CN115522207A - A preparation method for constructing PLL/CPP-ACP self-assembled multilayer film on the surface of sandblasted and acid-etched titanium - Google Patents

Abstract

The invention discloses a preparation method for constructing a PLL/CPP-ACP self-assembled multilayer film on a sand-blasting acid-etched titanium surface, and belongs to the field of materials. The method comprises the steps of grinding and polishing pure titanium, sand blasting, ultrasonic cleaning and drying; carrying out acid etching on the dried pure titanium at normal temperature by adopting hydrofluoric acid/nitric acid mixed solution, then carrying out ultrasonic cleaning and drying, then carrying out acid etching on the dried pure titanium at the water bath temperature of 75-85 ℃ by using concentrated hydrochloric acid/concentrated sulfuric acid mixed acid, carrying out ultrasonic cleaning and drying to form a micron structure on the titanium surface, thus obtaining an acid-etched titanium material; and adding the acid-etched titanium material into a sodium hydroxide solution for alkalization, placing the alkalized titanium sheet into a PLL solution, and then alternately and repeatedly immersing the titanium sheet pretreated by the PLL into the CPP-ACP solution and the PLL solution to obtain the target product. The method has simple process, can form the titanium surface nanostructure and load the bioactive calcium ions only by sodium hydroxide alkali heat treatment and self-assembly technology, has simple required equipment and low cost, and the titanium surface has the nanostructure and calcium element modification.

Description

A method for constructing PLL/CPP-ACP self-assembled multilayer film on the surface of sandblasted and acid-etched titanium preparation method

technical field

The invention belongs to the field of biological materials. Specifically relates to a preparation method for constructing a PLL/CPP-ACP self-assembled multilayer film on the surface of sandblasted and acid-etched titanium.

Background technique

Titanium and titanium alloy materials are widely used in orthopedics and dental medical fields, such as bone fixation devices and implants, due to their good corrosion resistance, biocompatibility and mechanical properties. However, titanium materials are biologically inert and lack significant bioactivity when implanted into bone tissue, so rapid osseointegration remains a great challenge. Numerous attempts have been made to modify titanium surfaces to induce rapid bone growth. Alkaline heat treatment is one of the most commonly used treatments, which can form a modified micron or nanometer titanium surface, promote osteoblast alkaline phosphatase and collagen synthesis on the titanium surface, and induce bone formation. Layer-by-layer self-assembly technology is one of the most efficient and promising approaches to prepare bioactive multilayer films with controllable thickness and molecular structure, and has great potential for commercialization [F.X.Xiao, M.Pagliaro, Y.J.Xu, B. Liu, Layer-by-layer assembly of versatile nanoarchitectures with diverse dimensionality: a new perspective for rational construction of multilayer assemblies, Chemical Society Reviews45(11)(2016)3088-3121].

The -ser(P)-ser(P)-ser(P)-glu-glu-sequence contained in CPP has a high affinity with divalent metal ions, so it can stabilize ACP to form a CPP-ACP complex, which is a stable calcium-phosphorus remineralization system. As a storehouse of calcium and phosphorus, CPP-ACP can significantly increase the bioavailability of these two ions, and maintain a higher concentration of calcium ions, phosphate ions or ion pairs in the solution [N.L.Huq, K.J.Cross, E.C. Reynolds, Molecular modeling of the multiphosphorylated casein phosphopeptide alphaS1-casein (59-79) based on NMR constraints, The Journal of dairy research 71(1)(2004)28-32.]. Calcium is an important trace element that can promote cell proliferation and differentiation, has a direct impact on osteoblast differentiation and bone tissue development, and is an effective regulator of various cell activities such as osteocalcin and collagen expression [E.Gabusi ,C.Manferdini,F.Grassi,A.Piacentini,L.Cattini,G.Filardo,E.Lambertini,R.Piva,N.Zini,A.Facchini,G.Lisignoli,Extracellular calcium chronically induced human osteoblastseffects:Specific modulation of osteocalcin and collagen type XV, Journal of cellular physiology 227(8)(2012) 3151-3161].

In the prior art, the methods for forming micro- and nano-scale microstructures and depositing calcium ions are relatively complicated, the required equipment is relatively complicated, the technology sensitivity is high, the processing time is long, and the surface particle size of the preparation is relatively coarse.

Contents of the invention

Technical problem to be solved: the present invention aims at the deficiencies of the above-mentioned prior art, and provides a preparation method for constructing a PLL/CPP-ACP self-assembled multilayer film on the surface of sandblasted and acid-etched titanium, which does not require complex equipment and has low technical sensitivity. The titanium sample prepared by the invention has nano structure and calcium element modification, simple process, easy processing and low preparation cost.

Technical solutions:

A preparation method for constructing a PLL/CPP-ACP self-assembled multilayer film on a sandblasted acid-etched titanium surface, the method comprising the steps of:

Step A): grinding and polishing the pure titanium, sandblasting, ultrasonic cleaning and drying;

Step B): The pure titanium after step A) is acid-etched with hydrofluoric acid/nitric acid mixture at room temperature for 5-15 minutes, then ultrasonically cleaned and dried, and then washed with concentrated hydrochloric acid/concentrated sulfuric acid mixed acid at a water bath temperature of 75-85°C Under acid etching for 25-35 minutes, ultrasonic cleaning and drying, so that the micron structure is formed on the titanium surface, and the acid-etched titanium material is obtained;

Step C): Add the acid-etched titanium material into the sodium hydroxide solution, perform an alkalization reaction in a water bath at 60-80°C for 10-14 hours, and then wash;

Step D): Place the alkalized titanium sheet in step C) in a PLL (poly(L-lysine), poly-L-lysine) solution, and incubate at 4-6°C for 10-12h to obtain the PLL pretreated Titanium sheet;

Step E): Alternately and repeatedly immerse the PLL pretreated titanium sheet in CPP-ACP (casein phosphopeptides-amorphous calcium phosphate) and PLL solution, cycle 10 times, soak 10- After soaking for 20 minutes, rinse and dry at room temperature to obtain calcium-containing nano-titanium material.

In the technical solution of the present invention: in step A), the pure titanium sample is polished using sandpaper with labels of 600#, 800#, 1200#, and 1500# in order to grind and polish the pure titanium step by step from coarse sand to fine sand.

In the technical solution of the present invention: in step A), the pure titanium sample is blasted with 80 mesh Al ₂ O ₃ sand, the pressure is 0.4 MPa, the distance is 1.5 cm, and the sand blasting time is 15 seconds.

In the technical scheme of the present invention: in the hydrofluoric acid/nitric acid mixed solution in step B), the volume ratio of water: hydrofluoric acid solution: nitric acid solution is 1000:1～3:3～5; the volume concentration of hydrofluoric acid solution is 0.22 %, the volume concentration of nitric acid solution is 0.57%.

In the technical solution of the present invention: in the step B) concentrated hydrochloric acid/concentrated sulfuric acid mixed acid medium water: concentrated hydrochloric acid solution: concentrated sulfuric acid solution volume ratio is 4～7:0.5～1.5:0.5～1.5, the volume concentration of hydrochloric acid solution is 37% , the volume concentration of sulfuric acid solution is 98%.

In the technical solution of the present invention: the concentration of the sodium hydroxide solution in step C) is 1-3 mol/L.

In the technical solution of the present invention: the concentration of PLL poly-L-lysine described in step D) and step E) is 2.5 mg/mL, and the mass concentration of CPP-ACP solution in step E) is 0.5-3.0%.

Beneficial effects: the process of the present invention is simple, only the sodium hydroxide alkali heat treatment and self-assembly technology can form titanium surface nanostructures and load bioactive calcium ions, the required equipment is simple, the cost is low, and the titanium surface has both nanostructures and calcium element modification . The PLL/CPP-ACP titanium surface can enhance the compatibility and bonding ability of titanium and bone, which is beneficial to the proliferation and differentiation of osteoblasts on the titanium surface, so as to improve the osseointegration ability of titanium. Among them, when the concentration of CPP-ACP was 3.0%, the surface structure of titanium was denser, the hydrophilicity was better, and the proliferation and adhesion of osteoblasts on the surface were stronger, which was more conducive to the expression of osteoblast-related proteins in osteoblasts.

Description of drawings

Fig. 1 is the sample scanning electron microscope picture (40000 times) that the embodiment of the present invention 1 makes;

Fig. 2 is the sample scanning electron microscope picture (40000 times) that the embodiment of the present invention 2 makes;

Fig. 3 is the XPS collection of illustrative plates that the embodiment of the present invention 1,2 records;

Fig. 4 is taking embodiment 1,2 of the present invention as experimental group, and the surface of sandblasted and acid-etched (SLA) titanium is a control group, and the water contact angles of 3 groups of titanium sheets measured;

Figure 5 is the image of cell adhesion taken after the MC3T3-E1 osteoblast cell line was inoculated on the surface and cultured for 8 hours, taking Examples 1 and 2 of the present invention as the experimental group, and the sandblasted and acid-etched (SLA) titanium surface as the control group. ;

Fig. 6 is with embodiment 1, 2 of the present invention as experimental group, sandblasting and acid etching (SLA) titanium surface is as control group, MC3T3-E1 osteoblast cell line is inoculated on its surface and cultivated for 1, 3, 5 days, measured Get the histogram of cell proliferation CCK-8 value, * indicates that there is a significant difference;

Fig. 7 is with embodiment 1, 2 of the present invention as experimental group, sandblasting and acid etching (SLA) titanium surface is as control group, after inoculating MC3T3-E1 osteoblast cell line on its surface and culturing for 7 days, measured Runx2, Protein expression levels of OPN and OCN.

detailed description

The following examples can enable those skilled in the art to understand the present invention more fully, but do not limit the present invention in any way.

Example 1

A preparation method for constructing a PLL/CPP-ACP self-assembled multilayer film on a sandblasted acid-etched titanium surface, comprising the following steps:

Step A) Grinding and polishing the pure titanium (the polishing of the pure titanium sample uses sandpaper with the labels of 600#, 800#, 1200#, and 1500# in order to grind and polish the pure titanium step by step from coarse sand to fine sand), sandblasting (pure titanium Titanium samples were sandblasted with 80 mesh Al ₂ O ₃ sand, pressure 0.4MPa, distance 1.5cm, sandblasting 15s), ultrasonic cleaning and drying for use;

Step B) The pure titanium material treated in step A) is treated with hydrofluoric acid/nitric acid mixed solution (the volume ratio of double distilled water:hydrofluoric acid solution:nitric acid solution in the hydrofluoric acid/nitric acid mixed solution is 1000:2:4; The volume concentration of the hydrofluoric acid solution is 0.22%, the volume concentration of the nitric acid solution is 0.57%) acid etching at normal temperature for 10 minutes, ultrasonic cleaning and drying with double distilled water, and then through concentrated hydrochloric acid/concentrated sulfuric acid mixed acid solution (concentrated hydrochloric acid/concentrated sulfuric acid mixed acid The volume ratio of double distilled water:hydrochloric acid solution:sulfuric acid solution is 6:1:1, the volume concentration of hydrochloric acid solution is 37%, the volume concentration of sulfuric acid solution is 98%) acid etching at 80℃ water bath temperature for 30min, double distilled water ultrasonic wash and dry;

Step C) adding the titanium material treated by sandblasting and acid etching in step B) into a sodium hydroxide solution (the concentration of the sodium hydroxide solution is 2 mol/L), performing an alkalization reaction in a water bath at 70° C. for 12 hours, and then cleaning;

Step D) Put the alkalized titanium sheet in step C) in PLL (poly(L-lysine), poly-L-lysine, concentration: 2.5 mg/mL) solution, and incubate at 5° C. for 11 h.

Step E) Alternately immerse the titanium material treated with PLL in step D) into CPP-ACP and PLL (concentration: 2.5mg/mL) solutions with a mass concentration of 0.5%. Rinse with distilled water for 2 minutes, and dry at room temperature for later use.

Example 2

A preparation method for constructing a PLL/CPP-ACP self-assembled multilayer film on a sandblasted acid-etched titanium surface, comprising the following steps:

Step A) Grinding and polishing the pure titanium (the polishing of the pure titanium sample uses sandpaper with the labels of 600#, 800#, 1200#, and 1500# in order to polish the pure titanium step by step from coarse sand to fine sand), sandblasting (blasting) Sand using 80 mesh Al ₂ O ₃ sand, pressure 0.4MPa, distance 1.5cm, sand blasting 15s), ultrasonic cleaning and drying for use;

Step B) The pure titanium material treated in step A) is treated with hydrofluoric acid/nitric acid mixed solution (the volume ratio of double distilled water:hydrofluoric acid solution:nitric acid solution in the hydrofluoric acid/nitric acid mixed solution is 1000:2:4; The volume concentration of the hydrofluoric acid solution is 0.22%, the volume concentration of the nitric acid solution is 0.57%) acid etching at normal temperature for 10 minutes, ultrasonic cleaning and drying with double distilled water, and then through concentrated hydrochloric acid/concentrated sulfuric acid mixed acid solution (concentrated hydrochloric acid/concentrated sulfuric acid mixed acid The volume ratio of double distilled water:hydrochloric acid solution:sulfuric acid solution is 6:1:1, the volume concentration of hydrochloric acid solution is 37%, the volume concentration of sulfuric acid solution is 98%) acid etching at 80℃ water bath temperature for 30min, double distilled water ultrasonic wash and dry;

Step C) adding the titanium material treated by sandblasting and acid etching in step B) into a sodium hydroxide solution (the concentration of the sodium hydroxide solution is 2 mol/L), performing an alkalization reaction in a water bath at 70°C for 12 hours, and then cleaning;

Step D) Put the alkalized titanium sheet in step C) in PLL (poly(L-lysine), poly-L-lysine, concentration: 2.5 mg/mL) solution, and incubate at 5° C. for 11 h.

Step E) Submerge step D) the PLL-treated titanium material alternately in CPP-ACP with a mass concentration of 3.0% and a PLL concentration of 2.5 mg/mL) solution, the number of cycles is 10 times, soaking for 15 minutes each time, double steaming after soaking Rinse with water for 2 minutes, and dry at room temperature for later use.

Observing Examples 1 and 2 with a scanning electron microscope makes scanning electron microscope pictures as shown in Figures 1 and 2 (40000 times). The results show that both Examples 1 and 2 have nanostructures, and their surface microstructures are in the form of nano-plexes, and the nanostructures of Example 2 are denser, and a thin film covers the surface of the nanostructures.

Examples 1 and 2 are the experimental groups, and the sandblasting and acid etching (SLA) titanium surface is the control group. The main elements contained in the three groups are determined by X-ray energy spectrometer, and the XPS spectra are obtained as shown in Figure 3. The result shows that embodiment 1, 2 all contain calcium element, wherein the content of embodiment 2 is higher. In Example 1 and 2, compared with the control group, the content of Ti element decreased, and the content of P element and C element increased.

Embodiment 1 and 2 are experimental groups, and the surface of sandblasting and acid etching (SLA) titanium is a control group, and the water contact angle and surface energy of 3 groups of titanium sheets are detected, and the results in Fig. 4 show that the water contact angle of embodiment 1 and 2 is less than the contrast Group, show that embodiment 1,2 has better surface hydrophilicity than control group, and example 2 hydrophilicity is the best.

Examples 1 and 2 are experimental groups, and the surface of sandblasting and acid etching (SLA) titanium is a control group. After culturing MC3T3-E1 osteoblast cell line on its surface for 8 hours, confocal laser microscope observation shows (Fig. 5), the control group Adhesive cells were less, and their shape was relatively narrow. The number of adherent cells in Example 1 and Group 2 was the largest, and they spread fully, with many and long pseudopodia. The results show that Example 1 and Group 2 have the effect of promoting the adhesion of MC3T3-E1 cells, and Example 2 has a better effect.

Examples 1 and 2 are the experimental groups, and the sandblasting and acid etching (SLA) titanium surface is the control group. After culturing MC3T3-E1 osteoblast cell lines on the surface for 1, 3, and 5 days, the cell proliferation is detected by CCK-8 kit . The results in Figure 6 show that the surface of Example 2 has a better effect of promoting cell proliferation than the control group, and the effect of Example 1 is not obvious.

Examples 1 and 2 are experimental groups, and the surface of sandblasting and acid etching (SLA) titanium is a control group. After inoculating MC3T3-E1 osteoblast cell line on its surface and culturing it for 7 days, use RIPA lysate to lyse the cells and extract the total cell protein , the protein expression levels of Runx2, OPN, and OCN in the cells were detected by Western Blot, and GAPDH was used as an internal reference. The results in Figure 7 show that the surfaces of Examples 1, 2, and 3 can significantly increase the expression of osteogenic differentiation-related proteins compared with the surfaces of the control group, and the effect of Example 2 is more obvious.

Claims (7)

1. A preparation method for constructing a PLL/CPP-ACP self-assembly multilayer film on the surface of sand blasting acid etching titanium is characterized by comprising the following steps:

step A): polishing, sand blasting, ultrasonic cleaning and drying pure titanium;

step B): carrying out acid etching on the pure titanium dried in the step A) for 5-15 min at normal temperature by adopting a hydrofluoric acid/nitric acid mixed solution, then carrying out ultrasonic cleaning and drying, then carrying out acid etching on the pure titanium for 25-35 min at a water bath temperature of 75-85 ℃ by using a concentrated hydrochloric acid/concentrated sulfuric acid mixed acid, carrying out ultrasonic cleaning and drying to form a micron structure on the surface of the titanium, thus obtaining an acid-etched titanium material;

step C): adding the acid-etched titanium material into a sodium hydroxide solution, carrying out an alkalization reaction for 10-14h under the water bath condition of 60-80 ℃, and then cleaning;

step D): placing the titanium sheet after alkalization in the step C) in a PLL solution, and incubating for 10-12h at 4-6 ℃ to obtain a titanium sheet after PLL pretreatment;

step E): alternately and repeatedly immersing the titanium sheet pretreated by the PLL into the CPP-ACP solution and the PLL solution for 10 times, immersing for 10-20min each time, washing after immersing, and drying at room temperature to obtain the target product.

2. The method for preparing the PLL/CPP-ACP self-assembled multilayer film constructed on the sand blasting acid etching titanium surface according to claim 1, wherein the method comprises the following steps: and D) polishing the pure titanium sample in the step A), and gradually grinding and polishing the pure titanium by using sand paper with the serial number of 600#,800#,1200#,1500#, from coarse sand to fine sand.

3. The method for preparing the PLL/CPP-ACP self-assembled multilayer film constructed on the sand blasting acid etching titanium surface according to claim 1, wherein the method comprises the following steps: the pure titanium sample in the step A) is blasted with 80-mesh Al ₂ O ₃ Sand, pressure 0.4MPa, distance 1.5cm, sand blasting 15s.

4. The method for preparing the PLL/CPP-ACP self-assembly multilayer film constructed on the surface of the sand blasting acid etching titanium according to claim 1, wherein the method comprises the following steps: water in the hydrofluoric acid/nitric acid mixed solution in the step B): hydrofluoric acid solution: the volume ratio of the nitric acid solution is 1000:1 to 3:3 to 5; the volume concentration of the hydrofluoric acid solution was 0.22%, and the volume concentration of the nitric acid solution was 0.57%.

5. The method for preparing PLL/CPP-ACP self-assembled multilayer film constructed on the surface of sand blasting acid etching titanium according to claim 1, is characterized in that: water in the concentrated hydrochloric acid/concentrated sulfuric acid mixed acid in the step B): concentrated hydrochloric acid solution: the volume ratio of the concentrated sulfuric acid solution is 4-7: 0.5 to 1.5: 0.5-1.5, the volume concentration of the hydrochloric acid solution is 37 percent, and the volume concentration of the sulfuric acid solution is 98 percent.

6. The method for preparing PLL/CPP-ACP self-assembled multilayer film constructed on the surface of sand blasting acid etching titanium according to claim 1, wherein the concentration of the sodium hydroxide solution in the step C) is 1-3 mol/L.

7. The method for preparing the PLL/CPP-ACP self-assembled multilayer film constructed on the titanium surface by sand blasting and acid etching according to claim 1, wherein the concentration of the PLL solution in the step D) and the step E) is 2.5mg/mL, and the mass concentration of the CPP-ACP solution in the step E) is 0.5-3.0%.

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