CN111821506A

CN111821506A - Preparation of strontium/silver nano-coating modified bone bionic titanium implant

Info

Publication number: CN111821506A
Application number: CN202010549171.5A
Authority: CN
Inventors: 王海燕; 屈海忠; 姚李韬; 胡荣党; 史春玲
Original assignee: SCHOOL & HOSPITAL OF STOMATOLOGY WENZHOU MEDICAL UNIVERSITY
Current assignee: SCHOOL & HOSPITAL OF STOMATOLOGY WENZHOU MEDICAL UNIVERSITY
Priority date: 2020-06-16
Filing date: 2020-06-16
Publication date: 2020-10-27

Abstract

The invention discloses a preparation method of a strontium/silver nano coating modified bone bionic titanium implant, which comprises the steps of modifying the surface of a titanium material by a magnetron sputtering method, performing thermokalite treatment, and then sequentially coating two layers of nano-sized films containing silver element and strontium element on the surface to obtain the bone bionic titanium implant with good biocompatibility, osteogenesis promotion induction function and good antibacterial property. The magnetron sputtering method has the advantages of simple process, small damage to the matrix and the like.

Description

Preparation of strontium/silver nano-coating modified bone bionic titanium implant

Technical Field

The invention relates to the field of titanium material modification, in particular to preparation of a strontium/silver nano-coating modified bone bionic titanium implant.

Background

Titanium is a silvery white transition metal characterized by light weight, high strength, metallic luster, and resistance to wet chlorine corrosion. Titanium and titanium alloy are the most common biomaterials for orthodontic implantation nails, prosthetic implants, bone repair and the like because of excellent biocompatibility, chemical stability, mechanical property and better osseointegration capability, and titanium implants are the gold standard for implant repair.

However, the titanium implant has no antibacterial performance, and has certain phenomena of sensitization, inflammation, loosening and shedding and the like, so that the application of the titanium implant in the biological field is limited. In addition, patients with osteoporosis are often encountered in clinical application, the healing time and the osseointegration rate of the implant are seriously influenced by the density and the quantity of trabeculae caused by the osteoporosis condition, and the conventional titanium implant has difficult anti-osteoporosis effect. The effective material modification method is sought to ensure that the material has better biocompatibility and better osteogenic differentiation promoting effect and bone resorption resisting capability so as to improve the osseointegration capability of the material, and the improvement of the planting success rate is a target which needs to be explored and pursued in clinic all the time. Peri-implantitis is a major cause of implant failure in implant surgery, in addition to the severe effects of osteoporotic conditions on implant osseointegration. The oral cavity is a microenvironment with coexistence of multiple floras, and infection may be caused by operation, aseptic control and the like in the implantation operation, and infection around the implant is also caused by personal care and the like after the implant is implanted. The formation of the bacterial plaque biofilm on the implant surface not only causes inflammation around the implant and new bone formation obstacle, but also causes the falling of the implant, and in severe cases, bacteria enter the systemic blood circulation to cause more serious systemic diseases.

Silver has been demonstrated to have optimal antibacterial ability in metallic elements, however its significant biotoxicity limits its clinical use. The biological toxicity mainly comes from the quick release of silver ions and the contact killing effect of the nano silver particles on the surface. Therefore, it is very important to promote bone union and prevent peri-implant inflammation, and it is a major challenge in clinical research to prepare an implant that has the multifunctional effects of promoting bone formation, inhibiting osteoclasts, resisting bacteria and inflammation.

Disclosure of Invention

Aiming at the defects in the prior art, the invention provides a preparation method of a strontium/silver nano coating modified bone bionic titanium implant, which can be used for obtaining the titanium implant respectively coated with nano-sized two films containing strontium and silver elements, has good biocompatibility, osteogenesis induction promotion function and good antibacterial performance, and has the advantages of simple process and small damage to a matrix.

The invention specifically adopts the technical scheme that:

the method comprises the following steps: and (4) preprocessing. Grinding and polishing the titanium material by using sand paper, and then washing and drying;

step two: and (4) alkali treatment. Placing the titanium material treated in the first step into an alkali solution for reaction for 20-30 h, and then washing, drying and vacuum-storing to obtain a standby titanium material;

step three: and constructing the nano silver coating. Taking metal silver as a target source, and sputtering the surface of the standby titanium material by utilizing magnetron sputtering to construct a nano silver coating;

step four: further build up strontium titanate coating. And further sputtering a strontium titanate coating on the surface of the nano-silver coating by using magnetron sputtering by taking strontium titanate as a target source to obtain the strontium/silver double-layer film coating.

Preferably, the specific method of grinding and polishing is to grind and polish the surface of the titanium material by using 600-mesh, 800-mesh, 1500-mesh and 2000-mesh sandpaper in sequence.

Preferably, the washing method comprises the step of sequentially carrying out ultrasonic cleaning by using acetone, ethanol and deionized water.

Preferably, the temperature of the alkali treatment is 70-90 ℃.

Preferably, the working conditions of magnetron sputtering in the third step are as follows: firstly, pre-sputtering is carried out, wherein parameters are set to be pressure intensity of 1Pa, power of 80W, temperature of 20-25 ℃ and time of 5 min; and then, carrying out working sputtering, wherein the parameters are set to be 0.5Pa, 80W of power, 20-25 ℃ and 40-80 s of time.

Preferably, the working conditions of the magnetron sputtering in the fourth step are as follows: firstly, pre-sputtering is carried out, wherein parameters are set to be pressure intensity of 1Pa, power of 80W, temperature of 20-25 ℃ and time of 5 min; and then, carrying out working sputtering, wherein the parameters are set to be 0.5Pa of pressure, 80W of power, 20-25 ℃ of temperature and 80-100 min of time.

The invention has the beneficial effects that:

the natural bone tissue not only contains a micron-scale structure but also contains a nano-scale structure, and the microenvironment of the cells in the bone also contains a large amount of micron-scale and nano-scale information, so that from the bionic angle, the material with the bionic micro-nano interface of the trabecular bone can simulate the natural bone tissue structure vividly, thereby being beneficial to the formation of new bone and improving the bone combination rate.

According to the invention, through hot alkali treatment, a sponge-like net structure is formed on the surface of the titanium material, so that osteoblasts can be mineralized more favorably, and the implant can be endowed with better osteogenesis induction promoting capability. And then a nano-sized double-layer coating containing strontium and silver elements is successfully constructed on the surface of the prepared micron material by magnetron sputtering equipment.

Namely: the surface of the bone bionic titanium implant modified by the method is of a multi-stage micro-nano structure, so that more extracellular matrix deposition can be generated, the micro-nano structure can obviously change the surface appearance, increase the surface roughness, improve the torque, improve the stability of the implant in vivo and enhance the mechanical locking between the implant and the tissue, and meanwhile, the increase of the surface roughness can enhance fibronectin adsorption and cell attachment by inducing the assembly of membrane receptor tissue and cytoskeleton. In addition, the nano-structured surface can also directly influence the interaction between the implant and the cells, thereby remarkably promoting osteoblast adhesion, proliferation and osteogenic differentiation, and compared with a single micro-morphology, the multi-level micro-nano structured surface is more effective in inducing cell differentiation and promoting osteointegration, can effectively enhance fixation and attachment of living tissue, and induces initial cell adhesion and osteointegration, and the multi-level micro-nano structured morphology can accurately reflect the structure of a natural extracellular matrix, and can provide a more appropriate interface for cell growth.

With respect to the introduced elements: the strontium element has good osteogenic differentiation promoting ability and bone combination enhancing ability. The antibacterial effect of the nano silver particles is obviously superior to that of silver materials with mesoscopic and macroscopic scales. Therefore, the surface appearance of the multi-level micro-nano structure is constructed by carrying out magnetron sputtering silver on the titanium sheet after the thermokalite treatment and then carrying out magnetron sputtering on the strontium titanate coating so as to obtain the bone bionic structure, and the bone bionic titanium implant subjected to surface modification has excellent performances in the aspects of osteogenesis, antibiosis, anti-inflammation, osteoporosis resistance and bone combination promotion. The concrete expression is as follows: the coating of the silver coating and the strontium coating further improves the biocompatibility of the material, promotes the osteogenesis induction function and has good antibacterial capacity. The covering of the strontium titanate layer on the surface of the silver coating can reduce the early quick release of silver and improve the biocompatibility. Strontium ions and the surface microstructure can synergistically promote osteogenesis induction capability, and the nano silver coating can release silver ions for a long time and exert remarkable antibacterial capability under the protection of the strontium titanate coating.

In addition, compared with other coating preparation processes, the magnetron sputtering technology has wide application range on the target and the substrate, and is convenient for introducing new elements. Under the magnetron sputtering process, the formed coating has the advantages of strong bonding strength, uniform and compact film forming thickness and the like, the process method is simple, the damage to the matrix is small, the coating with the nano-scale size can be formed on the surface of the matrix material, and target particles can be deposited on the surface of the substrate material through high-energy sputtering under the action of an external high-energy magnetic field, so that the coating and the substrate have very high bonding strength, and the good bonding strength also provides a powerful guarantee for the coating to be implanted into a body to play a biological role.

Drawings

The invention will be further explained with reference to the drawings.

FIG. 1 shows SEM characterization results of modified titanium materials of example 1, comparative example 2, comparative example 6, and comparative examples 8 to 9 (comparative example 9 is a blank control);

fig. 2 shows the cell activity (/ p <0.05,/p <0.01) of the modified titanium materials of examples 1, 2, 6 and 8 to 9 (comparative example 9 is a blank) after 5 days of MC3T3-E1 cell culture;

fig. 3 is a data statistics of the dead and alive staining of surface bacteria of the modified titanium materials of example 1, comparative example 2, comparative example 6, and comparative examples 8 to 9 (comparative example 9 is a blank control) (. p <0.05,. p < 0.01);

FIG. 4 shows the alkaline phosphatase activity test of the modified titanium materials of example 1, comparative example 2, comparative example 6 and comparative examples 8 to 9 (comparative example 9 is a blank control) on preosteoblasts MC3T3-E1 ([ p ] 0.05, [ p ] 0.01);

FIG. 5 is a plot of the results of the spectral surface scan of the surfaces of the modified titanium materials of examples 1, 2, 6, and 8-9 (comparative example 9 is a blank control) showing the distribution of different elements.

FIG. 6 is a SEM characterization result of the nanometer silver coating thermokalite titanium material of comparative examples 1-4;

FIG. 7 shows the cell activity of MC3T3-E1 cells cultured for 5 days on the surface of the nano silver coated thermokalite titanium material of comparative examples 1-4 and 8-9 (comparative example 9 is blank control);

FIG. 8 shows the antibacterial activity of the nano-silver coated thermokalite titanium material of comparative examples 1 to 4 and 8 to 9 (comparative example 9 is a blank control) against Staphylococcus aureus in 24 hours;

FIG. 9 shows SEM and AFM characterization results of strontium titanate coated hot-alkali titanium materials of comparative examples 5 to 7;

FIG. 10 shows fluorescence and SEM images of growth of MC3T3-E1 cells cultured for 5 days on the surface of strontium titanate coated hot alkali titanium material of comparative examples 5-7;

FIG. 11 shows the activity and osteogenic differentiation of MC3T3-E1 cells cultured for 5 days on the surface of strontium titanate coated thermokalite titanium material of comparative examples 5-7;

FIG. 12 is a three-dimensional image of osseointegration of the modified titanium materials of comparative example 6 and comparative example 8 in osteoporosis and a common rat model;

FIG. 13 is a result of statistical analysis of data on the osseointegration of the modified titanium materials of comparative example 6 and comparative example 8 in osteoporosis and common rat models;

FIG. 14 shows the new bone formation of the modified titanium materials of comparative example 6 and comparative example 8 in the HE staining and MASSON staining of osteoporosis and common rat models;

Detailed Description

The present invention will be further described with reference to the following embodiments.

Example 1:

the method comprises the following steps: and (4) preprocessing. Sequentially grinding and polishing the surface of a titanium material by 600-mesh, 800-mesh, 1500-mesh and 2000-mesh sand paper, sequentially ultrasonically cleaning by acetone, ethanol and deionized water, and drying;

step two: and (4) alkali treatment. Placing the titanium material treated in the first step in an alkali solution at the temperature of 80 ℃ for reaction for 24 hours, taking out a sample after the reaction is finished, and then washing, drying and storing in vacuum to obtain a spare titanium material;

step three: and constructing the nano silver coating. And sputtering the surface of the standby titanium material by using magnetron sputtering to construct a nano silver coating by taking the metal silver as a target source. Firstly, pre-sputtering is carried out, and parameters are set to be 1Pa, 80W of power, 23 ℃ and 5 min; then, working sputtering is carried out, and parameters are set to be 0.5Pa, 80W of power, 23 ℃ and 1min of time.

Step four: further build up strontium titanate coating. Further sputtering a strontium titanate coating on the surface of the nano-silver coating by using magnetron sputtering by taking strontium titanate as a target source, firstly carrying out pre-sputtering, setting parameters to be pressure 1Pa, power 80W, temperature 23 ℃ and time 5 min; and then, carrying out working sputtering, wherein the parameters are set to be 0.5Pa, 80W of power, 23 ℃ and 90min of time, and finally obtaining the strontium/silver double-layer film coating.

Comparative example 1:

step three: and constructing the nano silver coating. And sputtering the surface of the standby titanium material by using magnetron sputtering to construct a nano silver coating by taking the metal silver as a target source. Firstly, pre-sputtering is carried out, and parameters are set to be 1Pa, 80W of power, 23 ℃ and 5 min; working sputtering was then carried out with the parameters set to a pressure of 0.5Pa, a power of 80W, a temperature of 23 ℃ and a time of 15 s.

Comparative example 2:

step three: and constructing the nano silver coating. And sputtering the surface of the standby titanium material by using magnetron sputtering to construct a nano silver coating by taking the metal silver as a target source. Firstly, pre-sputtering is carried out, and parameters are set to be 1Pa, 80W of power, 23 ℃ and 5 min; subsequently, working sputtering was carried out with the parameters set to a pressure of 0.5Pa, a power of 80W, a temperature of 23 ℃ and a time of 60 s.

Comparative example 3:

step three: and constructing the nano silver coating. And sputtering the surface of the standby titanium material by using magnetron sputtering to construct a nano silver coating by taking the metal silver as a target source. Firstly, pre-sputtering is carried out, and parameters are set to be 1Pa, 80W of power, 23 ℃ and 5 min; working sputtering was then carried out with the parameters set to a pressure of 0.5Pa, a power of 80W, a temperature of 23 ℃ and a time of 120 s.

Comparative example 4:

step three: and constructing the nano silver coating. And sputtering the surface of the standby titanium material by using magnetron sputtering to construct a nano silver coating by taking the metal silver as a target source. Firstly, pre-sputtering is carried out, and parameters are set to be 1Pa, 80W of power, 23 ℃ and 5 min; working sputtering was then carried out with the parameters set to a pressure of 0.5Pa, a power of 80W, a temperature of 23 ℃ and a time of 300 s.

Comparative example 5:

step three: and constructing a strontium titanate coating. Further sputtering a strontium titanate coating on the surface of the nano-silver coating by using magnetron sputtering by taking strontium titanate as a target source, firstly carrying out pre-sputtering, setting parameters to be pressure 1Pa, power 80W, temperature 23 ℃ and time 5 min; then, working sputtering is carried out, and parameters are set to be 0.5Pa, 80W of power, 23 ℃ and 30min of time.

Comparative example 6:

step three: and constructing a strontium titanate coating. Further sputtering a strontium titanate coating on the surface of the nano-silver coating by using magnetron sputtering by taking strontium titanate as a target source, firstly carrying out pre-sputtering, setting parameters to be pressure 1Pa, power 80W, temperature 23 ℃ and time 5 min; then, working sputtering is carried out, and the parameters are set to be 0.5Pa, 80W of power, 23 ℃ and 90min of time.

Comparative example 7:

step three: and constructing a strontium titanate coating. Further sputtering a strontium titanate coating on the surface of the nano-silver coating by using magnetron sputtering by taking strontium titanate as a target source, firstly carrying out pre-sputtering, setting parameters to be pressure 1Pa, power 80W, temperature 23 ℃ and time 5 min; then, working sputtering is carried out, and parameters are set to be 0.5Pa, 80W of power, 23 ℃ and 150min of time.

Comparative example 8:

step two: and (4) alkali treatment. And (3) placing the titanium material treated in the step one in an alkali solution at the temperature of 80 ℃ for reaction for 24h, taking out a sample after the reaction is finished, and then washing, drying and storing in vacuum.

Comparative example 9:

the method comprises the following steps: the surface of the titanium material is sequentially polished by 600-mesh, 800-mesh, 1500-mesh and 2000-mesh sandpaper, and then is sequentially ultrasonically cleaned by acetone, ethanol and deionized water, and dried.

Next, the modified titanium materials of example 1 and comparative examples 1 to 9 (comparative example 9 is a blank) were characterized and examined.

Detecting the topography of the sample surface using a Scanning Electron Microscope (SEM); detecting the crystalline phase structure of the surfaces of different materials by X-ray diffraction (XRD); detecting the element composition of different sample surfaces by an energy spectrometer (EDS); detecting the hydrophilic change of the surfaces of the materials with different coating thicknesses by using the water contact angle; and detecting the surface roughness of the material by an Atomic Force Microscope (AFM).

The performance of the products obtained in the examples and the comparative examples was evaluated by in vitro cytology.

The samples were incubated for 4h and 24h in a cell culture incubator by inoculating MC3T3-E1 cells and Staphylococcus aureus on the sterilized samples, after removal of the samples, rinsed with PBS, and analyzed by staining DEAD and LIVE cells and bacteria with LIVE/DEAD staining kit (Thermo, American).

TABLE 1 comparative table of example 1 and comparative examples 1 to 9

Wherein "AH" represents "hot alkali treatment".

The strontium titanate coating is successfully constructed on the surface of the nano-silver coating by magnetron sputtering equipment, so that AH-Ti/Ag/Sr with the silver and strontium double coatings is prepared. The shapes of the titanium materials obtained by each group are observed through a scanning electron microscope, and as shown in figures 1 and 2, the bone bionic micro-nano interface is successfully constructed. After further in vitro cell activity experiments, it was found that: the silver coating is still less conducive to cell proliferation than titanium alone, whereas the coverage of the strontium coating significantly reduces the early cell activity inhibition of the silver coating and contributes to cell proliferation. The phenomenon is probably related to the slow release of silver ions caused by the coverage of the strontium titanate coating and the synergistic osteogenesis promoting effect of the surface bionic micro-nano interface and the strontium ions.

In order to further verify the superiority of the process, firstly, the inventor makes the surface of the titanium material form a sponge-like reticular structure through hot alkali treatment, so that osteoblasts are more favorably mineralized, and the implant is endowed with better osteogenesis promotion induction capability. And then, successfully constructing nano silver coatings with different thicknesses on the surfaces of the prepared micron materials by magnetron sputtering equipment, controlling the power to be 80W, changing the thicknesses by utilizing time change, setting the working sputtering time to be 15s, 60s, 120s and 300s respectively, referring to the comparative examples 1-4 for specific parameters, respectively representing the obtained modified titanium materials as AH-Ti/Ag15, AH-Ti/Ag60, AH-Ti/Ag120 and AH-Ti/Ag300, and observing the morphology structures of the titanium materials obtained in each group by a scanning electron microscope, referring to FIG. 6. Further, in vitro cell activity experiments confirmed that the strontium titanate coating layer sputtered for 60s had good antibacterial activity without significant cytotoxicity, see fig. 7, fig. 8. From the above experimental results, it can be seen that AH-Ti/Ag60 has significant antibacterial ability without significantly affecting cell activity; the AH-Ti/Ag120 and AH-Ti/Ag300 have obvious activity inhibition effect on cells, and the AH-Ti/Ag15 group has weak antibacterial activity and the antibacterial rate is only about 40 percent although no obvious cytotoxicity is generated.

In addition, the inventor successfully constructs nano strontium titanate coatings with different thicknesses on the surface of the prepared micron material by magnetron sputtering equipment. The power is controlled to be stabilized at 80W, the thickness is changed by utilizing time change, the working sputtering time is respectively set to be 30min, 90min and 150min, specific parameters are shown in comparative examples 5-7, the obtained modified titanium materials are respectively expressed as AH-Ti/Sr30, AH-Ti/Sr90 and AH-Ti/Sr150, and the shape and structure of each group of the obtained titanium materials are observed by a scanning electron microscope, and the figure 9 is shown. Fluorescence topography it was observed that the surface of the strontium titanate (AH-Ti/Sr90) coating sputtered for 90 minutes had better growth with more filopodia and larger spreading area, see fig. 10. In vitro cell experiments show that the strontium titanate coating sputtered for 90 minutes has the optimal cell activity and ALP activity, promotes the osteogenesis mineralization level, remarkably promotes the expression of osteogenesis related genes such as ALP, RUNX2, OCN and COL-1 (figure 11), and confirms that the coating (AH-Ti/Sr90) prepared for 90 minutes shows the optimal bone promotion capability. In vivo animal experiments, in MASSON staining, a thick and continuous band of new bone formation (shown by arrows in FIG. 14, second row) was present around the AH-Ti/Sr90 group. HE staining showed that new bone formation of the AH-Ti/Sr90 group was significantly thickened and continuous (fig. 14, arrows in the first row). Thus, the AH-Ti/Sr90 group was further confirmed to have significant in vivo osteogenesis-promoting effects by Micro-CT (fig. 12, 13) and tissue section (fig. 14).

In summary, the application combines the nano-silver coating with the strontium titanate micro-nano coating through thermokalite treatment and secondary magnetron sputtering, so as to construct a multi-level micro-nano structure surface morphology on the pure titanium surface to obtain a bone bionic structure (AH-Ti/Ag/Sr, embodiment 1). The biocompatibility of the material is improved by coating the silver coating and the strontium coating, the osteogenesis inducing function is promoted, and the antibacterial material has good antibacterial capacity. The covering of the strontium titanate layer on the surface of the silver coating can reduce the early quick release of silver and improve the biocompatibility. Strontium ions and the surface microstructure can synergistically promote osteogenesis induction capability, and the nano silver coating can release silver ions for a long time and exert remarkable antibacterial capability under the protection of the strontium titanate coating.

Other embodiments of the present invention than the preferred embodiments described above, and those skilled in the art can make various changes and modifications according to the present invention without departing from the spirit of the present invention, should fall within the scope of the present invention defined in the claims.

Claims

1. The preparation method of the bone bionic titanium implant modified by the strontium/silver nano coating is characterized by comprising the following steps: the method comprises the following steps:

2. The preparation method of the strontium/silver nano-coating modified bone biomimetic titanium implant according to claim 1, characterized in that: the specific method for grinding and polishing comprises the step of grinding and polishing the surface of the titanium material by using 600-mesh, 800-mesh, 1500-mesh and 2000-mesh sandpaper in sequence.

3. The preparation method of the strontium/silver nano-coating modified bone biomimetic titanium implant according to claim 1, characterized in that: the washing method comprises the step of sequentially carrying out ultrasonic cleaning by using acetone, ethanol and deionized water.

4. The preparation method of the strontium/silver nano-coating modified bone biomimetic titanium implant according to claim 1, characterized in that: the temperature of the alkali treatment is 70-90 ℃.

5. The preparation method of the strontium/silver nano-coating modified bone biomimetic titanium implant according to claim 1, characterized in that: the working conditions of the magnetron sputtering in the third step are as follows: firstly, pre-sputtering is carried out, wherein parameters are set to be pressure intensity of 1Pa, power of 80W, temperature of 20-25 ℃ and time of 5 min; and then, carrying out working sputtering, wherein the parameters are set to be 0.5Pa, 80W of power, 20-25 ℃ and 40-80 s of time.

6. The preparation method of the strontium/silver nano-coating modified bone biomimetic titanium implant according to claim 1, characterized in that: the working conditions of the magnetron sputtering in the fourth step are as follows: firstly, pre-sputtering is carried out, wherein parameters are set to be pressure intensity of 1Pa, power of 80W, temperature of 20-25 ℃ and time of 5 min; and then, carrying out working sputtering, wherein the parameters are set to be 0.5Pa of pressure, 80W of power, 20-25 ℃ of temperature and 80-100 min of time.