A time-gradient controlled bone implant device coating with anti-infection-cell recruitment-osteogenesis functions and its preparation method

Abstract

This invention discloses a time-gradient regulated coating for bone implants with anti-infection, cell recruitment, and osteogenic functions, and its preparation method. The coating comprises, from the inside out, a microporous base layer, a hydroxyapatite / silver nanolayer, and a composite gel layer. The preparation process is as follows: 1) A microporous base layer is formed on the surface of the bone implant; 2) A hydroxyapatite / silver nanolayer is sprayed onto the microporous base layer; 3) Chitosan is dual-modified using methacrylic anhydride and aldehyde-containing bisphosphonates to obtain ChiMA-BP; 4) A ChiMA-BP gel layer is prepared on the surface of the hydroxyapatite / silver nanolayer; 5) The above materials are sequentially immersed in a bone-promoting metal ion solution and a solution containing peptides with stem cell recruitment functions to achieve chelation and loading of metal ions and peptides, resulting in the composite gel layer. This bone implant simultaneously possesses anti-infection, cell recruitment, and osteogenic functions, and each function has its own time-dependent gradient, achieving a synergistic effect in bone defect repair.

Description

Technical Field

[0001] This invention relates to the field of bone implant device fabrication, specifically to a time-gradient regulated bone implant device coating with anti-infection, cell recruitment, and osteogenic functions, and its preparation method. Background Technology

[0002] Currently, the main treatment for traumatic fractures involves using bone implants for fixation or to assist in recovery. However, the high incidence of infection and slow fracture healing rates during treatment severely impact postoperative recovery. Patients with severe infections may require surgical debridement, secondary surgery, or even amputation, placing a significant psychological and financial burden on them. Furthermore, the recovery period for fractures typically ranges from several months to several years, which, in today's fast-paced society, has a considerable impact on patients' lives.

[0003] Currently, many research institutions and medical device companies are developing bone implants with anti-infection functions, and some studies have reported the successful introduction of coatings that promote bone formation onto the surface of medical devices. However, the situation after a fracture in the human body is extremely complex, and single-function bone implants may be insufficient. Moreover, the human body has a time-controlled order for injury repair; simply adding the required functions to the implant may disrupt this internal repair process. Therefore, a bone implant that simultaneously possesses infection prevention and bone-promoting functions, with each function operating independently, urgently needs to be developed. Summary of the Invention

[0004] The purpose of this invention is to address the problems of high incidence of postoperative infection and low fracture healing rate by providing a time-gradient-regulated bone implant device with anti-infection, cell recruitment, and osteogenic functions, as well as its preparation method. To address the aforementioned problems, this invention combines sandblasting, acid etching, plasma spraying, dual chemical grafting, ion chelation, impregnation adsorption, and in-situ ultraviolet curing processes to sequentially introduce a microporous substrate layer, a hydroxyapatite / nanosilver layer, and a composite gel layer structure onto the surface of a titanium / titanium alloy, thereby obtaining a time-gradient-regulated bone implant device with anti-infection, cell recruitment, and osteogenic functions. This device can continuously release silver ions to combat infection and sequentially recruit cells, promoting osteogenic differentiation and ultimately improving bone healing efficiency, achieving the goal of high-quality and efficient fracture treatment.

[0005] This invention is achieved using the following technical solution:

[0006] A time-gradient regulated bone implant coating with anti-infection, cell recruitment, and osteogenic functions is disclosed. The coating comprises, from the inside out, a microporous base layer, a hydroxyapatite / nanosilver layer, and a composite gel layer. The coating is prepared by first using a sandblasting and acid etching process to obtain a two-level microporous structure, then using a plasma spraying process to introduce the hydroxyapatite / nanosilver layer, followed by a cyclic in-situ ultraviolet curing process to introduce a gel coating on its surface, and finally using an impregnation method to achieve metal ion chelation and loading of active peptides, thus obtaining a time-gradient regulated bone implant coating with anti-infection, cell recruitment, and osteogenic functions.

[0007] The specific preparation steps of the time-gradient-regulated bone implant coating with anti-infection, cell recruitment, and osteogenic functions are as follows:

[0008] 1) First, the surface of the bone implant device is polished, and then a sandblasting process is used to form a primary pore structure with a diameter of about 20-30 micrometers on the surface of the bone implant device. Then, an acid etching process is used to form a secondary pore structure with a diameter of about 1-4 micrometers to obtain a microporous base layer; the surface material of the bone implant device is titanium / titanium alloy.

[0009] 2) Hydroxyapatite and nano-silver are mixed in a specific ratio to form a powder with a particle size of about 40-80 micrometers. A hydroxyapatite / nano-silver layer with a thickness of about 100-300 micrometers is deposited on the surface of a microporous substrate using a plasma spraying process.

[0010] 3) Methacrylic anhydride was used to graft chitosan molecules to obtain methacrylic anhydride-modified chitosan (ChiMA); then bisphosphonates with aldehyde groups were used to graft ChiMA to obtain ChiMA-BP.

[0011] 4) Under the conditions of water bath at 50-70℃ and protection from light, dissolve ChiMA-BP in PBS solution containing photoinitiator, ultrasonically impregnate the bone implant device with hydroxyapatite / nanosilver layer deposited in step 2) in the above ChiMA-BP solution, remove it and perform rotation-in-situ UV curing. Repeat the above impregnation-curing process multiple times until the gel layer completely covers the hydroxyapatite / nanosilver layer.

[0012] 5) The sample obtained in step 4) is sequentially immersed in a solution containing osteogenic metal ions and a solution containing peptides with stem cell recruitment function to achieve chelation of metal ions with ChiMA-BP and loading of peptides to obtain a composite gel layer; after irradiation sterilization, a time gradient-regulated bone implant device with anti-infection-cell recruitment-osteogenic function is finally obtained.

[0013] In the above technical solution, further, in step 1), the grinding process involves sequentially grinding the surface of the bone implant device with 400#, 800#, and 1200# silicon carbide sandpaper; the sandblasting process involves sandblasting with 200-350 micrometer aluminum oxide particles at a pressure of 0.3-0.5 MPa, a sandblasting time of 30-60 seconds, and a spray angle of 70-80°; after sandblasting, ultrasonic cleaning is performed sequentially with ethanol and deionized water.

[0014] The process involves one ethanol cleaning followed by two deionized water cleanings, each lasting 15 minutes. The acid etching process consists of first etching with 0.3-0.6 wt% hydrofluoric acid for 30-60 minutes, then etching with a 10-15 wt% hydrochloric acid / 35-55 wt% sulfuric acid aqueous solution (using deionized water) at 60-70°C for 60-120 minutes. Finally, the surface is thoroughly washed with a large amount of deionized water and dried for later use. The micro / nano structures prepared in this process, by increasing the mechanical interlocking area, facilitate high-strength bonding of subsequent coatings.

[0015] Further, in step 2), the mass ratio of hydroxyapatite to nano-silver is between 90:10 and 95:5, wherein the diameter of hydroxyapatite is 40-80 micrometers; the particle size of nano-silver is approximately 60-90 nanometers; the parameters of the plasma spraying process are as follows: the plasma gas is a hydrogen / oxygen mixture containing argon, with a volume ratio of 15-35% argon, 15-20% hydrogen, and the remainder being oxygen; the gas flow rate is controlled at 40-80 liters / minute; the spraying distance is 10-25 centimeters; the spraying power is 40-70 watts; and the powder feeding rate is 10-20 grams / minute.

[0016] Further, in step 3), the ChiMA synthesis process is as follows: Chitosan with a viscosity-average molecular weight of 600,000-1,200,000 and a degree of deacetylation of 70-80% is dissolved in 1-2 wt% acetic acid water under a 60°C water bath and magnetic stirring at 300-500 rpm to obtain 3-6 wt% chitosan. Then, 5-10 wt% methacrylic anhydride is added dropwise. After the addition is complete, the reaction continues for 2-3 hours. After the reaction is complete, an equal volume of PBS preheated to 60°C is added, and the reaction continues for 15-25 minutes. Dialysis is performed using deionized water and a dialysis bag with a molecular weight of 8000-14000 for 5-7 days. The precipitate is removed by centrifugation at 8000-10000 rpm, and the ChiMA is obtained by freeze-drying. The ChiMA-BP synthesis process is as follows: a 5-15 wt% ChiMA solution is prepared under light-protected and 50°C water bath conditions, and then 0.5-1 wt% bisphosphonate BP-CHO with aldehyde groups is added. After reacting for 12-24 hours under magnetic stirring at 300-500 rpm, the mixture is dialyzed for 3-5 days using deionized water and a dialysis bag with a molecular weight of 8000-14000. The precipitate is removed by centrifugation at 8000-10000 rpm, and the ChiMA-BP is obtained by freeze-drying.

[0017] Further, in step 4), the concentration of ChiMA-BP is 5-10 wt%; during rotation-in-situ UV curing, the photoinitiator used is one or more of LAP, I2959, Irgacure, etc., with a concentration of 0.25-2.5 wt%; the bone implant device with the deposited hydroxyapatite / nanosilver layer is immersed in the ChiMA-BP solution for 3-5 minutes, and the ultrasonic power is 80-100 watts; the rotation-UV curing process parameters are: rotation speed of 15 rpm, wavelength of 405 nm, and curing time of 5-15 minutes. The immersion-curing process is repeated 5-10 times. The roughness generated by spraying the hydroxyapatite / nanosilver layer serves as the physical binding site for gel layer curing, and the immersion-rotation-UV curing process and the cyclic immersion-curing process are cleverly designed to achieve good coating of the device surface and good formation of the gel layer.

[0018] Further, in step 5), the bone-promoting metal ions are one or more of magnesium ions, zinc ions, calcium ions, and copper ions, with a concentration of 30-70 mg / mL, an immersion time of 30-80 minutes, and rinsing with deionized water 5-10 times after immersion; the polypeptides with stem cell recruitment function are one or more of DOPA-E7, DOPA-Y5, LL-37, etc., with a concentration of 5-20 mg / mL, and an immersion time of 15-30 minutes.

[0019] A time-gradient regulated bone implant device with anti-infection, cell recruitment, and osteogenic functions, wherein the surface of the bone implant device is coated as described above; the bone implant device continuously releases silver ions for infection prevention and control, and simultaneously releases polypeptides with stem cell recruitment functions in a time gradient to recruit stem cells, dechelates and releases metal ions to achieve the first stage of bone formation, ChiMA-BP degradation further releases metal ions and BP to achieve the second stage of bone formation, and after ChiMA-BP degradation is completed, hydroxyapatite is exposed to achieve the third stage of bone formation.

[0020] Compared with the prior art, the present invention has the following advantages:

[0021] 1) The bone implant device coating in this invention uses titanium / titanium alloy as the base and hydroxyapatite, nano silver, methacrylic anhydride, aldehyde-containing bisphosphonates, chitosan, bone-promoting metal ions, and peptides with stem cell recruitment function as raw materials. It is prepared by a combination of processes such as sandblasting, acid etching, plasma spraying, double chemical grafting, ion chelation, impregnation adsorption, and in-situ ultraviolet curing. The above processes and raw materials are selected by this invention according to actual needs and are original to this invention.

[0022] 2) The bone implant device coating of this invention comprises, from the inside out, a microporous base layer, a hydroxyapatite / nanosilver layer, and a composite gel layer. The composite gel layer utilizes double bonds and metal ion chelation to form a double network structure, and further loads active peptides within the gel matrix. The microporous base layer increases surface roughness, which is beneficial for improving the adhesion of the subsequent coating. The hydroxyapatite / nanosilver layer allows the nanosilver to continuously release silver ions, thus achieving a long-lasting antibacterial / anti-infection effect, while the presence of hydroxyapatite enhances the subsequent osteogenic effect. The introduction of the composite gel layer, on the one hand, encapsulates the nanosilver, slowing down the release rate of silver ions and prolonging the antibacterial / anti-infection period; on the other hand, the gel layer can act as a carrier to load metal ions with bone-promoting function and peptides that recruit stem cells, and also has a sustained-release effect on metal ions and peptides. The structure of the above materials is original to this invention.

[0023] 3) The bone implant device coating of this invention simultaneously possesses multiple functions, including long-term antibacterial / anti-infection, stem cell recruitment, triple osteogenic effect, bone ingrowth, and high biocompatibility. These functions work synergistically to promote fracture recovery: the gel layer, made using ChiMA-BP molecules, exhibits high biocompatibility; the nano-silver in the bone implant device coating continuously releases silver ions, providing antibacterial / anti-infection effects throughout the process; the polypeptides in the gel layer are slowly released through the sustained-release effect provided by the dual-network structure, recruiting stem cells in the early stages of fracture; the metal ions in the gel layer are released through dechelation and degradation of ChiMA-BP molecules, with the metal ions released during dechelation playing a first osteogenic role; the degradation of ChiMA-BP releases metal ions and bisphosphonates, playing a second osteogenic role; and when ChiMA-BP is fully degraded and hydroxyapatite is exposed, hydroxyapatite plays a third osteogenic role and promotes bone ingrowth.

[0024] 4) The bone implant coating of this invention possesses multiple functions, including long-term antibacterial / anti-infection, stem cell recruitment, triple osteogenic enhancement, and bone ingrowth. Its action is time-gradient regulated. The long-term antibacterial / anti-infection effect is continuous, providing ongoing infection prevention and control. Stem cell recruitment occurs in the early stages of fracture recovery, attracting a large number of stem cell sources for later osteogenic differentiation. Triple osteogenic enhancement occurs sequentially through the dechelation of metal ions, the release of metal ions and bisphosphonates during ChiMA-BP degradation, and the exposure of hydroxyapatite after degradation, promoting osteogenic differentiation of stem cells from multiple aspects. Furthermore, after gel degradation, the exposed hydroxyapatite also promotes bone ingrowth, enabling cell ingrowth onto the device surface, resulting in better fracture fixation, reduced device loosening, and improved fracture repair. Through the selection of materials and structural design, the organic synergy and time-gradient efficacy of these multiple functions are ultimately achieved, making this invention unique. Detailed Implementation

[0025] The invention will be further illustrated below with specific examples.

[0026] Example 1:

[0027] 1) First, use 400#, 800#, and 1200# silicon carbide sandpaper to polish the TC4 titanium alloy in sequence. Then, use a sandblasting process (using 200-micron aluminum oxide particles, a pressure of 0.3 MPa, a sandblasting time of 30 seconds, and a spray angle of 70°) to form a primary pore structure with a diameter of about 20 microns on the surface of the TC4 titanium alloy. Then, use an acid etching process (first, use 0.3 wt% hydrofluoric acid for acid etching for 30 minutes, then use a 10 wt% hydrochloric acid / 35 wt% sulfuric acid aqueous solution at 60°C for acid etching for 60 minutes, and finally use a large amount of deionized water to wash completely) to form a secondary pore structure with a diameter of about 1 micron, thus obtaining a microporous substrate layer.

[0028] 2) A hydroxyapatite / nanosilver layer with a particle size of approximately 40 micrometers was prepared by mixing 40-micrometer diameter hydroxyapatite and 60-nanometer diameter nanosilver in a 95:5 ratio. This mixture was then deposited on a microporous substrate using a plasma spraying process (the plasma gas consisted of a hydrogen / oxygen mixture containing argon, with a volume ratio of 15% argon, 15% hydrogen, and the remainder oxygen; the gas flow rate was controlled at 40 liters / minute; the spraying distance was 25 centimeters; the spraying power was 40 watts; and the powder feed rate was 10 grams / minute). 3) Under the conditions of 60℃ water bath and 300 rpm stirring, chitosan molecules (600,000 molecular weight, 70% degree of deacetylation) were dissolved in 1 wt% acetic acid aqueous solution to prepare a 3 wt% chitosan solution; 5 wt% methacrylic anhydride was added dropwise to the above solution, and the reaction was continued for 2 hours after the addition was completed. Then, an equal volume of PBS solution was added and reacted for 15 minutes. Dialysis was performed for 7 days using deionized water and a dialysis bag with a molecular weight of 8000-14000. The precipitate was removed by centrifugation at 10000 rpm, and the ChiMA was obtained by freeze drying. Under light-protected and 50°C water bath conditions, a 10wt% ChiMA aqueous solution was prepared, and then 0.5wt% of aldehyde-containing bisphosphonate was added to perform secondary grafting modification of ChiMA. After reacting for 12 hours under magnetic stirring at 300 rpm, the mixture was dialyzed for 5 days using deionized water and a dialysis bag with a molecular weight of 8000-14000. The precipitated impurities were removed by centrifugation at 10000 rpm, and the mixture was lyophilized to obtain the desired ChiMA-BP.

[0029] 4) Under 60°C water bath and light-protected conditions, 7 wt% ChiMA-BP was dissolved in PBS solution containing 0.3 wt% LAP until completely dissolved. The TC4 titanium alloy with hydroxyapatite / nanosilver layer deposited in step 2) was ultrasonically impregnated in the above ChiMA-BP solution (impregnation time was 3 minutes, ultrasonic power was 100 watts). After removal, it was subjected to rotation-in-situ UV curing (rotation speed was 15 rpm, wavelength was 405 nm, curing time was 5 minutes). The above impregnation-curing process was repeated 10 times until the gel layer completely covered the hydroxyapatite / nanosilver layer.

[0030] 5) Immerse the sample obtained in step 4) in a magnesium chloride solution of 50 mg / mL for 30 minutes. After immersion, rinse with deionized water 10 times. Then immerse the sample in a DOPA-E7 solution of 5 mg / mL for 15 minutes to achieve chelation of metal ions with ChiMA-BP and loading of peptides, thus obtaining a composite gel layer. After irradiation sterilization, a time-gradient regulated bone implant coating with anti-infection, cell recruitment and osteogenic functions is finally obtained.

[0031] The bone implant device coating prepared above has good biocompatibility. The CCK-8 test results after 7 days showed that the cell survival rate was 91%. The bending strength of the device reached 383.5 MPa, the bonding strength between hydroxyapatite / nanosilver and the substrate was about 18.4 MPa, the in vitro antibacterial rate was greater than 90%, and the osteogenic differentiation experiment proved that the material has a good bone differentiation promoting function. The hydrogel inside the scaffold was completely degraded in about 48 days.

[0032] Example 2:

[0033] 1) First, use 400#, 800#, and 1200# silicon carbide sandpaper to polish the TC4 titanium alloy in sequence. Then, use a sandblasting process (using 200-micron aluminum oxide particles, a pressure of 0.3 MPa, a sandblasting time of 30 seconds, and a spray angle of 70°) to form a primary pore structure with a diameter of about 20 microns on the surface of the TC4 titanium alloy. Then, use an acid etching process (first, use 0.3 wt% hydrofluoric acid for acid etching for 30 minutes, then use a 10 wt% hydrochloric acid / 35 wt% sulfuric acid aqueous solution at 60°C for acid etching for 60 minutes, and finally use a large amount of deionized water to wash completely) to form a secondary pore structure with a diameter of about 1 micron, thus obtaining a microporous substrate layer.

[0034] 2) A hydroxyapatite / nanosilver mixture with a particle size of approximately 40 micrometers was prepared by mixing 40-micrometer diameter hydroxyapatite and 60-nanometer diameter nanosilver in a 90:10 ratio. This mixture was then deposited on a microporous substrate surface using a plasma spraying process (the plasma gas consisted of a hydrogen / oxygen mixture containing argon, with a volume ratio of 15% argon, 15% hydrogen, and the remainder oxygen; the gas flow rate was controlled at 40 liters / minute; the spraying distance was 25 centimeters; the spraying power was 40 watts; and the powder feed rate was 10 grams / minute). 3) Under the conditions of 60℃ water bath and 300 rpm stirring, chitosan molecules (600,000 molecular weight, 70% degree of deacetylation) were dissolved in 1 wt% acetic acid aqueous solution to prepare a 3 wt% chitosan solution; 5 wt% methacrylic anhydride was added dropwise to the above solution, and the reaction was continued for 2 hours after the addition was completed. Then, an equal volume of PBS solution was added to the above solution and reacted for 15 minutes. Dialysis was performed for 7 days using deionized water and a dialysis bag with a molecular weight of 8000-14000. The precipitate was removed by centrifugation at 10000 rpm and the ChiMA was obtained by freeze drying. A 10wt% ChiMA aqueous solution was prepared under light-protected and 50℃ water bath conditions. Then, 0.5wt% of bisphosphonate with aldehyde group was added to perform secondary grafting modification on ChiMA. After reacting for 12 hours under magnetic stirring at 300 rpm, the mixture was dialyzed for 5 days using deionized water and a dialysis bag with a molecular weight of 8000-14000. The precipitate was removed by centrifugation at 10000 rpm, and the ChiMA-BP was obtained by freeze drying.

[0035] 4) Under 60°C water bath and light-protected conditions, 7 wt% ChiMA-BP was dissolved in PBS solution containing 0.3 wt% LAP until completely dissolved. The TC4 titanium alloy with hydroxyapatite / nanosilver layer deposited in step 2) was ultrasonically impregnated in the above ChiMA-BP solution (impregnation time was 3 minutes, ultrasonic power was 100 watts). After removal, it was subjected to rotation-in-situ UV curing (rotation speed was 15 rpm, wavelength was 405 nm, curing time was 5 minutes). The above impregnation-curing process was repeated 10 times until the gel layer completely covered the hydroxyapatite / nanosilver layer.

[0036] 5) Immerse the sample obtained in step 4) in a magnesium chloride solution of 50 mg / mL for 30 minutes. After immersion, rinse with deionized water 10 times. Then immerse the sample in a DOPA-E7 solution of 5 mg / mL for 15 minutes to achieve chelation of metal ions with ChiMA-BP and loading of peptides to obtain a composite gel layer. After irradiation sterilization, a time-gradient regulated bone implant coating with anti-infection, cell recruitment and osteogenic functions is finally obtained.

[0037] Compared to Example 1, the amount of nanosilver used was increased. The bone implant device coating prepared above has good biocompatibility. The CCK-8 test results after 7 days showed that the cell survival rate was 87%. The bending strength of the device reached 385.1 MPa, the bonding strength between hydroxyapatite / nanosilver and the substrate was about 19.1 MPa, the in vitro antibacterial rate was greater than 99%, and the osteogenic differentiation experiment proved that the material has a good bone differentiation promoting function. The hydrogel inside the scaffold was completely degraded in about 52 days.

[0038] Example 3:

[0039] 1) First, use 400#, 800#, and 1200# silicon carbide sandpaper to polish the TC4 titanium alloy in sequence. Then, use a sandblasting process (using 350-micron aluminum oxide particles, a pressure of 0.5 MPa, a sandblasting time of 60 seconds, and a spray angle of 70°) to form a primary pore structure with a diameter of about 30 microns on the surface of the TC4 titanium alloy. Then, use an acid etching process (first, use 0.6 wt% hydrofluoric acid for acid etching for 60 minutes, then use a 15 wt% hydrochloric acid / 35 wt% sulfuric acid aqueous solution at 70°C for acid etching for 120 minutes, and finally use a large amount of deionized water to wash completely) to form a secondary pore structure with a diameter of about 3 microns, thus obtaining a microporous substrate layer.

[0040] 2) A hydroxyapatite / nanosilver mixture with a particle size of approximately 40 micrometers was prepared by mixing 40-micrometer diameter hydroxyapatite and 60-nanometer diameter nanosilver in a 90:10 ratio. This mixture was then deposited on a microporous substrate surface using a plasma spraying process (the plasma gas consisted of a hydrogen / oxygen mixture containing argon, with a volume ratio of 15% argon, 15% hydrogen, and the remainder oxygen; the gas flow rate was controlled at 40 liters / minute; the spraying distance was 25 centimeters; the spraying power was 40 watts; and the powder feed rate was 10 grams / minute). 3) Under the conditions of 60℃ water bath and 300 rpm stirring, chitosan molecules (600,000 molecular weight, 70% degree of deacetylation) were dissolved in 1 wt% acetic acid aqueous solution to prepare a 3 wt% chitosan solution; 5 wt% methacrylic anhydride was added dropwise to the above solution, and the reaction was continued for 2 hours after the addition was completed. Then, an equal volume of PBS solution was added to the above solution and reacted for 15 minutes. Dialysis was performed for 7 days using deionized water and a dialysis bag with a molecular weight of 8000-14000. The precipitate was removed by centrifugation at 10000 rpm and the ChiMA was obtained by freeze drying. A 10wt% ChiMA aqueous solution was prepared under light-protected and 50℃ water bath conditions. Then, 0.5wt% of bisphosphonate with aldehyde group was added to perform secondary grafting modification on ChiMA. After reacting for 12 hours under magnetic stirring at 300 rpm, the mixture was dialyzed for 5 days using deionized water and a dialysis bag with a molecular weight of 8000-14000. The precipitate was removed by centrifugation at 10000 rpm, and the ChiMA-BP was obtained by freeze drying.

[0041] 4) Under 60°C water bath and light-protected conditions, 7 wt% ChiMA-BP was dissolved in PBS solution containing 0.3 wt% LAP until completely dissolved. The TC4 titanium alloy with hydroxyapatite / nanosilver layer deposited in step 2) was ultrasonically impregnated in the above ChiMA-BP solution (impregnation time was 3 minutes, ultrasonic power was 100 watts). After removal, it was subjected to rotation-in-situ UV curing (rotation speed was 15 rpm, wavelength was 405 nm, curing time was 5 minutes). The above impregnation-curing process was repeated 10 times until the gel layer completely covered the hydroxyapatite / nanosilver layer.

[0042] 5) Immerse the sample obtained in step 4) in a magnesium chloride solution of 50 mg / mL for 30 minutes. After immersion, rinse with deionized water 10 times. Then immerse the sample in a DOPA-E7 solution of 5 mg / mL for 15 minutes to achieve chelation of metal ions with ChiMA-BP and loading of peptides to obtain a composite gel layer. After irradiation sterilization, a time-gradient regulated bone implant coating with anti-infection, cell recruitment and osteogenic functions is finally obtained.

[0043] Compared to Example 1, the sandblasting and acid etching processes are more stringent. The bone implant device prepared above has a coating with good biocompatibility. The CCK-8 test results after 7 days show a cell survival rate of 90%. The bending strength of the device reaches 380.2 MPa, the bonding strength between hydroxyapatite / nanosilver and the substrate is about 22.4 MPa, the in vitro antibacterial rate is greater than 92%, and the osteogenic differentiation experiment shows that the material has a good bone differentiation promoting function. The hydrogel inside the scaffold is completely degraded in about 49 days.

[0044] Example 4:

[0045] 1) First, use 400#, 800#, and 1200# silicon carbide sandpaper to polish the TC4 titanium alloy in sequence. Then, use a sandblasting process (using 200-micron aluminum oxide particles, a pressure of 0.3 MPa, a sandblasting time of 30 seconds, and a spray angle of 70°) to form a primary pore structure with a diameter of about 20 microns on the surface of the TC4 titanium alloy. Then, use an acid etching process (first, use 0.3 wt% hydrofluoric acid for acid etching for 30 minutes, then use a 10 wt% hydrochloric acid / 35 wt% sulfuric acid aqueous solution at 60°C for acid etching for 60 minutes, and finally use a large amount of deionized water to wash completely) to form a secondary pore structure with a diameter of about 1 micron, thus obtaining a microporous substrate layer.

[0046] 2) Hydroxyapatite with a diameter of 40 micrometers and nanosilver with a diameter of 60 nanometers are mixed in a ratio of 95:5 to form a mixed powder with a particle size of about 40 micrometers. Combined with plasma spraying process (the plasma gas is a hydrogen / oxygen mixture containing argon, with a volume ratio of 15% argon, 15% hydrogen, and the remainder oxygen, the gas flow rate is controlled at 40 liters / minute, the spraying distance is 25 cm, the spraying power is 40 watts, and the powder feeding rate is 10 g / minute), a hydroxyapatite / nanosilver layer with a thickness of about 100 micrometers is deposited on the surface of the microporous substrate. 3) Under the conditions of 60℃ water bath and 300 rpm stirring, chitosan molecules (molecular weight 1.2 million, degree of deacetylation 70%) were dissolved in 1 wt% acetic acid aqueous solution to prepare a 3 wt% chitosan solution; 10 wt% methacrylic anhydride was added dropwise to the above solution, and the reaction was continued for 2 hours after the addition was completed. Then, an equal volume of PBS solution was added to the above solution and the reaction was carried out for 15 minutes. Dialysis was performed for 7 days using deionized water and a dialysis bag with a molecular weight of 8000-14000. The precipitate was removed by centrifugation at 10000 rpm, and the ChiMA was obtained by freeze drying. A 10wt% ChiMA aqueous solution was prepared under light-protected and 50℃ water bath conditions. Then, 0.5wt% of bisphosphonate with aldehyde group was added to perform secondary grafting modification on ChiMA. After reacting for 12 hours under magnetic stirring at 300 rpm, the mixture was dialyzed for 5 days using deionized water and a dialysis bag with a molecular weight of 8000-14000. The precipitate was removed by centrifugation at 10000 rpm, and the ChiMA-BP was obtained by freeze drying.

[0047] 4) Under 60°C water bath and light-protected conditions, 10 wt% ChiMA-BP was dissolved in PBS solution containing 0.3 wt% LAP until completely dissolved. The TC4 titanium alloy with hydroxyapatite / nanosilver layer deposited in step 2) was ultrasonically impregnated in the above ChiMA-BP solution (impregnation time: 3 minutes, ultrasonic power: 100 W). After removal, it was subjected to rotation-in-situ UV curing (rotation speed: 15 rpm, wavelength: 405 nm, curing time: 5 minutes). The above impregnation-curing process was repeated 10 times until the gel layer completely covered the hydroxyapatite / nanosilver layer.

[0048] 5) Immerse the sample obtained in step 4) in a magnesium chloride solution of 50 mg / mL for 30 minutes. After immersion, rinse with deionized water 10 times. Then immerse the sample in a DOPA-E7 solution of 5 mg / mL for 15 minutes to achieve chelation of metal ions with ChiMA-BP and loading of peptides to obtain a composite gel layer. After irradiation sterilization, a time-gradient regulated bone implant coating with anti-infection, cell recruitment and osteogenic functions is finally obtained.

[0049] Compared to Example 1, the molecular weight of the chitosan raw material was increased, the amount of MA was increased to improve the degree of substitution, and the amount of ChiMA-BP in the gel layer was also increased. The bone implant device coating prepared above has good biocompatibility. The CCK-8 test results after 7 days showed that the cell survival rate was 94%. The bending strength of the device reached 386.4 MPa, the bonding strength between hydroxyapatite / nanosilver and the substrate was about 18.7 MPa, the in vitro antibacterial rate was greater than 86%, and the osteogenic differentiation experiment showed that the material has a good bone differentiation promoting function. The hydrogel inside the scaffold was completely degraded in about 57 days.

Claims

1. A method for preparing a time-gradient-regulated coating for bone implant devices with anti-infection, cell recruitment, and osteogenic functions, characterized in that: Here are the steps: 1) First, the surface of the bone implant device is polished, and then a sandblasting process is used to form a primary pore structure with a diameter of 20-30 micrometers on the surface of the bone implant device. Then, an acid etching process is used to form a secondary pore structure with a diameter of 1-4 micrometers to obtain a microporous base layer; the surface material of the bone implant device is titanium / titanium alloy. 2) Use hydroxyapatite and nano-silver to prepare a mixed powder with a particle size of 40-80 micrometers in a certain proportion, and deposit a hydroxyapatite layer / nano-silver layer with a thickness of 100-300 micrometers on the surface of the microporous substrate using plasma spraying process. 3) Methacrylic anhydride was used to graft chitosan molecules to obtain methacrylic anhydride-modified chitosan (ChiMA); then bisphosphonates with aldehyde groups were used to graft ChiMA to obtain ChiMA-BP. 4) Dissolve ChiMA-BP in a PBS solution containing a photoinitiator under water bath conditions of 50-70℃ and in the dark. Ultrasonically impregnate the bone implant device with hydroxyapatite / nanosilver layer deposited in step 2) in the ChiMA-BP solution. After removal, perform rotation-in-situ UV curing. Repeat the impregnation-curing process multiple times until the gel layer completely covers the hydroxyapatite / nanosilver layer. 5) The sample obtained in step 4) is sequentially immersed in a solution containing osteogenic metal ions and a solution containing peptides with stem cell recruitment function to achieve chelation of metal ions with ChiMA-BP and loading of peptides to obtain a composite gel layer; after irradiation sterilization, a time gradient-regulated bone implant device coating with anti-infection-cell recruitment-osteogenic function is finally obtained.

2. The method for preparing the time-gradient regulated bone implant coating with anti-infection-cell recruitment-osteogenic functions according to claim 1, characterized in that: In step 1), the polishing process involves sequentially polishing the surface of the bone implant with 400#, 800#, and 1200# silicon carbide sandpaper; the sandblasting process involves sandblasting with 200-350 micrometer aluminum oxide particles at a pressure of 0.3-0.5 MPa for 30-60 seconds at a spray angle of 70-80°; after sandblasting, ultrasonic cleaning is performed sequentially with ethanol and deionized water, once with ethanol and twice with deionized water, each time for 15 minutes; the acid etching process involves first acid etching with 0.3-0.6 wt% hydrofluoric acid for 30-60 minutes, then acid etching with an aqueous solution of 10-15 wt% hydrochloric acid / 35-55 wt% sulfuric acid at 60-70°C for 60-120 minutes, and finally washing with deionized water and drying.

3. The method for preparing the time-gradient regulated bone implant coating with anti-infection-cell recruitment-osteogenic functions according to claim 1, characterized in that: In step 2), the mass ratio of hydroxyapatite to nano-silver is between 90:10 and 95:5, wherein the diameter of hydroxyapatite is 40-80 micrometers and the particle size of nano-silver is 60-90 nanometers; the parameters of the plasma spraying process are as follows: the plasma gas is a hydrogen / oxygen mixture containing argon, with a volume ratio of 15-35% argon, 15-20% hydrogen, and the remainder being oxygen; the gas flow rate is controlled at 40-80 liters / minute; the spraying distance is 10-25 centimeters; the spraying power is 40-70 watts; and the powder feeding rate is 10-20 grams / minute.

4. The method for preparing the time-gradient regulated bone implant coating with anti-infection-cell recruitment-osteogenic functions according to claim 1, characterized in that: In step 3), the ChiMA synthesis method is as follows: Chitosan with a viscosity-average molecular weight of 600,000-1,200,000 and a degree of deacetylation of 70-80% is dissolved in 1-2 wt% acetic acid solution under a 60°C water bath and magnetic stirring at 300-500 rpm to obtain 3-6 wt% chitosan. Then, 5-10 wt% methacrylic anhydride is added dropwise, and the reaction continues for 2-3 hours after the addition is complete. After the reaction is complete, an equal volume of PBS preheated to 60°C is added, and the reaction continues for 15-25 minutes. Dialysis is performed for 5-7 days using deionized water and a dialysis bag with a molecular weight of 8000-14000. The ChiMA-BP synthesis method is as follows: a 5-15 wt% ChiMA solution is prepared under light-protected and 50°C water bath conditions, and then 0.5-1 wt% bisphosphonate BP-CHO with aldehyde groups is added. After reacting for 12-24 hours under magnetic stirring at 300-500 rpm, the mixture is dialyzed with deionized water and a dialysis bag with a molecular weight of 8000-14000 for 3-5 days. The precipitate is removed by centrifugation at 8000-10000 rpm, and the mixture is then freeze-dried to obtain the ChiMA-BP.

5. The method for preparing the time-gradient regulated bone implant coating with anti-infection-cell recruitment-osteogenic functions according to claim 1, characterized in that: In step 4), the concentration of ChiMA-BP is 5-10 wt%; during rotation-in-situ UV curing, the photoinitiator used is LAP, and the concentration of the photoinitiator is 0.25-2.5 wt%; the bone implant device with deposited hydroxyapatite / silver nanolayer is immersed in the ChiMA-BP solution for 3-5 minutes, and the ultrasonic power is 80-100 watts; the process parameters for rotation-UV curing are: rotation speed of 15 rpm, wavelength of 405 nm, and curing time of 5-15 minutes. The impregnation-curing process is repeated 5-10 times.

6. The method for preparing the time-gradient regulated bone implant coating with anti-infection-cell recruitment-osteogenic functions according to claim 1, characterized in that: In step 5), the bone-promoting metal ions are one or more of magnesium ions, zinc ions, calcium ions, and copper ions, with a concentration of 30-70 mg / mL, and an immersion time of 30-80 minutes. After immersion, the ions are rinsed with deionized water 5-10 times. The polypeptides with stem cell recruitment function are one or more of DOPA-E7, DOPA-Y5, and LL-37, with a concentration of 5-20 mg / mL, and an immersion time of 15-30 minutes.

7. A time-gradient-regulated bone implant coating with anti-infection, cell recruitment, and osteogenic functions, characterized in that: The bone implant device coating, prepared by the method described in any one of claims 1-6, comprises, from the inside out, a microporous base layer, a hydroxyapatite / nanosilver layer, and a composite gel layer.

8. A time-gradient-regulated bone implant device with anti-infection, cell recruitment, and osteogenic functions, characterized in that: The surface of the bone implant device is provided with the coating as described in claim 7.