CN114949374B

CN114949374B - Antibacterial bone-promoting difunctional titanium metal orthopedic implant and preparation method thereof

Info

Publication number: CN114949374B
Application number: CN202210570697.0A
Authority: CN
Inventors: 陈鹏鹏; 苏世兴; 周艺峰; 聂王焰; 徐颖; 曾少华
Original assignee: Anhui University
Current assignee: Anhui University
Priority date: 2022-05-24
Filing date: 2022-05-24
Publication date: 2023-06-13
Anticipated expiration: 2042-05-24
Also published as: CN114949374A

Abstract

The invention discloses an antibacterial bone-promoting difunctional titanium metal orthopedic implant and a preparation method thereof. The titanium metal obtained by the invention has enhanced antibacterial property and osteogenesis activity, and can be widely used as clinical medical materials.

Description

Antibacterial bone-promoting difunctional titanium metal orthopedic implant and preparation method thereof

Technical Field

The invention relates to the field of antibacterial orthopedic implants, in particular to an antibacterial bone-promoting difunctional titanium orthopedic implant and a preparation method thereof.

Background

Open fracture is a common and frequently-occurring disease in orthopaedics trauma, which means that the skin and soft tissue of the fracture part are broken due to direct or indirect violent factors, the fracture end is directly communicated with the outside, and the open fracture caused by frequent traffic accidents is also becoming serious and complex, and the treatment is also becoming more difficult. In the treatment of open fractures, effective control of infection is critical in determining success. The bone substance is directly communicated with the outside and contacts bacteria to pollute the skin due to the damage of the soft tissue of the skin. As bacteria proliferate within the wound, serious infections are increasingly caused. Even if the patient can get timely and correct debridement, irrigation, fixation, closure, anti-infection and other treatments, infection still occurs. Eventually, the fracture nonunion, traumatic osteomyelitis and the like can be caused, the healing time is prolonged, the operation frequency is increased, and even the amputation or death is caused by serious patients, so that great mental and physical burden is brought to the patients. The infection rates of various types of open fractures are reported to be 0% to 2% for type I, 2% to 5% for type II, 5% to 10% for type IIIA, 10% to 50% for type IIIB, and 25% to 50% for type iiic, respectively. Thus, how to more effectively control infections is the most interesting issue for clinicians. In recent years, with the development of research on auxiliary treatment of open fracture by more and more antibiotic slow-release systems, it is now widely considered that the antibiotic local slow-release system has important significance in the treatment of open fracture. On one hand, the local antibiotic slow-release system can accurately generate higher drug concentration at the focus part, and simultaneously reduces the serum drug concentration, thereby reducing the toxic and side effects of the drug. However, topical antibiotic treatment has problems with overuse of antibiotics resulting in the emergence of multiple resistant bacteria, which in turn creates a greater risk.

Titanium (Ti) and its alloys are widely used as implantable materials for orthopedic, orthopedic and dental applications due to their excellent mechanical properties, corrosion resistance and biocompatibility. However, the original Ti surface is a good breeding ground for opportunistic pathogens. Furthermore, due to its bio-inertness, the original Ti is not able to sufficiently induce or stimulate specific cellular behaviour, eventually leading to poor integration with the surrounding bone tissue and hindering new bone formation. These drawbacks of original Ti can lead to implant failure and even tissue necrosis, and in severe cases even require removal of the implant and repeated surgery, giving the patient pain and economic burden. Thus, a Ti-based implant having enhanced antibacterial activity and facilitating osteoblast growth is highly desired.

Methods that have been reported to date to improve the bioactivity of Ti-based implants are hydrothermal treatment, anodic oxidation, micro-arc oxidation, plasma impregnation, and the like. Among them, the anodic oxidation method is widely used for etching Ti substrates and forming titanium dioxide nanotubes (TNT) on the surfaces thereof due to a simple preparation process and low cost. The tubular array structure of TNT can enlarge the surface area of Ti and improve the surface roughness of Ti. In addition, TNT having an appropriate diameter (about 70 nm) is effective for improving the bioactivity of Ti substrates. Specifically, TNT can promote differentiation of preosteoblasts by affecting adsorption of fibrinogen and fibronectin, modulating cell signaling pathways and affecting cellular responses through its nano-roughness. In addition, tubular structures are often loaded with bactericides to impart antimicrobial capabilities to the TNT. Heavy metal based bactericides such as silver are typically loaded into the TNT and then the TNT is released to exert biocidal effects. However, the potential health risks due to the dose dependency, cytotoxicity, etc. of heavy metal ions/nanoparticles limit the scale-up of the use of this method. Likewise, loading antibiotics into TNT is also effective in preventing the occurrence of BAI, but excessive use of antibiotics leads to the emergence of multiple resistant bacteria, thereby creating a greater risk. In recent years, a method of introducing molecules having antibacterial activity on the surface of a Ti substrate by covalent grafting to thereby form a non-leachable coating on the surface thereof has been attracting attention due to its high stability and long-lasting antibacterial property.

The Ti substrate can be introduced into a plurality of antibacterial molecules in a covalent grafting mode, and how to select proper antibacterial molecules and graft the proper antibacterial molecules on the Ti substrate is the key of successful implementation of the strategy, so that the implant with high antibacterial activity, favorable for the growth of osteoblasts and high stability is obtained.

In recent years, guanidine antibacterial agents are often reported as a new generation disinfectant at home and abroad, and common guanidine antibacterial agents include polyhexamethylene monoguanidine, polyhexamethylene biguanide, polyguanidine derivatives thereof and the like. The special high polymer structure reduces toxicity, has more excellent sterilization effect, is widely applied to various fields of medical and health, family life, food industry and the like, and is continuously developed along with research on the properties and the efficacy of guanidine disinfectants.

At present, according to research on monoguanidine and biguanide at home and abroad, biguanide is generally considered to have higher safety and lower irritation than monoguanidine in terms of safety, and biguanide has been listed in a national permissible bill of raw materials, so biguanide is more advantageous than polyhexamethylene monoguanidine in terms of production and application of orthopedic implant materials. Guanidine groups in PHMB have high activity, are positively charged in aqueous solution, can be adsorbed by negatively charged bacterial cell membranes, and are subjected to ion exchange with calcium ions and magnesium ions in the membranes to destroy the charge balance of the cell membranes and finally destroy the membrane system, so that substances in bacteria body outflow to cause bacterial lysis, and the PHMB can block respiratory passages of microorganisms to inhibit bacterial reproduction. Guanidine polymers do not substantially produce target-target specific binding to bacteria when they achieve antibacterial action, and therefore are not prone to drug resistance. Meanwhile, as the outer layer of the cell membrane of the mammal is electrically neutral and the outer layer of the cell membrane of the bacteria/fungi is negatively charged, the positively charged guanidine polymer has strong selectivity on the cell membrane of the bacteria or fungi, and further the toxicity of the guanidine polymer on the cells of the mammal is reduced.

Currently, methods for preparing calcium-phosphorus coatings on porous surfaces of metal implants are disclosed in the prior art that can improve their osseointegration properties. But the surface of the biological material which can promote the adhesion and growth of bone cells is also suitable for the colonization of bacteria, in particular to a porous titanium orthopedic implant which has the characteristics of strong osseointegration capability and individuation adaptation to complex-shaped parts, but the porous characteristic of the titanium implant greatly increases the integration surface area of bones and prostheses, which also increases the infection probability. The related infection of the orthopaedics endophyte is a common complication of the orthopaedics operation, which often causes delayed healing of bones, even nonunion of bones and loosening of endophytes, thereby causing prolonged use of antibiotics and even failure of the operation. Therefore, the titanium-based antibacterial agent is necessary to be further endowed with antibacterial function on the basis of titanium metal, so that the antibacterial agent can effectively avoid related infection of orthopedics endophytes, and is better applied to clinic.

Disclosure of Invention

Aiming at the problems that the metal implant in the prior art can promote osseointegration and increase the probability of bacterial infection, the invention provides an antibacterial bone-promoting difunctional titanium metal orthopaedics implant which has enhanced antibacterial property and bone-forming activity and can be widely used as clinical medical materials.

In order to solve the technical problems, the invention adopts the following technical scheme:

a preparation method of an antibacterial bone-promoting difunctional titanium metal orthopedic implant, which comprises the following steps:

(1) Performing surface treatment on medical titanium metal by using an anodic oxidation method, and forming a titanium dioxide nanotube array film on the surface of the titanium metal;

(2) Immersing the titanium metal treated in the step (1) into a phosphoric acid solution for room-temperature activation;

(3) Washing and drying the activated titanium metal, and immersing the titanium metal into CoCl ₂ ·6H ₂ Performing room temperature treatment in O aqueous solution, taking out and drying, and performing heat treatment in air at 300 ℃ for 1 hour to form a metal-phosphate double-layer film;

(4) And (3) placing the titanium metal treated in the step (3) into succinic anhydride aqueous solution to react for 0.5-1 h at the constant temperature of 20-40 ℃, adding EDC and NHS to activate surface carboxyl for 0.5-1 h, adding PHMB to react for 6-12 h, and cleaning and drying the obtained sample to obtain the antibacterial bone-promoting difunctional titanium metal orthopedic implant.

Further, in the step (1), the electrolyte used in the anodic oxidation method is NH ₄ F-glycol system, the voltage applied to the anode is 20-50V, and the anodic oxidation time is 1-4h.

Further, in the step (1), the pipe diameter of the prepared titanium dioxide nanotube array is 60nm-90nm.

Further, in the step (2), the mass concentration of the phosphoric acid solution is 0.5-5%, and the activation time is 1-4h.

Further, in step (3), the CoCl ₂ ·6H ₂ The concentration of the O aqueous solution is 0.1M-0.3M, and the room temperature treatment time is 1-3h.

Further, in the step (4), the mass ratio of succinic anhydride, EDC, NHS and PHMB is 1:1:1:0.1.

further, in the step (4), the concentration of the succinic anhydride aqueous solution is 0.01g/mL.

The antibacterial bone-promoting difunctional titanium metal orthopedic implant obtained by the invention has enhanced antibacterial property and bone-forming activity, and can be widely used as clinical medical materials.

The beneficial effects of the invention are as follows:

1. the invention grafts metal-phosphate double-layer film and PHMB on the surface of titanium implant with anodic oxidation coating, which are combined together, PHMB is firmly adsorbed and fixed on the surface of metal implant, thus the metal implant provided by the invention has stronger antibacterial ability, bacteria can be effectively killed after 3min of surface contact, and traditional endophytes can achieve the same effect after longer contact time.

2. According to the invention, PHMB is grafted on the surface of the metal-phosphate double-layer film of the metal implant subjected to anodic oxidation treatment, so that the metal implant can promote osseointegration, kill bacteria on and around the implant surface, and prevent the bacteria from adhering to the implant surface to form a biological film in a long time.

3. The proportion of the raw materials and the reaction temperature selected by the invention can ensure that the performance of titanium metal, especially the sterilization performance is optimal.

Drawings

FIG. 1 is an SEM image of a TNT-Co-PHMB sample obtained in example 1 of the present invention, wherein it can be seen that the TNT nanotube array has a PHMB coating on the surface.

FIG. 2 is an SEM image of a TNT-Co-PHMB sample obtained in example 2 of the present invention;

FIG. 3 is an SEM image of a TNT-Co-PHMB sample obtained in example 3 of the present invention;

FIG. 4 is an SEM image of the original TNT nanotube array obtained in comparative example 2, and TNT tube diameter was measured to be 70nm.

FIG. 5 is a WCA image of the sample obtained in each step of example 2.

FIG. 6 is an EDS mapping graph of the TNT-Co-PHMB sample obtained in example 2, wherein the distribution of three elements Co, N and P on the surface of the sample can be seen.

FIG. 7 is a photograph of a plate coated with the samples obtained in comparative examples 1 and 2 and TNT-Co-PHMB samples obtained in example 2 according to the present invention for sterilizing effect against Staphylococcus aureus at the same time.

Detailed Description

The following describes in detail the examples of the present invention, which are implemented on the premise of the technical solution of the present invention, and detailed embodiments and specific operation procedures are given, but the scope of protection of the present invention is not limited to the following examples.

Comparative example 1

The titanium sheet was cut into 10mm 1mm square pieces using high purity titanium having a purity of 99.99%. Sequentially polishing with 400# water-based abrasive paper, 600# water-based abrasive paper, 800# water-based abrasive paper, 1000# water-based abrasive paper and 1200# water-based abrasive paper until the surface is smooth, sequentially ultrasonically cleaning with acetone, absolute ethyl alcohol and pure water for 15min to remove oil stains on the surface, and then using the volume ratio HNO ₃ :HF:H ₂ And (3) ultrasonically polishing the titanium sheet by using chemical polishing solution with O=4:1:5 for 3min to obtain a titanium sheet with a smooth surface, and finally washing the titanium sheet with deionized water and drying the titanium sheet to obtain a pure Ti sheet serving as a control.

Comparative example 2

The titanium sheet was cut into 10mm 1mm square pieces using high purity titanium having a purity of 99.99%. Sequentially polishing with 400# water-based abrasive paper, 600# water-based abrasive paper, 800# water-based abrasive paper, 1000# water-based abrasive paper and 1200# water-based abrasive paper until the surface is smooth, sequentially ultrasonically cleaning with acetone, absolute ethyl alcohol and pure water for 15min to remove oil stains on the surface, and then using the volume ratio HNO ₃ :HF:H ₂ And (3) ultrasonically polishing the titanium sheet by using chemical polishing solution with O=4:1:5 for 3min to obtain a titanium sheet with a smooth surface, and finally washing the titanium sheet with deionized water, and drying the titanium sheet for later use.

Will be 0.887g NH ₄ F. 97mL of ethylene glycol and 3mL of deionized water were mixed to prepare an electrolyte, and then the electrolyte was slowly stirred with a magnetic stirrer at room temperature for 1h, and then a titanium sheet was immersed in the electrolyte as an anode and a platinum electrode as a cathode. Subsequently, a voltage of 30V was applied at 30 ℃ for 60 minutes to perform an oxidation process. After the sample was annealed at 450 ℃ for 1 hour, the sample was ultrasonically washed with deionized water and dried to obtain a TNT sheet as a control.

Example 1

The titanium sheet was cut into 10mm 1mm square pieces using high purity titanium having a purity of 99.99%. Sequentially polishing with 400# water-based abrasive paper, 600# water-based abrasive paper, 800# water-based abrasive paper, 1000# water-based abrasive paper and 1200# water-based abrasive paper until the surface is smooth, sequentially ultrasonically cleaning with acetone, absolute ethyl alcohol and pure water for 15min to remove oil stains on the surface, and then using the volume ratio HNO ₃ :HF:H ₂ O=4:1:5, to obtain a smooth-surface titanium sheet,and finally, washing with deionized water, and drying for later use.

Will be 0.887gNH ₄ F. 97mL of ethylene glycol and 3mL of deionized water were mixed to prepare an electrolyte, the electrolyte was slowly stirred with a magnetic stirrer at room temperature for 1h, and then a titanium sheet was immersed in the electrolyte as an anode and a platinum electrode as a cathode. Subsequently, a voltage of 30V was applied at 30 ℃ for 60 minutes to perform an oxidation process. After annealing the sample at 450 ℃ for 1h, the sample is ultrasonically cleaned by deionized water and dried for later use.

The sample was immersed in a solution containing 5mL of 0.5% H at room temperature ₃ PO ₄ The solution was sealed in a glass bottle for 60 minutes. The sample was then rinsed with deionized water and dried with nitrogen. The sample was then immersed in 5mL of CoCl at a concentration of 0.1M ₂ ·6H ₂ O aqueous solution for 120min. After removal from the solution, the sample was thoroughly rinsed with deionized water and dried with nitrogen. The sample was heat treated in air at 300 ℃ for 1 hour to form a metal-phosphate bilayer film.

The sample was placed in 10mL of 0.01g/mL succinic anhydride aqueous solution at a constant temperature of 30℃and reacted for 0.5h, then 0.1g of EDC and 0.1g of NHS were added to activate the surface carboxyl groups for 0.5h, and finally 0.01g of PHMB was added to react for 6h. After washing the sample with pure water, drying at 60 ℃ for 24 hours to obtain TNT of PHMB covalent grafted metal-phosphate double-layer film, which is named TNT-Co-PHMB.

Example 2

Will be 0.887gNH ₄ F. 97mL of ethylene glycol and 3mL of deionized water were mixed to prepare an electrolyte, the electrolyte was slowly stirred with a magnetic stirrer at room temperature for 1h, and then a titanium sheet was immersed in the electrolyte as an anode and a platinum electrode as a cathode. Subsequently, 3 is applied at 30 DEG CThe oxidation process was performed at 0V for 60 minutes. After annealing the sample at 450 ℃ for 1h, the sample is ultrasonically cleaned by deionized water and dried for later use.

The sample was immersed in a solution containing 5mL of 1% H at room temperature ₃ PO ₄ The solution was sealed in a glass bottle for 60 minutes. The sample was then rinsed with deionized water and dried with nitrogen. The sample was then immersed in 5mL of CoCl at a concentration of 0.1M ₂ ·6H ₂ O aqueous solution for 120min. After removal from the solution, the sample was thoroughly rinsed with deionized water and dried with nitrogen. The sample was heat treated in air at 300 ℃ for 1 hour to form a metal-phosphate bilayer film (noted TNT-Co).

Example 3

The sample was immersed in a solution containing 5mL of 2% H at room temperature ₃ PO ₄ The solution was sealed in a glass bottle for 60 minutes. Then, usingThe sample was rinsed with deionized water and dried with nitrogen. The sample was then immersed in 5mL of CoCl at a concentration of 0.1M ₂ ·6H ₂ O aqueous solution for 120min. After removal from the solution, the sample was thoroughly rinsed with deionized water and dried with nitrogen. The sample was heat treated in air at 300 ℃ for 1 hour to form a metal-phosphate bilayer film.

SEM images of the TNT-Co-PHMB samples obtained in examples 1, 2 and 3 are shown in fig. 1, 2 and 3, respectively, and SEM images of the titanium metal surface titanium dioxide nanotube array obtained in comparative example 2 are shown in fig. 4. It can be seen from the figure that the titanium dioxide nanotubes of the products obtained in examples 1 and 2 have no significant change in morphology compared to comparative example 2, while example 3 has a significant change in morphology due to the higher concentration of phosphoric acid used in the treatment.

FIG. 5 is a WCA image of the sample obtained in the steps of example 2, in which the hydrophilicity of the surface of the anodized material is significantly improved, while the hydrophilicity after grafting is slightly reduced. Therefore, the titanium dioxide nanotube array morphology can greatly improve the hydrophilicity of titanium metal, and is beneficial to improving the biocompatibility of the titanium metal. The morphology of the sample obtained in example 2 is not greatly changed compared with that of the sample obtained in comparative example 2, and the hydrophilicity is not greatly different from that of the sample obtained in comparative example 2; whereas the sample of example 3 resulted in a large change in the morphology of the tube due to the higher concentration of phosphoric acid, which was detrimental to the biocompatibility of the material.

As shown in FIG. 6, the TNT-Co-PHMB sample obtained in example 2 contains N, co and P elements, and is uniformly distributed, which indicates that the grafting of the metal-phosphate double-layer film and PHMB on the titanium metal surface is successful.

The bactericidal effect of the samples obtained in the examples above was tested as follows:

a. preparation before experiment: preparing a plurality of 15mL glass test tubes, a plurality of 5mL glass test tubes, a plurality of 1.5mL centrifuge tubes, lactose bile salt fermentation medium (LB), lactose bile salt agar fermentation solid medium and PBS buffer solution, performing high-pressure steam sterilization (121 ℃ for 40 min) completely, standing for later use, cooling the lactose bile salt agar fermentation solid medium to 60 ℃, pouring the flat plate, and waiting for solidification to form a solid culture dish.

b. Shaking: adding 10-12mL of lactose bile salt fermentation medium (LB) into 15mL glass test tube, inoculating Escherichia coli or Staphylococcus aureus, culturing at 37deg.C under 160r/min for 18-20 hr with constant temperature shaker to obtain colony count of about 5×10 ⁸ CFU/mL。

c. 3 mu L of the bacterial liquid obtained in the step b is sucked by a pipette, added into 4mL of deionized water and uniformly shaken.

d. Taking 70 mu L of the bacterial suspension obtained in the step c, and uniformly mixing in 70mLPBS buffer solution to obtain a mixed solution; samples prepared in each example were placed in clean dishes.

e. mu.L of the bacterial dilution obtained in step c was taken and added dropwise to the surface of the samples prepared in each example. Placing the sample into a centrifuge tube after 3min, 5min and 7min respectively, ultrasonically collecting bacterial liquid, coating a flat plate, placing a culture dish into a constant temperature incubator for culture (the culture temperature is 30-37 ℃ C., and the culture time is 24-36 h), and counting.

f. After the experiment is finished, the used sample of the example 2 is recovered, deionized water is used for washing the surface of the sample, the experiment steps are repeated after the sample is naturally air-dried, and the colony number is recorded.

The results of the sterilization performance test of the samples obtained in each example are shown in tables 1 and 2.

TABLE 1 Sterilization test on E.coli

Sample of	0min	3min	5min	7min	Sterilization rate
						TNT-Co-PHMB of example 1	277	97	0	0	100％
TNT-Co of example 2	322	298	283	275	14.6％
						TNT-Co-PHMB of example 2	254	76	0	0	100％
Ti of comparative example 1	324	320	313	308	4.9％
						TNT of comparative example 2	289	277	255	254	11％
Example 2 (post recovery secondary test)	314	110	0	0	100％
						Example 2 (three trials after recovery)	300	121	1	0	100％

TABLE 2 Sterilization test on Staphylococcus aureus

Comparison shows that the TNT-Co-PHMB sample obtained in each example has significantly better performance than TNT obtained in comparative example 2. The material can completely kill bacteria within 5 min. The TNT obtained in comparative example 2 has a poor sterilizing effect compared with the TNT-Co-PHMB sample obtained in example 2 because of the lack of PHMB, which does not have a rapid and efficient sterilizing effect. Meanwhile, the TNT-Co-PHMB has no reduction of antibacterial performance after repeating the experiment for three times, and has excellent stability.

It is apparent that the above examples are given by way of illustration only and are not limiting of the embodiments. Any person skilled in the art may make variations or modifications to the equivalent embodiments using the teachings disclosed above. However, any simple modification, equivalent variation and variation of the above embodiments according to the technical substance of the present invention still fall within the protection scope of the technical solution of the present invention.

Claims

1. The preparation method of the antibacterial bone-promoting difunctional titanium metal orthopedic implant is characterized by comprising the following steps of:

(1) Performing surface treatment on medical titanium metal by using an anodic oxidation method, and forming a titanium dioxide nanotube array film on the surface of the titanium metal; the electrolyte used in the anodic oxidation method is NH ₄ F-glycol system, wherein the voltage applied to the anode is 20-50V, and the anodic oxidation time is 1-4h;

(2) Immersing the titanium metal treated in the step (1) into a phosphoric acid solution with the mass concentration of 0.5% -5% and activating for 1-4h at room temperature;

(3) Washing and drying the activated titanium metal, and immersing the titanium metal into CoCl with the concentration of 0.1M-0.3M ₂ ·6H ₂ Performing room temperature treatment for 1-3h in O aqueous solution, taking out and drying, and performing heat treatment in air at 300 ℃ for 1h to form a metal-phosphate double-layer film;

2. The method of manufacturing according to claim 1, characterized in that: in the step (1), the pipe diameter of the prepared titanium dioxide nanotube array is 60nm-90nm.

3. The method of manufacturing according to claim 1, characterized in that: in the step (4), the mass ratio of succinic anhydride, EDC, NHS and PHMB is 1:1:1:0.1.

4. the method of manufacturing according to claim 1, characterized in that: in the step (4), the concentration of the succinic anhydride aqueous solution is 0.01g/mL.

5. An antibacterial bone-promoting bifunctional titanium orthopedic implant obtained by the method of any one of claims 1 to 4.