CN113198043B - Electroactive titanium implant with immune response and preparation method thereof - Google Patents
Electroactive titanium implant with immune response and preparation method thereof Download PDFInfo
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- 238000002360 preparation method Methods 0.000 title claims abstract description 9
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- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/02—Inorganic materials
- A61L27/04—Metals or alloys
- A61L27/06—Titanium or titanium alloys
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B23—MACHINE TOOLS; METAL-WORKING NOT OTHERWISE PROVIDED FOR
- B23K—SOLDERING OR UNSOLDERING; WELDING; CLADDING OR PLATING BY SOLDERING OR WELDING; CUTTING BY APPLYING HEAT LOCALLY, e.g. FLAME CUTTING; WORKING BY LASER BEAM
- B23K26/00—Working by laser beam, e.g. welding, cutting or boring
- B23K26/36—Removing material
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B23—MACHINE TOOLS; METAL-WORKING NOT OTHERWISE PROVIDED FOR
- B23K—SOLDERING OR UNSOLDERING; WELDING; CLADDING OR PLATING BY SOLDERING OR WELDING; CUTTING BY APPLYING HEAT LOCALLY, e.g. FLAME CUTTING; WORKING BY LASER BEAM
- B23K26/00—Working by laser beam, e.g. welding, cutting or boring
- B23K26/70—Auxiliary operations or equipment
- B23K26/702—Auxiliary equipment
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- B23—MACHINE TOOLS; METAL-WORKING NOT OTHERWISE PROVIDED FOR
- B23P—METAL-WORKING NOT OTHERWISE PROVIDED FOR; COMBINED OPERATIONS; UNIVERSAL MACHINE TOOLS
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- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2400/00—Materials characterised by their function or physical properties
- A61L2400/18—Modification of implant surfaces in order to improve biocompatibility, cell growth, fixation of biomolecules, e.g. plasma treatment
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- A61L2430/00—Materials or treatment for tissue regeneration
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Abstract
The invention discloses an electroactive titanium implant with immune response and a preparation method thereof. The method comprises the following steps: (1) polishing the surface of the medical titanium implant, ultrasonically cleaning and drying; (2) and fixing the dried implant on a stepping motor of a laser marking machine for laser irradiation treatment. The pure titanium has different electrical properties from the pure titanium due to the phase transition of the pure titanium under the irradiation of the laser, so that a micro-area electric field is formed to make the pure titanium electrically active. The electroactive titanium implant has good biocompatibility, can effectively enhance the proliferation and differentiation of rat bone marrow mesenchymal stem cells, and can regulate and control the immune response of macrophages to form an immune microenvironment for promoting the differentiation of the rat bone marrow mesenchymal stem cells. The preparation method is simple and easy to operate, and is suitable for large-scale production.
Description
Technical Field
The invention belongs to the technical field of medical materials, and particularly relates to an electroactive titanium implant with immune response and a preparation method thereof.
Background
With the aging of the world population, the incidence of bone-related diseases such as fracture, bone tumor and the like is gradually increased, and the corresponding treatment cost is increased. Although bone has a certain ability to regenerate and repair itself, a large bone defect caused by severe trauma, tumor resection, cancer or congenital disease can only be repaired by bone grafting. Titanium and titanium alloys have been widely used in the field of bone implants because of their advantages of Young's modulus similar to that of bones, good corrosion resistance, and the like. However, the biological inertia of the pure titanium surface is not good for the fast and effective osseointegration, so that the realization of the fast and effective osseointegration by improving the biological activity of the pure titanium surface through the surface modification technology has important significance. Based on the piezoelectric property of bones, stem cells and osteoblasts in bones are in an electrophysiological microenvironment, and the electrophysiological microenvironment which is beneficial to proliferation and differentiation of the stem cells in the bones is constructed on the surface of a pure titanium implant, so that the method has important practical significance for promoting osseointegration. Meanwhile, macrophages play an indispensable role therein, considering that osteointegration is a dynamic and complex physiological process. Therefore, the immune regulation of macrophages by the constructed electrophysiological microenvironment forms an immune microenvironment favorable for osseointegration, which cannot be ignored as well.
In recent years, studies have shown that rapid repair of bone defects can be promoted by constructing an electrophysiological microenvironment on the surface of an implant that is suitable for the differentiation of stem cells in bone. However, the studies on macrophage immune regulation by the constructed electrophysiological microenvironment are very limited.
Disclosure of Invention
In order to overcome the defects and shortcomings in the prior art, the invention aims to provide a preparation method of an electroactive titanium implant with immune response.
Another object of the present invention is to provide an electro-active titanium implant with immune response prepared by the above method. The osseointegration efficiency is improved through the constructed electrophysiological microenvironment and the immune microenvironment formed by macrophage regulation.
The purpose of the invention is realized by the following technical scheme:
a preparation method of an electroactive titanium implant with immune response comprises the following steps:
and (3) polishing, cleaning and drying the titanium implant by using abrasive paper, and then placing the titanium implant on a laser marking machine to perform selective laser irradiation treatment to obtain the periodic semiconductor-metal heterojunction titanium implant with electric activity.
Preferably, the sanding is specifically: and sequentially grinding the titanium implant by using 200#, 500#, 1000#, 3000# and 5000# sandpaper to remove the surface oxide layer.
Preferably, the cleaning is ultrasonic cleaning with acetone, absolute ethyl alcohol and deionized water for 10-20 min in sequence.
Preferably, the drying is conventional drying.
Preferably, the conditions of the laser irradiation treatment are as follows: the laser irradiation power is 2.4-3W, the marking speed is 100-200 mm/s, and the scanning width is 15-100 mu m.
More preferably, the scanning width is 30 to 50 μm.
Preferably, the step motor is arranged on the laser marking machine, and the cleaned titanium implant is fixed on the step motor of the laser marking machine.
Preferably, the titanium implant is a pure titanium or titanium-based bone implant.
The electroactive titanium implant with the immune response is prepared by the method.
Compared with the prior art, the invention has the following advantages and beneficial effects:
(1) the titanium implant is subjected to surface treatment by selective laser irradiation, and the method is low in cost, high in speed and suitable for large-scale production.
(2) The micro-area electric field formed by the semiconductor-metal heterojunction obtained by the selective laser irradiation treatment conforms to the electrophysiological microenvironment of bone tissues, and has the effects of promoting proliferation and differentiation on the mesenchymal stem cells.
(3) The micro-area electric field formed by the semiconductor-metal heterojunction obtained by selective laser irradiation treatment can carry out immune regulation on macrophages, so that an immune microenvironment beneficial to differentiation of mesenchymal stem cells is formed.
Drawings
FIG. 1 is an SEM image of an electro-active titanium implant (labeled 30-MEF) with immune response according to example 1 of the present invention. The laser irradiated area is marked LT and the unirradiated area is Ti.
FIG. 2 is an SEM image of an electro-active titanium implant (labeled 50-MEF) with immune response according to example 1 of the present invention. The laser irradiated area is marked LT and the unirradiated area is Ti.
FIG. 3 is an SEM image of an electro-active titanium implant (labeled 100-MEF) with immune response according to example 1 of the present invention. The laser irradiated area is marked LT and the unirradiated area is Ti.
FIG. 4 is an XRD pattern of the laser irradiated area (LT) and the non-irradiated area (Ti) of the 30-MEF sample of example 2.
FIG. 5 is a Mott-Schottky plot of laser irradiated area (LT) versus non-irradiated area (Ti) for the 30-MEF sample of example 2.
FIG. 6 is a statistical chart of the carrier density of the laser irradiated region (LT) and the non-irradiated region (Ti) of the 30-MEF sample in example 2.
FIG. 7 is a graph showing the results of CCK-8 measurement of cell proliferation in the sample groups (30-MEF, 50-MEF, 100-MEF) having electrical activity and pure titanium group cultured rat bone marrow mesenchymal stem cells (rBMSCs) for 1, 3, and 5 days in example 3.
FIG. 8 is a graph showing the results of measuring the alkaline phosphatase activity of the cells in the sample groups (30-MEF, 50-MEF, 100-MEF) having electrical activity and pure titanium group-cultured rat bone marrow mesenchymal stem cells (rBMSCs) in example 4 for 7 days and 14 days.
FIG. 9 is a graph of the results of 3 days detection of the anti-inflammatory phenotypic marker CD206 expression in the sample groups with electrical activity of example 5 (30-MEF, 50-MEF, 100-MEF) and RAW264.7 macrophage cultured in pure titanium group.
FIG. 10 is a graph of the results of measuring the expression of CCR7, a pro-inflammatory phenotypic marker, in example 5 in the groups of samples with electrical activity (30-MEF, 50-MEF, 100-MEF) and pure titanium group cultured RAW264.7 macrophages for 3 days.
FIG. 11 is a graph showing the results of 3-day qT-PCR detection of the proinflammatory gene iNOS expression in the sample groups (30-MEF, 50-MEF, 100-MEF) having electrical activity and pure titanium group-cultured RAW264.7 macrophages in example 6.
FIG. 12 is a graph showing the results of 3-day qT-PCR detection of IL-10, an anti-inflammatory gene, in the sample groups (30-MEF, 50-MEF, 100-MEF) having electrical activity in example 6, and RAW264.7 macrophages cultured in pure titanium groups.
FIG. 13 is a graph showing the results of measuring the alkaline phosphatase activity of cells in the sample group having electric activity (30-MEF, 50-MEF, 100-MEF) of example 7 after culturing RAW264.7 macrophages for 3 days and collecting the culture medium thereof and after culturing rat bone marrow mesenchymal stem cells (rBMSCs) using the culture medium for 7 days.
Detailed Description
The present invention will be described in further detail with reference to examples and drawings, but the embodiments of the present invention are not limited thereto.
Those who do not specify specific conditions in the examples of the present invention follow conventional conditions or conditions recommended by the manufacturer. The raw materials, reagents and the like which are not indicated for manufacturers are all conventional products which can be obtained by commercial purchase.
Example 1
Preparation of electroactive titanium implant with immune response
(1) Pretreatment of pure titanium implants: sequentially grinding 200#, 500#, 1000#, 3000# and 5000# sandpaper to remove surface oxide layer, sequentially ultrasonically cleaning with acetone, anhydrous alcohol and deionized water for 20min, and conventionally drying.
(2) Construction of electroactive implants: fixing the pure titanium implant obtained in the step (1) on a stepping motor of a laser marking machine, and performing selective laser irradiation treatment, wherein the laser irradiation power is 3W, the scanning speed is 100mm/s, and the scanning widths are respectively 30 micrometers, 50 micrometers and 100 micrometers; the resulting electroactive implants were designated 30-MEF, 50-MEF and 100-MEF, respectively.
Comparative example 1
Pretreatment of pure titanium implants: sequentially polishing the implant by using 200#, 500#, 1000#, 3000# and 5000# sandpaper to remove a surface oxide layer, sequentially ultrasonically cleaning the implant by using acetone, absolute ethyl alcohol and deionized water for 20min, and finally conventionally drying to obtain the pretreated pure titanium.
Example 2
Electroactive titanium implants with immune response were prepared according to the process conditions of example 1.
FIGS. 1 to 3 are scanning electron microscope pictures of electroactive titanium implants, in which laser irradiated regions are marked as LT, unirradiated regions are marked as Ti, and the spacing between adjacent LT is set to 30 μm, 50 μm, 100 μm. Fig. 4 is the X-ray diffraction analysis results of LT and Ti, showing that the laser irradiated region phase-transformed into TiO. Fig. 5 is a mott-schottky curve of LT and Ti, and the carrier density calculated by the curve in combination with the mott-schottky formula is shown in fig. 6, indicating that LT and Ti have significantly different electrical properties.
Example 3
The electroactive implants 30-MEF, 50-MEF and 100-MEF prepared in example 1, and the pure titanium group (Ti) of comparative example 1 were used as a control group. After the disinfection treatment, third generation rat bone marrow mesenchymal stem cells (rBMSCs) are inoculated on the surface of the material according to the cell density of 10000 cells/100 mul, the cell proliferation condition is tested by a CCK-8 kit after 1, 3 and 5 days of culture, the test result is shown in figure 7, and the light absorption value of the 30-MEF group with electric activity is increased compared with that of the pure titanium group after 3 days of culture; after 5 days of culture, the light absorption values of the 30-MEF, 50-MEF and 100-MEF groups with electric activity are significantly different compared with the pure titanium group, which shows that the electric activity implant can significantly promote cell proliferation.
Example 4
The electroactive implants 30-MEF, 50-MEF and 100-MEF prepared in example 1, and the pure titanium group (Ti) of comparative example 1 were used as a control group. After the sterilization treatment, third-generation rat bone marrow mesenchymal stem cells (rBMSCs) are inoculated on the surface of the material according to the cell density of 50000 cells/100 mu l, the cell differentiation condition is tested by an alkaline phosphatase kit after the culture for 7 and 14 days, the test result is shown in figure 8, and after the culture for 7 and 14 days, compared with a pure titanium group, the alkaline phosphatase activity of the 30-MEF, 50-MEF and 100-MEF groups with the electrical activity is obviously improved, which indicates that the electrically active implant can obviously promote the cell differentiation.
Example 5
The electroactive implants 30-MEF, 50-MEF and 100-MEF prepared in example 1, and the pure titanium group (Ti) of comparative example 1 were used as a control group. After disinfection treatment, third generation RAW264.7 macrophages are inoculated on the surface of the material according to the cell density of 10000 cells/100 mu l, and the macrophage typing condition is detected by flow cytometry after 3 days of culture, the test result is shown in figures 9 and 10, compared with the pure titanium group, the macrophage of the 30-MEF, 50-MEF and 100-MEF groups with electric activity expresses the anti-inflammatory phenotype marker CD206 which is obviously higher than the pure titanium group, and expresses the proinflammatory phenotype marker CCR7 which is obviously lower than the pure titanium group, which indicates that the electric activity implant can regulate the inflammatory reaction of the macrophages to be anti-inflammatory at the protein level.
Example 6
The electroactive implants 30-MEF, 50-MEF and 100-MEF prepared in example 1, and the pure titanium group (Ti) of comparative example 1 were used as a control group. After disinfection treatment, third generation RAW264.7 macrophage is inoculated on the surface of the material according to the cell density of 10000 cells/100 mul, the expression condition of macrophage inflammation related gene is detected by qT-PCR after 3 days of culture, the test result is shown in figure 11 and 12, compared with the pure titanium group, the 30-MEF, 50-MEF and 100-MEF group with electric activity can lower the expression of the proinflammatory gene iNOS and simultaneously up-regulate the expression of the anti-inflammatory gene IL-10, which shows that the electric activity implant can up-regulate the expression of the anti-inflammatory gene at the gene level.
Example 7
The electroactive implants 30-MEF, 50-MEF and 100-MEF prepared in example 1, and the pure titanium group (Ti) of comparative example 1 were used as a control group. After disinfection treatment, third generation RAW264.7 macrophage is inoculated on the surface of the material according to the cell density of 10000 cells/100 mul, two groups of sample surface macrophage culture mediums are respectively collected after 3 days of culture, and the two groups of sample surface macrophage culture mediums and a new culture medium are prepared into a conditioned medium according to the proportion of 1: 1. Third generation rat bone marrow mesenchymal stem cells (rBMSCs) were seeded at a cell density of 50000 cells/100. mu.l in a well plate, and after culturing for 7 days with this conditioned medium, cell differentiation was tested using an alkaline phosphatase kit. The test results are shown in FIG. 13, and after 7 days of culture, the alkaline phosphatase activity of the 30-MEF, 50-MEF and 100-MEF groups with electrical activity is remarkably increased compared with that of the pure titanium group, which indicates that the electrically active implant can form an immune microenvironment favorable for rBMSCs differentiation by regulating the immune response of macrophages.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Claims (7)
1. A preparation method of an electroactive titanium implant with immune response is characterized by comprising the following steps:
after being polished by abrasive paper, cleaned and dried, the titanium implant is placed on a stepping motor of a laser marking machine to be subjected to selective laser irradiation treatment, so that a periodic semiconductor-metal heterojunction titanium implant with electric activity is obtained;
the conditions of the laser irradiation treatment are as follows: the laser irradiation power is 2.4-3W, the marking speed is 100-200 mm/s, and the scanning width is 15-100 mu m;
2. the method of claim 1, wherein the scan width is 30-50 μm.
3. The method of claim 1, wherein the titanium implant is a titanium-based bone implant.
4. The method of claim 1, wherein the titanium implant is pure titanium.
5. The method for preparing an electro-active titanium implant with immune response as claimed in claim 1, wherein the sand paper polishing is specifically: and sequentially grinding the titanium implant by using 200#, 500#, 1000#, 3000# and 5000# sandpaper to remove the surface oxide layer.
6. The method for preparing an electro-active titanium implant with immune response as claimed in claim 1, wherein the cleaning is performed by sequentially ultrasonic cleaning with acetone, absolute ethyl alcohol and deionized water for 10-20 min.
7. An electro-active titanium implant with immune response prepared by the method of any one of claims 1 to 6.
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| JPS63160662A (en) * | 1986-12-23 | 1988-07-04 | 川崎製鉄株式会社 | Surface treatment of metal material excellent in bone compatibility |
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| SE0004854D0 (en) * | 2000-12-22 | 2000-12-22 | Haakan Nygren | Surface modification of implants for bone and soft tissue healing |
| JP2003105557A (en) * | 2001-10-01 | 2003-04-09 | Matsumoto Shika Univ | Method of forming oxide film of metal member |
| US8268340B2 (en) * | 2002-09-26 | 2012-09-18 | Advanced Bio Prosthetic Surfaces, Ltd. | Implantable materials having engineered surfaces and method of making same |
| US8545559B2 (en) * | 2007-10-05 | 2013-10-01 | Washington State University | Modified metal materials, surface modifications to improve cell interactions and antimicrobial properties, and methods for modifying metal surface properties |
| CN100581708C (en) * | 2008-04-25 | 2010-01-20 | 河北工业大学 | Application of femtosecond laser in titanium or titanium alloy implantation material surface treatment |
| JP2013221023A (en) * | 2012-04-19 | 2013-10-28 | Yuichiro Kawahara | Functional implant exhibiting photocatalytic effect |
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