Integrin nano-silver chelating peptide composite coating, preparation method and application thereof

Abstract

The present application relates to the technical field of bone external fixation screw coating material, and is a kind of integrin nano silver chelating peptide composite coating and its preparation method and application, titanium sheet or / and titanium nail is cleaned by distilled water, then immersed in anhydrous ethanol, then treated by oxygen plasma, immersed in PBS solution containing (DOPA) 4-G5-GRGDS to carry out first layer coating, then immersed in medical alcohol for disinfection, and (DOPA) 4-G5-GRGDS not attached is washed off, dried, then immersed in AgNO3 solution and placed to carry out second layer coating, then sterilized and dried, and integrin nano silver chelating peptide composite coating is obtained on the surface of titanium sheet or / and titanium nail. The integrin nano silver chelating peptide composite coating of the present application can be used as a bone external fixation support when the bone is damaged by modifying the medical titanium-based material screw, not only has strong antibacterial property, but also can promote bone integration around the nail channel and effectively prevent screw loosening.

Description

Technical Field

[0001] This invention relates to the field of coating materials for external fixation screws, specifically an integrin nanosilver chelate peptide composite coating, its preparation method, and its application. Background Technology

[0002] With the development of modern society, bone injuries caused by accidents are increasing year by year. More and more patients are undergoing surgical treatment for bone and joint diseases, trauma and spinal diseases. External fixation devices are flexible in structure, easy to master and have low invasiveness, which has made them popular among orthopedic surgeons. They are especially advantageous in treating limb fractures with poor soft tissue conditions and play an irreplaceable role.

[0003] However, while external fixation devices treat patients, they also bring many complications, especially for those requiring long-term wear. Screw loosening and pin tract infection are common complications of external fixation. The nail-bone interface is the weakest link in the external fixation system. Titanium-based screws, due to the presence of a bio-inert oxide layer on their surface, cannot form a chemically bonded structure that allows for stable bonding with bone tissue. This leads to the formation of fibrous septa at the nail-bone interface, causing screw loosening and resulting in surgical failure. Furthermore, a series of studies have indicated that the incidence of pin tract infection in patients treated with external fixation devices can reach 100%, meaning that pin tract infection is almost unavoidable when using external fixation devices. If the infection is limited to the skin surface, it often does not require much treatment. However, if it invades deeper bone and soft tissue, it can cause screw loosening, leading to loss of fracture reduction and even osteomyelitis. The existence of these two major complications—screw loosening and pin tract infection—significantly increases the risk of surgical failure and limits the clinical application of external fixation devices.

[0004] To improve screw stability and reduce complications, researchers have explored novel materials and surface modifications of medical screws, leading to the development of various coated screws, such as those using hydroxyapatite and bioactive peptides. Among these external fixation screw coatings, hydroxyapatite-coated screws, with their ability to promote osseointegration and improve bone-interface mechanical anchoring, are widely used in clinical trials and treatments for external fixation screws. However, hydroxyapatite-coated screws are relatively expensive, and the antibacterial properties of the coating are not ideal. In cases of severe wound contamination where thorough debridement is impossible, the probability of screw tract infection is extremely high. Furthermore, the gradual loosening of hydroxyapatite-coated implants is a critical issue in orthopedic practice. Additionally, the poor mechanical strength of the hydroxyapatite coating makes it prone to breakage, resulting in a high implant failure rate.

[0005] With the rapid development of materials science in recent years, various new materials and bio-coatings have been applied to the surface modification engineering of titanium-based materials. Among them, the integrin-targeting peptide biomimetic polypeptide (DOPA-RGD), which combines the advantages of both RGD sequence and DOPA group, has come into the view of scholars due to its unique advantages of high biomimicry and natural affinity. It has been proven that it can be used to modify the surface of titanium implants and improve the bone mass around the implants.

[0006] Therefore, the research and development of screw coatings that promote osseointegration, accelerate new bone formation, enhance the integration of implants with surrounding bone, and effectively control infection while being low-toxic or non-toxic is a key issue that urgently needs to be addressed. Summary of the Invention

[0007] This invention provides an integrin nanosilver chelate peptide composite coating, its preparation method, and its application, which overcomes the shortcomings of the prior art and can effectively solve the problems of easy nail tract infection and screw loosening in existing external fixation devices.

[0008] One of the technical solutions of this invention is achieved through the following measures: an integrin nanosilver chelate peptide composite coating, the chemical structural formula of which is: .

[0009] The following are further optimizations and / or improvements to one of the above-mentioned technical solutions: The aforementioned integrin nano-silver chelate peptide composite coating was prepared according to the following method: First, titanium sheets and / or titanium nails were washed with distilled water, then soaked in anhydrous ethanol, and then treated with oxygen plasma; Second, the treated titanium sheets and / or titanium nails were immersed in a PBS solution containing (DOPA)4-G5-GRGDS to form a first coating layer; Third, the titanium sheets and / or titanium nails after the first coating layer were immersed in medical alcohol for disinfection, and the unattached (DOPA)4-G5-GRGDS were washed away, and then dried; Fourth, the dried titanium sheets and / or titanium nails were immersed in AgNO3 solution and allowed to stand before forming a second coating layer, then disinfected by immersion in medical alcohol and air-dried, thus obtaining an integrin nano-silver chelate peptide composite coating on the surface of the titanium sheets and / or titanium nails.

[0010] In the first step above, the soaking time in anhydrous ethanol is 2 hours, and the oxygen plasma treatment time is 60 seconds.

[0011] In the second step above, the PBS solution containing (DOPA)4-G5-GRGDS contains 10 μg of (DOPA)4-G5-GRGDS per 1 mL of PBS solution, and the first coating time is 2 h.

[0012] In the third step above, the disinfection temperature is 25℃, the disinfection time is 1 hour, and the volume fraction of medical alcohol is 75%.

[0013] In the fourth step above, the second coating time is 10 min, the concentration of AgNO3 solution is 2 μg / mL, the disinfection time is 10 min, and the volume fraction of medical alcohol is 75%.

[0014] The second technical solution of the present invention is achieved through the following measures: a method for preparing an integrin nano-silver chelate peptide composite coating, which is carried out according to the following steps: First, titanium sheets and / or titanium nails are washed with distilled water, then soaked in anhydrous ethanol, and then treated with oxygen plasma; Second, the treated titanium sheets and / or titanium nails are immersed in a PBS solution containing (DOPA)4-G5-GRGDS to form a first coating layer; Third, the titanium sheets and / or titanium nails after the first coating layer are immersed in medical alcohol for disinfection, and the unattached (DOPA)4-G5-GRGDS are washed away, and then dried; Fourth, the dried titanium sheets and / or titanium nails are immersed in AgNO3 solution and allowed to stand before forming a second coating layer, then disinfected by soaking in medical alcohol and air-dried, thus obtaining an integrin nano-silver chelate peptide composite coating on the surface of the titanium sheets and / or titanium nails.

[0015] The third technical solution of the present invention is achieved through the following measures: the application of an integrin nanosilver chelate peptide composite coating in a medical device for inhibiting infection around the external fixator pin track during the treatment of bone injury.

[0016] The fourth technical solution of the present invention is achieved through the following measures: the application of an integrin nanosilver chelate peptide composite coating in a medical device for preventing loosening of medical screws during the treatment of bone injuries.

[0017] The integrin nano-silver chelate peptide composite coating of the present invention modifies medical titanium-based screws and can be used as an external fixation scaffold in cases of bone injury. It not only has strong antibacterial properties, but also promotes bone integration around the screw channel and effectively prevents screw loosening. Attached Figure Description

[0018] Appendix Figure 1 This is a graph showing the Ag⁺ release curve in the integrin nanosilver chelate peptide composite coating of the present invention.

[0019] Appendix Figure 2 The present invention provides a composite coating of silver nanoparticles and chelated peptides in the form of a laser power density of 1 W / cm². 2 The cyclic temperature rise curve under irradiation.

[0020] Appendix Figure 3 This is a qualitative test diagram of the antibacterial performance of different composite coatings in this invention after 24 hours of cultivation.

[0021] Appendix Figure 4This is a graph showing the quantitative data of the antibacterial performance of different composite coatings after 24 hours of cultivation in this invention.

[0022] Appendix Figure 5 The images show SEM images of BMSCs cultured on different composite coating surfaces for 7 days in this invention.

[0023] Appendix Figure 6 The bar chart shows the activity of CCK-8 cells cultured on different composite coating surfaces in this invention.

[0024] Appendix Figure 7 The images show DAPI staining of BMSCs nuclei cultured on different composite coating surfaces in this invention.

[0025] Appendix Figure 8 This is a peak pull-out force diagram of the external fixation screw for the rat femur in this invention.

[0026] Appendix Figure 9 These are histological images of the microenvironment of the screw track 6 weeks after different screws were implanted in this invention.

[0027] Appendix Figure 10 This is a histogram showing the percentage of Von kossa hard tissue staining deposition parameters in this invention. Detailed Implementation

[0028] This invention is not limited to the following embodiments; specific implementation methods can be determined based on the technical solution of this invention and actual circumstances. Unless otherwise specified, all chemical reagents and chemical products mentioned in this invention are well-known and commonly used chemical reagents and chemical products in the prior art.

[0029] The present invention will be further described below with reference to embodiments: Example 1: The chemical structural formula of the integrin nano-silver chelate peptide composite coating is as follows: .

[0030] Example 2: As an optimization of the above example, the integrin nano-silver chelate peptide composite coating was prepared according to the following method: First, the titanium sheet or / and titanium nail were washed with distilled water, soaked in anhydrous ethanol, and then treated with oxygen plasma; Second, the treated titanium sheet or / and titanium nail were immersed in (DOPA)4-G5-GRGDS in PBS solution to form a first coating layer; Third, the titanium sheet or / and titanium nail after the first coating layer were immersed in medical alcohol for disinfection, and the unattached (DOPA)4-G5-GRGDS were washed away and dried; Fourth, the dried titanium sheet or / and titanium nail were immersed in AgNO3 solution and allowed to stand before forming a second coating layer, then disinfected by soaking in medical alcohol and air-dried, thus obtaining the integrin nano-silver chelate peptide composite coating on the surface of the titanium sheet or / and titanium nail.

[0031] Example 3: As an optimization of the above example, in the first step, the soaking time in anhydrous ethanol is 2 hours, and the oxygen plasma treatment time is 60 seconds.

[0032] Example 4: As an optimization of the above example, in the second step, the PBS solution containing (DOPA)4-G5-GRGDS contains 10 μg of (DOPA)4-G5-GRGDS per 1 mL of PBS solution, and the time for the first coating layer is 2 h.

[0033] Example 5: As an optimization of the above example, in the third step, the disinfection temperature is 25°C, the disinfection time is 1 hour, and the volume fraction of medical alcohol is 75%.

[0034] Example 6: As an optimization of the above example, in the fourth step, the second coating time is 10 min, the concentration of AgNO3 solution is 2 μg / mL, the disinfection time is 10 min, and the volume fraction of medical alcohol is 75%.

[0035] Example 7: The preparation method of the integrin nano-silver chelate peptide composite coating is as follows: First, the titanium sheet or / and titanium nail are washed with distilled water, soaked in anhydrous ethanol, and then treated with oxygen plasma; Second, the treated titanium sheet or / and titanium nail are immersed in (DOPA)4-G5-GRGDS in PBS solution to form a first coating layer; Third, the titanium sheet or / and titanium nail after the first coating layer is immersed in medical alcohol for disinfection, and the unattached (DOPA)4-G5-GRGDS is washed away and dried; Fourth, the dried titanium sheet or / and titanium nail are immersed in AgNO3 solution and allowed to stand before forming a second coating layer, then disinfected by soaking in medical alcohol and air-dried, thus obtaining the integrin nano-silver chelate peptide composite coating on the surface of the titanium sheet or / and titanium nail.

[0036] Example 8: Application of the integrin nanosilver chelate peptide composite coating in a medical device for inhibiting peri-fixation infection during the treatment of bone injury.

[0037] Example 9: Application of the integrin nanosilver chelate peptide composite coating in a medical device for preventing loosening of medical screws during the treatment of bone injuries.

[0038] Example 10: After washing with distilled water, titanium sheets and / or titanium nails were soaked in anhydrous ethanol for 2 hours, followed by oxygen plasma treatment for 60 seconds. They were then immersed in a PBS solution of (DOPA)4-G5-GRGDS at a concentration of 10 μg / mL for 2 hours. Afterwards, the titanium sheets and / or titanium nails were disinfected by immersing them in 75% medical alcohol at 25°C for 1 hour, and any unadhered (DOPA)4-G5-GRGDS was washed away. After drying in a laminar flow hood, they were immersed in a 2 μg / mL AgNO3 solution, allowed to stand for 10 minutes, and then disinfected by immersion in 75% medical alcohol for 30 minutes. An integrin nano-silver chelate peptide (hereinafter referred to as Ti@DOPA-RGD-nAg) composite coating was obtained on the surface of the titanium sheets and / or titanium nails, and then air-dried in a laminar flow hood for later use.

[0039] I. According to Example 10 above, an integrin nano-silver chelate peptide composite coating was obtained on the surface of titanium (Ti) sheets and titanium (Ti) nails, and its stability and antibacterial properties were determined: (a) Materials and reagents (1) Pure titanium (Sichuan Altair Medical Devices Co., Ltd.) (2) Blood agar plate culture medium (Changde Bickman Biotechnology Co., Ltd.) (3) CCK-8 reagent kit (Beijing Bio-Sens) (4) 1,3-Diphenylisobenzofuran (Shanghai Baishun Biotechnology Co., Ltd.) (5)(DOPA)4-G5-GRGDS (Jiangsu Shenlang Biotechnology Co., Ltd.) (6) AgNO3 (Shanghai Institute of Fine Chemical Materials) (7) 4% Paraformaldehyde (Beijing Regen Biotechnology Co., Ltd.) (ii) Instruments (1) Scanning electron microscope (SEM, Hitachi SU8010, Japan) (2) Constant temperature incubator (PBH-9082, China Yiheng) (3) Inductively Coupled Plasma Emission Spectrometer (ICP, Beijing Purkinje General Instrument Co., Ltd.) (4) 808nm NIR laser (Beijing Honglan Optoelectronic Technology) (5) Fourier transform infrared spectrometer (BRUKER, Germany) (6) Biosafety cabinet (AnTai Air Technology Co., Ltd., Suzhou, China) (7) Vertical Pressure Steam Sterilizer (Medical Equipment Factory, Shanghai Boxun Industrial Co., Ltd., China) (8) Electric heating blower drying (Tianjin Tester Instrument Co., Ltd.) (9) NaCl (Tianjin Zhiyuan Chemical Reagent Co., Ltd.) (10) Solid-state UV-Vis spectrophotometer (Hitachi High Technology Corporation, Japan) (III) Physiological stability test of integrin nanosilver chelate peptide composite coating In an integrin nano-silver chelate peptide composite coating obtained on the surface of titanium sheets and / or titanium nails, Ag + Release curve as shown Figure 1 As shown, Figure 1 Chinese Ag + Release results showed that Ag was observed within the first two days. + The sudden release of Ag in the composite coating, and with further extension of immersion time, by day 10, the Ag in the composite coating... + The release amount was 0.24 mg·L⁻¹. -1 Below low cell concentrations, this may be due to the effect of DOPA's catechol groups on Ag. + It has a chelating effect and will form DOPA-Ag + The coordination bonds reduce the amount released. Therefore, the integrin nano-silver chelate peptide composite coating of this invention exhibits good physiological stability.

[0040] (iv) Photothermal performance analysis of integrin nano-silver chelate peptide composite coating Five heating and cooling cycles were performed on the integrin nano-silver chelate peptide composite coating obtained on the surface of titanium sheets and / or titanium nails. The results are as follows: Figure 2 As shown. By Figure 2 It is known that the photothermal conversion performance of the integrin nano-silver chelate peptide composite coating remains stable during 5 heating-cooling cycles. Therefore, the integrin nano-silver chelate peptide composite coating of the present invention has good photothermal conversion stability.

[0041] (v) Antibacterial performance test of integrin nano-silver chelate peptide composite coating Antibacterial properties of composite coatings of Ti, Ti@DOPA-RGD, Ti@DOPA-RGD-nAg, and light-treated Ti@DOPA-RGD-nAg were tested: E. coli and S. aureus were inoculated into LB medium, and inoculation was repeated three times to obtain relatively pure colonies. Each group of materials was pre-sterilized by UV irradiation, and then the materials were fixed in petri dishes. Single colonies of both strains were scraped and further prepared into 0.5 McFarland unit bacterial suspensions using physiological saline. 50 μL of bacterial suspension was dropped onto the surface of each group of materials and incubated in a constant temperature incubator for 1 day. 10 μL of bacterial suspension was transferred from each composite coating surface, diluted to 2 mL with physiological saline, and 50 μL was transferred to a plate. The plates were incubated again in a constant temperature incubator for 1 day before being removed. The plates were photographed, and the results are shown below. Figure 3 As shown.

[0042] according to Figure 3Qualitative antibacterial analysis was performed on the growth of *E. coli* and *S. aureus* on the culture plate surface. After 24 hours of incubation, the control group (pure Ti) showed no antibacterial activity, while numerous colonies of *E. coli* and *S. aureus* formed on the pure Ti surface, exhibiting strong proliferation and vigorous metabolism. Compared to the control group, Ti@DOPA-RGD showed some antibacterial ability, with a significant reduction in the number of *E. coli* and *S. aureus* colonies. The number of *E. coli* and *S. aureus* on the unlit Ti@DOPA-RGD-nAg surface was significantly reduced compared to the Ti@DOPA-RGD group. However, no *E. coli* or *S. aureus* were found on the illuminated Ti@DOPA-RGD-nAg surface.

[0043] right Figure 3 The number of colonies in the culture plate was used for quantitative analysis of antibacterial activity, and the results are as follows: Figure 4 As shown. Figure 4 It can be seen that Ti@DOPA-RGD has an antibacterial rate of 42% against Staphylococcus aureus and 35% against Escherichia coli. Ti@DOPA-RGD-nAg without light has an antibacterial rate of 85% against Staphylococcus aureus and 97% against Escherichia coli. Ti@DOPA-RGD-nAg with light has an antibacterial rate of 100% against both bacteria. Post-hoc pairwise comparisons showed no statistically significant difference between the Ti@DOPA-RGD-nAg group and the Ti@DOPA-RGD-nAg+Light group (P>0.05), while all other groups showed statistically significant differences (P<0.05). These results indicate that Ti@DOPA-RGD-nAg and Ti@DOPA-RGD-nAg+Light have good antibacterial activity against both Escherichia coli and Staphylococcus aureus. The formula for calculating the average antibacterial rate is as follows: Antibacterial rate = (Number of colonies in control group & Number of colonies in experimental group) / Number of colonies in control group Therefore, the integrin nanosilver chelate peptide composite coating of the present invention has good physiological stability and good rapid bactericidal ability under near-infrared light excitation.

[0044] II. Based on Example 10 above, an integrin nano-silver chelate peptide composite coating was obtained on the surface of titanium (Ti) sheets and titanium (Ti) nails. The effect of this coating on cell activity and osteogenic capacity was determined. (a) Materials and reagents (1) SPF-grade male SD rats (Experimental Animal Center of Xinjiang Medical University) (2) Phosphate buffer (Thermo, USA) (3) Fetal bovine serum (Hyclone, USA) (4) α-MEM medium (BI, Israel) (5) Penicillin and streptomycin (Gibco, Australia) (6) Trypsin / EDTA solution (BI, Israel) (7) CCK-8 (Beijing Boaosen) (8) Integrin-targeting peptide (DOPA) 4-G5-GRGDS (Jiangsu Shenlang Biotechnology Co., Ltd.) (9) BCA kit (Thermo, USA) (10) 4% Paraformaldehyde (Beijing Regen Biotechnology Co., Ltd.) (11) Rats bone marrow mesenchymal stem cell osteogenic differentiation induction kit (Guangzhou Cyagen) (12) DAPI solution (Beijing Solarbio) (13) Anhydrous ethanol (Shandong Zhuojian Medical Technology Co., Ltd.) (14) 95% ethanol (Shandong Zhuojian Medical Technology Co., Ltd.) (15) 75% ethanol (Shandong Zhuojian Medical Technology Co., Ltd.) (16) Alizarin Red Staining Solution (Beijing Solarbio) (17) ALP staining solution (Beijing Solarbio) (18)PageRulerPrestainedProteinLadder (Thermo, United States) (19) SDS-PAGE Gel Preparation Kit (Beijing Solarbio) (20) 5x Tris-glycine electrophoresis buffer (Solomon Syrup, Beijing) (21) AP colorimetric solution (Invitrogen, USA) (22) RIPA lysis buffer (Sigma, USA) (23) Rabbit-Anti-GAPDH-antibody (Abcam, USA) (24) Rabbit-Anti-RUNX2-antibody (Beijing Boson) (25) Rabbit-Anti-Osterix-antibody (Abcam, USA) (26) Rabbit-Anti-Osteopontin-antibody (Abcam, USA) (ii) Instruments (1) Flow cytometer (BD, USA) (2) Cell incubator (Thermo, USA) (3) Microplate reader (Thermo, USA) (4) Inverted phase contrast microscope (Zeiss, Germany) (5) Ultrapure water system (Zerab Corporation, USA) (6) Vertical Automatic Double Pure Water Dispenser (Zerab Corporation, USA) (7) 20℃ refrigerator (Siemens, Germany) (8) 4℃ refrigerator (Siemens, Germany) (9) ImageProPlus 4.5 image analysis software (MediaCybernetics, USA) (10) Pipettes 0.2μL-1000μL (Eppendorf, Germany) (11) Low-temperature ultracentrifuge (Eppendorf, Germany) (12) Western blot electrophoresis apparatus (Bio-Rad, USA) (13) Constant temperature decolorizing shaker (IKA, Germany) (14) Electronic balance (IKA, Germany) (15) Optical microscope (Zeiss, Germany) (16) Upright optical microscope (Nikon, Japan) (III) Isolation and culture of bone marrow mesenchymal stem cells from SD rats SD rats were euthanized by cervical dislocation. They were immersed in 75% ethanol solution for 1 minute, placed on a sterile sheet, and the skin and muscles were incised along the femoral surface with a scalpel blade, reaching the bone surface. The hip and knee joints were released, and the soft tissue was completely removed. The femur was then extracted, and the remaining soft tissue on the bone surface was further cleaned with sterile gauze. The rats were repeatedly rinsed with phosphate-buffered saline containing 10% penicillin and antibiotics. The epiphyses at both ends were cut open with tissue scissors. α-MEM complete culture medium was drawn into the bone marrow cavity using a 10 mL syringe and injected to flush out the bone marrow. The bone marrow fluid was then repeatedly aspirated and mixed using 10 mL, 5 mL, and 1 mL syringes until the fluid was generally homogeneous in color. The collected bone marrow fluid was centrifuged (1500 rpm, 5 min). The culture medium was changed, and the rats were resuspended and centrifuged twice more. After the final resuspension, the cell suspension was seeded into culture flasks and placed in a CO2 cell incubator. The medium was completely changed every 3 days thereafter.

[0045] When the cells proliferated to 90%, they were passaged, the culture medium was discarded, the cells were washed with an appropriate amount of phosphate buffer, the cells were digested with 0.25% trypsin + 0.02% EDTA, passaged at a ratio of 1:2, the cell morphology was observed under a microscope, and the images were recorded.

[0046] (iv) Cell compatibility test of integrin nanosilver chelate peptide composite coating The experiment consisted of three titanium-based coating culture groups: Ti, Ti@DOPA-RGD, and Ti@DOPA-RGD-nAg. The Ti, Ti@DOPA-RGD, and Ti@DOPA-RGD-nAg culture plates were placed in 6-well plates, with a density of 1×10⁻⁶. 4 Cells at a density of / mL were seeded onto the composite coating surface, and 1.0mL of α-MEM culture medium was added to each culture plate. The plates were then incubated at a constant temperature of 37℃ in a CO2 incubator for cell morphology, viability, and proliferation detection. The procedure is as follows: (1) Cell morphology: After 7 days of culture, the samples were washed with phosphate buffer, blocked with serum, and fixed with glutaraldehyde solution (2.5%). Cells were then dehydrated and de-alcoholized with ethanol and ethyl acetate, respectively. The morphology of cells growing on Ti, Ti@DOPA-RGD, and Ti@DOPA-RGD-nAg coatings was observed by SEM. Human bone is formed by the proliferation and differentiation of bone marrow stromal cells (hereinafter referred to as BMSCs) into osteoblasts. SEM images of BMSCs on day 7 are shown below. Figure 5 As shown.

[0047] Figure 5 It can be seen that BMSCs spread well on the surface of Ti@DOPA-RGD and Ti@DOPA-RGD-nAg composite coatings and cover the coating.

[0048] Therefore, it can be concluded that, compared with the pure Ti group, the Ti@DOPA-RGD and Ti@DOPA-RGD-nAg composite coatings have better cell compatibility and are more conducive to the adhesion and proliferation of BMSCs.

[0049] Cell viability: Add 10% CCK-8 mixed medium to 24-well plates and incubate in a dark incubator for 2 hours. After incubation, transfer 100 μL of cell suspension from each well of the 24-well plate to a 96-well plate. Measure the absorbance using a microplate reader (wavelength: 450 nm). Cell viability and proliferation are assessed based on the absorbance values. CCK-8 cell viability results are shown below. Figure 6 As shown.

[0050] Figure 6It can be seen that at 1 day, the number of cells on the surface of the pure Ti group was significantly greater than that of the Control group and the Ti@DOPA-RGD-Ag group (P<0.01); the number of cells on the surface of the Ti@DOPA-RGD group was significantly greater than that of the Control group and the Ti@DOPA-RGD-Ag group (P<0.05), while there was no statistically significant difference between the pure Ti group and the Ti@DOPA-RGD group (P>0.05); when it reached 3 days, the number of cells on the surface of the Ti@DOPA-RGD-Ag group was significantly greater than that of the pure Ti group (P<0.01); the number of cells on the surface of the Ti@DOPA-RGD group was also significantly greater than that of the pure Ti group (P<0.01).

[0051] Therefore, it can be seen that the Ti@DOPA-RGD coating has a superior effect on promoting cell proliferation, and that Ag is chelated by the functional groups of DOPA-RGD. + Low cytotoxicity, and Ag released from the composite coating within a suitable concentration range. + It is beneficial to the growth of BMSCs cells.

[0052] Staining: BMSCs were co-cultured on titanium sheets with different coatings. After 1, 3, 5, and 7 days, the cells were washed three times with PBS; fixed with 4% paraformaldehyde for 15 min, washed twice with PBS for 5 min each time; DAPI solution (1:300) was added and incubated at room temperature in the dark for 10 min; washed three times with PBS for 5 min each time; and then photographed using an upright fluorescence microscope. The results are shown below. Figure 8 As shown.

[0053] Figure 7 As can be seen, the DAPI staining of BMSCs nuclei after 1, 3, 5, and 7 days of culture on the three material surfaces showed a trend that was basically consistent with that shown by cck-8. Compared with the cell proliferation on Ti, Ti@DOPA-RGD and Ti@DOPA-RGD-nAg maintained excellent cell proliferation-promoting ability at all time points, especially at 3 and 5 days.

[0054] III. Study on the anti-loosening properties of integrin-targeted nanosilver chelate peptide modified screws in external fixation systems (a) Materials and reagents (1) Male SD rats (Experimental Animal Center of Xinjiang Medical University) (2) Simple External Fixation System (Key Laboratory of Engineering and Technology College, Xinjiang University) (3) Virbac 50 (France) (4) 4% Paraformaldehyde (Beijing Baishayi) (5) Von-Kossa stain (Wuhan Sewell) (6) Neutral resin (Sinopharm Group) (7) Phosphate buffer solution (Thermo, USA) (8) Xylene (Sinopharm Group) (9) 95% ethanol (Shandong Zhuojian Medical Technology Co., Ltd.) (10) 75% ethanol (Shandong Zhuojian Medical Technology Co., Ltd.) (11) Anhydrous ethanol (Sinopharm Group) (12) Von-Kossa stain (Wuhan Sewell) (13) Differentiation solution (Wuhan Sewell) (14) Blue Reversion Liquid (Wuhan Sewell) (15) Ethylene glycol ethyl ether acetate (Shanghai Maclean Biochemical Technology Co., Ltd.) (ii) Instruments (1) Surgical instruments (Shanghai Jinzhong) (2) X-ray machine (Kodak DR7500, USA) (3) MicroCT machine (Bruker SkyScan 1176, Germany) (4) Hard tissue microtome (LEICA HistoCoreAUTOCUT, Germany) (5) Miniature oscillating saw (Suzhou Baolikang) (6) Microcomputer-controlled fully digital electronic universal material testing machine (Shenzhen Ruigeer Instrument Co., Ltd.) (7) Dehydrator (Wuhan Junjie Electronics Co., Ltd.) (8) Embedding machine (Wuhan Junjie Electronics Co., Ltd.) (9) Pathology slide machine (Shanghai Leica Instruments Co., Ltd.) (10) Freezing station (Wuhan Junjie Electronics Co., Ltd.) (11) Organizing sheet spreader (Jinhua Kedi Instrument Equipment Co., Ltd.) (12) Oven (Tianjin Laiborui Instrument Equipment Co., Ltd.) (13) Upright optical microscope (Nikon, Japan) (14) Imaging system (Nikon, Japan) (III) Animal grouping and establishment of a rat external fixator screw efficacy verification model Forty-five healthy adult male Sprague-Dawley rats weighing 400 g to 450 g, of clean grade, were provided by the Animal Experiment Center of Xinjiang Medical University, with animal license number: SYXK(Xin)2013-0001. They were all housed individually in cages at a room temperature of 18°C to 20°C, a humidity of 50% to 60%, with good ventilation. They had free access to food and water, and humane care was given in accordance with the 3R principles of laboratory animals. This study was carried out in strict accordance with the animal medical ethics standards of China and Xinjiang Medical University (ethical approval number: LACUC-20170222054). Ti group (control group) (15 rats); Ti@DOPA-RGD group (15 rats); Ti@DOPA-RGD-nAg (15 rats).

[0055] The rats were fasted and watered for 6 to 8 hours before surgery, and anesthetized by intraperitoneal injection of Zoletil 50 at a dose of 50 mg / kg. The right hind limb of the rats was shaved and prepared for surgery. The rats were placed in the left lateral position, and the right hind limb was repeatedly disinfected with iodophor and alcohol. A longitudinal incision of about 30 mm was made above the femur of the right hind limb of the rats. After the skin was incised, a white line could be seen, which was the marker of the intermuscular septum. The vastus lateralis and biceps femoris were bluntly dissected until the bone surface was exposed, and the femur was exposed as completely as possible while keeping the minimally invasive approach. During this period, the surrounding soft tissues were pulled by a retractor to protect the surrounding nerves, blood vessels, muscles, etc. Four injection heads of 20 mL syringes were assembled on the simple external fixator as sleeves, with a slider spacing of 5 mm, and four titanium Kirschner wires were drilled sequentially from the proximal end to the distal end. After drilling, the surrounding soft tissues were retracted, and the periosteum should be as little damaged as possible. At the equal division points between the two sliders and the two groups of screws, an oscillating saw was used for osteotomy. During osteotomy, the osteotomy area was continuously irrigated to avoid thermal injury. The femur was tractioned at both ends by rotating the end nut to adjust the slider, and it could be seen that the bone segment was completely transected. The nut was adjusted in the reverse direction to close and compress the fracture ends, and the rat femoral external fixator screw efficacy verification model was successfully established. The wound was rinsed successively with hydrogen peroxide, iodophor, and normal saline, the wound surface was closed, covered with gauze and sutured to prevent the rats from biting and falling off, and the modeling was completed.

[0056] (IV) Postoperative care After surgery, the rats were placed on a warming blanket. After recovery, they were fasted and watered for 6 hours, and then had normal diet. Antibiotics were given continuously for 3 days after surgery to prevent infection and painkillers to relieve pain. The wound surface was disinfected and the pin tracts were cared for with iodophor cotton balls every 2 days after surgery, and the sterile gauze was replaced until the wound surface healed.

[0057] (V)取材及处理 of rat femoral specimens Six weeks after surgery when the fracture had healed, the experimental animals were euthanized by intraperitoneal injection of an overdose of pentobarbital sodium, and the specimens of the right femur with screws were collected. The external fixator and Kirschner wires were carefully separated, and mechanical injuries such as traction, contusion, and extrusion were minimized. After carefully removing the soft tissues on the bone surface of the fresh specimens, they were stored in an ice bag, and 6 specimens of femur with screws were randomly selected by the random sampling method for biomechanical testing on the same day.

[0058] (vi) Biomechanical measurements Screw pull-out force is the most significant indicator of the stability of experimental external fixation screws. The peak pull-out force of rat femoral external fixation screws is as follows: Figure 8 As shown.

[0059] Figure 8 It can be seen that in the external fixation rat femoral fracture model, the mean pull-out force of the Ti@DOPA-RGD group and the Ti@DOPA-RGD-nAg group was significantly higher than that of the Ti group (P<0.05). After 2 and 4 weeks, the Ti@DOPA-RGD-nAg group was significantly higher than that of the Ti group (P<0.05), while there was no significant difference between the Ti@DOPA-RGD group and the Ti@DOPA-RGD-nAg group (P>0.05). At week 6, both the Ti@DOPA-RGD group and the Ti@DOPA-RGD-nAg group were significantly higher than that of the Ti group (P<0.05), while there was no significant difference between the Ti@DOPA-RGD group and the Ti@DOPA-RGD-nAg group (P>0.05).

[0060] (vii) Histological staining images Histological analysis of the screw tract microenvironment 6 weeks after screw implantation; histological images show as follows: Figure 9 As shown, where A: Ti, B: Ti@DOPA-RGD, C: Ti@DOPA-RGD-nAg.

[0061] Figure 9 It can be seen that extensive black calcium salt deposits can be found between the threads on Ti@DOPA-RGD and Ti@DOPA-RGD-nAg. The histological appearance of the screw channels shows that the black color of the screw channels is due to the newly formed calcium salt deposits, which appear as new bone forming around the screws.

[0062] Hard tissue staining revealed differences in the nascent calcium salt deposition around the screws across the three groups. Histograms of the percentages of deposition parameters from Vonkossa hard tissue staining are shown below. Figure 10 As shown. Among them, P<0.05 P<0.01, P<0.001.

[0063] Figure 10 It can be seen that, compared with Ti screws, Ti@DOPA-RGD screws have more calcium salt deposition around them, with a statistically significant difference (P<0.01); the calcium salt deposition around Ti@DOPA-RGD-nAg screws is significantly higher than that around Ti@DOPA-RGD screws (P<0.05). This indicates that there is more neobondation on the surface of Ti@DOPA-RGD-nAg screws.

[0064] Therefore, it can be seen that Ti@DOPA-RGD-nAg screws and Ti@DOPA-RGD screws integrate into the bone better than Ti screws and have better stability. Among them, the integrin nano-silver chelate peptide composite coating screw of the present invention has the best stability.

[0065] In summary, the integrin nano-silver chelate peptide composite coating of the present invention, by modifying medical screws, can serve as an external fixation scaffold in cases of bone injury. It not only has strong antibacterial properties, but also promotes bone integration around the screw track and effectively prevents medical screw loosening.

[0066] The above technical features constitute the embodiments of the present invention, which have strong adaptability and implementation effect. Unnecessary technical features can be added or removed according to actual needs to meet the needs of different situations.

Claims

1. An integrin nanosilver chelate peptide composite coating, characterized in that... The titanium sheet and / or titanium nail were prepared according to the following method: First, the titanium sheet and / or titanium nail were washed with distilled water, then soaked in anhydrous ethanol, and then treated with oxygen plasma. Second, the treated titanium sheet and / or titanium nail were immersed in a PBS solution containing (DOPA)4-G5-GRGDS for a first coating layer. Third, the titanium sheet and / or titanium nail after the first coating layer were immersed in medical alcohol for disinfection, and the unattached (DOPA)4-G5-GRGDS were washed away, followed by drying. Fourth, the dried titanium sheet and / or titanium nail were immersed in AgNO3 solution and allowed to stand before a second coating layer was applied. The coating was then disinfected with medical alcohol and air-dried, resulting in an integrin nano-silver chelate peptide composite coating on the surface of the titanium sheet and / or titanium nail. The chemical structural formula of the integrin nano-silver chelate peptide composite coating material is as follows: ； In the second step, the PBS solution containing (DOPA)4-G5-GRGDS contains 10 μg of (DOPA)4-G5-GRGDS per 1 mL of PBS solution, and the first coating time is 2 h. In the fourth step, the second coating time is 10 min, and the concentration of AgNO3 solution is 2 μg / mL.

2. The integrin nano-silver chelate peptide composite coating according to claim 1, characterized in that... In the first step, the immersion time in anhydrous ethanol is 2 hours, and the oxygen plasma treatment time is 60 seconds.

3. The integrin nano-silver chelate peptide composite coating according to claim 1, characterized in that... In the third step, the disinfection temperature is 25℃, the disinfection time is 1 hour, and the volume fraction of medical alcohol is 75%.

4. The integrin nano-silver chelate peptide composite coating according to claim 3, characterized in that, In the fourth step, the disinfection time is 10 minutes, and the volume fraction of medical alcohol is 75%.

5. A method for preparing an integrin nano-silver chelate peptide composite coating according to any one of claims 2 to 4, characterized in that... The following steps were performed: First, the titanium sheet and / or titanium nails were washed with distilled water, then soaked in anhydrous ethanol, and then treated with oxygen plasma. Second, the treated titanium sheet and / or titanium nails were immersed in a PBS solution containing (DOPA)4-G5-GRGDS to form a first coating layer. Third, the titanium sheet and / or titanium nails with the first coating layer were immersed in medical alcohol for disinfection, and the unattached (DOPA)4-G5-GRGDS was washed away and dried. Fourth, the dried titanium sheet and / or titanium nails were immersed in AgNO3 solution and allowed to stand before forming a second coating layer. The second coating layer was then disinfected by immersion in medical alcohol and air-dried, resulting in an integrin nano-silver chelate peptide composite coating on the surface of the titanium sheet and / or titanium nails.

6. The application of the integrin nanosilver chelate peptide composite coating according to any one of claims 1 to 4 in the preparation of an instrument for treating bone injuries by inhibiting infection around the external fixator pin tract, characterized in that... The integrin nanosilver chelate peptide composite coating is used to prepare an external fixation scaffold by modifying medical screws.

7. The application of the integrin nanosilver chelate peptide composite coating according to any one of claims 1 to 4 in the preparation of an instrument for preventing loosening of medical screws during the treatment of bone injuries, characterized in that... The integrin nanosilver chelate peptide composite coating is used to prepare an external fixation scaffold by modifying medical screws.

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