure 8 The MTT detection result diagram of the cell adhesion force in the present invention.

[0028] Figure 9 The pseudopod detail diagram of cell growth for 24 h on the SGM sample and BMN2 sample in the present invention. Detailed Description of the Invention

[0029] To make the objectives, technical solutions, and advantages of the present technical solution clearer and more understandable, the present technical solution will be further described in detail below in conjunction with specific implementation manners. It should be understood that these descriptions are only exemplary and are not intended to limit the scope of the present technical solution.

[0030] Example 1

[0031] The present invention provides a technical solution: a preparation method for the surface of a bionic composite micro-nano structure metal for promoting bone fusion, comprising the following steps:

[0032] S1. Using a nanosecond laser double-beam interference direct writing system, a periodic groove structure is prepared on the metal surface. The groove structure effectively improves the surface performance compared with the blank substrate.

[0033] S2. Using a laser marking machine to perform secondary processing on the metal surface with the periodic groove structure to prepare blind hole structures, and a bionic composite micro-structure is prepared on the metal surface. Compared with the groove structure, the structural complexity is increased, which helps to enhance the surface hydrophilicity.

[0034] S3. Thermally oxidizing the surface of the obtained bionic composite micro-structure with a quartz tube furnace at a set temperature of 700 °C, effectively improving the surface roughness and hydrophilic-hydrophobic properties of the titanium alloy, generating micro-nano structures on the surface that are beneficial to cell growth and adhesion. After 4 h, it is cooled to room temperature, then washed with deionized water and dried to obtain the metal bionic composite micro-nano structure, which is sterilized at high temperature and by ultraviolet light for subsequent cell attachment.

[0035] As Figure 1 shown, the nanosecond laser double-beam interference direct writing system used in the present invention is a typical system, including a nanosecond pulsed laser, a high reflector M, a semi-reflective semi-transmissive lens BS, a half-wave plate W, a polarizer P, with a laser frequency of 10 Hz, an exposure time of 10 s, a pulse number of 70 - 90, a laser wavelength of 1064 nm, and a pulse duration of 7 - 9 ns. Then, the light intensity energy distribution of the groove pattern is simulated by MATLAB software to determine the interference parameters. According to the simulation results, a double-beam laser interference system is built. The relative positions and angles of the high reflector and the semi-reflective semi-transmissive lens are calculated according to the incident angle. A beam of laser emitted by the nanosecond pulsed laser is divided into two beams of light after passing through the high reflector M and the semi-reflective semi-transmissive lens BS, and the two beams of light reach the interference field after passing through the combination of the half-wave plate W and the polarizer P. The combination of the half-wave plate W and the polarizer P is placed between the high reflector and the sample. By rotating the angle of the half-wave plate W, the double-light intensity energy density ratio is made 1:1, and by measuring with a power meter, the light intensity energy density of each beam of light is the same at 35 mJ / mm2. By rotating the angle of the polarizer P, the polarization state of the double-beam interference light is made TE-TE; the metal surface is directly etched using the double-beam laser interference direct writing system, and a micron-level periodic groove structure with a period of 600 μm is prepared on the titanium alloy surface.

[0036] Then, secondary processing is performed on the surface of the periodic groove structure using laser scanning technology. The laser scanning speed is 2000 mm / s, the laser power is 4 W, and a bionic composite micro-structure is generated on the surface of the periodic groove structure, with a structure height of 8.7 μm and a spacing of 49 μm, as Figure 2 shown. The laser output power is 20 W, the laser rated power is 0.5 KW, and the laser pulse width is 20 - 100 ns.

[0037] Then they were respectively placed in a 700 °C quartz tube furnace (the temperature of the tube furnace was raised to 700 °C within 700 s), heat-treated for 4 h and then cooled to room temperature, and nanostructures were formed on the surface of the bionic composite microscale structure, denoted as the BMN2 sample, as Figure 3 shown. Finally, they were washed with deionized water and sterilized at 126 °C for 2 hours for subsequent cell attachment.

[0038] Prepare the minimum essential medium (10% FBS + MEM medium) for culturing osteoblasts and maintain it under incubation conditions of 37 °C and 5% CO 2 to achieve the generation of bone cells.

[0039] This method has the significant advantages of high precision and high flexibility, can accurately control the size, shape and spacing of the grooves, is more controllable than other technologies when preparing groove structures with complex patterns, and can be realized on a variety of metal materials, greatly expanding the application range. The groove structure effectively improves the surface performance compared with the blank substrate.

[0040] In addition, the used marking machine has high processing efficiency and convenient operation, can quickly and accurately construct blind holes on the basis of the existing groove structure. The formed composite microscale structure has significantly improved structural complexity compared with the single groove structure. This increase in structural complexity has a positive promoting effect on the improvement of surface hydrophilicity, can effectively reduce the surface contact angle, and enhance the interaction efficiency between the surface and the liquid medium.

[0041] In this embodiment, a 700 °C quartz tube furnace was used, and the temperature of the tube furnace was raised to 700 °C within 700 s. Under this temperature condition, an oxidation reaction occurred on the metal surface and nanostructures were formed, effectively improving the surface roughness of the titanium alloy. The improvement of this roughness is beneficial to biological behaviors such as cell adhesion, proliferation and differentiation on its surface. After 4 h, it was cooled to room temperature, then washed with deionized water and dried to obtain the metal bionic composite micro-nano structure, which was sterilized at high temperature and by ultraviolet for subsequent cell attachment. Thermal oxidation treatment can promote the formation of surface nanostructures under relatively mild conditions, effectively improve the biocompatibility of the material, and this process is mature and stable, and is easy to realize large-scale preparation in industrial production.

[0042] Example 2

[0043] The difference from Example 1 is that the laser power for secondary processing of the groove structure surface using laser scanning technology is 2 W, and the blind hole structure formed on the groove structure surface is denoted as the BMN sample.

[0044] Example 3

[0045] It is different from Example 1 in that the laser power for secondary processing of the surface of the groove structure using laser scanning technology is 6W, and the blind hole structure generated on the surface of the groove structure is denoted as the BMN4 sample.

[0046] Example 4

[0047] It is different from Example 1 in that the laser power for secondary processing of the surface of the groove structure using laser scanning technology is 8W, and the blind hole structure generated on the surface of the groove structure is denoted as the BMN6 sample.

[0048] Cell regulation behavior detection includes cell morphology detection and cell adhesion ability detection. Untreated samples (US), groove micro-structure samples (SGM), BMN samples, BMN2 samples, BMN4 samples, and BMN6 samples were respectively taken for surface characterization and detection of their cell behavior regulation ability according to the conventional methods in the art.

[0049] Figure 4 The roughness and depth measurement results are shown. The roughness of the BMN2 sample is 7.73μm, the structure depth is 154.6μm, and the blind hole diameter size is 49μm, which most conforms to the structure size of the plant Nepenthes. In the natural environment, the lid of Nepenthes secretes a special smell to attract insects. Due to the superhydrophilicity of the lip, insects are very likely to slide into the cage. In this example, the hydrophilic characteristic structure of the lip of Nepenthes is imitated on the metal surface. The surface structure has a large contact area and a relatively high surface energy angle, with strong hydrophilicity, which can improve the spreading property of osteoblasts.

[0050] Figure 5 The hydrophilicity measurement results of the US sample, SGM sample, and BMN2 sample are shown. The contact angles of the samples are all within the range of 90°, showing hydrophilicity. The BMN2 sample has a higher roughness. When 5 mL of water droplets drop on the surface of the sample instantaneously, the water droplets "disappear", and the contact angle of the sample is 0°, showing superhydrophilicity, which improves the fusion of cells with the surface.

[0051] Figure 6 The quantitative analysis diagram of the deflection angle of cells on the SGM sample and BMN2 sample is shown. In order to quantitatively analyze the arrangement of the cell skeleton on the sample surface, ImageJ (NIH) was used to analyze the included angle between the cell arrangement direction and the direction of different micro-nano structure substrates, and Origin software was used to organize and plot the results. Generally, when the deflection angle is less than 15°, it is considered that the cells are arranged orderly in the structure direction. The number of cells with a deflection angle in the range of 0° - 15° in the SGM sample is significantly larger, indicating that the cells grow orderly along the groove structure in the SGM sample; most of the cell deflection angles in the BMN2 sample are in the range of 45° - 90°. At the same time, combined with Figure 7The length and width of the cells when attached to the sample indicate that the cells spread well in the BMN2 sample, with uniform spreading and a large surface area, which is beneficial to the life activities of the cells. Compared with the aspect ratio of the cells in the US sample, the aspect ratio of the cells in the BMN2 sample is much better, indicating that BMN2 can better present the stretched state of the cells.

[0052] As Figure 8 shown, the OD values of the cells cultured on the US sample, SGM sample, and BMN2 sample for different days. With the increase in the number of culture days, both the cell number and density showed a stable increase. Among them, the BMN2 substrate exhibited the best proliferation performance, indicating that the cells have good adhesion and proliferation abilities on the BMN2 sample.

[0053] Figure 9 Shown are the details of cell pseudopodia in the SGM sample and BMN2 sample. In the BMN2 sample, the cell antennae in some areas are connected to the surface while the main part of the cells spreads over a large area on the substrate. The pseudopodia are interconnected, and most of the cells have more lamellipodia. At the same time, in addition to filopodia and lamellipodia, stress fiber bundles are distributed at the cell edge. The cells overlap and cover the substrate surface in large numbers, which is beneficial to cell adhesion and migration. In the SGM sample, filopodia account for the majority, and the length range of the filopodia is within 1.17 - 12.26 μm. There may be a phenomenon of pseudopod breakage and a relatively small adhesion force, resulting in a lower adhesion ability than the BMN2 sample with more lamellipodia.

[0054] In summary, the sample prepared in Example 1 significantly promotes the proliferation and adhesion ability of osteoblasts, shows superhydrophilicity on the surface, and the surface roughness is significantly increased by 4 times compared with the untreated surface. The aspect ratio of the cells is greater than 2, the cells are in a stretched state and the spreading ability is enhanced, which is 2 times higher than that of the untreated surface.

[0055] Although the embodiments of the present invention have been shown and described, for those of ordinary skill in the art, it can be understood that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present invention. The scope of the present invention is defined by the appended claims and their equivalents.

[0056] The above content is only the preferred embodiment of the present invention. For those of ordinary skill in the art, according to the idea of the present technical content, many changes can be made in the specific implementation manner and application scope. As long as these changes do not depart from the concept of the present invention, they all belong to the protection scope of this patent.

Claims

1. A method for preparing a bionic composite micro-nanostructure metal surface for promoting bone fusion, characterized in that: The steps include: S1. Using a dual-beam laser device to prepare a periodic groove structure on a metal surface; S2. Using laser scanning equipment to secondary process blind hole structures on the metal surface of the periodic groove structure and form a bionic composite microstructure; S3. The surface of the bionic composite microstructure is subjected to thermal oxidation treatment. The bionic composite microstructure after thermal oxidation treatment is cooled to room temperature, and then washed with deionized water and dried to obtain a metal bionic composite micro-nanostructure.

2. The method for preparing a bionic composite micro-nanostructure metal surface for promoting bone fusion according to claim 1, characterized in that: In S3, during the thermal oxidation treatment of the surface of the obtained biomimetic composite microstructure, the temperature is set at 700°C, and the temperature is lowered to room temperature after the thermal oxidation treatment time of 4 hours.

3. The method for preparing a bionic composite micro-nanostructure metal surface for promoting bone fusion according to claim 1, characterized in that: The dual-beam laser equipment is a nanosecond laser dual-beam interference direct writing system; the secondary processing equipment is a laser marking machine; and the thermal oxidation treatment of the surface of the bionic composite microstructure is carried out in a quartz tube furnace.

4. The method for preparing the bionic composite micro-nanostructure metal surface for promoting bone fusion according to claim 3, characterized in that: In S1, the interference beam of the dual-beam laser interference direct writing system has a central symmetric spatial angle, a TE-TE polarization state, and a 1:1 ratio of the interference beam intensity energy density; the laser wavelength is 1064nm, the repetition frequency is 10Hz, the pulse duration is 7-9ns, and the period is 200μm.

5. The method for preparing the bionic composite micro-nanostructure metal surface for promoting bone fusion according to claim 3, characterized in that: In S2, when the laser marking machine is used for scanning, the laser power is 4W, the scanning speed is 2000mm / s, the structure height is 8.7μm and the spacing is about 49μm, the laser output power is 20W, the laser rated power is 0.5KW, and the laser pulse width is 20-100ns.

6. A metal surface prepared by the method according to any one of claims 1 to 5.

7. A method for preparing human osteoblastic osteosarcoma cells that react to implanted metal surface structures, characterized in that: In the use of metal surface attached cells as claimed in claim 6, the preparation method of the human osteoblastic osteosarcoma cell response to implantation of metal surface structure comprises: The minimum essential medium for culturing osteoblasts was prepared. The osteoblasts were attached to the metal surface and maintained at an ambient temperature of 37°C and an ambient air CO2 content of 5%.

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