WO2022268035A1 - Multidirectional photofunctionalization instrument for surface modification of titanium alloy implant and method of use thereof - Google Patents
Multidirectional photofunctionalization instrument for surface modification of titanium alloy implant and method of use thereof Download PDFInfo
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- WO2022268035A1 WO2022268035A1 PCT/CN2022/099903 CN2022099903W WO2022268035A1 WO 2022268035 A1 WO2022268035 A1 WO 2022268035A1 CN 2022099903 W CN2022099903 W CN 2022099903W WO 2022268035 A1 WO2022268035 A1 WO 2022268035A1
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- titanium alloy
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61F—FILTERS IMPLANTABLE INTO BLOOD VESSELS; PROSTHESES; DEVICES PROVIDING PATENCY TO, OR PREVENTING COLLAPSING OF, TUBULAR STRUCTURES OF THE BODY, e.g. STENTS; ORTHOPAEDIC, NURSING OR CONTRACEPTIVE DEVICES; FOMENTATION; TREATMENT OR PROTECTION OF EYES OR EARS; BANDAGES, DRESSINGS OR ABSORBENT PADS; FIRST-AID KITS
- A61F2/00—Filters implantable into blood vessels; Prostheses, i.e. artificial substitutes or replacements for parts of the body; Appliances for connecting them with the body; Devices providing patency to, or preventing collapsing of, tubular structures of the body, e.g. stents
- A61F2/02—Prostheses implantable into the body
- A61F2/28—Bones
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61F—FILTERS IMPLANTABLE INTO BLOOD VESSELS; PROSTHESES; DEVICES PROVIDING PATENCY TO, OR PREVENTING COLLAPSING OF, TUBULAR STRUCTURES OF THE BODY, e.g. STENTS; ORTHOPAEDIC, NURSING OR CONTRACEPTIVE DEVICES; FOMENTATION; TREATMENT OR PROTECTION OF EYES OR EARS; BANDAGES, DRESSINGS OR ABSORBENT PADS; FIRST-AID KITS
- A61F2/00—Filters implantable into blood vessels; Prostheses, i.e. artificial substitutes or replacements for parts of the body; Appliances for connecting them with the body; Devices providing patency to, or preventing collapsing of, tubular structures of the body, e.g. stents
- A61F2/02—Prostheses implantable into the body
- A61F2/30—Joints
- A61F2/3094—Designing or manufacturing processes
- A61F2/30942—Designing or manufacturing processes for designing or making customized prostheses, e.g. using templates, CT or NMR scans, finite-element analysis or CAD-CAM techniques
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B22—CASTING; POWDER METALLURGY
- B22F—WORKING METALLIC POWDER; MANUFACTURE OF ARTICLES FROM METALLIC POWDER; MAKING METALLIC POWDER; APPARATUS OR DEVICES SPECIALLY ADAPTED FOR METALLIC POWDER
- B22F10/00—Additive manufacturing of workpieces or articles from metallic powder
- B22F10/30—Process control
- B22F10/38—Process control to achieve specific product aspects, e.g. surface smoothness, density, porosity or hollow structures
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B22—CASTING; POWDER METALLURGY
- B22F—WORKING METALLIC POWDER; MANUFACTURE OF ARTICLES FROM METALLIC POWDER; MAKING METALLIC POWDER; APPARATUS OR DEVICES SPECIALLY ADAPTED FOR METALLIC POWDER
- B22F10/00—Additive manufacturing of workpieces or articles from metallic powder
- B22F10/60—Treatment of workpieces or articles after build-up
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B33—ADDITIVE MANUFACTURING TECHNOLOGY
- B33Y—ADDITIVE MANUFACTURING, i.e. MANUFACTURING OF THREE-DIMENSIONAL [3-D] OBJECTS BY ADDITIVE DEPOSITION, ADDITIVE AGGLOMERATION OR ADDITIVE LAYERING, e.g. BY 3-D PRINTING, STEREOLITHOGRAPHY OR SELECTIVE LASER SINTERING
- B33Y40/00—Auxiliary operations or equipment, e.g. for material handling
- B33Y40/20—Post-treatment, e.g. curing, coating or polishing
-
- C—CHEMISTRY; METALLURGY
- C22—METALLURGY; FERROUS OR NON-FERROUS ALLOYS; TREATMENT OF ALLOYS OR NON-FERROUS METALS
- C22F—CHANGING THE PHYSICAL STRUCTURE OF NON-FERROUS METALS AND NON-FERROUS ALLOYS
- C22F3/00—Changing the physical structure of non-ferrous metals or alloys by special physical methods, e.g. treatment with neutrons
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61F—FILTERS IMPLANTABLE INTO BLOOD VESSELS; PROSTHESES; DEVICES PROVIDING PATENCY TO, OR PREVENTING COLLAPSING OF, TUBULAR STRUCTURES OF THE BODY, e.g. STENTS; ORTHOPAEDIC, NURSING OR CONTRACEPTIVE DEVICES; FOMENTATION; TREATMENT OR PROTECTION OF EYES OR EARS; BANDAGES, DRESSINGS OR ABSORBENT PADS; FIRST-AID KITS
- A61F2/00—Filters implantable into blood vessels; Prostheses, i.e. artificial substitutes or replacements for parts of the body; Appliances for connecting them with the body; Devices providing patency to, or preventing collapsing of, tubular structures of the body, e.g. stents
- A61F2/02—Prostheses implantable into the body
- A61F2/30—Joints
- A61F2/30767—Special external or bone-contacting surface, e.g. coating for improving bone ingrowth
- A61F2002/3093—Special external or bone-contacting surface, e.g. coating for improving bone ingrowth for promoting ingrowth of bone tissue
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P10/00—Technologies related to metal processing
- Y02P10/25—Process efficiency
Definitions
- the invention belongs to the field of photofunctionalization, and in particular relates to a multi-directional photofunctionalization instrument aimed at modifying the surface of titanium alloy inner plants and a use method thereof.
- Photofunctionalization refers to the technology of modifying the surface of metal implants (especially titanium alloys) by using ultraviolet rays of specific wavelength, intensity, and irradiation time to enhance their physical and chemical properties and biological effects. After the titanium alloy implant is exposed to the environment after fabrication, it will gradually lose its biological activity, which can be reactivated by exposure to ultraviolet light.
- the existing irradiation treatment can only treat the surface of the structure, and cannot treat the inner surface of the porous structure.
- the existing treatment uses a high-pressure mercury lamp, and the ultraviolet wavelength emitted by it is fixed.
- the structure of the light source is bulky, usually used in a fixed position, and can only irradiate the structural parts from one angle, lacking suitable photofunctional instruments. It is necessary to have holes in the interior to facilitate the entry of cells into the colonization and solve the problem of bone fusion after implantation; fourth, the structural pores of 3D printed porous structural parts or porous implants are complex, and the existing technical means cannot be used everywhere. hole.
- the first aspect of the present invention provides a method for modifying a titanium alloy structure.
- the method uses an ultraviolet light source to irradiate the titanium alloy structure from at least two different angles.
- the titanium alloy structure to be modified The interior of the part contains holes.
- the titanium alloy structural part is a porous titanium alloy bracket manufactured by 3D printing.
- the titanium alloy structural part includes any product processed from titanium alloy.
- the titanium alloy structural part contains holes, which means There can be holes in any proportion, including but not limited to hollow structures or structures processed by 3D printing.
- the different angles in the present invention refer to the origin of the three-dimensional coordinate system as a reference, and irradiate from directions including at least two different directions.
- the movement of multiple light sources can also be realized by setting multiple light sources at different positions.
- the second aspect of the present invention provides another method for modifying titanium alloy structural parts, the method uses at least two ultraviolet rays of different wave bands to irradiate titanium alloy structural parts; further, at least one of the ultraviolet rays is 320 - UV-A at 400 nm, and another UV-C at 10-290 nm.
- 365 ⁇ 20nm ultraviolet A band (365nm is the peak, hereinafter referred to as 365nm band) and 270 ⁇ 20nm ultraviolet C band (270nm is the peak, hereinafter referred to as 270nm band) are used for irradiation.
- the ultraviolet irradiation using two different wavelength bands can be carried out simultaneously, separately, or alternately, for example, using ultraviolet A and ultraviolet C to irradiate simultaneously for 15 minutes, or first using ultraviolet A to irradiate for 15 minutes and then using ultraviolet C to irradiate 15 minutes, or ultraviolet A and ultraviolet C alternately irradiate for 1 minute, 3 minutes, and 5 minutes, and the irradiation time can be selected arbitrarily.
- other wavelength bands of light may also exist at the same time.
- the irradiation can last for any time needed, for example, the irradiation of ultraviolet A and ultraviolet C lasts at least 1 second, 5 seconds, 15 seconds, 30 seconds, 45 seconds, 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 11 minutes, 12 minutes, 13 minutes, 14 minutes, 15 minutes, 30 minutes, 60 minutes; UVA and UVC irradiation The times can be the same or different.
- the third aspect of the present invention provides a titanium alloy structural part, which has been treated by the method of the first aspect or the second aspect of the present invention, and has improved properties, such as improved biocompatibility, surface resistance One or more of aging, increasing protein absorption, antibacterial, antirust.
- the fourth aspect of the present invention provides a photofunctionalization instrument, which includes: a housing, the housing is provided with a cavity for accommodating titanium alloy structural parts to be processed; an ultraviolet lamp, the ultraviolet lamp is arranged on the inner wall of the housing, Capable of emitting ultraviolet light from at least two angles; a control panel for setting and displaying the wavelength and treatment time of the ultraviolet light.
- the housing has an openable part for placing the structural member to be irradiated (that is, processed);
- the control panel includes a controller and a display, the controller is used to control the ultraviolet wavelength and time of irradiation, and the display is used to The user sets and reads the operating conditions of the program, and the display can be a touch screen.
- the ultraviolet light source includes at least two ultraviolet light sources, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more, further, the ultraviolet light source can be led ultraviolet light source.
- At least one of the ultraviolet light sources can emit ultraviolet A, such as but not limited to an ultraviolet band with a peak value of 365nm, and at least another ultraviolet light source can emit ultraviolet light C, such as but not limited to an ultraviolet band with a peak value of 270nm.
- the housing can be in any suitable shape, as long as it can realize ultraviolet irradiation on the cavity from at least two angles, multiple angles or all directions.
- the housing is a cuboid, and the ultraviolet light source The intervals are arranged on the inner surface of the casing; in other examples of the present invention, the casing is a cylinder, and the ultraviolet light sources are arranged on the inner surface of the casing according to a certain interval.
- the fifth aspect of the present invention provides a titanium alloy structural part, and the treatment of the light-functionalized instrument described in the fourth aspect.
- the titanium alloy structural parts can be various suitable titanium alloy structural parts, such as dental implants, orthopedic implants, and porous titanium alloy brackets (prostheses) manufactured by 3D printing.
- the optical functionalization instrument is built by using LED ultraviolet light source.
- the optical functionalization instrument is equipped with multiple LED ultraviolet light sources, which can perform uniform and multi-directional optical functionalization on the structure in the cavity.
- the existing conventional ultraviolet light The volume of the light source is large, and it is impossible to set up intensive integration of multiple light sources;
- the optical functionalization instrument of the present invention can perform optical functionalization treatment on the exterior and interior of the porous titanium alloy structural part at the same time, and is very suitable for the treatment of implanting the porous titanium alloy in 3D printing.
- the surface of the titanium alloy treated by the light-functionalized instrument of the present invention has an obvious bone-promoting effect, and the bone ingrowth in the porous metal is mainly in the form of contact osteogenesis, which is successful for the fixation of the bone graft material in the porous metal It is of great significance to the prognosis; it can significantly promote the formation of collagen fibers in bone tissue, which provides a strong condition for the formation of stronger osseointegration between bone tissue and the surface of the prosthesis.
- Fig. 1 is the front photo of photofunctionalized instrument in one embodiment of the present invention
- Fig. 2 is the operation display screen of the optical functionalization instrument in an embodiment of the present invention
- Fig. 3 is the internal structure of the photofunctionalized instrument in an embodiment of the present invention
- the black arrow (lowest) indicates the prosthesis
- the red arrow (middle position) indicates the 365nm band UV lamp
- the white arrow (uppermost) indicates the 270nm band UV lamp .
- Figures 4A, 4B, 4C, 4D, and 4E are the contact angle test results of GC, GL, G270, G365, and GU, respectively, and Figure 5F is the statistical result of the contact angles of the five groups.
- Figures 5A, 5B, 5C, 5D, and 5E are imaging images of five groups of GC, GL, G270, G365, and GU under a scanning electron microscope (SEM), respectively.
- Figures 6A, 6B, 6C, 6D, and 6E are the full spectrum of element composition determined by EDS of GC, GL, G270, G365, and GU, respectively; 6F, 6G, 6H, 6I, and 6J are GC, GL, G270, and G365, respectively. , Comparison of the contents of different elements on the inner surface of GU 5 groups and 5 groups.
- Figure 7A, 7D, 7G, 7J, 7M are the XPS full spectrum of GC, GL, G270, G365, GU 5 groups respectively;
- Figure 7B, 7E, 7H, 7K, 7N are GC, GL, G270, G365, GU 5 respectively;
- Figure 7C, 7F, 7I, 7L, and 7O are the peak fitting figures of O element in 5 groups GC, GL, G270, G365, and GU, respectively.
- Figures 8A, 8B, 8C, 8D, and 8E are the peak fittings of the GC group, GL group, G270 group, G365 group, and GU group, respectively.
- Figure 9A Comparison of cell proliferation rates in GC group, GL group, G270 group, G365 group, and GU group after co-culture of SD rat bone marrow mesenchymal stem cells with 3D printed metal scaffolds for 1 day, 3 days, and 7 days;
- Figure 9B.SD Comparison of ALP activity of rat bone marrow mesenchymal stem cells co-cultured with 3D printed metal scaffolds for 7 days and 14 days after GC group, GL group, G270 group, G365 group, and GU group;
- Figure 9C SD rat bone marrow mesenchymal stem cells Cell attachment and collagen fibers on the porous metal surface of scaffolds co-cultured with 3D printed metal scaffolds observed by electron microscopy;
- Figure 9D Comparison of cell proliferation rates in GC group, GL group, G270 group, G365 group, and GU group after co-culture of SD rat bone marrow mesenchymal stem cells with 3D printed metal scaffolds for 1 day, 3 days, and
- SD rat bone marrow mesenchyme The F-Acting cytoskeleton imaging of GC group, GL group, G270 group, G365 group and GU group after 3 days of co-culture of stem cells and 3D printed metal scaffold; Live-Dead fluorescence results after 3 days of culture.
- Figure 10 shows the 10,000-fold magnification of collagen fiber attachment in the GU group.
- Figures 11A, 11B, and 11C are the comparisons of elastic modulus, yield strength, and compressive strength between GC, GL, G270, G365, and GU 5 groups respectively;
- Figure 11D is the stress-strain of GC, GL, G270, G365, and GU 5 groups curve.
- Figure 12A, 12B, 12C, 12D, and 12E respectively show the bone ingrowth of GC, GL, G270, G365, and GU 5 groups;
- Figure 12F shows the internal bone volume of porous metal in GC, GL, G270, G365, and GU 5 groups Score comparison.
- Figures i to V in Fig. 13A show respectively the bone ingrowth situation of tissue sections of GC, GL, G270, G365, GU 5 groups; Ingrowth percentage) (left figure) and BICR (bone and prosthesis interface percentage); Shown in Fig. 13C: Osteoid and mineralized bone percentage of GC, GL, G270, G365, GU 5 groups; Fig. 13D : Figures i to V show the results of sequential fluorescence (tetracycline-calcein) of GC, GL, G270, G365, and GU 5 groups, respectively.
- Figure 14A shows the comparison of the maximum pushing force of GC, GL, G270, G365, GU 5 groups; 14B shows the pushing force-displacement curves of GC, GL, G270, G365, GU 5 groups; 14C shows the porous titanium alloy pseudo The experimental model diagram of the built-in and push-out of the rabbit femoral condyle.
- Ethanol Beijing Chemical Plant; 24-well Ultra Low Cluster Plate Corning; 48-well Ultra Low Cluster Plate, Corning; culture bottle (74cm 2 , 24cm 2 ), Corning; gradient freezer box, Nalgene; cell counter, Nucleocounter; microplate reader, Thermo; SD rat bone marrow-derived mesenchymal stem cells (BMSC); self-extracted basal medium (BM), Gibco; osteogenic differentiation induction medium (OM), CYAGEN, USA; FBS, fetal bovine serum, Gibco.
- BMSC bone marrow-derived mesenchymal stem cells
- BM self-extracted basal medium
- OM osteogenic differentiation induction medium
- FBS fetal bovine serum
- Basal medium (BM, 500mL);
- Cyagen Mesenchymal stem cell basal medium (PT-3238) 440mL;
- Osteogenic induction medium (OM, 200mL);
- the porous titanium alloy stent used in the embodiment of the present invention is prepared by EBM (electron beam melting) rapid prototyping technology, including 3 kinds of specifications for different test and detection, respectively: (1) a cylinder with a diameter of 10mm and a height of 5mm , used for cell experiments; (2) A cylinder with a diameter of 5 mm and a height of 6 mm, used for material characterization, in vitro mechanical testing, cell culture, implantation experiments, etc.; (3) EBM technology was used to prepare pictures with a diameter of 10 mm and a thickness of 1 mm Ti6A14V-like material was used for characterization. See Table 1 below.
- EBM electron beam melting
- the prosthesis was designed based on CAD image software (CATIA and INCAT USA), with an internal pore diameter of 640 ⁇ m, of which the pillar diameter is 400 ⁇ m, which is a cylinder with diamond-like lattice pores.
- CAD image software CAA and INCAT USA
- Arcam EBM S12 Arcam AB, Sweden
- the raw material of the sample is: Ti6A14V ELI Arcam standard powder, the diameter of the powder is between 45-100 ⁇ m, and the electronic In a vacuum environment, the powder starts to be melted and printed layer by layer, with a layer thickness of 0.1mm. After forming, it is filled with helium and naturally cooled to 100°C, and then filled with air to complete the printing process.
- Embodiment 1 Structure and parameter design of photofunctionalized instrument
- the light source is composed of two groups of wave bands, one group is 365 ⁇ 20nm (365nm is the peak value, hereinafter referred to as the 365nm band), and the other group is 270 ⁇ 20nm (270nm is the peak value, hereinafter referred to as the 270nm band);
- the light source is an LED light source, which is small in size and convenient Compactly arranged, the ultraviolet wavelength emitted by the light source is usually dominated by a certain wavelength, supplemented by adjacent wavelengths, so only the peak value is used to describe the light source;
- the irradiation energy in the 270nm band is greater than 2mw/cm 2
- the irradiation energy in the 365nm band is greater than 30mw/cm 2
- the uniformity is greater than 80%; from at least two angles, multiple angles, preferably all Ultraviolet rays are emitted at angles to achieve a relatively uniform energy distribution of ultraviolet rays in the cavity, and in the most optimal case, it can be irradiated from all angles; ultraviolet rays of different bands can be irradiated separately or at the same time;
- the inner cavity has a volume of at least 1 cubic centimeter, can withstand the pressure of a certain weight object, and is provided with an opening; this size is only exemplary, and can be adjusted according to the size of the structure to be processed. In some embodiments, a hexahedron Lumen size ⁇ 3x3x4 cubic centimeters.
- the ultraviolet light source model 365nm light source: CUN66B1B (Seoul); 270nm light source: CUD7GF1A (Seoul).
- the light source is arranged, and the distance between all 270nm and 365nm lamp beads is 5mm, covering the irradiated surface evenly, as shown in Figure 1 to Figure 3.
- Figure 1 shows a real photo of the light functionalized instrument, the inside of which is a hexahedral cavity, and the hatch is opened, showing the regular arrangement of led ultraviolet lamp beads inside and on the hatch, the light source arrangement is only Exemplarily, there is no requirement on the spacing of the lamp beads, as long as the target energy density can be achieved.
- Figure 3 shows the state of use of the photofunctionalized instrument, showing the internal structure of the photofunctionalized instrument, the black arrow indicates the prosthesis; the red arrow indicates the 365nm band UV lamp, and the white arrow indicates the 270nm band UV lamp;
- the structure of the light-functionalized instrument is only exemplary, and other structures of the shell, such as cylinder, cube, and irregular shapes are also feasible.
- the photofunctionalized instrument includes: a housing, the housing is provided with a cavity for accommodating titanium alloy structural parts to be processed; an ultraviolet lamp, the ultraviolet lamp is arranged on the inner wall of the housing; a control panel, the control panel is used to set and display the wavelength and processing time.
- the shell has a part that can be opened, and the interior is used to place the structure to be irradiated;
- the control panel includes a controller and a display, the controller is used to control the ultraviolet wavelength and time of irradiation, and the display is used for the user to set and read the program
- the display is a touch screen, and the user can perform input settings.
- the control panel can control the switch and timing of the UV lamp.
- two wavelengths of UV lamps can be turned on at the same time, or a UV lamp of one band can be turned on separately, or UV lamps of different bands can be controlled to irradiate alternately;
- Embodiment 2 Using the optical functionalization instrument of the present invention to carry out optical functionalization treatment on titanium alloy materials and experimental detection of treated titanium alloy materials
- the experiment was divided into 5 groups, namely GC (control group), GL, G270, G365 and GU.
- GC did not accept light treatment
- G270, G365 and GU groups were instrument treatment groups of the present invention, which were respectively 270nm, 365nm, 270nm and 365nm dual-wavelength irradiation, and the irradiation time was 15 minutes. minute.
- GC control group treatment process: after 4 weeks in a laboratory environment (normal temperature, normal pressure, air environment), place it on a sterile operating table for 15 minutes (without irradiation), and keep it sealed for 24 hours During the period, high-pressure steam sterilization and drying were carried out. During the operation, metal instruments were used to avoid contact with possible contamination, and subsequent processing was started.
- GL treatment process After the prosthesis is prepared, it is placed in a laboratory environment (normal temperature, normal pressure, and air environment) for 4 weeks, and then it is irradiated by a high-pressure mercury lamp (multi-wavelength peaks, including 270nm and 365nm) on a sterile operating table 15 minutes, and sealed for storage, high-pressure steam sterilization and drying within 24 hours, using metal instruments during the operation to avoid contact with possible contamination, and start subsequent processing.
- a high-pressure mercury lamp multi-wavelength peaks, including 270nm and 365nm
- G270, G365, and GU instrument irradiation treatment group standard procedure: After the prosthesis is prepared, it is placed in the laboratory environment (normal temperature, normal pressure, and air environment) for 4 weeks, and then the functionalized instrument is used with ultraviolet light at 270nm or/and Ultraviolet rays with two wavelengths of 365nm irradiated the prosthesis for 15 minutes at the same time, and sealed the prosthesis. Within 24 hours, it was sterilized and dried by high-pressure steam. During the operation, metal instruments were used to avoid contact with possible contamination, and the follow-up treatment was started.
- the contact angle was measured on the surface of 3D printed titanium alloy (10 pieces in each group) by Sessile Drop method.
- X-ray photoelectron spectroscopy (XPS; 250XI, Thermo escalab, USA) was used to analyze the chemical composition of the sample surface.
- X-ray diffraction technique (XRD; D8, ADVANCE, Bruker) was used to analyze the phase of the coating, using Cu-K ⁇ ray source, the tube voltage was 40kV, the tube current was 50mA, continuous scanning mode, and the scanning speed was 4°/min , the diffraction angle 2 ⁇ is 10-80°.
- EDS energy dispersive X-ray spectrometer
- Preparation of cell growth medium 10% fetal calf serum and double antibody, usually serum, should be added to the medium before use.
- the culture medium was divided into vials (100-200mL) for use, and the cap was turned over to seal the bottle mouth tightly.
- the general cell growth medium is culture medium + 10% fetal bovine serum, and finally add double antibody stock solution (penicillin + streptomycin) according to 1% volume fraction, so that the final concentrations of penicillin and streptomycin are 100U/mL and 100U/mL respectively. 100U/mL.
- SD rats were taken, killed by cervical dislocation, immersed in 75% ethanol for about 5-10 minutes (min), bilateral femurs and tibias were taken out with sterile surgical instruments, attached muscles were removed, and they were soaked in an appropriate amount of medium. Take a 10mL syringe, and make 2-3 holes at both ends of the femur and tibia. The syringe absorbs an appropriate amount of medium and inserts it into one end of the metaphysis.
- the cells in the bone marrow are washed into the culture dish, and repeated several times until the femur and tibia Turn white, collect the cell suspension, filter with a 200-mesh metal filter to remove slightly larger impurities, collect the filtered cell suspension into a centrifuge tube, centrifuge at 800r/min for 5min, discard the supernatant, and add an appropriate amount of ⁇ - Mix the MEM medium evenly, inoculate it in a 10cm plastic culture dish, incubate in an incubator with a volume fraction of 5% CO 2 and a saturated humidity at 37°C for 48-72 hours (h), and then replace the medium. Change the medium once a day, and after about 9 to 12 days, multiple cell colonies are formed, which can be subcultured after about 70% to 80% of the bottom of the bottle is covered.
- Subculture after the cells reach 80% confluence we first remove the culture medium in the culture bottle, then add an appropriate amount of phosphate buffered saline (PBS) to slowly wash and discard the liquid, and then add the inside of the culture bottle An appropriate amount of trypsin at a concentration of 0.125% covers the cells, and the cells are digested at room temperature. When we observe that the cells gradually become round under an inverted microscope, and then separate from the culture flask, we add an appropriate amount of basal medium to neutralize the cells.
- PBS phosphate buffered saline
- a cell counting plate for cell quantification: first wipe the counting plate and the cover slip clean, and cover the cover slip on the counting plate; the cell digestion step is the same as before, after the cells are resuspended, suck 100 ⁇ L of the cell suspension into the Ep tube, Dilute a certain number of times with the medium, then suck out a little of the cell suspension with a pipette gun, and drop it on the edge of the cover slip, so that the suspension fills the space between the cover slip and the counting plate, and let it stand for 3 minutes (note that there are no air bubbles under the cover slip) Finally, calculate the total number of cells in the four major grids on the plate, and only count the cells on the left and upper sides of the plate.
- the pre-treated 3D porous titanium alloy inserts into the basal medium and soak for 30 minutes. After soaking, place them inside a 48-well ultra-low attachment culture plate, and then drop 600 ⁇ L of cell suspension on each bracket. Use a pipette gun to repeatedly suck back and drop, so that the cell suspension is in uniform contact with the scaffold, and cultivate for 24 hours to further attach the cells.
- Cell proliferation we tested the cell proliferation activity (Cell proliferation) at 1 day, 3 days and 7 days after inoculation.
- PBS phosphate buffered saline
- CCK-8 reagent we placed the culture plate at 37°C and incubated for 2 hours. After the reaction was over, suck out 100 ⁇ L from each well and add it to a 96-well plate, and then use a microplate reader to measure the absorbance at a wavelength of 450 nm.
- the osteogenic differentiation medium was replaced to continue culturing, and the medium was changed every 3 days.
- the alkaline phosphatase Alkaline phosphatase, ALP
- the alkaline phosphatase activity of the cells on the 3D porous titanium alloy insert was detected, and the total protein content of the cells was measured for correction.
- Alkaline phosphatase (ALP) activity detection first, we use phosphate buffered saline (PBS) to wash the sample to be tested twice, and then use 1mL 0.2% TritonX-100 to lyse the cells on the 3D porous titanium alloy built-in; Cell lysate was used to detect the activity of ALP; we prepared the detection buffer, chromogenic substrate solution (para-nitrophenyl phosphate, pNPP) and the working solution of the standard according to the instructions of the experiment, and then diluted the standard (p-nitrophenol) to 0.002 ⁇ moL; According to the instructions of the experiment, we added the sample to be tested, the substrate solution, the detection buffer and the standard to each well, incubated at 37°C for 5min, and then read the absorbance value at a wavelength of 405nm, and Calculate the DEA activity unit of alkaline phosphatase (ALP), and the DEA activity unit of alkaline phosphatase (ALP) is defined as
- a scanning electron microscope was used to observe the adhesion of the cells on the 3D porous titanium alloy built-in.
- a general mechanical testing machine (Landmark, MTS Inc. MN, USA) was used to perform static compression tests on the 3D porous titanium alloy scaffolds before and after treatment.
- the tested sample is a 3D printed cylindrical metal bracket with a diameter of 5 mm and a height of 6 mm.
- the deformation rate during compression is 1.8 mm/min.
- the number of samples tested in each group is 5.
- the test data is automatically generated as a stress-strain curve ( Stress-strain curve), thus, analyze the obtained elastic modulus (Elastic modulus), yield strength (Yield strength) and compression strength (Compression strength).
- Implantation experiments in the rabbit femoral condyle to evaluate the in vivo biocompatibility and osseointegration properties of ultraviolet photofunctionalization were all provided by Beijing Weitong Lihua Company. All the experimental plans have been approved by the Ethics Committee of Peking University Health Science Center (approved by LA2014214, and the operation and feeding of animal experiments are in accordance with the national experimental animal standards. Care and use guidelines.
- Surgical method male New Zealand white rabbits were randomly divided into groups, anesthetized by intramuscular injection of amiodarone 50 mg/kg, skin preparation of bilateral lateral femoral condyles, disinfection with 0.5% povidone iodine, and then longitudinal incision of the skin subcutaneously to On the periosteum surface, then use a sharp blade to scrape the attached periosteum, and, to locate the femoral bone, we use a 5mm diameter electric drill to create a 5mm diameter bone defect in the femoral condyle under normal saline cooling, and then use normal saline to After washing the local area, the prosthesis was implanted in the bone defect, and then the muscle layer, fascia layer and skin were sutured layer by layer with 4# antibacterial Vicryl suture. Three days after the operation, 400,000 units of penicillin was injected intramuscularly to prevent infection.
- Figure 5 shows the changes in the surface topography of the four control groups (GL, G270, G365, GU) before and after UV photofunctionalization. Compared with the control group (GC), we observed more complex topography and Surface micropore formation; further, the most surface micropore formation was observed in the GU group relative to GL, G270 and G365.
- the EDS selection area determined inside the metal stent suggests that the elemental composition of the titanium alloy surface consists of Ti, O and a small amount of Al, V, and C, as shown in Figure 6A-E.
- Figure 6G also shows The single-band irradiation G365 performed by the illuminator has a significant advantage over GL inside the prosthesis, and the multi-angle irradiation has obvious benefits for the photofunctionalization of the internal aperture surface of the prosthesis.
- Figure 7 shows the chemical composition and state before and after ultraviolet light functionalization treatment after XPS detection.
- the main surface elements are O, Ti, and C.
- the chemical environment of the two elements and the relative proportions of elements in different chemical environments can be obtained as shown in the table.
- the narrow-scan spectrum of C element is fitted with peaks (as shown in Figures 7B, 7E, 7H, 7K, and 7N) to obtain three peaks: the C-C peak corresponds to a binding energy of 284.82eV, the C-O peak corresponds to a binding energy of 286.41eV, and the C-O peak corresponds to a binding energy of 286.41eV.
- the peak positions corresponding to different samples will be slightly different.
- the relative proportions of C elements in the three chemical environments in different samples are shown in Table 3.
- the peak positions corresponding to different samples will be slightly different.
- the relative proportions of O elements in the three chemical environments in the five groups of samples are shown in Table 4.
- the surface of 3D printed porous titanium alloy is composed of titanium dioxide and carbon oxides before and after ultraviolet treatment.
- the XRD results show the phase composition of the 3D printed titanium alloy stent before and after UV photofunctionalization. Multiple diffraction peaks (1 peak, Ti6AL4V) and a small amount of carbon (C) produced by the matrix titanium can be seen in all five groups, but the four photofunctionalized treatment groups (GL, G270, G365, GU) all The formation of more oxides (AL2O3) was found.
- the compatibility of the cells was evaluated by measuring the cell activity (proliferation rate) on the porous metal surface.
- the difference in cell proliferation rate was consistent: the GU group was significantly higher than the GC, GL, and G365 groups, and the difference was statistically significant (P ⁇ 0.01 ); GU group was significantly higher than G270, and the difference was statistically significant (P ⁇ 0.05); GL and G270 also had significant statistical difference (P ⁇ 0.05), indicating that dual-band UV treatment had a significant difference compared with single-band treatment.
- the ALP activity test (as shown in Figure 9B) showed that at 7 days, there was no significant statistical difference among the five groups (P>0.05); at 14 days, the GU group had higher alkaline phosphatase activity than the GC and GL groups, The difference was statistically significant (P ⁇ 0.01); the GU group also had higher alkaline phosphatase activity compared to the G365 group, the difference was statistically significant (P ⁇ 0.05); the GL group was significantly statistically different from the G270 group ( P ⁇ 0.01); while G270 and GU group had no significant statistical difference (P>0.05).
- Fig. 9D The results of the cell morphology experiment (Fig. 9D) can be seen visually, on the 3D printed scaffolds, compared with the GC group, the cells in the GL, G270, G365 and GU groups proliferated and attached more, had a larger spreading area, and more Stretching of cell pseudopodia; while cells in photofunctionalized groups (G270, G365, GU) with omnidirectional irradiation had more attachment inside the 3D printed scaffold.
- Figure 11 shows the performance of the mechanical properties before and after the UV light functionalization treatment, through the compression test, the yield strength (Figure 11A), the elastic modulus (Figure 11B), the compressive strength (Figure 11C) are compared, GC, GL, G270 , G365, GU) There was no significant statistical difference among the 5 groups (p>0.05); the stress-strain curve (Fig. 11D) was basically the same among the 5 groups.
- the 3D printed titanium alloy scaffold is white, and the bone tissue is green. According to Fig. 12A-E, it can be seen intuitively that the photofunctionalized treatment group (GU) has more bone tissue ingrowth than the control group (GC), almost covering the internal pores of the prosthesis.
- the bone volume fraction (bone ingrowth rate BIR) of GU, GC group, GL group, G270 group, G365 group, GU group were (43.33 ⁇ 5.08)%, (61.03 ⁇ 3.44)%, (74.44 ⁇ 1.90)%, (66.16 ⁇ 1.52)%, (75.53 ⁇ 1.40)%, there were significant statistical differences between GC and photofunctionalized treatment group (GL, G270, G365, GU), GL and GU, G365 and GU ( P ⁇ 0.05).
- the single-band irradiation of the instrument of the present invention achieves a better effect, and the dual-band ultraviolet irradiation effect is significantly better than the single-band ultraviolet irradiation.
- the GC and GL groups Eight weeks after implantation into the rabbit femoral condyle, the GC and GL groups only had bone ingrowth in the periphery, and the generated bone tissue was not in close contact with the porous scaffold; on the contrary, the porous titanium alloy scaffolds of the G270, G365 and GU groups It can be found that more extensive bone ingrowth, the new bone tissue is tightly combined with the titanium alloy scaffold, and mineralized to form bone, as shown in Figure 13A.
- the BI of the five groups were (14.56 ⁇ 1.15)%, (10.58 ⁇ 0.91)%, (24.7 ⁇ 1.74)%, (19.22 ⁇ 3.26)%, (25.04 ⁇ 1.29)% respectively %, GC, GL and GU group had significant statistical difference (P ⁇ 0.01); G270 and GU had no significant statistical difference (P>0.05); G365 and GU group had significant statistical difference (P ⁇ 0.05), as shown in 13B.
- the BICR of the five groups were (15.14 ⁇ 0.67)%, (10.26 ⁇ 1.00)%, (32.04 ⁇ 1.12)%, (18.56 ⁇ 1.65)%, (41.66 ⁇ 1.64) %, GC, GL and GU groups have significant statistical differences (P ⁇ 0.01); G270 and GU have significant statistical differences (P ⁇ 0.05); G365 and GU groups have significant statistical differences (P ⁇ 0.01), as shown in the figure 13B.
- the proportions of osteoid in the five groups were (55.08 ⁇ 4.06)%, (63.3 ⁇ 10.85)%, (29.42 ⁇ 4.02)%, (40.94 ⁇ 8.61)%, (20.62 ⁇ 1.59)%, there was significant statistical difference between GC, GL and GU group (P ⁇ 0.01); there was no significant statistical difference between G270 and GU (P>0.05); there was significant statistical difference between G365 and GU group (P ⁇ 0.05) , as shown in Figure 13C.
- the proportions of mineralized bone in the 5 groups were (44.68 ⁇ 4.64)%, (35.02 ⁇ 10.41)%, (70.58 ⁇ 4.02)%, (59.06 ⁇ 8.61)%, (79.38 ⁇ 1.59)%, there was significant statistical difference between GC, GL and GU group (P ⁇ 0.01); there was no significant statistical difference between G270 and GU (P>0.05); there was significant statistical difference between G365 and GU group (P ⁇ 0.05) , as shown in Figure 13C.
- the single-band irradiation of the instrument of the present invention achieves a better effect, and the dual-band ultraviolet irradiation effect is significantly better than the single-band ultraviolet irradiation.
- Sequential fluorescent labeling in vivo revealed different osseointegration patterns over time points after implantation of prostheses functionalized with or without UV light.
- the color of the titanium alloy prosthesis is white, the green fluorescence represents the 3-week osteogenesis, and the red represents the 8-week osteogenesis. According to the fluorescent labeling, it can be seen intuitively that in the titanium alloy prosthesis, the osteogenesis at 3 weeks was more common in the G270, G365 and GU groups. The GC and GL groups showed more osteogenesis at 8 weeks.
- Figure 14 shows the results of the push-out experiment of the control group GC and the experimental group GU at 8 weeks after implantation of the rabbit femoral condyle prosthesis.
- the maximum pushing forces of GC, GL, G270, G365, and GU groups were: 250.3 ⁇ 16.6, 271.7 ⁇ 19.9, 391.2 ⁇ 16.7, 301.7 ⁇ 10.2, 450.5 ⁇ 18.5; GC, GL, G365 compared with GU
- G270 and G270 groups P ⁇ 0.01
- G270 and GU group P ⁇ 0.05
- there was a significant statistical difference between GL and G270 group P ⁇ 0.01
- the axial pushing force Compared with the GC, GL, and G365 groups, the G270 and GU groups need a larger push-out force in order to produce the same displacement.
- ultraviolet C (270nm band) and ultraviolet A (365nm band) ultraviolet rays have unique effects in different directions on the photofunctionalization of titanium alloy prostheses, and can be obtained by using dual-band ultraviolet treatment.
- the optical functionalized instrument of the present invention can realize uniform treatment of the titanium alloy surface through multiple ultraviolet light sources, and the effect is far superior to the existing mercury lamp; the single-band multi-angle ultraviolet light source in the optical functionalized instrument and uniform
- the high power can realize the external and internal light functionalization of the structure with holes inside, and the effect is far superior to the existing mercury lamp.
- each group uses three prostheses, the first group, using 270nm ultraviolet band irradiation treatment for 30 minutes; the second group, irradiated with 365nm ultraviolet band for 30 minutes; the third group, alternately irradiated with 270nm ultraviolet band and 365nm ultraviolet band for 1 minute, and continued to irradiate for 30 minutes in total; the fourth group, used 270nm ultraviolet band and 365nm ultraviolet band
- the bands were irradiated alternately for 5 minutes, a total of 30 minutes of continuous irradiation; the fifth group, irradiated with 270nm ultraviolet band and 365nm ultraviolet band for 15 minutes at the same time; the sixth group, irradiated with 270nm ultraviolet band for 60 minutes; the second group, used 365nm ultraviolet band Irradiation treatment for 60 minutes;
- Example 3 It can be known from Example 3 that how to set the irradiation time of ultraviolet rays using two wavebands has no significant impact on the results, that is, simultaneous irradiation of UVA and UVC, alternate irradiation, or even sequential irradiation have little effect on modification;
- the results of the 15-minute processing of the wave band compared the prosthesis processed for 30 minutes and 60 minutes, and found that after 15 minutes, the single-band processing basically no longer continues to have an impact, and the surface shape of the prosthesis basically does not change.
Abstract
A multidirectional photofunctionalization instrument for surface modification of a titanium alloy implant and a method of use thereof. The instrument comprises at least two ultraviolet light sources which can emit ultraviolet light, such that modification processing can be performed on a titanium alloy structure which is located in the instrument. Furthermore, the ultraviolet light source may comprise ultraviolet lamps which emit at least two types of ultraviolet light having different wavelengths. By means of the method, irradiation is performed from different angles, or titanium alloy is treated by using at least two types of ultraviolet light having different wavebands, such that the performance can be significantly improved on a surface of a titanium alloy structural member and both an outer surface and an inner surface of a porous titanium alloy structure.
Description
相关申请的交叉引用Cross References to Related Applications
本申请要求2021年06月21日提交的中国申请号2021106874841的权益。所述申请号2021106874841据此全文以引用方式并入本文。This application claims the benefit of Chinese application number 2021106874841 filed on June 21, 2021. Said application number 2021106874841 is hereby incorporated by reference in its entirety.
本发明属于光功能化领域，具体涉及一种针对钛合金内植物表面改性的多方向光功能化仪器及其使用方法。The invention belongs to the field of photofunctionalization, and in particular relates to a multi-directional photofunctionalization instrument aimed at modifying the surface of titanium alloy inner plants and a use method thereof.
光功能化技术(Photofunctionalization)是指利用特定波长、强度、照射时间的紫外线对金属内植物(特别是钛合金)进行表面改性以增强其理化性质和生物学效应的技术。钛合金内植物制作后在环境中暴露，会逐渐失去生物活性，可通过暴露在紫外线下重新激活生物活性。但是目前仍有多个问题尚未解决，第一，现有的照射处理只能处理结构表面，对于多空结构内部表面无法处理，第二，现有处理使用高压汞灯，其发射的紫外线波长固定，光源结构体积大，通常固定使用，只能从一个角度对结构件进行照射，缺乏合适的光功能化仪器，第三，现有的处理集中在对牙科产品的照射处理，而骨科内植物结构需要在内部有孔，方便细胞进入其中定植，解决植入后的骨融合问题；第四，3D打印的多孔结构件或多孔内植物的结构孔复杂，现有技术手段无法无处不规则的内部孔。Photofunctionalization refers to the technology of modifying the surface of metal implants (especially titanium alloys) by using ultraviolet rays of specific wavelength, intensity, and irradiation time to enhance their physical and chemical properties and biological effects. After the titanium alloy implant is exposed to the environment after fabrication, it will gradually lose its biological activity, which can be reactivated by exposure to ultraviolet light. However, there are still many problems that have not been resolved. First, the existing irradiation treatment can only treat the surface of the structure, and cannot treat the inner surface of the porous structure. Second, the existing treatment uses a high-pressure mercury lamp, and the ultraviolet wavelength emitted by it is fixed. , the structure of the light source is bulky, usually used in a fixed position, and can only irradiate the structural parts from one angle, lacking suitable photofunctional instruments. It is necessary to have holes in the interior to facilitate the entry of cells into the colonization and solve the problem of bone fusion after implantation; fourth, the structural pores of 3D printed porous structural parts or porous implants are complex, and the existing technical means cannot be used everywhere. hole.
发明内容Contents of the invention
本发明的第一个方面提供了一种对钛合金结构件进行改性的方法，该方法使用紫外线光源从至少两个不同角度对所述钛合金结构件进行照射，待改性的钛合金结构件内部含有孔，优选地钛合金结构件为3D打印制造的多孔钛合金支架，本发明中，钛合金结构件包括任何使用钛合金加工而成的产品，钛合金结构件内部含有孔，是指其中可以有任意比例的空洞，包括但不限于镂空结构或者是3D打印加工而成的结构。本发明中的不同角度是指以三维坐标系原点为参考，从包括至少两个不同方向的方向进行照射，可选地，照射方向可以有任意多个，例如3至无穷多；照射可以通过一个或多个光源的移动实现，也可以通过设置多个不同位置的光源实现。The first aspect of the present invention provides a method for modifying a titanium alloy structure. The method uses an ultraviolet light source to irradiate the titanium alloy structure from at least two different angles. The titanium alloy structure to be modified The interior of the part contains holes. Preferably, the titanium alloy structural part is a porous titanium alloy bracket manufactured by 3D printing. In the present invention, the titanium alloy structural part includes any product processed from titanium alloy. The titanium alloy structural part contains holes, which means There can be holes in any proportion, including but not limited to hollow structures or structures processed by 3D printing. The different angles in the present invention refer to the origin of the three-dimensional coordinate system as a reference, and irradiate from directions including at least two different directions. Optionally, there can be any number of irradiating directions, such as 3 to infinity; Or the movement of multiple light sources can also be realized by setting multiple light sources at different positions.
本发明的第二个方面提供了另一种对钛合金结构件进行改性的方法，该方法使用至少两种不同波段的紫外线照射钛合金结构件；进一步地，紫外线中的至少一种为320-400纳米的紫外线A，并且另一种紫外线为10-290纳米的紫外线C。The second aspect of the present invention provides another method for modifying titanium alloy structural parts, the method uses at least two ultraviolet rays of different wave bands to irradiate titanium alloy structural parts; further, at least one of the ultraviolet rays is 320 - UV-A at 400 nm, and another UV-C at 10-290 nm.
在一个实施例中，使用了365±20nm紫外线A波段(365nm为峰值，下文简称365nm波段)，和270±20nm紫外线C波段(270nm为峰值，下文简称270nm波段)进行照射。In one embodiment, 365±20nm ultraviolet A band (365nm is the peak, hereinafter referred to as 365nm band) and 270±20nm ultraviolet C band (270nm is the peak, hereinafter referred to as 270nm band) are used for irradiation.
本发明的方法中，使用两种不同波段波段的紫外线照射可以是同时、分别、或者交替进行，例如使用紫外线A和紫外线C同时照射15分钟，或者先使用紫外线A照射15分钟再使用紫外线C照射15分钟，或者紫外线A与紫外线C依次交替照射1分钟、3分钟、5分钟，照射时间可以任意选择。In the method of the present invention, the ultraviolet irradiation using two different wavelength bands can be carried out simultaneously, separately, or alternately, for example, using ultraviolet A and ultraviolet C to irradiate simultaneously for 15 minutes, or first using ultraviolet A to irradiate for 15 minutes and then using ultraviolet C to irradiate 15 minutes, or ultraviolet A and ultraviolet C alternately irradiate for 1 minute, 3 minutes, and 5 minutes, and the irradiation time can be selected arbitrarily.
可选的，在一些实施方案中，还可以同时存在其他波段的光线。Optionally, in some embodiments, other wavelength bands of light may also exist at the same time.
前述方法中，可选地，照射可以持续需要的任何时间，例如紫外线A和紫外线C的照射至少持续1秒、5秒、15秒、30秒、45秒、1分钟、2分钟、3分钟、4分钟、5分钟、6分钟、7 分钟、8分钟、9分钟、10分钟、11分钟、12分钟、13分钟、14分钟、15分钟，30分钟、60分钟；其中紫外线A和紫外线C的照射时间可以相同也可以不同。In the aforementioned method, optionally, the irradiation can last for any time needed, for example, the irradiation of ultraviolet A and ultraviolet C lasts at least 1 second, 5 seconds, 15 seconds, 30 seconds, 45 seconds, 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 11 minutes, 12 minutes, 13 minutes, 14 minutes, 15 minutes, 30 minutes, 60 minutes; UVA and UVC irradiation The times can be the same or different.
本发明的第三方面提供了一种钛合金结构件，该钛合金结构件经过了本发明第一方面或第二方面的方法处理，得到了改善的性质，例如提高生物相容性、抗表面老化、增加蛋白吸收、抗菌、防锈中的一个或更多个。The third aspect of the present invention provides a titanium alloy structural part, which has been treated by the method of the first aspect or the second aspect of the present invention, and has improved properties, such as improved biocompatibility, surface resistance One or more of aging, increasing protein absorption, antibacterial, antirust.
本发明的第四方面提供了一种光功能化仪器，其包括：壳体，壳体设置有容纳待处理钛合金结构件的腔体；紫外线灯，紫外线灯设置在所述壳体的内壁，能够从至少两个角度发射紫外线；控制面板，控制面板用于设置和显示紫外线的波长和处理时间。进一步地，所述壳体具有可以打开的部分，用于将待照射(即处理)的结构件放置其中；控制面板包括控制器和显示器，控制器用于控制照射的紫外线波长和时间，显示器用于使用者设置和读取程序的运行情况，显示器可以是触摸屏。The fourth aspect of the present invention provides a photofunctionalization instrument, which includes: a housing, the housing is provided with a cavity for accommodating titanium alloy structural parts to be processed; an ultraviolet lamp, the ultraviolet lamp is arranged on the inner wall of the housing, Capable of emitting ultraviolet light from at least two angles; a control panel for setting and displaying the wavelength and treatment time of the ultraviolet light. Further, the housing has an openable part for placing the structural member to be irradiated (that is, processed); the control panel includes a controller and a display, the controller is used to control the ultraviolet wavelength and time of irradiation, and the display is used to The user sets and reads the operating conditions of the program, and the display can be a touch screen.
可选地，紫外线光源包括至少两个紫外线光源，例如可以是2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30个或更多个，进一步地，紫外线光源可以是led紫外线光源。Optionally, the ultraviolet light source includes at least two ultraviolet light sources, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more, further, the ultraviolet light source can be led ultraviolet light source.
优选地，紫外线光源中至少一种紫外线光源可以发射紫外线A，例如但不限于峰值365nm的紫外线波段，并且至少另一种紫外线光源可以发射紫外线C，例如但不限于峰值270nm的紫外线波段。Preferably, at least one of the ultraviolet light sources can emit ultraviolet A, such as but not limited to an ultraviolet band with a peak value of 365nm, and at least another ultraviolet light source can emit ultraviolet light C, such as but not limited to an ultraviolet band with a peak value of 270nm.
壳体可以是任意合适的形状，只要能实现从至少两个角度、多个角度或者全方向对腔内进行紫外线照射即可，在本发明的一些实例中，壳体是长方体，紫外线光源按照一定的间隔排列设置在壳体内部表面；在本发明的另一些实例中，壳体是圆柱体，紫外线光源按照一定的间隔排列设置在壳体内部表面。The housing can be in any suitable shape, as long as it can realize ultraviolet irradiation on the cavity from at least two angles, multiple angles or all directions. In some examples of the present invention, the housing is a cuboid, and the ultraviolet light source The intervals are arranged on the inner surface of the casing; in other examples of the present invention, the casing is a cylinder, and the ultraviolet light sources are arranged on the inner surface of the casing according to a certain interval.
本发明的第五方面提供了一种钛合金结构件，其第四方面描述的光功能化仪器的处理。进一步地，钛合金结构件可以为各种合适的钛合金结构件，例如牙科内植物、骨科内植物、以及3D打印制造的多孔钛合金支架(假体)等。The fifth aspect of the present invention provides a titanium alloy structural part, and the treatment of the light-functionalized instrument described in the fourth aspect. Further, the titanium alloy structural parts can be various suitable titanium alloy structural parts, such as dental implants, orthopedic implants, and porous titanium alloy brackets (prostheses) manufactured by 3D printing.
本发明的优点至少在于：The advantages of the present invention are at least:
1、使用LED紫外线光源构件了光功能化仪器，该光功能化仪器设置有多个LED紫外线光源，可以对其腔体内的结构进行均匀的多个方向的光功能化处理，现有的常规紫外线光源体积大，无法进行多个光源的密集集成设置；1. The optical functionalization instrument is built by using LED ultraviolet light source. The optical functionalization instrument is equipped with multiple LED ultraviolet light sources, which can perform uniform and multi-directional optical functionalization on the structure in the cavity. The existing conventional ultraviolet light The volume of the light source is large, and it is impossible to set up intensive integration of multiple light sources;
2、使用多角度的紫外线照射，实现对钛合金结构件(特别是含有孔的结构)的均匀光功能化处理，能够实现对钛合金结构件整体进行良好的改性，提高一些列相关的特性，例如提高生物相容性、抗表面老化、增加蛋白吸收、抗菌、防锈中的一个或更多个；2. Use multi-angle ultraviolet radiation to achieve uniform photofunctionalization of titanium alloy structural parts (especially structures containing holes), which can achieve good modification of titanium alloy structural parts as a whole and improve a series of related characteristics , such as one or more of improving biocompatibility, anti-aging on the surface, increasing protein absorption, antibacterial, and antirust;
3、双波段紫外线光功能化处理方便且高效，仅仅需要短时间的照射处理，与单波段紫外光相比，发现了UVA和UVC联合使用的显著协同效应，出乎意料的了改进了钛合金结构件的表面结构和各项性能；3. The functional treatment of dual-band ultraviolet light is convenient and efficient, and only requires short-term irradiation treatment. Compared with single-band ultraviolet light, a significant synergistic effect of combined use of UVA and UVC was found, which unexpectedly improved the titanium alloy. The surface structure and various properties of structural parts;
4、本发明的光功能化仪器能够对多孔钛合金结构件的外部和内部同时进行光功能化处理，非常适用于3D打印多孔钛合金内植入的处理。4. The optical functionalization instrument of the present invention can perform optical functionalization treatment on the exterior and interior of the porous titanium alloy structural part at the same time, and is very suitable for the treatment of implanting the porous titanium alloy in 3D printing.
5、本发明的光功能化仪器处理过的钛合金表面有明显的促成骨效应，在多孔金属内部的骨长入方式主要以接触成骨方式，这对于孔隙金属内的植骨材料的固定成功和预后有重要意义；可以明显促进骨组织中胶原纤维的形成，这为骨组织与假体表面形成更牢固的骨结合提供了有力条件。5. The surface of the titanium alloy treated by the light-functionalized instrument of the present invention has an obvious bone-promoting effect, and the bone ingrowth in the porous metal is mainly in the form of contact osteogenesis, which is successful for the fixation of the bone graft material in the porous metal It is of great significance to the prognosis; it can significantly promote the formation of collagen fibers in bone tissue, which provides a strong condition for the formation of stronger osseointegration between bone tissue and the surface of the prosthesis.
附图用来提供对本发明的进一步理解，并且构成说明书的一部分，与本发明的实施例一起用于解释本发明，并不构成对本发明的限制。在附图中：The accompanying drawings are used to provide a further understanding of the present invention, and constitute a part of the description, and are used together with the embodiments of the present invention to explain the present invention, and do not constitute a limitation to the present invention. In the attached picture:
图1为本发明的一个实施例中光功能化仪器的正面照片；Fig. 1 is the front photo of photofunctionalized instrument in one embodiment of the present invention;
图2为本发明的一个实施例中光功能化仪器的操作显示屏；Fig. 2 is the operation display screen of the optical functionalization instrument in an embodiment of the present invention;
图3为本发明的一个实施例中光功能化仪器的内部结构，黑色箭头(最下)指示假体；红色箭头(中间位置)指示365nm波段紫外灯，白色箭头(最上)指示270nm波段紫外灯。Fig. 3 is the internal structure of the photofunctionalized instrument in an embodiment of the present invention, the black arrow (lowest) indicates the prosthesis; the red arrow (middle position) indicates the 365nm band UV lamp, and the white arrow (uppermost) indicates the 270nm band UV lamp .
图4A、4B、4C、4D、4E分别为GC、GL、G270、G365、GU五组的接触角测试结果，图5F为5组的接触角的统计结果。Figures 4A, 4B, 4C, 4D, and 4E are the contact angle test results of GC, GL, G270, G365, and GU, respectively, and Figure 5F is the statistical result of the contact angles of the five groups.
图5A、5B、5C、5D、5E分别为GC、GL、G270、G365、GU五组在扫描电子显微镜(SEM)下的成像图。Figures 5A, 5B, 5C, 5D, and 5E are imaging images of five groups of GC, GL, G270, G365, and GU under a scanning electron microscope (SEM), respectively.
图6A、6B、6C、6D、6E分别是GC、GL、G270、G365、GU 5组的EDS确定的元素组成全谱；6F、6G、6H、6I、6J分别是GC、GL、G270、G365、GU 5组5组内表面不同元素含量的比较。Figures 6A, 6B, 6C, 6D, and 6E are the full spectrum of element composition determined by EDS of GC, GL, G270, G365, and GU, respectively; 6F, 6G, 6H, 6I, and 6J are GC, GL, G270, and G365, respectively. , Comparison of the contents of different elements on the inner surface of GU 5 groups and 5 groups.
图7A、7D、7G、7J、7M分别为GC、GL、G270、G365、GU 5组的XPS全谱；图7B、7E、7H、7K、7N分别为GC、GL、G270、G365、GU 5组的C元素分峰拟合图；图7C、7F、7I、7L、7O分别为GC、GL、G270、G365、GU 5组的O元素分峰拟合图。Figure 7A, 7D, 7G, 7J, 7M are the XPS full spectrum of GC, GL, G270, G365, GU 5 groups respectively; Figure 7B, 7E, 7H, 7K, 7N are GC, GL, G270, G365, GU 5 respectively Figure 7C, 7F, 7I, 7L, and 7O are the peak fitting figures of O element in 5 groups GC, GL, G270, G365, and GU, respectively.
图8A、8B、8C、8D、8E分别为GC组、GL组、G270组、G365组、GU组的分峰拟合。Figures 8A, 8B, 8C, 8D, and 8E are the peak fittings of the GC group, GL group, G270 group, G365 group, and GU group, respectively.
图9A.SD大鼠骨髓间充质干细胞与3D打印金属支架共培养1天、3天、7天后GC组、GL组、G270组、G365组、GU组的细胞增值率比较；图9B.SD大鼠骨髓间充质干细胞与3D打印金属支架共培养7天、14天后GC组、GL组、G270组、G365组、GU组的ALP活性的比较；图9C.SD大鼠骨髓间充质干细胞与3D打印金属支架共培养电子显微镜观察到的GC组、GL组、G270组、G365组、GU组的支架共培养孔隙金属表面的细胞附着与胶原纤维；图9D.SD大鼠骨髓间充质干细胞与3D打印金属支架共培养3天GC组、GL组、G270组、G365组、GU组的F-Acting细胞骨架显像；图9E.SD大鼠骨髓间充质干细胞与3D打印金属支架共培养3天后Live-Dead荧光结果。Figure 9A.Comparison of cell proliferation rates in GC group, GL group, G270 group, G365 group, and GU group after co-culture of SD rat bone marrow mesenchymal stem cells with 3D printed metal scaffolds for 1 day, 3 days, and 7 days; Figure 9B.SD Comparison of ALP activity of rat bone marrow mesenchymal stem cells co-cultured with 3D printed metal scaffolds for 7 days and 14 days after GC group, GL group, G270 group, G365 group, and GU group; Figure 9C. SD rat bone marrow mesenchymal stem cells Cell attachment and collagen fibers on the porous metal surface of scaffolds co-cultured with 3D printed metal scaffolds observed by electron microscopy; Figure 9D. SD rat bone marrow mesenchyme The F-Acting cytoskeleton imaging of GC group, GL group, G270 group, G365 group and GU group after 3 days of co-culture of stem cells and 3D printed metal scaffold; Live-Dead fluorescence results after 3 days of culture.
图10是GU组10000倍放大的胶原纤维附着。Figure 10 shows the 10,000-fold magnification of collagen fiber attachment in the GU group.
图11A、11B、11C分别是GC、GL、G270、G365、GU 5组间弹性模量、屈服强度、压缩强度的比较；图11D为GC、GL、G270、G365、GU 5组的应力-应变曲线。Figures 11A, 11B, and 11C are the comparisons of elastic modulus, yield strength, and compressive strength between GC, GL, G270, G365, and GU 5 groups respectively; Figure 11D is the stress-strain of GC, GL, G270, G365, and GU 5 groups curve.
图12A、12B、12C、12D、12E分别显示了GC、GL、G270、G365、GU 5组的骨长入情况；图12F为GC、GL、G270、G365、GU 5组的孔隙金属内部骨体积分数比较。Figure 12A, 12B, 12C, 12D, and 12E respectively show the bone ingrowth of GC, GL, G270, G365, and GU 5 groups; Figure 12F shows the internal bone volume of porous metal in GC, GL, G270, G365, and GU 5 groups Score comparison.
图13A中i至V图分别显示了GC、GL、G270、G365、GU 5组组织切片的骨长入情况；图13B中示出：GC、GL、G270、G365、GU 5组的BI(骨长入百分比)(左图)和BICR(骨与假体接触面百分比)；图13C中示出：GC、GL、G270、G365、GU 5组的类骨质和矿化骨百分比；图13D中：i至V图分别显示了GC、GL、G270、G365、GU 5组的序贯荧光(四环素-钙黄绿素)的结果。Figures i to V in Fig. 13A show respectively the bone ingrowth situation of tissue sections of GC, GL, G270, G365, GU 5 groups; Ingrowth percentage) (left figure) and BICR (bone and prosthesis interface percentage); Shown in Fig. 13C: Osteoid and mineralized bone percentage of GC, GL, G270, G365, GU 5 groups; Fig. 13D : Figures i to V show the results of sequential fluorescence (tetracycline-calcein) of GC, GL, G270, G365, and GU 5 groups, respectively.
图14A中示出GC、GL、G270、G365、GU 5组最大推出力的对比；14B示出GC、GL、G270、G365、GU 5组的推出力-位移曲线；14C示出多孔钛合金假体在兔股骨髁的内置与推出实验模式图。Figure 14A shows the comparison of the maximum pushing force of GC, GL, G270, G365, GU 5 groups; 14B shows the pushing force-displacement curves of GC, GL, G270, G365, GU 5 groups; 14C shows the porous titanium alloy pseudo The experimental model diagram of the built-in and push-out of the rabbit femoral condyle.
以下结合附图对本发明的优选实施例进行说明，应当理解，此处所描述的优选实施例仅用于说明和解释本发明，并不用于限定本发明。The preferred embodiments of the present invention will be described below in conjunction with the accompanying drawings. It should be understood that the preferred embodiments described here are only used to illustrate and explain the present invention, and are not intended to limit the present invention.
实验材料及主要试剂的来源与配制Source and preparation of experimental materials and main reagents
乙醇，北京化工厂；24孔超低贴附培养皿(Ultra Low Cluster Plate)Corning；48孔超低贴附培养皿，Corning；培养瓶(74cm
2，24cm
2)，Corning；梯度冻存盒，Nalgene；细胞计数仪，Nucleocounter；酶标仪，Thermo；SD大鼠骨髓来源间充质干细胞(BMSC)；自提基础培养基(BM)，Gibco；成骨分化诱导培养基(OM)，CYAGEN，美国；FBS，胎牛血清，Gibco。胰蛋白酶(0.25％)，Gibco磷酸盐缓冲液(PBS)Gibco；DMSO，Sigma-Aldrich；CCK-8 Dojindo，日本；BCA蛋白浓度定量试剂盒Beyotime；Dapi溶液Solarbio；Tritc Phalloidin罗丹明标记鬼笔环肽Solarbio；Goldner三色染色液Solarbio；活、死细胞活力毒性检测试剂盒，keyGen；ALP活性测定试剂盒Beyotime；Triton X-100 Biotopped；多聚甲醛谷歌生物；二甲苯北京化工厂；TECHNOVIT7200树脂EXAKT，德国；钙黄绿素Sigma；四环素Sigma；甲苯胺蓝染液谷歌生物；品红国药集团；
Ethanol, Beijing Chemical Plant; 24-well Ultra Low Cluster Plate Corning; 48-well Ultra Low Cluster Plate, Corning; culture bottle (74cm 2 , 24cm 2 ), Corning; gradient freezer box, Nalgene; cell counter, Nucleocounter; microplate reader, Thermo; SD rat bone marrow-derived mesenchymal stem cells (BMSC); self-extracted basal medium (BM), Gibco; osteogenic differentiation induction medium (OM), CYAGEN, USA; FBS, fetal bovine serum, Gibco. Trypsin (0.25%), Gibco Phosphate Buffered Saline (PBS) Gibco; DMSO, Sigma-Aldrich; CCK-8 Dojindo, Japan; BCA Protein Concentration Quantification Kit Beyotime; Dapi Solution Solarbio; Peptide Solarbio; Goldner trichrome staining solution Solarbio; live and dead cell viability toxicity detection kit, keyGen; ALP activity assay kit Beyotime; Triton X-100 Biotopped; , Germany; Calcein Sigma; Tetracycline Sigma; Toluidine Blue Staining Solution Google Bio; Magenta Sinopharm Group;
实验仪器laboratory apparatus
EBM S12电子束熔融设备；Arcam，德国便携式微弧氧化电源(JHMAO-220/6 A)金弧绿宝，中国；扫描电子显微镜(SEM)Hitachi，日本；Micro-CT扫描仪Siemens USA；X-射线光电子谱分析仪(XPS)Kratos，UK；能量色散X射线谱仪(EDS)Philips，Netherlands X线衍射仪(XRD)Focus,Bruker；通用力学测试仪，MTS Inc，USA；电感耦合等离子体原子发射光谱分析，ICP-AES；硬组织切磨片机EXAKT，德国；脱水浸透仪EXAKT，德国；光固化包埋仪，EXAKT，德国；平行载片粘合压片装置，EXAKT，德国；干燥渗透再聚合装置，EXAKT，德国；塑料载玻片EXAKT，德国；甲基丙烯酸甲酯光聚合树脂EXAKT，德国；TECHNOVIT 7210精密粘合剂EXAKT，德国；TECHNOVIT 4000黏合剂EXAKT，德国；砂纸EXAKT，德国；防冷却型外径千分尺EXAKT，德国；荧光显微镜Leica，德国；EBM S12 Electron Beam Melting Equipment; Arcam, Germany Portable Micro-arc Oxidation Power Supply (JHMAO-220/6 A) Golden Arc Emerald, China; Scanning Electron Microscope (SEM) Hitachi, Japan; Micro-CT Scanner Siemens USA; X- Ray Photoelectron Spectrometer (XPS) Kratos, UK; Energy Dispersive X-ray Spectrometer (EDS) Philips, Netherlands X-ray Diffraction Instrument (XRD) Focus, Bruker; General Mechanical Tester, MTS Inc, USA; Inductively Coupled Plasma Atom Emission Spectroscopy, ICP-AES; Hard Tissue Cutting and Polishing Machine EXAKT, Germany; Dehydration and Permeation Apparatus EXAKT, Germany; Photocuring and Embedding Apparatus, EXAKT, Germany; Parallel Slide Bonding Device, EXAKT, Germany; Repolymerization apparatus, EXAKT, Germany; plastic glass slides EXAKT, Germany; methyl methacrylate photopolymerization resin EXAKT, Germany; TECHNOVIT 7210 precision adhesive EXAKT, Germany; TECHNOVIT 4000 adhesive EXAKT, Germany; sandpaper EXAKT, Germany ; Anti-cooling outer micrometer EXAKT, Germany; Fluorescence microscope Leica, Germany;
主要试剂的配制：Preparation of main reagents:
基础培养基(BM，500mL)；Basal medium (BM, 500mL);
Cyagen Mesenchymal stem cell basal medium(PT-3238)440mL；Cyagen Mesenchymal stem cell basal medium (PT-3238) 440mL;
Cyagen Mesenchymal cell growth supplement(PT-4106E)50mL；Cyagen Mesenchymal cell growth supplement (PT-4106E) 50mL;
Cyagen GA-100(PT-4504E)0.5mL；Cyagen GA-100 (PT-4504E) 0.5mL;
Cyagen L-glutamine(PT-4107E)10mL；Cyagen L-glutamine (PT-4107E) 10mL;
成骨诱导培养基(OM，200mL)；Osteogenic induction medium (OM, 200mL);
Cyagen Osteogenic basal medium 175mL；Cyagen Osteogenic basal medium 175mL;
Cyagen Mesenchymal cell growth supplement 20mL；Cyagen Mesenchymal cell growth supplement 20mL;
Cyagen Penicillin/Streptomycin 2mL；Cyagen Penicillin/Streptomycin 2mL;
Cyagen L-glutamine 4mL；Cyagen L-glutamine 4mL;
CyagenAscorbate 400μL； Cyagen Ascorbate 400 μL;
Cyagen Dexamethasone 20μL；Cyagen Dexamethasone 20μL;
Cyagenβ-glycerophosphate 2mL；Cyagenβ-glycerophosphate 2mL;
0.25％胰蛋白酶5mL；0.25% trypsin 5mL;
细胞冻存液(10mL)；Cell Freezing Solution (10mL);
基础培养基7mL；Basal medium 7mL;
FBS胎牛血清2mL；FBS fetal bovine serum 2mL;
DMSO 1mL；DMSO 1mL;
蛋白标准液(1mg/mL，10mL)；Protein standard solution (1mg/mL, 10mL);
白蛋白10mg；Albumin 10mg;
磷酸盐缓冲剂10mg；Phosphate buffer 10mg;
十二烷基硫酸钠溶液(SDS，1％，10mL)；Sodium dodecyl sulfate solution (SDS, 1%, 10mL);
十二烷基硫酸钠1g；Sodium lauryl sulfate 1g;
磷酸盐缓冲液10mL；Phosphate buffer 10mL;
多孔Ti6A4V假体的制备Preparation of porous Ti6A4V prosthesis
本发明实施例采用的多孔钛合金支架为EBM(electron beam melting)快速成型技术制备而成，包括3种规格分别用于不同的试验检测，分别为：(1)直径10mm、高度5mm的圆柱体，用于细胞实验；(2)直径5mm、高度6mm的圆柱体，用于材料表征、体外力学测试、细胞培养、植入实验等；(3)采用EBM技术制备直径10mm、厚度1mm的图片片状Ti6A14V材料用于表征。参见下表1。The porous titanium alloy stent used in the embodiment of the present invention is prepared by EBM (electron beam melting) rapid prototyping technology, including 3 kinds of specifications for different test and detection, respectively: (1) a cylinder with a diameter of 10mm and a height of 5mm , used for cell experiments; (2) A cylinder with a diameter of 5 mm and a height of 6 mm, used for material characterization, in vitro mechanical testing, cell culture, implantation experiments, etc.; (3) EBM technology was used to prepare pictures with a diameter of 10 mm and a thickness of 1 mm Ti6A14V-like material was used for characterization. See Table 1 below.
假体依据CAD图像软件(CATIA以及INCAT USA)设计，内部孔径为640μm，其中支柱直径400μm，为具有钻石状晶格孔隙的圆柱体。设计完成导出CAD设计存为STL格式后使用Arcam EBM S12(Arcam AB，Sweden)设备进行样品的制备，样品的原材料为：Ti6A14V ELI Arcam标准粉末，粉粒的直径在45-100μm之间，采用电子束在真空环境下对粉末开始进行逐层的熔融打印，层厚0.1mm，成型后，充入氦气后自然冷却至100℃，然后充入空气，并完成打印过程。The prosthesis was designed based on CAD image software (CATIA and INCAT USA), with an internal pore diameter of 640 μm, of which the pillar diameter is 400 μm, which is a cylinder with diamond-like lattice pores. After the design is completed, export the CAD design and save it in STL format, use Arcam EBM S12 (Arcam AB, Sweden) equipment to prepare the sample. The raw material of the sample is: Ti6A14V ELI Arcam standard powder, the diameter of the powder is between 45-100 μm, and the electronic In a vacuum environment, the powder starts to be melted and printed layer by layer, with a layer thickness of 0.1mm. After forming, it is filled with helium and naturally cooled to 100°C, and then filled with air to complete the printing process.
实施例1：光功能化仪器的结构与参数设计Embodiment 1: Structure and parameter design of photofunctionalized instrument
参见附图1，提供了本发明的光功能化照射设备的一个实例，其参数满足：Referring to accompanying drawing 1, an example of the photofunctionalized irradiation device of the present invention is provided, and its parameter satisfies:
1)光源由两组波段组成，一组365±20nm(365nm为峰值，下文简称365nm波段)，一组270±20nm(270nm为峰值，下文简称270nm波段)；光源是LED光源，体积小，方便紧凑排列，光源发射的紫外线波长通常以某一波长为主，相邻波长为辅，所以仅以峰值对光源进行描述；1) The light source is composed of two groups of wave bands, one group is 365±20nm (365nm is the peak value, hereinafter referred to as the 365nm band), and the other group is 270±20nm (270nm is the peak value, hereinafter referred to as the 270nm band); the light source is an LED light source, which is small in size and convenient Compactly arranged, the ultraviolet wavelength emitted by the light source is usually dominated by a certain wavelength, supplemented by adjacent wavelengths, so only the peak value is used to describe the light source;
2)照射时，腔任意位置能量分布：270nm波段照射能量大于2mw/cm
2，365nm波段照射能量大于30mw/cm
2，且均匀性大于80％；从至少两个角度、多个角度、优选全向角度发射紫外线，实现腔体内紫外线的能量分布较为均匀，最优选的情况下能够实现从所有角度照射；不 同波段的紫外线可以单独照射，亦可以同时照射；
2) When irradiating, the energy distribution at any position of the cavity: the irradiation energy in the 270nm band is greater than 2mw/cm 2 , the irradiation energy in the 365nm band is greater than 30mw/cm 2 , and the uniformity is greater than 80%; from at least two angles, multiple angles, preferably all Ultraviolet rays are emitted at angles to achieve a relatively uniform energy distribution of ultraviolet rays in the cavity, and in the most optimal case, it can be irradiated from all angles; ultraviolet rays of different bands can be irradiated separately or at the same time;
3)内腔具有至少1立方厘米的体积，能承受一定重量物体压迫，设置有开口；这个尺寸仅仅是示例性的，可以根据需要处理的结构大小进行适应新调整，在一些实施例中，六面体内腔尺寸≥3x3x4立方厘米。3) The inner cavity has a volume of at least 1 cubic centimeter, can withstand the pressure of a certain weight object, and is provided with an opening; this size is only exemplary, and can be adjusted according to the size of the structure to be processed. In some embodiments, a hexahedron Lumen size ≥ 3x3x4 cubic centimeters.
一个优选实施例中，紫外线光源型号：365nm光源：CUN66B1B(Seoul)；270nm光源：CUD7GF1A(Seoul)。光源排列，所有270nm与365nm灯珠的间距为5mm，均匀覆盖于照射面，如图1至图3所示。图1中示出了光功能化仪器的一个实物照片，其内部为六面体内腔，并打开了舱门，显示出内部和舱门上规则设置的led紫外灯珠，所述的光源排列仅仅是示例性的，灯珠的间距没有要求，只要能实现目标能量密度即可。In a preferred embodiment, the ultraviolet light source model: 365nm light source: CUN66B1B (Seoul); 270nm light source: CUD7GF1A (Seoul). The light source is arranged, and the distance between all 270nm and 365nm lamp beads is 5mm, covering the irradiated surface evenly, as shown in Figure 1 to Figure 3. Figure 1 shows a real photo of the light functionalized instrument, the inside of which is a hexahedral cavity, and the hatch is opened, showing the regular arrangement of led ultraviolet lamp beads inside and on the hatch, the light source arrangement is only Exemplarily, there is no requirement on the spacing of the lamp beads, as long as the target energy density can be achieved.
图3给出了光功能化仪器的使用状态，示出了光功能化仪器的内部结构，黑色箭头指示假体；红色箭头指示365nm波段紫外灯，白色箭头指示270nm波段紫外灯；Figure 3 shows the state of use of the photofunctionalized instrument, showing the internal structure of the photofunctionalized instrument, the black arrow indicates the prosthesis; the red arrow indicates the 365nm band UV lamp, and the white arrow indicates the 270nm band UV lamp;
该光功能化仪器的结构仅仅是示例性，壳体的其他结构，例如圆柱体、正方体、非规则形状也是可行的。The structure of the light-functionalized instrument is only exemplary, and other structures of the shell, such as cylinder, cube, and irregular shapes are also feasible.
光功能化仪器包括：壳体，壳体设置有容纳待处理钛合金结构件的腔体；紫外线灯，紫外线灯设置在所述壳体的内壁；控制面板，控制面板用于设置和显示紫外线的波长和处理时间。壳体具有可以打开的部分，内部用于将待照射的结构件放置其中；控制面板包括控制器和显示器，控制器用于控制照射的紫外线波长和时间，显示器用于使用者设置和读取程序的运行情况，一个优选实施例中，显示器是触摸屏，使用者可以进行输入设定。控制面板可以控制紫外灯的开关和时序，例如在六面体内腔的情况下，可以同时开启两种波长的紫外灯，也可以单独开启一个波段的紫外灯，或者控制不同波段的紫外灯交替照射；又例如，可以开启一个面，两个面或更多个面上的紫外灯，而不开启其他面上的紫外灯，还可以按照需求加上时序控制。The photofunctionalized instrument includes: a housing, the housing is provided with a cavity for accommodating titanium alloy structural parts to be processed; an ultraviolet lamp, the ultraviolet lamp is arranged on the inner wall of the housing; a control panel, the control panel is used to set and display the wavelength and processing time. The shell has a part that can be opened, and the interior is used to place the structure to be irradiated; the control panel includes a controller and a display, the controller is used to control the ultraviolet wavelength and time of irradiation, and the display is used for the user to set and read the program In terms of operation, in a preferred embodiment, the display is a touch screen, and the user can perform input settings. The control panel can control the switch and timing of the UV lamp. For example, in the case of a hexahedral cavity, two wavelengths of UV lamps can be turned on at the same time, or a UV lamp of one band can be turned on separately, or UV lamps of different bands can be controlled to irradiate alternately; For another example, it is possible to turn on the UV lamps on one surface, two or more surfaces, but not to turn on the UV lamps on other surfaces, and it is also possible to add timing control according to the requirements.
实施例2：使用本发明的光功能化仪器对钛合金材料进行光功能化处理及经处理钛合金材料实验检测Embodiment 2: Using the optical functionalization instrument of the present invention to carry out optical functionalization treatment on titanium alloy materials and experimental detection of treated titanium alloy materials
2.1实验分组与对应的处理2.1 Experimental grouping and corresponding processing
实验共分为5组，分别是为GC(对照组)、GL、G270、G365、GU。其中GC未接受光照处理，GL接受15分钟的紫外线高压汞灯照射；G270、G365和GU组为本发明仪器处理组，分别为接受270nm、365nm、270nm与365nm双波长照射，照射时间均为15分钟。The experiment was divided into 5 groups, namely GC (control group), GL, G270, G365 and GU. Among them, GC did not accept light treatment, and GL accepted 15 minutes of ultraviolet high-pressure mercury lamp irradiation; G270, G365 and GU groups were instrument treatment groups of the present invention, which were respectively 270nm, 365nm, 270nm and 365nm dual-wavelength irradiation, and the irradiation time was 15 minutes. minute.
GC(对照组)处理流程：在实验室环境下(常温、常压、空气环境下)放置4周后，在无菌操作台上放置15分钟(不接受照射)，并进行密封保存，24小时之内进行高压蒸汽灭菌及烘干，期间操作均使用金属器械，避免接触可能污染，并开始后续处理。GC (control group) treatment process: after 4 weeks in a laboratory environment (normal temperature, normal pressure, air environment), place it on a sterile operating table for 15 minutes (without irradiation), and keep it sealed for 24 hours During the period, high-pressure steam sterilization and drying were carried out. During the operation, metal instruments were used to avoid contact with possible contamination, and subsequent processing was started.
GL处理流程：假体制备后，在实验室环境下(常温、常压、空气环境下)放置4周后，在无菌操作台上接受高压汞灯(多波长峰值，包括270nm和365nm)照射15分钟，并进行密封保存，24小时之内进行高压蒸汽灭菌及烘干，期间操作均使用金属器械，避免接触可能污染，并开始后续处理。GL treatment process: After the prosthesis is prepared, it is placed in a laboratory environment (normal temperature, normal pressure, and air environment) for 4 weeks, and then it is irradiated by a high-pressure mercury lamp (multi-wavelength peaks, including 270nm and 365nm) on a sterile operating table 15 minutes, and sealed for storage, high-pressure steam sterilization and drying within 24 hours, using metal instruments during the operation to avoid contact with possible contamination, and start subsequent processing.
G270、G365和GU(仪器照射处理组)标准流程：假体制备后，在实验室环境下(常温、常压、空气环境下)放置4周后，使用紫外线光功能化仪器用270nm或/与365nm两种波长的紫外线同时照射假体15分钟，并进行密封保存，24小时之内进行高压蒸汽灭菌及烘干，期间操作均使用金属器械，避免接触可能污染，并开始后续处理。G270, G365, and GU (instrument irradiation treatment group) standard procedure: After the prosthesis is prepared, it is placed in the laboratory environment (normal temperature, normal pressure, and air environment) for 4 weeks, and then the functionalized instrument is used with ultraviolet light at 270nm or/and Ultraviolet rays with two wavelengths of 365nm irradiated the prosthesis for 15 minutes at the same time, and sealed the prosthesis. Within 24 hours, it was sterilized and dried by high-pressure steam. During the operation, metal instruments were used to avoid contact with possible contamination, and the follow-up treatment was started.
后续实验检测如下：Subsequent experimental tests are as follows:
2.2物理化学测试：接触角测试、XPS、XRD等；2.2 Physical and chemical tests: contact angle test, XPS, XRD, etc.;
2.3体外细胞培养：细胞培养、ALP活性测试、细胞形态测试等；2.3 In vitro cell culture: cell culture, ALP activity test, cell morphology test, etc.;
2.4体内实验：处理后的假体进行兔股骨髁上假体植入术。2.4 In vivo experiment: the treated prosthesis was implanted into supracondylar prosthesis of rabbit femur.
2.2物理化学表征测试2.2 Physical and chemical characterization test
2.2.1亲水性测试接触角测试2.2.1 Hydrophilicity Test Contact Angle Test
采用座滴法(Sessile Drop)对3D打印钛合金(每组各10个)表面进行接触角测The contact angle was measured on the surface of 3D printed titanium alloy (10 pieces in each group) by Sessile Drop method.
试并照相，并利用直接测量法测量出角度，后进行统计分析。Tested and photographed, and used direct measurement method to measure the angle, after statistical analysis.
2.2.2扫描电子显微镜2.2.2 Scanning Electron Microscope
经过处理后，使用金刚石锯从正中间切开多孔钛合金支架，用场发射的扫描电镜After processing, use a diamond saw to cut the porous titanium alloy scaffold from the middle, and use a field emission scanning electron microscope
(FE-SEM：S-4800，hITACHI)分析3D打印多空金属支架内外部的表面形态。(FE-SEM: S-4800, hITACHI) Analysis of surface morphology inside and outside of 3D printed porous metal stents.
2.2.3 X射线光电子能谱2.2.3 X-ray photoelectron spectroscopy
X-射线光电子能谱(XPS；250XI，Thermo escalab，USA)用于分析样品表面的化学组成。单色Al Kα(1486.6eV)为射线源，功率150W，500μm束斑，电荷校正采用污染碳C1s＝284.8eV进行校正。X-ray photoelectron spectroscopy (XPS; 250XI, Thermo escalab, USA) was used to analyze the chemical composition of the sample surface. The monochromatic Al Kα (1486.6eV) is the radiation source, the power is 150W, the beam spot is 500μm, and the charge correction is performed by polluted carbon C1s=284.8eV.
2.4 X射线衍射2.4 X-ray diffraction
采用X射线衍射技术(XRD；D8，ADVANCE，Bruker)对涂层进行物相分析，使用Cu-Kα射线源，管电压为40kV，管电流为50mA，连续扫描模式，扫描速度为4°/min，衍射角2θ为10-80°。X-ray diffraction technique (XRD; D8, ADVANCE, Bruker) was used to analyze the phase of the coating, using Cu-Kα ray source, the tube voltage was 40kV, the tube current was 50mA, continuous scanning mode, and the scanning speed was 4°/min , the diffraction angle 2θ is 10-80°.
2.2.5能量色散X-射线谱仪2.2.5 Energy dispersive X-ray spectrometer
采用能量色散X射线能谱仪(EDS：DX-4，Phililps，the Netherlands)来分析3D打印钛合金表面的元素组成(Elemental Composition)。An energy dispersive X-ray spectrometer (EDS: DX-4, Phililps, the Netherlands) was used to analyze the elemental composition (Elemental Composition) of the 3D printed titanium alloy surface.
2.3细胞培养2.3 Cell culture
细胞生长培养基的制备：培养基在使用前需加入10％的胎牛血清和双抗，一般为血清。培养基分装成小瓶(100～200mL)以便使用，翻帽塞塞紧瓶口。按细胞生长需求，制备相应的生长培养基。一般的细胞生长培养基为培养基+10％胎牛血清，最后按1％体积分数加入双抗贮存液(青霉素+链霉素)，使青霉素和链霉素的终浓度分别为100U/mL和100U/mL。Preparation of cell growth medium: 10% fetal calf serum and double antibody, usually serum, should be added to the medium before use. The culture medium was divided into vials (100-200mL) for use, and the cap was turned over to seal the bottle mouth tightly. According to the needs of cell growth, prepare the corresponding growth medium. The general cell growth medium is culture medium + 10% fetal bovine serum, and finally add double antibody stock solution (penicillin + streptomycin) according to 1% volume fraction, so that the final concentrations of penicillin and streptomycin are 100U/mL and 100U/mL respectively. 100U/mL.
2.3.1 SD大鼠骨髓来源间充质干细胞的培养与鉴定2.3.1 Culture and identification of SD rat bone marrow-derived mesenchymal stem cells
BMSCs分离培养BMSCs isolation and culture
取SD大鼠，颈椎脱臼法处死，浸泡于75％乙醇中约5-10分钟(min)，无菌手术器械取出双侧股骨及胫骨，去除附着肌肉，将其浸泡于适量培养基中。取10mL针筒，在股骨及胫骨两端打各打2～3个孔，针筒吸取适量培养基插入一端干骺端，将骨髓中细胞冲至培养皿中，反复几次，直至股骨及胫骨发白，收集此细胞悬液，用200目金属滤网过滤去除稍大的杂质，将过滤后细胞悬液收集至离心管中，800r/min，5min离心，弃上清，加人适量α-MEM培养基混匀，接种于10cm塑料培养皿中，于37℃，体积分数5％CO
2饱和湿度孵育箱中培48-72小时(h)后更换培养基，注意动作轻柔，此后每隔3天更换培养基1次，约9～12天后形成多个细胞集落，长满瓶底约70％～80％后可进行传代培养。
SD rats were taken, killed by cervical dislocation, immersed in 75% ethanol for about 5-10 minutes (min), bilateral femurs and tibias were taken out with sterile surgical instruments, attached muscles were removed, and they were soaked in an appropriate amount of medium. Take a 10mL syringe, and make 2-3 holes at both ends of the femur and tibia. The syringe absorbs an appropriate amount of medium and inserts it into one end of the metaphysis. The cells in the bone marrow are washed into the culture dish, and repeated several times until the femur and tibia Turn white, collect the cell suspension, filter with a 200-mesh metal filter to remove slightly larger impurities, collect the filtered cell suspension into a centrifuge tube, centrifuge at 800r/min for 5min, discard the supernatant, and add an appropriate amount of α- Mix the MEM medium evenly, inoculate it in a 10cm plastic culture dish, incubate in an incubator with a volume fraction of 5% CO 2 and a saturated humidity at 37°C for 48-72 hours (h), and then replace the medium. Change the medium once a day, and after about 9 to 12 days, multiple cell colonies are formed, which can be subcultured after about 70% to 80% of the bottom of the bottle is covered.
细胞传代cell inheritance
等待细胞达到80％的汇合度后进行再传代培养，我们首先要将培养瓶中培养液移出，接着加入适量的磷酸盐缓冲液(PBS)缓慢的清洗后弃液，再之后将培养瓶内部加入适量的0.125％浓度的胰蛋白酶覆盖住细胞，在室温下来进行细胞消化，当我们在倒置显微镜下面观察细胞逐渐变为圆形，接着从培养瓶分离时，我们加入适量的基础培养基来中和胰蛋白酶，然后轻轻的 敲打培养瓶从而使细胞脱离后，接着将细胞悬液在为800转/分钟(rpm)的条件下来离心5分钟，移出液体后，我们加入适量基础培养基，轻轻的吹打细胞团块重悬细胞，最后，我们将等量搅拌均匀的细胞悬液来分至培养瓶内部，补充适量的基础培养基后，再置于培养箱内部培养，培养环境需为温度37℃，二氧化碳浓度为5％，细胞培养到第3～5代时使用。Subculture after the cells reach 80% confluence, we first remove the culture medium in the culture bottle, then add an appropriate amount of phosphate buffered saline (PBS) to slowly wash and discard the liquid, and then add the inside of the culture bottle An appropriate amount of trypsin at a concentration of 0.125% covers the cells, and the cells are digested at room temperature. When we observe that the cells gradually become round under an inverted microscope, and then separate from the culture flask, we add an appropriate amount of basal medium to neutralize the cells. Trypsin, then gently tap the culture bottle to detach the cells, then centrifuge the cell suspension at 800 revolutions per minute (rpm) for 5 minutes, remove the liquid, add an appropriate amount of basal medium, gently Finally, we divide the same amount of uniformly stirred cell suspension into the inside of the culture flask, add an appropriate amount of basal medium, and then place it in the incubator for culture. The culture environment needs to be at a temperature of 37 ℃, the carbon dioxide concentration is 5%, and it is used when the cells are cultured to the 3rd to 5th passage.
细胞鉴定cell identification
通过流式表面标记鉴定：成骨、成软骨、成脂肪三系的分化实验确定CD105、CD166、CD29和CD44的表达为阳性，CD14、CD34以及CD45的表达为阴性。Identification by flow cytometry surface markers: The differentiation experiments of osteogenic, chondrogenic and adipogenic lines confirmed that the expression of CD105, CD166, CD29 and CD44 was positive, and the expression of CD14, CD34 and CD45 was negative.
细胞计数cell counts
采用细胞计数板来进行细胞定量：首先将计数板及盖片擦拭干净，并将盖片盖在计数板；细胞消化步骤同前，在细胞重悬后，吸入100μL细胞悬液放置于Ep管内，用培养基稀释一定的倍数，然后移液枪将细胞悬液吸出少许，滴加在盖片边缘，使悬液充满盖片和计数板之间，静置3min(注意盖片下不要有气泡)最后计算板四大格细胞总数，压线细胞只计左侧和上方，根据公式计算(细胞数/mL＝四大格细胞总数/4×10
4×稀释倍数/mL)细胞数量，重复3次，取平均值。根据测定的细胞数量，再加入适量的基础培养基调整细胞浓度至5×10
4/600μL。
Use a cell counting plate for cell quantification: first wipe the counting plate and the cover slip clean, and cover the cover slip on the counting plate; the cell digestion step is the same as before, after the cells are resuspended, suck 100 μL of the cell suspension into the Ep tube, Dilute a certain number of times with the medium, then suck out a little of the cell suspension with a pipette gun, and drop it on the edge of the cover slip, so that the suspension fills the space between the cover slip and the counting plate, and let it stand for 3 minutes (note that there are no air bubbles under the cover slip) Finally, calculate the total number of cells in the four major grids on the plate, and only count the cells on the left and upper sides of the plate. Calculate the number of cells according to the formula (number of cells/mL=total number of cells in the four major grids/4×10 4 ×dilution factor/mL), repeat 3 times ,take the average. According to the determined number of cells, an appropriate amount of basal medium was added to adjust the cell concentration to 5×10 4 /600 μL.
2.3.2细胞接种2.3.2 Cell Seeding
将预先处理好的3D多孔钛合金内置物置于基础培养基中先浸泡30min，在浸泡完成后放置于48孔超低贴附的培养板内部，然后在每个支架上滴加600μL细胞悬液，用移液枪反复回吸和滴加，以便细胞悬液与支架均匀接触，培养至24小时使细胞的进一步贴附。Put the pre-treated 3D porous titanium alloy inserts into the basal medium and soak for 30 minutes. After soaking, place them inside a 48-well ultra-low attachment culture plate, and then drop 600 μL of cell suspension on each bracket. Use a pipette gun to repeatedly suck back and drop, so that the cell suspension is in uniform contact with the scaffold, and cultivate for 24 hours to further attach the cells.
接种24h后换液，用于CKK8、细胞骨架、live/Dead Viability，每孔分别加入600μL基础培养基，在用于成骨分化的检测实验的样品中，加入1mL的成骨诱导培养基，再每3天进行一次换液。Change the medium 24 hours after inoculation. For CKK8, cytoskeleton, and live/Dead Viability, add 600 μL of basal medium to each well, add 1 mL of osteogenic induction medium to the samples used for the detection experiment of osteogenic differentiation, and then Change the medium every 3 days.
2.3.3细胞增殖2.3.3 Cell Proliferation
我们在接种后1天、3天和7天，分别对细胞的增殖活性(Cell proliferation)来进行检测。首先我们将培养板内的培养液吸出，加入1mL磷酸盐缓冲液(PBS)来清洗剩余的培养基1次，然后将支架分别移至24孔板内，每孔加入1mL的新鲜基础培养基以及100μL CCK-8的试剂，我们将培养板放置于37℃条件下来孵育2h，待反应结束后，每孔吸出100μL加入96孔板，再使用酶标仪在450nm的波长下来测定吸光度。We tested the cell proliferation activity (Cell proliferation) at 1 day, 3 days and 7 days after inoculation. First, we sucked out the culture medium in the culture plate, added 1 mL of phosphate buffered saline (PBS) to wash the remaining medium once, then moved the scaffolds to 24-well plates, and added 1 mL of fresh basal medium and For 100 μL of CCK-8 reagent, we placed the culture plate at 37°C and incubated for 2 hours. After the reaction was over, suck out 100 μL from each well and add it to a 96-well plate, and then use a microplate reader to measure the absorbance at a wavelength of 450 nm.
活、死细胞活力毒性检测Live and Dead Cell Viability Toxicity Assay
细胞在支架接种3天后，对细胞的增殖活性(Cell proliferation)来进行观察。将培养板内的培养基吸出，加入1mL PBS液清洗支架上剩余的培养基，然后将支架分别移至24孔板内，每孔加入1mL Live/Dead工作液(2μM Calcein AM和8μM PI)，室温孵育30min后，吸出工作液，终止孵育。用PBS对支架清洗2遍(操作过程要轻柔)，然后使用激光共聚焦显微镜观察标记细胞。Three days after the cells were seeded on the scaffold, the cell proliferation was observed. Aspirate the medium in the culture plate, add 1mL PBS solution to wash the remaining medium on the bracket, and then move the bracket to a 24-well plate, add 1mL Live/Dead working solution (2μM Calcein AM and 8μM PI) to each well, After incubating at room temperature for 30 min, the working solution was aspirated and the incubation was terminated. Wash the scaffold twice with PBS (the operation process should be gentle), and then observe the labeled cells with a laser confocal microscope.
细胞骨架Cytoskeleton
细胞在支架接种3天后，对细胞的增殖活性(Cell proliferation)来进行观察。将培养板内的培养基吸出，加入1mL PBS液清洗支架上剩余的培养基，然后将支架分别移至24孔板内，吸掉培养液，在37℃条件下预热的1×PBS(PH7.4)清洗细胞2次；使用溶于PBS的4％甲醛溶液进行细胞固定，室温固定10min；室温条件下，用PBS清洗细胞2次，每次10min；取1mL配置好的TRITC标记鬼笔环肽工作液，室温避光孵育30min；然后用PBS清洗支架3次，每次5min；使用1mL DAPI溶液(浓度：100nM)对细胞核进行复染10min；最后吸出染色液，用 PBS清洗3遍，终止染色，使用激光共聚焦显微镜观察标记细胞。Three days after the cells were seeded on the scaffold, the cell proliferation was observed. Aspirate the medium in the culture plate, add 1mL PBS solution to wash the remaining medium on the bracket, then move the brackets to 24-well plates respectively, suck off the culture medium, and preheat 1×PBS (PH7 .4) Wash the cells twice; fix the cells with 4% formaldehyde solution dissolved in PBS, and fix at room temperature for 10 minutes; at room temperature, wash the cells twice with PBS, each time for 10 minutes; take 1 mL of prepared TRITC-labeled phalloid ring Peptide working solution, incubate at room temperature in the dark for 30 minutes; then wash the scaffold 3 times with PBS, 5 minutes each time; use 1mL DAPI solution (concentration: 100nM) to counterstain the cell nucleus for 10 minutes; finally suck out the staining solution, wash 3 times with PBS, and stop After staining, the labeled cells were visualized using a laser confocal microscope.
2.3.4细胞碱性磷酸酶活性2.3.4 Cellular alkaline phosphatase activity
细胞在3D打印支架接种24h后，更换成骨分化培养基继续培养，再每3天进行换液。在成骨分化培养基培养第7天与14天，检测3D多孔钛合金内置物上细胞的碱性磷酸酶(Alkaline phosphatase，ALP)的活性，并且测定细胞的总蛋白含量进行校正。碱性磷酸酶(ALP)的活性检测：首先我们使用磷酸盐缓冲液(PBS)来清洗待测样品两次，然后用1mL 0.2％TritonX-100来裂解3D多孔钛合金内置物上的细胞；吸取细胞裂解液来检测ALP的活性；我们依照实验的说明来配制检测缓冲液、显色底物溶解(para-nitrophenyl phosphate，pNPP)以及标准品的工作液，然后稀释标准品(p-nitrophenol)为0.002μmoL；依照实验的说明我们将每孔分别加入待测的样品，底物溶液、检测缓冲液以及标准品，在37℃的条件下孵育5min，然后在405nm的波长下读取吸光值，并且计算碱性磷酸酶(ALP)的DEA活性单位，碱性磷酸酶(ALP)的DEA活性单位来定义为在pH9.8的diethanoamine(DEA)缓冲液中，37℃的条件下，每分钟的水解pNPP显色底物生成1μmnol p-nitrophenol所需ALP的量定义为一个酶活力的单位，也被称作为一个DEA的酶活力单位。After the cells were inoculated on the 3D printed scaffold for 24 hours, the osteogenic differentiation medium was replaced to continue culturing, and the medium was changed every 3 days. On the 7th and 14th day of culture in the osteogenic differentiation medium, the alkaline phosphatase (Alkaline phosphatase, ALP) activity of the cells on the 3D porous titanium alloy insert was detected, and the total protein content of the cells was measured for correction. Alkaline phosphatase (ALP) activity detection: first, we use phosphate buffered saline (PBS) to wash the sample to be tested twice, and then use 1mL 0.2% TritonX-100 to lyse the cells on the 3D porous titanium alloy built-in; Cell lysate was used to detect the activity of ALP; we prepared the detection buffer, chromogenic substrate solution (para-nitrophenyl phosphate, pNPP) and the working solution of the standard according to the instructions of the experiment, and then diluted the standard (p-nitrophenol) to 0.002μmoL; According to the instructions of the experiment, we added the sample to be tested, the substrate solution, the detection buffer and the standard to each well, incubated at 37°C for 5min, and then read the absorbance value at a wavelength of 405nm, and Calculate the DEA activity unit of alkaline phosphatase (ALP), and the DEA activity unit of alkaline phosphatase (ALP) is defined as the hydrolysis per minute in diethanoamine (DEA) buffer at pH 9.8 at 37°C The amount of ALP required for pNPP chromogenic substrate to generate 1 μmnol p-nitrophenol is defined as a unit of enzyme activity, also known as a unit of enzyme activity of DEA.
细胞总蛋白定量，首先我们使用磷酸盐缓冲液(PBS)来清洗待测样品两次，然后用1mL0.2％TritonX-100裂解3D多孔钛合金内置物上的细胞，将细胞裂解液稀释至15倍后进行检测：按照实验说明来配制蛋白的定量工作液(Micro BCA Woriking Reagent)，并且，我们将标准品稀释至200μg/mL、20μg/mL、10μg/mL、5μg/mL、2.5μg/mL、1μg/mL以及0.5μg/mL，来建立300μL的反应体系，我们将每孔分别加入150μL的标准品或者待测样品，然后向每孔分别加入150μL的工作液，搅拌均匀后进行封孔膜密封，在37℃条件下来孵育2小时，然后在562nm的波长下，再读取吸光值，并用BCA测得的总蛋白含量对于ALP活性行标准化处理。For the quantification of total cell protein, first we use phosphate buffered saline (PBS) to wash the sample to be tested twice, then use 1mL0.2% TritonX-100 to lyse the cells on the 3D porous titanium alloy built-in, and dilute the cell lysate to 15 Detection after doubling: Prepare protein quantitative working solution (Micro BCA Working Reagent) according to the experimental instructions, and we dilute the standard to 200μg/mL, 20μg/mL, 10μg/mL, 5μg/mL, 2.5μg/mL , 1 μg/mL, and 0.5 μg/mL to establish a 300 μL reaction system, we add 150 μL of standard or sample to each well, and then add 150 μL of working solution to each well, stir well and then seal the membrane Seal and incubate at 37°C for 2 hours, then read the absorbance at a wavelength of 562nm and normalize the total protein content measured by BCA to the ALP activity.
2.3.5细胞形态分析2.3.5 Cell morphology analysis
细胞的接种在3天后，采用扫描电镜(SEM)来观察细胞在3D多孔钛合金内置物上的粘附情况，具体的步骤如下：细胞在支架接种3天后，将培养板内的培养基吸出，加入1mLPBS液清洗支架上剩余的培养基，然后将支架分别移至24孔板内，吸掉培养液，用PBS(PH7.4)清洗细胞2次；首先，用3％戊二醛在4℃的条件下来固定2h；0.18M的蔗糖冲洗液在4℃条件下固定2h；1％的锇酸在4℃的条件下固定2小时；接着，梯度乙醇(30％，50％，70％，90％，100％)脱水3次，每次15min；然后将乙醇异戊脂置换20min；在临界点干燥；对待测样品进行喷金，采用SEM观察。After 3 days of inoculation of the cells, a scanning electron microscope (SEM) was used to observe the adhesion of the cells on the 3D porous titanium alloy built-in. Add 1 mL of PBS solution to wash the remaining medium on the scaffold, then move the scaffolds to 24-well plates, suck off the culture medium, and wash the cells twice with PBS (pH7.4); first, wash the cells with 3% glutaraldehyde at 4°C 0.18M sucrose washing solution was fixed at 4°C for 2h; 1% osmic acid was fixed at 4°C for 2 hours; then, gradient ethanol (30%, 50%, 70%, 90 %, 100%) dehydration for 3 times, 15min each time; then replace amyl alcohol with isoamyl ester for 20min; dry at the critical point; spray gold on the sample to be tested, and observe with SEM.
2.3.6力学特性测试2.3.6 Mechanical properties test
为了测试紫外线光功能化处理对3D多孔钛合金支架压缩强度造成的影响，运用通用力学测试机(Landmark，MTS Inc.MN，USA)来对处理前后的3D多孔钛合金支架进行静态压缩的测试。测试的样品为直径5mm，高度为6mm的3D打印圆柱金属支架，压缩过程中的形变率为1.8mm/分，每组测试的样品数量为5个，测试数据自动的生成为应力-应变曲线(Stress-strain curve)，从而，分析得到的弹性模量(Elastic modulus)、屈服强度(Yield strength)以及压缩强度(Compression strength)。In order to test the effect of ultraviolet light functionalization on the compressive strength of 3D porous titanium alloy scaffolds, a general mechanical testing machine (Landmark, MTS Inc. MN, USA) was used to perform static compression tests on the 3D porous titanium alloy scaffolds before and after treatment. The tested sample is a 3D printed cylindrical metal bracket with a diameter of 5 mm and a height of 6 mm. The deformation rate during compression is 1.8 mm/min. The number of samples tested in each group is 5. The test data is automatically generated as a stress-strain curve ( Stress-strain curve), thus, analyze the obtained elastic modulus (Elastic modulus), yield strength (Yield strength) and compression strength (Compression strength).
2.4体内实验2.4 In vivo experiments
兔股骨髁植入的实验，评价紫外线光功能化的体内生物的相容性，以及骨整合的性能。本次实验所采用的实验动物，均有由北京维通利华公司所提供，所有的实验方案均已通过北京大学医学部的伦理委员会审批(LA2014214通过，动物实验的操作和饲养均符合国家实验动物的 饲养和使用指南。Implantation experiments in the rabbit femoral condyle to evaluate the in vivo biocompatibility and osseointegration properties of ultraviolet photofunctionalization. The experimental animals used in this experiment were all provided by Beijing Weitong Lihua Company. All the experimental plans have been approved by the Ethics Committee of Peking University Health Science Center (approved by LA2014214, and the operation and feeding of animal experiments are in accordance with the national experimental animal standards. Care and use guidelines.
2.4.1兔股骨骼植入实验2.4.1 Rabbit Femoral Bone Implantation Experiment
实验设计：考虑到实验的成本效益和实验动物“3R”的原则(Reduction，Replacement，Refinement)，所以，我们选择新西兰的大白兔作为本次实验动物的初步评估紫外线光功能化对3D打印多孔钛合金内置物骨整合性能造成的影响，尽管这样，我们根据兔股骨骼的解剖特点(兔股骨骼的前后径大约为12-15mm)，再将兔股骨的内侧骼所造的骨缺损大小为5mm，以及模拟极限骨缺损的模型(Critical bone defect)。据文献统计，正常的兔骨愈合的骨重塑发生于3周至8周，8周时骨重塑已经大部分都完成，从而，在本研究中，我们观察的时间点选择为植入术后8周，而且为了动态反应的3D打印多孔钛合金植入物植入术后骨整合的过程，将分别在术后第3周及第7周分别皮下注射钙黄绿素和四环素来标记不同的时期骨生成的情况。Experimental design: Considering the cost-effectiveness of the experiment and the "3R" principle (Reduction, Replacement, Refinement) of experimental animals, we chose New Zealand white rabbits as the experimental animals for the preliminary evaluation of ultraviolet light functionalization on 3D printing porous titanium. The impact of the osseointegration performance of the alloy implants, despite this, according to the anatomical characteristics of the rabbit femoral bone (the anteroposterior diameter of the rabbit femoral bone is about 12-15mm), and then the size of the bone defect created by the medial bone of the rabbit femur is 5mm , and a model for simulating critical bone defects. According to literature statistics, the bone remodeling of normal rabbit bone healing occurs from 3 weeks to 8 weeks, and most of the bone remodeling has been completed at 8 weeks. Therefore, in this study, the time point we observed was post-implantation 8 weeks, and in order to dynamically respond to the process of osseointegration after implantation of 3D printed porous titanium alloy implants, calcein and tetracycline will be injected subcutaneously at the 3rd and 7th weeks after the operation to mark the bone at different stages. generated situation.
手术方法：雄性新西兰大白兔，随机分组，采用胺碘酮50mg/kg肌肉来注射麻醉，双侧的股骨外侧髁备皮，用0.5％的碘伏进行消毒，然后，纵行切开皮肤皮下至骨膜面，接着采用尖刀片来刮涂附着的骨膜，并且，定位股骨骼部，我们采用5mm直径的电钻，在生理盐水冷却下在股骨髁造一个直径为5mm的骨缺损，然后用生理盐水来冲洗局部，再将假体植入骨缺损的地方，接着采用4#的抗菌薇乔线逐涂层间断缝合肌肉层、筋膜层和皮肤。术后3天再采用青霉素40万单位肌肉来注射预防感染。Surgical method: male New Zealand white rabbits were randomly divided into groups, anesthetized by intramuscular injection of amiodarone 50 mg/kg, skin preparation of bilateral lateral femoral condyles, disinfection with 0.5% povidone iodine, and then longitudinal incision of the skin subcutaneously to On the periosteum surface, then use a sharp blade to scrape the attached periosteum, and, to locate the femoral bone, we use a 5mm diameter electric drill to create a 5mm diameter bone defect in the femoral condyle under normal saline cooling, and then use normal saline to After washing the local area, the prosthesis was implanted in the bone defect, and then the muscle layer, fascia layer and skin were sutured layer by layer with 4# antibacterial Vicryl suture. Three days after the operation, 400,000 units of penicillin was injected intramuscularly to prevent infection.
2.4.2序贯荧光素标记2.4.2 Sequential fluorescein labeling
为了能够动态的观察3D多孔钛合金内置物骨整合的详细过程，我们分别采用钙黄绿素(Calcein)和四环素(Tetracycline)序贯荧光标记(Sequential fluorescent labeling)3周以及7周的骨生成的情况，我们采用试剂的配制和给药时间(如表2)，给药前的试剂采用为0.2μm的过滤器来进行消毒，给药的途径采用为皮下注射的方式，手术后3周和7周的生成骨分别被标记为绿色和黄色。In order to dynamically observe the detailed process of osseointegration of 3D porous titanium alloy implants, we used Calcein and Tetracycline sequential fluorescent labeling (Sequential fluorescent labeling) for 3 weeks and 7 weeks of bone formation, respectively, We used the preparation and administration time of the reagent (as shown in Table 2). The reagent before administration was sterilized with a filter of 0.2 μm, and the route of administration was subcutaneous injection. Generated bone is marked in green and yellow, respectively.
表2：荧光素标记的试剂配制和给药计划Table 2: Fluorescein-labeled reagent preparation and dosing schedule
2.4.3 Micro-CT扫描2.4.3 Micro-CT scanning
我们在术后8周后，首先以过量的苯巴比妥钠杀死实验的动物，接着剥离股骨骼，然后将植入的标本分为两部分分别进行处理，我们将标本置于-80℃的冷冻保存，Eight weeks after the operation, we first killed the experimental animals with an excessive amount of phenobarbital sodium, then stripped the femoral bones, and then divided the implanted specimens into two parts for processing separately. We placed the specimens at -80°C cryopreservation,
2.5实验结果2.5 Experimental results
2.5.1亲水性测试：接触角测量2.5.1 Hydrophilicity test: contact angle measurement
在接触角测试中可以直观的看到(图4)，实验组(GU)的亲水性较对照组(GC)有明显升高。经测量，GU组的接触角θ(θ＝60.3±0.05)明显低于用GC组(θ＝90.1±2.41)，且p<0.05，有明显统计学差异。GL、G270、G365与GC组相比，有明显统计学差异(p<0.05)；GL、G270、G365与GU组相比，GU组的接触角最小，有明显统计学差异(p<0.05)。In the contact angle test, it can be seen intuitively (Figure 4), that the hydrophilicity of the experimental group (GU) was significantly higher than that of the control group (GC). After measurement, the contact angle θ (θ=60.3±0.05) of the GU group was significantly lower than that of the GC group (θ=90.1±2.41), and p<0.05, there was a significant statistical difference. Compared with GC group, GL, G270, G365 had significant statistical difference (p<0.05); compared with GU group, GL, G270, G365 had the smallest contact angle, with significant statistical difference (p<0.05) .
2.5.2 SEM与EDS分析涂层的形貌特征和元素比例2.5.2 SEM and EDS analysis of the morphology and element ratio of the coating
图5显示了经过紫外线光功能化处理前后，4个对照组(GL、G270、G365、GU)的表面形貌的变化，相对于对照组(GC)，我们观察到更多的复杂形貌和表面微孔形成；进一步地， 相对于GL、G270和G365，在GU组，观察到最多的表面微孔形成。Figure 5 shows the changes in the surface topography of the four control groups (GL, G270, G365, GU) before and after UV photofunctionalization. Compared with the control group (GC), we observed more complex topography and Surface micropore formation; further, the most surface micropore formation was observed in the GU group relative to GL, G270 and G365.
在金属支架内部确定的EDS选择区域提示了钛合金表面的元素组成由Ti、O以及少量Al、V、C组成，如图6A～E。而且值得注意的是，如图6F～J，EDS确定的对照组(GC)与4个光功能化处理组(GL、G270、G365、GU)内表面不同化学元素(C、O、AL、Ti、V)的原子质量百分比，相对于对照组(GC)，光功能化处理过的4组(GL、G270、G365、GU)的碳(元素C)含量减少，且p<0.05，有明显统计学差异(图6F)；GC与GU组有明显统计学差异(p<0.01)；且GL、G270、G365与GU组相比，GU组的碳含量最少，有明显统计学差异(p<0.05)；而其他元素如Ti、O、Al|、V均无明显差异(p>0.05)。The EDS selection area determined inside the metal stent suggests that the elemental composition of the titanium alloy surface consists of Ti, O and a small amount of Al, V, and C, as shown in Figure 6A-E. And it is worth noting that, as shown in Figure 6F~J, the control group (GC) determined by EDS and the four photofunctionalized treatment groups (GL, G270, G365, GU) with different chemical elements (C, O, AL, Ti) on the inner surface , V), compared with the control group (GC), the carbon (element C) content of the four photofunctionalized groups (GL, G270, G365, GU) decreased, and p<0.05, there was a significant statistical Compared with GL, G270, G365 and GU group, the carbon content of GU group was the least, and there was a significant statistical difference (p<0.05 ); while other elements such as Ti, O, Al|, V had no significant difference (p>0.05).
总体而言，经过紫外线光功能化处理的实验组，出现了表面形貌变化和C元素的减少；且GU组的碳含量最少、表面形貌变化最大，另外图6G还显示了使用本发明的照射仪所进行的单波段照射G365在假体内部形成了相对于GL的显著优势，多角度照射对于假体内部孔径表面的光功能化有明显益处。In general, the experimental group treated with ultraviolet light functionalization showed changes in surface morphology and a reduction in C elements; and the GU group had the least carbon content and the largest change in surface morphology. In addition, Figure 6G also shows The single-band irradiation G365 performed by the illuminator has a significant advantage over GL inside the prosthesis, and the multi-angle irradiation has obvious benefits for the photofunctionalization of the internal aperture surface of the prosthesis.
2.5.3 XPS分析钛合金表面的化学组成2.5.3 XPS analysis of the chemical composition of the titanium alloy surface
图7显示经XPS检测后紫外线光功能化处理前后的化学组成以及状态。根据全谱及分峰拟合的结果，主要表面的元素为O、Ti、C。Figure 7 shows the chemical composition and state before and after ultraviolet light functionalization treatment after XPS detection. According to the results of full spectrum and peak fitting, the main surface elements are O, Ti, and C.
对5组中的C元素和O元素进行分析，可以得到两种元素所处的化学环境及不同化学环境的元素所占的相对比例如表所示。对C元素的窄扫谱进行分峰拟合(如图7B、7E、7H、7K、7N)可以得到三个峰：C-C峰对应结合能为284.82eV，C-O峰对应结合能为286.41eV，C＝O峰对应结合能为288.41eV。不同样品对应的峰位会略有差异。不同样品中三种化学环境的C元素的相对比例如表3所示。对O元素的窄扫谱进行分峰拟合(如图7C、7F、7I、7L、7O)可以得到三个峰：O-Ti(TiO2)峰对应的结合能为529.99eV，O-C峰对应的结合能为531.89eV，O＝C峰对应的结合能为532.99eV。不同样品对应的峰位会略有差异。5组样品中三种化学环境的O元素的相对比例如表4所示。By analyzing the C elements and O elements in the five groups, the chemical environment of the two elements and the relative proportions of elements in different chemical environments can be obtained as shown in the table. The narrow-scan spectrum of C element is fitted with peaks (as shown in Figures 7B, 7E, 7H, 7K, and 7N) to obtain three peaks: the C-C peak corresponds to a binding energy of 284.82eV, the C-O peak corresponds to a binding energy of 286.41eV, and the C-O peak corresponds to a binding energy of 286.41eV. The =0 peak corresponds to a binding energy of 288.41 eV. The peak positions corresponding to different samples will be slightly different. The relative proportions of C elements in the three chemical environments in different samples are shown in Table 3. The narrow-scan spectrum of the O element is fitted with peaks (as shown in Figures 7C, 7F, 7I, 7L, and 7O) to obtain three peaks: the binding energy corresponding to the O-Ti(TiO2) peak is 529.99eV, and the O-C peak corresponds to The binding energy is 531.89eV, and the binding energy corresponding to the O=C peak is 532.99eV. The peak positions corresponding to different samples will be slightly different. The relative proportions of O elements in the three chemical environments in the five groups of samples are shown in Table 4.
表3：5组样本不同化学环境的C元素所占相对比例Table 3: The relative proportion of C elements in different chemical environments of the 5 groups of samples
表4：5组样本不同化学环境的O元素所占相对比例Table 4: The relative proportion of O elements in different chemical environments of the 5 groups of samples
综上所述，提示3D打印多孔钛合金表面在紫外线处理前后均由二氧化钛以及碳氧化合物组成。In summary, it is suggested that the surface of 3D printed porous titanium alloy is composed of titanium dioxide and carbon oxides before and after ultraviolet treatment.
2.5.4 XRD物相构成的测试2.5.4 Test of XRD phase composition
如图8，XRD结果显示了3D打印钛合金支架经过紫外线光功能化处理前后的物相组成。5个组均可以看到基体钛产生的多个衍射峰(1峰，即Ti6AL4V)和少量碳元素(C)的存在，但 是4个光功能化处理组(GL、G270、G365、GU)均发现了更多氧化物的形成(AL2O3)。Ti6AL4V为主相，代表的衍射峰有2θ＝35.921゜，2θ＝39.010゜，2θ＝41.166゜，2θ＝53.921゜，2θ＝64.677゜，2θ＝72.030゜，2θ＝77.923゜和2θ＝79.549゜分别对应于(200)晶面、(002)晶面、(201)晶面、(202)晶面、(220)晶面、(203)晶面、(222)晶面和(401)晶面。As shown in Figure 8, the XRD results show the phase composition of the 3D printed titanium alloy stent before and after UV photofunctionalization. Multiple diffraction peaks (1 peak, Ti6AL4V) and a small amount of carbon (C) produced by the matrix titanium can be seen in all five groups, but the four photofunctionalized treatment groups (GL, G270, G365, GU) all The formation of more oxides (AL2O3) was found. Ti6AL4V is the main phase, and the representative diffraction peaks are 2θ=35.921゜, 2θ=39.010゜, 2θ=41.166゜, 2θ=53.921゜, 2θ=64.677゜, 2θ=72.030゜, 2θ=77.923゜ and 2θ=79.549゜ respectively Corresponding to (200) crystal plane, (002) crystal plane, (201) crystal plane, (202) crystal plane, (220) crystal plane, (203) crystal plane, (222) crystal plane and (401) crystal plane.
2.5.5细胞相容性CCK8活性测试与Live-Dead荧光测试2.5.5 Cytocompatibility CCK8 activity test and Live-Dead fluorescence test
在5组3D打印金属支架上进行骨髓间充质干细胞的培养1天、3天、7天后，通过测定孔隙金属表面的细胞活性(增殖率)来评估细胞的相容性。如图9A，在共培养3天和7天两个时间点，细胞增殖率比较差别具有一致性：GU组较GC、GL、G365组有明显升高，且差异有统计学意义(P<0.01)；GU组较G270有明显升高，且差异有统计学意义(P<0.05)；GL和G270也存在明显统计学差异(P<0.05)，说明双波段紫外线处理相对于单波段处理具有显著优势。而在Live-Dead荧光测试中(图9E)，可以直观看到G270、G365、GU组相对GC和GL组在3D打印金属支架表面的活细胞数目更多，证明本发明仪器和方法提供了更加均匀的多角度照射，比现有的汞灯照射效果好。After culturing bone marrow mesenchymal stem cells on 5 groups of 3D printed metal scaffolds for 1 day, 3 days, and 7 days, the compatibility of the cells was evaluated by measuring the cell activity (proliferation rate) on the porous metal surface. As shown in Figure 9A, at the two time points of co-culture 3 days and 7 days, the difference in cell proliferation rate was consistent: the GU group was significantly higher than the GC, GL, and G365 groups, and the difference was statistically significant (P<0.01 ); GU group was significantly higher than G270, and the difference was statistically significant (P<0.05); GL and G270 also had significant statistical difference (P<0.05), indicating that dual-band UV treatment had a significant difference compared with single-band treatment. Advantage. In the Live-Dead fluorescence test (Fig. 9E), it can be directly seen that the G270, G365, and GU groups have more living cells on the surface of the 3D printed metal stent than the GC and GL groups, which proves that the instrument and method of the present invention provide more Uniform multi-angle irradiation, better than the existing mercury lamp irradiation.
2.5.6 ALP活性2.5.6 ALP activity
ALP活性测试(如图9B)显示，在7天时，5组间无明显统计学差异(P>0.05)；在14天时，GU组相对于GC和GL组具备更高的碱性磷酸酶活性，差异具备统计学意义(P＜0.01)；GU组相对于G365也具备更高的碱性磷酸酶活性，差异具备统计学意义(P＜0.05)；GL与G270组相对，有明显统计学差异(P＜0.01)；而G270与GU组无明显统计学差异(P>0.05)。The ALP activity test (as shown in Figure 9B) showed that at 7 days, there was no significant statistical difference among the five groups (P>0.05); at 14 days, the GU group had higher alkaline phosphatase activity than the GC and GL groups, The difference was statistically significant (P<0.01); the GU group also had higher alkaline phosphatase activity compared to the G365 group, the difference was statistically significant (P<0.05); the GL group was significantly statistically different from the G270 group ( P<0.01); while G270 and GU group had no significant statistical difference (P>0.05).
2.5.7细胞形态2.5.7 Cell Morphology
细胞形态学实验结果(图9D)可以直观看到，在3D打印支架上，相对于GC组，GL、G270、G365和GU组的细胞的增殖和附着更多，铺展面积更大，更多的细胞伪足的伸展；而使用全方位照射的光功能化组(G270、G365、GU)细胞在3D打印支架内部的附着更多。The results of the cell morphology experiment (Fig. 9D) can be seen visually, on the 3D printed scaffolds, compared with the GC group, the cells in the GL, G270, G365 and GU groups proliferated and attached more, had a larger spreading area, and more Stretching of cell pseudopodia; while cells in photofunctionalized groups (G270, G365, GU) with omnidirectional irradiation had more attachment inside the 3D printed scaffold.
支架细胞共培养的SEM结果显示光功能化处理组(GL、G270、G365、GU)在假体表面出现更多的胶原纤维附着，如图9C。值得注意的是，在10000倍电子显微镜下，GU组观察到大量的胶原纤维附着(图10)。The SEM results of scaffold cell co-culture showed that photofunctionalized treatment groups (GL, G270, G365, GU) had more collagen fibers attached to the surface of the prosthesis, as shown in Figure 9C. It is worth noting that under a 10,000X electron microscope, a large number of collagen fibers were observed in the GU group (Fig. 10).
2.5.8紫外线光功能化对于多孔钛合金支架力学特性的影响2.5.8 The effect of ultraviolet light functionalization on the mechanical properties of porous titanium alloy scaffolds
图11显示了紫外线光功能化处理前后的力学特性的表现，通过压缩测试，在屈服强度(图11A)、弹性模量(图11B)、压缩强度(图11C)进行比较，GC、GL、G270、G365、GU)5组间均没有明显统计学差异(p＞0.05)；应力应变曲线(图11D)5组也基本一致。Figure 11 shows the performance of the mechanical properties before and after the UV light functionalization treatment, through the compression test, the yield strength (Figure 11A), the elastic modulus (Figure 11B), the compressive strength (Figure 11C) are compared, GC, GL, G270 , G365, GU) There was no significant statistical difference among the 5 groups (p>0.05); the stress-strain curve (Fig. 11D) was basically the same among the 5 groups.
2.5.9骨缺损修复实验结果2.5.9 Experimental results of bone defect repair
所有实验动物在术中未出现异常，术后恢复情况良好，无明显严重并发症出现，实施麻醉安乐死后，切开手术部位，大体观察见局部无炎症的迹象，手术部位无感染及植入物脱出等。All experimental animals had no abnormalities during the operation, recovered well after the operation, and had no obvious serious complications. After euthanasia with anesthesia, the surgical site was incised, and there were no signs of local inflammation, and there was no infection or implant at the surgical site Escape etc.
2.5.10 Micro-CT分析2.5.10 Micro-CT analysis
其中3D打印的钛合金支架为白色，骨组织为绿色。根据图12A～E直观可见，光功能化处理组(GU)相对于对照组(GC)有更多的骨组织长入，几乎长满了假体的内部孔隙。如图12F，GU的骨体积分数(骨长入率BIR)，GC组、GL组、G270组、G365组、GU组分别为(43.33±5.08)％、(61.03±3.44)％、(74.44±1.90)％、(66.16±1.52)％、(75.53±1.40)％，GC与光功能化处理组(GL、G270、G365、GU)、GL与GU、G365和GU之间有明显统计学差异(P<0.05)。相比于汞灯照射，本发明的仪器的单波段照射实现了更好的效果，双波段紫外线比单波段紫外线照射效果显著更好。Among them, the 3D printed titanium alloy scaffold is white, and the bone tissue is green. According to Fig. 12A-E, it can be seen intuitively that the photofunctionalized treatment group (GU) has more bone tissue ingrowth than the control group (GC), almost covering the internal pores of the prosthesis. As shown in Figure 12F, the bone volume fraction (bone ingrowth rate BIR) of GU, GC group, GL group, G270 group, G365 group, GU group were (43.33±5.08)%, (61.03±3.44)%, (74.44± 1.90)%, (66.16±1.52)%, (75.53±1.40)%, there were significant statistical differences between GC and photofunctionalized treatment group (GL, G270, G365, GU), GL and GU, G365 and GU ( P<0.05). Compared with mercury lamp irradiation, the single-band irradiation of the instrument of the present invention achieves a better effect, and the dual-band ultraviolet irradiation effect is significantly better than the single-band ultraviolet irradiation.
2.5.11组织学分析2.5.11 Histological analysis
我们采用Goldner三色染色对多孔支架的骨长入进行组织学分析，染色后在光镜下多孔支架的支柱是黑色的，而矿化的骨小梁绿色的，类骨质为黄色。We used Goldner's trichrome staining to analyze the bone ingrowth of the porous scaffold histologically. After staining, under the light microscope, the pillars of the porous scaffold were black, while the mineralized bone trabeculae were green, and the osteoid was yellow.
植入兔股骨髁8周后，GC和GL两组仅在周边有骨长入，并且生成的骨组织与多孔支架没有紧密的接触；相反，在G270、G365和GU组的多孔钛合金支架上可以发现更广泛的骨长入，新生骨组织与钛合金支架紧密结合，并且矿化形成骨质，如图13A。Eight weeks after implantation into the rabbit femoral condyle, the GC and GL groups only had bone ingrowth in the periphery, and the generated bone tissue was not in close contact with the porous scaffold; on the contrary, the porous titanium alloy scaffolds of the G270, G365 and GU groups It can be found that more extensive bone ingrowth, the new bone tissue is tightly combined with the titanium alloy scaffold, and mineralized to form bone, as shown in Figure 13A.
骨长入的定量分析结果，如图13B和13C，分别比较了5组在植入8周后的骨长入(BI)、骨/假体接触率(BICR)，类骨质和矿化骨比例。The results of quantitative analysis of bone ingrowth, as shown in Figure 13B and 13C, compared the bone ingrowth (BI), bone/prosthesis contact rate (BICR), osteoid and mineralized bone in the 5 groups after 8 weeks of implantation. Proportion.
5组(GC、GL、G270、G365、GU)的BI分别为(14.56±1.15)％、(10.58±0.91)％、(24.7±1.74)％、(19.22±3.26)％、(25.04±1.29)％，GC、GL与GU组有明显统计学差异(P<0.01)；G270与GU无明显统计学差异(P>0.05)；G365与GU组有明显统计学差异(P<0.05)，如图13B。The BI of the five groups (GC, GL, G270, G365, GU) were (14.56±1.15)%, (10.58±0.91)%, (24.7±1.74)%, (19.22±3.26)%, (25.04±1.29)% respectively %, GC, GL and GU group had significant statistical difference (P<0.01); G270 and GU had no significant statistical difference (P>0.05); G365 and GU group had significant statistical difference (P<0.05), as shown in 13B.
5组(GC、GL、G270、G365、GU)的BICR分别为(15.14±0.67)％、(10.26±1.00)％、(32.04±1.12)％、(18.56±1.65)％、(41.66±1.64)％，GC、GL与GU组有明显统计学差异(P<0.01)；G270与GU有明显统计学差异(P<0.05)；G365与GU组有明显统计学差异(P<0.01)，如图13B。The BICR of the five groups (GC, GL, G270, G365, GU) were (15.14±0.67)%, (10.26±1.00)%, (32.04±1.12)%, (18.56±1.65)%, (41.66±1.64) %, GC, GL and GU groups have significant statistical differences (P<0.01); G270 and GU have significant statistical differences (P<0.05); G365 and GU groups have significant statistical differences (P<0.01), as shown in the figure 13B.
5组(GC、GL、G270、G365、GU)的类骨质比例分别为(55.08±4.06)％、(63.3±10.85)％、(29.42±4.02)％、(40.94±8.61)％、(20.62±1.59)％，GC、GL与GU组有明显统计学差异(P<0.01)；G270与GU无明显统计学差异(P>0.05)；G365与GU组有明显统计学差异(P<0.05)，如图13C。The proportions of osteoid in the five groups (GC, GL, G270, G365, GU) were (55.08±4.06)%, (63.3±10.85)%, (29.42±4.02)%, (40.94±8.61)%, (20.62 ±1.59)%, there was significant statistical difference between GC, GL and GU group (P<0.01); there was no significant statistical difference between G270 and GU (P>0.05); there was significant statistical difference between G365 and GU group (P<0.05) , as shown in Figure 13C.
5组(GC、GL、G270、G365、GU)的矿化骨比例分别为(44.68±4.64)％、(35.02±10.41)％、(70.58±4.02)％、(59.06±8.61)％、(79.38±1.59)％，GC、GL与GU组有明显统计学差异(P<0.01)；G270与GU无明显统计学差异(P>0.05)；G365与GU组有明显统计学差异(P<0.05)，如图13C。The proportions of mineralized bone in the 5 groups (GC, GL, G270, G365, GU) were (44.68±4.64)%, (35.02±10.41)%, (70.58±4.02)%, (59.06±8.61)%, (79.38 ±1.59)%, there was significant statistical difference between GC, GL and GU group (P<0.01); there was no significant statistical difference between G270 and GU (P>0.05); there was significant statistical difference between G365 and GU group (P<0.05) , as shown in Figure 13C.
相比于汞灯照射，本发明的仪器的单波段照射实现了更好的效果，双波段紫外线比单波段紫外线照射效果显著更好。Compared with mercury lamp irradiation, the single-band irradiation of the instrument of the present invention achieves a better effect, and the dual-band ultraviolet irradiation effect is significantly better than the single-band ultraviolet irradiation.
2.5.12骨荧光标记2.5.12 Bone fluorescent labeling
体内的序贯荧光标记显示是否经过紫外线光功能化处理的假体植入后呈现随不同时间点表现出的不同的骨整合模式。Sequential fluorescent labeling in vivo revealed different osseointegration patterns over time points after implantation of prostheses functionalized with or without UV light.
图13D中，钛合金假体颜色为白色，绿色荧光为3周的成骨，而红色代表8周的成骨。根据荧光标记可以直观的看到，在钛合金假体内部，G270、G365和GU组中，3周的成骨更多见。而GC和GL组的呈现更多的是8周的成骨。In Fig. 13D, the color of the titanium alloy prosthesis is white, the green fluorescence represents the 3-week osteogenesis, and the red represents the 8-week osteogenesis. According to the fluorescent labeling, it can be seen intuitively that in the titanium alloy prosthesis, the osteogenesis at 3 weeks was more common in the G270, G365 and GU groups. The GC and GL groups showed more osteogenesis at 8 weeks.
2.5.13推出实验2.5.13 Launch experiment
图14显示对照组GC与实验组GU在兔股骨髁假体植入术后8周进行推出实验的结果。如图14A，GC、GL、G270、G365、GU 5组的最大推出力分别为：250.3±16.6、271.7±19.9、391.2±16.7、301.7±10.2、450.5±18.5；GC、GL、G365相比GU组有明显统计学差异(P<0.01)；G270与GU组有明显统计学差异(P<0.05)；GL与G270组有明显统计学差异(P<0.01)；如图14B，轴向推出力与位移曲线显示，G270和GU组相对于GC、GL、G365组，为了产生相同的位移，假体需要更大的推出力。Figure 14 shows the results of the push-out experiment of the control group GC and the experimental group GU at 8 weeks after implantation of the rabbit femoral condyle prosthesis. As shown in Figure 14A, the maximum pushing forces of GC, GL, G270, G365, and GU groups were: 250.3±16.6, 271.7±19.9, 391.2±16.7, 301.7±10.2, 450.5±18.5; GC, GL, G365 compared with GU There was a significant statistical difference between G270 and G270 groups (P<0.01); there was a significant statistical difference between G270 and GU group (P<0.05); there was a significant statistical difference between GL and G270 group (P<0.01); as shown in Figure 14B, the axial pushing force Compared with the GC, GL, and G365 groups, the G270 and GU groups need a larger push-out force in order to produce the same displacement.
根据以上实施例可知，紫外线C(270nm波段)和紫外线A(365nm波段)紫外线对钛合 金假体的光功能化各自具有不同方向的独特效果，通过使用双波段的紫外线处理可以获得相对于单独波段处理的显著效果；本发明的光功能化仪器能够通过多个紫外线光源实现对钛合金表面的均匀处理，效果远超现有的汞灯；光功能化仪器内单波段多角度的紫外线光源和均匀的功率可以对内部有孔的结构实现外部和内部光功能化处理，效果远超现有的汞灯。According to the above examples, it can be seen that ultraviolet C (270nm band) and ultraviolet A (365nm band) ultraviolet rays have unique effects in different directions on the photofunctionalization of titanium alloy prostheses, and can be obtained by using dual-band ultraviolet treatment. Remarkable effect of treatment; the optical functionalized instrument of the present invention can realize uniform treatment of the titanium alloy surface through multiple ultraviolet light sources, and the effect is far superior to the existing mercury lamp; the single-band multi-angle ultraviolet light source in the optical functionalized instrument and uniform The high power can realize the external and internal light functionalization of the structure with holes inside, and the effect is far superior to the existing mercury lamp.
实施例3Example 3
使用多孔钛合金假体，其他实验方法如前所述，本发明人还进行了不同照射方法和时间的处理实验对比，每组使用三个假体，第一组，使用270nm紫外线波段照射处理30分钟；第二组，使用365nm紫外线波段照射处理30分钟；第三组，使用270nm紫外线波段和365nm紫外线波段依次交替照射1分钟，一共持续照射30分钟；第四组，使用270nm紫外线波段和365nm紫外线波段依次交替照射5分钟一共持续照射30分钟；第五组，使用270nm紫外线波段和365nm紫外线波段同时照射15分钟；第六组，使用270nm紫外线波段照射处理60分钟；第二组，使用365nm紫外线波段照射处理60分钟；Using a porous titanium alloy prosthesis, other experimental methods are as described above, the inventors have also carried out a treatment experiment comparison of different irradiation methods and time, each group uses three prostheses, the first group, using 270nm ultraviolet band irradiation treatment for 30 minutes; the second group, irradiated with 365nm ultraviolet band for 30 minutes; the third group, alternately irradiated with 270nm ultraviolet band and 365nm ultraviolet band for 1 minute, and continued to irradiate for 30 minutes in total; the fourth group, used 270nm ultraviolet band and 365nm ultraviolet band The bands were irradiated alternately for 5 minutes, a total of 30 minutes of continuous irradiation; the fifth group, irradiated with 270nm ultraviolet band and 365nm ultraviolet band for 15 minutes at the same time; the sixth group, irradiated with 270nm ultraviolet band for 60 minutes; the second group, used 365nm ultraviolet band Irradiation treatment for 60 minutes;
扫描电子显微镜观察对比五组的处理结果，第三组、第四组、第五组之间差别不大，未见明显差别，第三组、第四组、第五组与第一组和第二组之间均有明显差别，第六组和第一组之间几乎没有差别，第七组和第二之间几乎没有差别，实验数据未示出。Scanning electron microscope observed and compared the treatment results of the five groups. There was little difference between the third group, the fourth group, and the fifth group, and no significant difference was found. There are obvious differences between the two groups, there is almost no difference between the sixth group and the first group, there is almost no difference between the seventh group and the second group, and the experimental data is not shown.
通过实施例3可以知晓，使用两个波段的紫外线的照射时间如何设置对于结果没有显著影响，即，UVA和UVC同时照射、交替照射、甚至先后照射对改性的影响很小；另外通过结合单波段15分钟处理结果，对比处理30分钟和60分钟的假体，发现15分钟之后，单一波段的处理已经基本不再继续产生影响，假体的表面形态基本不再变化。It can be known from Example 3 that how to set the irradiation time of ultraviolet rays using two wavebands has no significant impact on the results, that is, simultaneous irradiation of UVA and UVC, alternate irradiation, or even sequential irradiation have little effect on modification; The results of the 15-minute processing of the wave band, compared the prosthesis processed for 30 minutes and 60 minutes, and found that after 15 minutes, the single-band processing basically no longer continues to have an impact, and the surface shape of the prosthesis basically does not change.
实施例4Example 4
使用多孔钛合金假体，其他实验方法如前所述，本发明人还进行了UVA和UVC波段内不同波长的处理实验对比，每组使用3个假体，第1组，使用200±20nm紫外线波段和365±20nm紫外线波段同时照射15分钟；第2组，使用240±20nm紫外线波段和365±20nm紫外线同时照射15分钟；第3组，使用270±20nm紫外线波段和365±20nm紫外线波段同时照射15分钟；第4组，使用270±20nm紫外线波段和340±20nm紫外线波段同时照射15分钟；第5组，使用270±20nm紫外线波段和380±20nm紫外线波段同时照射15分钟；Using a porous titanium alloy prosthesis, other experimental methods are as described above, the inventor also carried out a comparison of treatment experiments with different wavelengths in the UVA and UVC bands, using 3 prostheses in each group, the first group, using 200 ± 20nm ultraviolet rays UV band and 365±20nm UV band were irradiated for 15 minutes at the same time; Group 2 was irradiated with 240±20nm UV band and 365±20nm UV band for 15 minutes at the same time; Group 3 was irradiated with 270±20nm UV band and 365±20nm UV band simultaneously 15 minutes; Group 4, irradiated with 270±20nm ultraviolet band and 340±20nm ultraviolet band for 15 minutes at the same time; Group 5, irradiated with 270±20nm ultraviolet band and 380±20nm ultraviolet band for 15 minutes at the same time;
扫描电子显微镜观察对比3组的处理结果，组之间未见明显差异，说明在波段之内的紫外线有类似的效果，可以根据需求选择该波段之内的合适波长。Scanning electron microscope observed and compared the treatment results of the three groups, and there was no significant difference between the groups, indicating that ultraviolet rays within the wavelength band have similar effects, and appropriate wavelengths within the wavelength range can be selected according to requirements.
显然，本领域的技术人员可以对本发明进行各种改动和变型而不脱离本发明的精神和范围。这样，倘若本发明的这些修改和变型属于本发明权利要求及其等同技术的范围之内，则本发明也意图包含这些改动和变型在内。Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and equivalent technologies thereof, the present invention also intends to include these modifications and variations.
Claims (10)
- 一种对钛合金结构件进行改性的方法，其特征在于，使用紫外线光源从至少两个不同角度对所述钛合金结构件进行照射，所述钛合金结构件内部含有孔。A method for modifying a titanium alloy structural part, characterized in that an ultraviolet light source is used to irradiate the titanium alloy structural part from at least two different angles, and the titanium alloy structural part contains holes inside.
- 一种对钛合金结构件进行改性的方法，其特征在于，使用至少两种不同波段的紫外线照射钛合金结构件。A method for modifying a titanium alloy structural part is characterized in that the titanium alloy structural part is irradiated with ultraviolet rays of at least two different wavebands.
- 如权利要求3所述的方法，其特征在于，所述紫外线中的至少一种为320-400纳米的紫外线A，并且另一种紫外线为10-290纳米的紫外线C。The method of claim 3, wherein at least one of the ultraviolet rays is ultraviolet A at 320-400 nm, and the other ultraviolet light is ultraviolet C at 10-290 nm.
- 如权利要求1或2所述的方法，其特征在于，所述照射持续至少1秒。The method of claim 1 or 2, wherein said irradiating lasts at least 1 second.
- 钛合金结构件，其特征在于，经过权利要求1或2所述的方法进行改性。The titanium alloy structural part is characterized in that it is modified by the method described in claim 1 or 2.
- 光功能化仪器，其特征在于，包括：The photofunctionalization instrument is characterized in that, comprising:壳体，所述壳体设置有容纳待处理钛合金结构件的腔体；A casing, the casing is provided with a cavity for accommodating titanium alloy structural parts to be processed;紫外线光源，所述紫外线光源设置在所述壳体的内壁，能够从至少两个角度发射紫外线；an ultraviolet light source, the ultraviolet light source is arranged on the inner wall of the housing and can emit ultraviolet rays from at least two angles;控制面板，所述控制面板用于设置和显示紫外线的波长和处理时间。A control panel for setting and displaying the wavelength and treatment time of the ultraviolet rays.
- 如权利要求6所述的光功能化仪器，其特征在于，所述紫外线光源包括至少两个紫外线光源。The photofunctionalization apparatus of claim 6, wherein the ultraviolet light source comprises at least two ultraviolet light sources.
- 如权利要求6所述的光功能化仪器，其特征在于，所述紫外线光源包括能够发射至少两种不同波长紫外光的紫外灯。The photofunctionalization instrument according to claim 6, wherein the ultraviolet light source comprises an ultraviolet lamp capable of emitting ultraviolet light of at least two different wavelengths.
- 如权利要求6所述的光功能化仪器，其特征在于，所述控制面板包括控制器和显示器。The light functionalization apparatus of claim 6, wherein the control panel includes a controller and a display.
- 钛合金结构件，其特征在于，经过权利要求6至9中任一项所述的光功能化仪器的处理。The titanium alloy structural part is characterized in that it has been processed by the photofunctionalization instrument according to any one of claims 6 to 9.
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| US20090283701A1 (en) * | 2005-03-07 | 2009-11-19 | Takahiro Ogawa | Medical Implants |
| US20080058921A1 (en) * | 2006-08-09 | 2008-03-06 | Lindquist Jeffrey S | Improved adhesion of a polymeric coating of a drug eluting stent |
| CN102245221A (en) * | 2008-11-25 | 2011-11-16 | 加州大学董事会 | Functionalized titanium implants and related regenerative materials |
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