A homogeneous and stable nanometal cell culture interface and its preparation method

Abstract

The present invention discloses a method for preparing a homogeneous and stable nanometal cell culture interface, comprising the following steps: S1, determining a metal material; S2, performing low-plasticity micro-grinding and polishing on the metal material using high-frequency ultrasonic signals and pulsed currents, so that a nanostructured layer is formed on the surface of the metal material; S3, grinding the metal material with the nanostructured layer formed on the surface, and separating the nanostructured layer, which is the nanometal cell culture interface. By coupling overclocking rolling with high current processing and non-destructive surface extraction technology, the present invention not only reduces process costs and waste, but also uses metal materials to replace certain toxic chemical reagents to construct a cell-matrix interface with a homogeneous and stable "net-like" nanostructure. This interface structure can effectively improve the efficiency of cell proliferation in vitro and enhance the comprehensive biological functions of cells.

Description

Technical Field

[0001] The present invention relates to the application fields of metal nanomaterial processing and preparation and in vitro culture of bioengineering biological cells, and in particular to a homogeneous and stable nanometal cell culture interface and a preparation method thereof. Background Art

[0002] There is currently a great deal of controversy surrounding the preparation of nanomaterials that can be applied in the fields of biochemistry and biomedicine. Physical methods such as grinding or impact etching require very complex operating procedures and sophisticated instruments to prepare nanomaterials. This is not only time-consuming, insensitive, and costly, but also cannot meet the needs of daily experiments and industrial production. Chemical methods for preparing nanomaterials require large amounts of reducing agents and even certain toxic chemical reagents, which not only wastes materials but also easily pollutes the environment during the preparation process. Moreover, these reagents have certain potential hazards to the production and life of animals, plants, and even humans. Combining with biology to prepare these nanomaterials with special properties is low-cost, reduces waste, and makes the preparation method more convenient and simple. The use of bioactive materials containing a large number of active sites and reducing groups to replace certain toxic chemical reagents as reducing agents, capping agents, and stabilizers in the preparation process has become a research hotspot for many scientific researchers.

[0003] Biologically, a high density of nanoscale boundaries promotes protein adsorption, a hallmark of human osteoblast biological function and the first step in the interface of biomedical devices with the physiological environment. It is well established that osteoblast function can be modulated by nanoscale structures such as nanopillars, nanotubes, and nanogrooves. Furthermore, the surface properties of the matrix, such as micro / nanoscale topography, grain size, and stiffness, play an important role in mediating cellular activity and significantly influence the optimal and rapid osseointegration of biomedical implants. Several studies have shown that surfaces containing nanoparticles / ultrafine particles exhibit enhanced biological properties, attributed to the increased surface energy resulting from the high density of grain boundaries.

[0004] The morphological characteristics of a material's surface are crucial factors influencing implant osseointegration. Appropriately rough surfaces are more conducive to osseointegration than smooth surfaces. Currently, most implant surfaces used clinically feature micron-scale topography, which favors osteoblast differentiation but may inhibit osteoblast proliferation, affecting the rate of bone formation on the implant surface. These nanoscale topography, either alone or in combination with micron-scale topography, can positively stimulate and guide the biological behavior of tissue cells, promoting cell culture on the material surface. Metal nanomaterials have several limitations and drawbacks in their biochemical preparation and biomedical applications. One limitation is process immaturity: most preparation methods struggle to control bionanosurface energy, resulting in poor biostructure stability. Furthermore, large-scale nanomaterial production is costly and time-consuming. Another limitation of these methods is the inability to simultaneously achieve optimal mechanical and biological properties. Furthermore, chemical methods (such as high-concentration hydrofluoric acid etching) to modify the cell-substrate interface can disrupt the matrix structure and introduce unwanted ions into the surface, which can be detrimental to tissue and reduce the fatigue life, corrosion, and wear resistance of bioimplant materials. In addition, these methods have difficulty in achieving the high levels of biological requirements in terms of cell number and cell coverage area at the interface of culturing human osteoblasts, and it is difficult to simultaneously achieve better attachment, wider diffusion and higher proliferation rate in vitro, which ultimately greatly reduces the effectiveness of biological functions. Summary of the Invention

[0005] To address the shortcomings of the above-mentioned prior art, the present invention aims to provide a homogeneous and stable nanometallic cell culture interface and its preparation method. This method, by coupling overclocking rolling with high-current processing and non-destructive surface extraction techniques, not only reduces process costs and waste but also uses metal materials to replace certain toxic chemicals. This creates a homogeneous and stable cell-matrix interface with a "net-like" nanostructure. This interface structure can effectively improve the efficiency of cell proliferation in vitro and enhance the comprehensive biological functions of cells.

[0006] To achieve the above object, the present invention is implemented through the following technical solutions:

[0007] A method for preparing a homogeneous and stable nanometal cell culture interface comprises the following steps:

[0008] S1. Determine the metal material;

[0009] S2. Using high-frequency ultrasonic signals and pulse current to perform low-plasticity micro-motion grinding and polishing on the metal material, so that a nanostructured layer is formed on the surface of the metal material;

[0010] S3. Grind the metal material with the nanostructured layer formed on the surface to separate the nanostructured layer, which is the nanometal cell culture interface.

[0011] Preferably, the metal material in step S1 is 316L stainless steel plate with a thickness of 3 mm and a size of 50×50 mm. 2 .

[0012] Preferably, step S2 is to grind and polish the metal material in a plasma aerosol medium, by subjecting the plate surface to infinitely ultra-high frequency low plasticity micro-motion impact in the plasma aerosol medium, causing intermittent friction corrosion of the material to accelerate the deterioration process.

[0013] Preferably, in step S2, a spot welding electric pulse generator is used to generate the pulse current, and the peak value of a single pulse current of the spot welding electric pulse generator satisfies the following formula:

[0014]

[0015] Where, I p is the current through the metal material, A is the cross-sectional area of ​​the material, ρ is the material density, c is the specific heat capacity, σ is the resistivity, is the heating rate.

[0016] Preferably, the pulse current in step S2 is on for 1 second.

[0017] Preferably, the overfrequency threshold of the high-frequency ultrasonic signal in step S2 is 20KHz, the maintenance time accounts for 80% of the total polishing time, and the rest time accounts for 20% of the total polishing time.

[0018] Preferably, in step S3, an automatic grinding machine with a friction coefficient detection function is used to grind the metal material with the nanostructured layer formed on the surface from bottom to top.

[0019] Preferably, during the grinding process, the center of the wear track of the automatic grinding machine is maintained at 1.59×10 -3 A constant speed of m / s.

[0020] Preferably, the grinding time in step S3 is determined by subtracting the wear thickness D from the original thickness D of the metal material. L Get the remaining thickness D R , when the remaining thickness D R Stop grinding when the friction coefficient is less than 100; or stop grinding when the friction coefficient is stably increased to the range of 0.35<μ<0.4.

[0021] Preferably, according to the wear volume V of the metal material w and wear surface area S e Get the wear thickness D L , where the wear volume and wear surface area of ​​metal materials are calculated by the following formulas:

[0022]

[0023] S e =c×c′

[0024] Where V w is the wear volume (mm 3 );S e is the wear surface area (mm 2 ); R and r are the radii of the wear track and the grinding ball pin, respectively; c and c′ are the width and length of the wear track, respectively.

[0025] The present invention also provides a nano-metal cell culture interface prepared by the above preparation method.

[0026] The present invention proposes a method for preparing a homogeneous and stable nanometal cell culture interface. Different from the "surface morphology modification" method of traditional biological culture interfaces, the method is based on metal materials and first uses high-density and high-frequency ultrasonic energy fields and pulse currents to perform low-plasticity micro-grinding and polishing on the metal materials to make the material surface homogenized and nano-sized. Then, an automatic grinding machine is used to grind the material from bottom to top by abrasion to regulate the residual thickness. The resulting nanocrystalline network layer has a high degree of fineness and surface integrity. The non-destructive surface extraction technology avoids irreversible damage such as corrosion and grain growth caused by local high temperature generated by electric spark wire cutting. At the same time, the efficiency and reliability of the matrix are verified through control experiments, detection and statistics of in vitro culture.

[0027] The beneficial effects of the present invention are:

[0028] The present invention provides a method for preparing a homogeneous and stable nanometal cell culture interface. By coupling overclocking rolling with high current processing and non-destructive surface extraction technology, not only the process cost is low and waste is reduced, but also metal materials are used to replace certain toxic chemical reagents to construct a cell-matrix interface with a homogeneous and stable "network" nanostructure. This interface structure can effectively improve the efficiency of cell proliferation in vitro and enhance the comprehensive biological functions of cells.

[0029] The method designed in the present invention can realize quantitative calculation of the effective culture area and thickness of the processed matrix, thereby providing the possibility of obtaining a stable, efficient, homogeneous and reliable cell culture interface. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings of the embodiments are briefly introduced below.

[0031] Figure 1 A diagram of the device for preparing a homogeneous and stable nanometal cell culture interface according to the present invention;

[0032] Figure 2 This is a schematic diagram of the present invention coupling ultrasonic energy field and high-voltage electric field to perform low-plasticity micro-grinding and polishing on metal materials;

[0033] Figure 3 This is a schematic diagram of the structure of a metal material after low-plasticity micro-grinding and polishing;

[0034] Figure 4 A diagram of an automatic sample grinding machine for grinding metal materials according to the present invention;

[0035] Figure 5 This is a graph showing the change of friction coefficient over time during the grinding process;

[0036] Figure 6 The in vitro physical and chemical environment of cells when they are cultured on the ultrafine nano-metal surface layer prepared by the present invention and the cell guidance process on the nano-metal substrate surface;

[0037] Figure 7 The cell coverage and cell density of human bone cells after culturing on two culture substrates for 2, 4, and 6 hours;

[0038] Figure 8 The figure shows the cell proliferation of human bone cells after 12 hours of proliferation culture on two culture matrices. DETAILED DESCRIPTION

[0039] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0040] Example 1

[0041] S1. Determine the metal material.

[0042] The metal material used in this embodiment is 316L stainless steel plate with a thickness D of 3 mm and a size of 50×50 mm. 2 .

[0043] S2. Use high-frequency ultrasonic signals and pulse current to perform low-plasticity micro-grinding and polishing on metal materials, so that a nanostructured layer is formed on the surface of the metal material.

[0044] Figure 1Diagram of the device for preparing a homogeneous and stable nanometal cell culture interface in this embodiment, including a boss 1 soaked in a plasma aerosol medium; a low-plasticity polishing grinding ball 2; a spot welding electric pulse generator 3; an ultrasonic generator 4 that drives the grinding ball to produce micro-motion back and forth vibration; a quantum wave white light interferometer thermal imager 5 for observing the Joule heating effect of eddy currents; and an ultra-frequency rolling variable amplitude frequency-cross-axis connecting rod 6 for amplifying the ultrasonic phase difference angle and transmitting it to the grinding ball.

[0045] The specific steps are as follows: 3 A 316L steel plate is fixed in the reaction tank, i.e., on the concave surface of the reaction tank between the boss 1 and the low-plasticity polished grinding ball 2. Polished and cleaned Al2O3 balls (6mm) are used as the grinding material. A 3.5% NaCl solution is added to the groove to enhance the conductivity and accelerate the deterioration process of the material by friction corrosion. The spot welding electric pulse generator 3 is adjusted to pass the sample through the electromagnetic defibrillator. The power-on time t is determined before the electromagnetic defibrillator is used. d and the peak value of a single pulse current I p When a large current is used to adaptively sinter the conductor, the peak value of a single pulse current I p It is mainly determined by the heating rate of the material and satisfies the following formula:

[0046]

[0047] Where Ip is the current passing through the metal material, A is the cross-sectional area of ​​the material, ρ is the material density, c is the specific heat capacity, and σ is the resistivity. is the heating rate.

[0048] The large-scale electrothermal cycle process is conducive to the growth of sintered necks and the enhancement of particle connection strength because the continuous iteration of the heating process generates a large amount of Joule heat. The sample obtains more Joule heating. The cyclic electrothermal effect provides more energy and electroplasticity than constant sintering, and the densification process is significantly enhanced. Therefore, under the action of electrothermal cycling, the calculation formula for the temperature increase ΔT of the material due to Joule heating is:

[0049]

[0050] Where: ρ e is the resistivity of the material; j is the current density; t d is the power-on time; c is the specific heat capacity of the material; d is the density of the material.

[0051] The present invention sets ΔT to not exceed the maximum operating temperature of 450°C recommended by the national standard GB150-98, so as to avoid the decrease of the strength and oxidation resistance of the obtained matrix. dThe spot welding electric pulse generator 3 is then started. The surface temperature of the sample is observed to be within 450°C by the quantum wave white light interference thermal imager 5. After the internal stress of the material is released and the material is homogenized and a certain degree of electroplasticity is preserved, the ultrasonic generator 4 is started and its overclocking threshold is set to 20KHz, the holding time is 800ms, and the rest time t step Finally, the ultra-frequency rolling variable amplitude frequency penetrating shaft connecting rod 6 is started, so that the ultrasonic phase difference angle is amplified and transmitted to the grinding ball, thereby achieving infinite ultra-frequency low plasticity micro-impact on the surface of the plate by coupling the ultrasonic energy field and the high voltage electric field (such as Figure 2 As shown in the figure, the material undergoes intermittent friction corrosion, which accelerates the degradation process and creates a nanostructured layer with strong plasticity matching on the surface of the metal material.

[0052] S3. Observe and count the metal materials after ultrasonic signal and pulse current processing.

[0053] First, the 316L stainless steel obtained in S2 was taken and its cross section was prepared into an electron backscatter diffraction sample for mechanical polishing. Then, it was etched in a corrosive solution at room temperature to remove the impurities in the deformation layer on the surface of the sample. The gradient layer of the material along the depth direction was observed by SEM scanning electron microscopy, and the morphology of the processed 316L stainless steel was observed as follows: Figure 3 The following images are shown: (a) shows the topmost surface layer of the sheet, which features a "net-like" nanostructure; (b) and (c), located 100μm and 200μm from the topmost surface, respectively, show short-range ordered grains, also at the nanometer scale. The observed images were imported into analysis and processing software such as ImageJ and OIM, which can extract phase boundaries in crystal structures, to generate crystal structure contours. The average grain size of the extremely fine nanostructured surface layer at a depth of 0μm was found to be 326nm.

[0054] S4. Grind the metal material with the nanostructured layer formed on the surface to separate the nanostructured layer, which is the nanometal cell culture interface.

[0055] According to the observation and statistical results of step S3, it can be concluded that Figure 3 This finest "net-like" structure in the middle (a) layer makes the material very strong, because the "net-like" nano-scale grain boundaries have the necessary conditions for cell proliferation in vitro, providing a good breeding ground for cell proliferation in vitro. Figure 3 The finest "net-like" nanostructured surface in the middle (a) layer was removed and used as the matrix interface for cell culture.

[0056] Since the nanomatrix prepared in the present invention is used for biological culture, high requirements are placed on the fineness and surface integrity of the nanocrystalline network layer. In order to avoid irreversible damage such as corrosion and grain growth caused by local high temperature generated by electric spark wire cutting, the embodiment of the present invention uses an automatic grinding machine with a friction coefficient detection function to grind the plate from bottom to top in S3 after the impurity layer is removed.

[0057] Combine Figure 4 The specific grinding process includes the following steps: fixing the plate obtained after processing in S3 on the grinding machine through the spring clip 41, and adjusting the height to just contact the sand plate; passing the weight 42 with a mass of 2000N through the pore and installing the pressure plate, thereby applying a certain positive pressure to the sample; connecting the wiring harness 43 of the pressure sensor to the computer display 46; adjusting the positive pressure in the initial state to zero through the mechanical zeroing scale 44; finally, presetting the grinding end time to 30 minutes and the temperature to room temperature 25℃ through the controller button 45, and the equipment running time and real-time temperature are displayed on the display screen corresponding to TI and LI respectively; after the preparation work is completed, the equipment is operated.

[0058] The following equation describes the heat transfer in stainless steel during grinding. Since the stirrer mill is fixed in the milling plate coordinates, the equation includes a convection term in addition to a conduction term.

[0059]

[0060] Where k is the thermal conductivity, ρ is the density, Cp is the specific heat capacity, and u is the speed of the friction head relative to the workpiece.

[0061] The heat generated at the interface between the pin of the stirring head and the workpiece is simulated as a surface heat source. The friction temperature rise of the sample is read and converted into the surface heat source heat q through the relationship with the specific heat capacity. s (T)

[0062]

[0063] Where μ is the friction coefficient, r p represents the radius of the rubbing ball pin, ω is the angular velocity of the rubbing ball pin (rad / s), is the average shear stress of the material, and the average shear stress in the sensor is a function of temperature.

[0064] The above heat transfer formula and surface heat calculation were used to evaluate the influence of wear heat on the sample, which proved that the temperature rise during the grinding process had little effect on the final matrix and the fineness was high.

[0065] Specifically, in the grinding process of this embodiment, the center of the wear track of the automatic grinding machine is maintained at u=1.59×10 -3 The grinding time is determined by subtracting the wear thickness D from the original thickness D of the metal material. L Get the remaining thickness D R , when the remaining thickness D R =DD L Stop grinding when the friction coefficient is less than 100μm; or stop grinding when the friction coefficient is stably increased to the range of 0.35<μ<0.4. The size of the plate in this embodiment is 50×50mm 2 , the original thickness D is 3 mm, combined with the wear volume V w and wear surface area S e Get the wear thickness D L , where the wear volume and wear surface area of ​​metal materials are calculated by the following formulas:

[0066]

[0067] S e =c×c′

[0068] Where V w is the wear volume (mm 3 );S e is the wear surface area (mm 2 ); R and r are the radii of the wear track and the grinding ball pin, respectively; c and c′ are the width and length of the wear track, respectively.

[0069] Under partial sliding rolling conditions, normal and shear surface tractions for circular and line contacts with a maximum Hertzian pressure of 1 GPa and a Hertzian semi-contact with a pressure of a = 100 μm were applied to the top surface of the workpiece, with a fretting displacement amplitude of δ = 0.7 μm. Based on the Hills and Herte elastic theory for the cyclic variation of the tangential force in partial sliding fretting wear of similar materials, c and c′ can be calculated as follows:

[0070]

[0071] Where, F max is the maximum normal force, T max is the maximum tangential force (T in partial slip state max <μF max ), F max and T max It can be read by the rolling head sensor measuring device, 0<t step <1s, t in this embodiment step is 200ms.

[0072] When D R =3000-D LStop the grinding machine when the friction coefficient is less than 100μm, that is, press the stop key when the friction coefficient is steadily increased to 0.35<μ<0.4 through the display. (The TXT document exported by the real-time pressure and time parameters detected by the sensor is copied to EXCEL and divided by the weight. Finally, it is plotted by ORIGIN image software, as shown in the figure. Figure 5 Finally, the sample was removed and gently grasped with tweezers and placed in a culture dish (denoted as NG-316L) to serve as the matrix interface for culturing cells.

[0073] Example 2

[0074] S1. A cell culture experiment was conducted using the nanometal material NG-316L prepared in Example 1 as the matrix interface for cell culture; another piece of initial material was taken as the matrix for culture in the control group (denoted as AR-316L).

[0075] S2. Implant cells into the matrix interface for in vitro culture and perform morphological statistics and analysis

[0076] S2.1 Use water, inorganic salts, CO2, etc. to prepare the necessary in vitro nutritional environment for cells. Figure 6 This figure shows the in vitro physicochemical environment of cells cultured on the ultrafine nano-surface layer of NG-316L metal prepared by the present invention, as well as the cell guidance process on the nano-metal surface. The figures include (I) hydrophilic groups, (II) recrystallized structure in the metal, (III) porous surface defects generated after processing, (IV) localized rough surface formed after processing, and (V) metal ion proteins.

[0077] S2.2 Human bone cells of a certain density were inoculated on the AR-316L surface and NG-316L surface matrix, respectively, to detect their respective biological function indicators of attachment, diffusion and reproduction.

[0078] Cells were cultured at a density of 400,000 cells / cm² in AR-316L and NG-316L culture media for 2, 4, and 6 hours, respectively. The cells were fixed with an ethanol solution for 30 minutes and then removed using a graded aerosol spray. Finally, the samples were incubated in reduced ATP for 1 hour at room temperature and processed using a Greenwich sputtering machine. The samples were then examined using a scanning electron microscope. Proliferating samples were observed using a fluorescence microscope.

[0079] S2.3 Observe and count the differences in cell spreading and attachment under the two culture media.

[0080] The cell samples were observed after being cultured on the two control matrix surfaces for 6 hours. The results showed that the cells gradually spread on the two matrix surfaces with the increase of culture time. Compared with AR-316L, the NG-316L group had a greater cell density in each time period, indicating better attachment to the NG-316L surface. It was observed that the cells spread more widely on the NG-316L surface than on the AR-316L surface. Further, ImageJ software was used to analyze the cell density and cell coverage area observation graphs to determine the effectiveness of cell attachment and spreading. The statistical analysis results of cell coverage and cell density are shown below. Figure 7 The combined observation results and the data from the image statistics software indicate that the cells cultured on the NG-316L surface prepared by the present invention exhibited better attachment and more extensive spreading than those on the untreated AR-316L surface.

[0081] S2.4 Observe and count the differences in cell proliferation ability under two culture media

[0082] Cells were cultured at a density of 400,000 cells / cm2 in AR-316L and NG-316L medium for 12 hours and then samples were collected to ensure that the cells had undergone a high level of proliferation before observation under a fluorescence microscope. The results of the samples of the two matrices were as follows: Figure 8 (a)(b), RH staining is used to indicate the cell cycle status. Figure 8 (a) and (b) show that RH positive cells are undergoing cell proliferation, while negative cells are in a silent state, respectively recording Figure 8 The number of RH-positive cells in the cell samples cultured under the two matrices in (a) and (b) are N r1 、N r2 , the number of RH negative cells is N b1 、N b2 , the cell ratio of the two groups of culture samples was calculated according to the following formula:

[0083]

[0084]

[0085] N in the formula r1 、N r2 N is the number of proliferating cells after culturing on the AR-316L matrix and NG-316L interface for 12 h; b1 、N b2 The number of static non-proliferating cells after culturing at the two interfaces for 12 hours; DAPI Ais the percentage of proliferating cells in human osteoblast samples cultured in AR-316L matrix; DAPI B is the percentage of proliferating cells in human osteoblast samples cultured in NG-316L matrix.

[0086] This indicator is used to evaluate the size of cell proliferation rate and to determine the rate of cell proliferation in vitro. The cell proliferation observation and statistical analysis results of the two samples are as follows: Figure 8 As shown in (c) and (d), the number of proliferating cells and the cell proliferation index (DAPI) in the samples cultured on the NG-316L surface matrix were significantly increased. B 87%), both of which were higher than those of the AR-316L culture matrix sample (DAPI A is 69%).

[0087] By comparing the observation and statistical results of in vitro cell culture on the surfaces of the two structures of the matrix, and using cell density and cell coverage to evaluate the morphological properties of attachment and diffusion, respectively, and using the proliferation index DAPI to evaluate the cell proliferation ability, it is shown that using the surface "network" nanostructured matrix NG-316L prepared and designed by the present invention as a cell culture interface can significantly improve the efficiency of cell proliferation in vitro and enhance the comprehensive biological functions of cells.

[0088] In summary, the present invention is different from the traditional "surface morphology modification" method of biological culture interface in that, based on 316L stainless steel plate, a preparation method of a mesh nanostructured matrix interface for in vitro cell culture is proposed. First, the matrix plate is subjected to low-plasticity micro-motion grinding and polishing by high-density and high-frequency ultrasonic energy field and pulse current to perform homogenized nano-surface modification. Secondly, the residual thickness is regulated by wear in an automatic grinding machine. The fineness and surface integrity of the obtained nanocrystalline mesh layer are of high quality. The non-destructive surface extraction technology avoids the irreversible damage such as corrosion and grain growth caused by local high temperature generated by electric spark wire cutting. At the same time, the efficiency and reliability of the matrix are verified by control experiments, detection and statistics of in vitro culture.

[0089] The present invention proposes a method for preparing a stable nanometal cell culture interface, based on the integration of advanced material nano-processing and manufacturing with bioengineering technology. The technical solution is different from the existing matrix morphology and production methods for osteoblast culture. From the processing method itself, the time and processing cost of producing the matrix are reduced, and the adverse effects of high-temperature electrochemical corrosion are avoided. The coupling of overclocking rolling with high current processing and non-destructive surface extraction technology, at the same time, the designed method can realize the quantitative calculation of the effective culture area and thickness of the processed matrix, thereby providing the possibility of obtaining a stable, efficient, homogeneous and reliable internal structure of the cell matrix.

[0090] It should be noted that the above embodiments all belong to the same inventive concept, and the description of each embodiment has its own focus. For any details not described in individual embodiments, reference may be made to the description in other embodiments.

[0091] The above-described embodiments merely illustrate the implementation methods of the present invention. While the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent. It should be noted that a person skilled in the art would be able to make various modifications and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the patent for this invention shall be determined by the appended claims.

Claims

1. A method for preparing a homogeneous and stable nanometal cell culture interface, characterized in that: The following steps are involved: S1. Determine the metal material; S2. performing low-plasticity micro-motion grinding and polishing on the metal material using a high-frequency ultrasonic signal and a pulse current, so that a nanostructured layer is formed on the surface of the metal material; S3, grinding the metal material with the nanostructured layer formed on the surface to separate the nanostructured layer, which is the nanometal cell culture interface; The metal material in step S1 is 316L stainless steel plate; Step S2 uses a spot welding electric pulse generator to generate the pulse current. The peak value of a single pulse current of the spot welding electric pulse generator satisfies the following formula: Where, I p is the current through the metal material, A is the cross-sectional area of ​​the material, ρ is the material density, c is the specific heat capacity, σ is the resistivity, is the heating rate; Step S3: grinding the metal material with the nanostructured layer formed on the surface from bottom to top using an automatic grinding machine; The grinding time in step S3 is determined by subtracting the wear thickness D from the original thickness D of the metal material. L Get the remaining thickness D R , when the remaining thickness D R Stop grinding when the friction coefficient is less than 100μm; or stop grinding when the friction coefficient is steadily increased to the range of 0.35<μ<0.4; According to the wear volume V of metal materials w and wear surface area S e The wear thickness D is obtained L , wherein the wear volume and wear surface area of ​​the metal material are calculated by the following formulas: Where V w is the wear volume (mm 3 );S e is the wear surface area (mm 2 ); R and r are the radii of the wear track and the grinding ball pin, respectively; c and c′ are the width and length of the wear track, respectively.

2. The method for preparing a homogeneous and stable nanometal cell culture interface according to claim 1, characterized in that: The metal material in step S1 has a thickness of 3 mm and a size of 50×50 mm. 2 .

3. The method for preparing a homogeneous and stable nanometal cell culture interface according to claim 1, characterized in that: Step S2 is to perform grinding and polishing on the metal material in a plasma aerosol medium.

4. The method for preparing a homogeneous and stable nanometal cell culture interface according to claim 1, characterized in that: The overfrequency threshold of the high-frequency ultrasonic signal in step S2 is 20KHz, the maintenance time accounts for 80% of the total polishing time, and the rest time accounts for 20% of the total polishing time.

5. The method for preparing a homogeneous and stable nanometal cell culture interface according to claim 1, characterized in that: During the grinding process of step S3, the wear track center of the automatic grinding machine is kept at 1.59×10 -3 A constant speed of m / s.

6. The nanometal cell culture interface prepared according to the method for preparing a homogeneous and stable nanometal cell culture interface according to any one of claims 1 to 5.

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