CN113440653A

CN113440653A - Titanium-based implant for promoting osseointegration and preparation method and application thereof

Info

Publication number: CN113440653A
Application number: CN202110742164.1A
Authority: CN
Inventors: 王兴; 李晖菲; 赵彬; 田春瑞
Original assignee: Stomatological Hospital Of Shanxi Medical University
Current assignee: Stomatological Hospital Of Shanxi Medical University
Priority date: 2021-07-01
Filing date: 2021-07-01
Publication date: 2021-09-28
Anticipated expiration: 2041-07-01
Also published as: CN113440653B

Abstract

The invention discloses a titanium-based implant for promoting osseointegration and a preparation method and application thereof, belonging to the field of biological implants; after a bovine pancreas beta cell exosome is loaded on the surface of a pure titanium-based implant by using a chemical immobilization method, TiO is deposited on the surface of an Exo-Ti implant by using an atomic layer deposition technology₂Forming a nano-layered film to form Ti-Exo-TiO₂A sandwich composite structure, which develops and prepares a titanium-based implant loaded with bovine pancreatic beta cell exosomes; Ti-Exo-TiO₂The nano-layered film is uniform and highly conformal, and has better hydrophilicity, antibacterial property and corrosion resistance. The titanium-based implant can remarkably increase the proliferation and osteogenic differentiation capacity of bone marrow mesenchymal stem cells, improve the osteogenic speed on the surface of the implant, and remarkably improve the bone-implant contact rate and the implant removal torque. The titanium-based implant prepared by the invention is suitable for industrial popularization and application, and provides a new product for the field of medical implanted human body materials.

Description

Titanium-based implant for promoting osseointegration and preparation method and application thereof

Technical Field

The invention relates to the field of biological implants, in particular to a titanium-based implant for promoting osseointegration and a preparation method and application thereof.

Background

Artificial dental implants have become the first choice for repairing defective or missing dentition. At present, a great problem of the implant is that the implant is not stable enough in the initial stage, and the continuous micromotion causes the osseointegration failure, and finally leads to the early falling of the implant. In the elderly with osteoporosis, diabetes, periodontal disease and smoking, the reduction of the osseointegration rate of the implant caused by various systemic diseases becomes the main risk of the failure of the existing artificial dental implant. Therefore, how to improve the osseointegration rate of the implant and expand the dental implant indications remains a problem to be solved urgently by artificial dental implant treatment.

In recent years, insulin has attracted attention from researchers in its bone-promoting action in a non-diabetic state. The insulin is periodically injected around the local part of the bone defect animal model, so that the bone formation in the defect area and around the implant can be remarkably accelerated. However, insulin has a short half-life in vivo, can enter the systemic circulation and be metabolized with the blood, and it is difficult to maintain a locally effective concentration. The local concentration of the insulin is too low, so that the effect of promoting bone differentiation is insufficient, the concentration is too high, so that systemic adverse reactions such as bone regeneration weakening, hypoglycemia or hyperinsulinemia are caused, and the research and application of the insulin in the implant osseointegration have technical barriers.

The exosome is an extracellular vesicle with the size of 20-140 nanometers, carries various bioactive molecules, and can induce various biological behaviors such as tissue regeneration, immunity, autophagy and the like. Research shows that pancreatic beta cells can secrete insulin, and secreted exosomes can generate an insulin-like bone-promoting effect and can avoid hyperinsulinemia and insulin resistance. The exosome is used as a natural intercellular information exchange carrier and has good biocompatibility; meanwhile, the molecular structure is small, the storage and the quantification are facilitated, and the method has great research and application values in the field of bone tissue regeneration.

At present, no relevant report that bovine pancreatic beta cell exosomes are loaded on the surface of an implant to form a Ti-Exo-TiO2 composite structure so as to improve the bone integration rate of the implant exists.

Disclosure of Invention

The invention aims to provide a titanium-based implant for promoting osseointegration and a preparation method and application thereof, which are used for solving the problems in the prior art and developing a titanium-based implant for loading bovine pancreatic beta cell exosomes so as to improve the osseointegration performance.

In order to achieve the purpose, the invention provides the following scheme:

according to one technical scheme, the titanium-based implant is provided, and bovine pancreatic beta cell exosomes are loaded on the titanium-based implant.

Preferably, the titanium-based implant surface is combined with bovine pancreas beta cell exosome to obtain Exo-Ti, and TiO is prepared on the Exo-Ti surface₂A film.

Preferably, the titanium dioxide film is prepared by an atomic layer deposition technology.

The second technical scheme provides a preparation method of the titanium-based implant, which comprises the following steps:

(1) fixing the bovine pancreas beta cell exosome on the titanium-based surface of the implant to obtain an Exo-Ti implant;

(2) depositing TiO on the surface of the Exo-Ti implant obtained in the step (1)₂And (4) nano film to obtain the titanium-based implant.

Preferably, the immobilization method in step (1) is a chemical immobilization method; the deposition method in the step (2) is an atomic layer deposition technology.

Preferably, the chemical immobilization method comprises the following specific steps:

(1) carrying out acid etching on a pure titanium implant to obtain an acid-etched implant, placing the acid-etched implant and a CD63 aptamer into an acetic acid buffer solution, taking tris (2-carboxyethyl) phosphine hydrochloride as a reducing agent, rotating under the condition of keeping out of the sun, and cleaning to obtain an implant loaded with the CD63 aptamer;

(2) and mixing and incubating the exosomes of the bovine pancreatic gland beta cells with the CD63 aptamer-loaded implant to obtain the bovine pancreatic gland beta cell exosome-loaded implant.

Preferably, the incubation is specifically: at 4 ℃ pH7.4, 5% CO₂And mixing the bovine pancreatic beta cell exosomes and the CD63 aptamer-loaded implant under the condition of 95% relative humidity, placing the mixture in a shaker at 100rpm, and incubating overnight to obtain the bovine pancreatic beta cell exosome-loaded implant.

Preferably, the metal precursor of the atomic layer deposition technique is tetrakis (dimethylamino) titanium, and the precursor of oxygen is deionized water.

The third technical proposal provides the application of the titanium-based implant in the preparation of medical implant human body materials.

Preferably, the medical implant body material is an artificial implant.

The invention discloses the following technical effects:

the invention develops and prepares a Titanium-based Implant (PES Implant for short) loaded with bovine pancreatic beta cell Exosomes by utilizing a chemical immobilization method and an atomic layer deposition technology, and the Ti-Exo-TiO2 nano layered film deposited on the Surface of the Titanium-based Implant is uniform and highly conformal, and has better hydrophilicity, antibacterial performance and corrosion resistance.

In vitro experiments, Bone Marrow Mesenchymal Stem Cells (BMSCs) cultured on the surface of PES implant have increased proliferation capacity (FIG. 3A), the expression of Bone formation key genes ALP (FIG. 3B) and OCN (FIG. 3C) is enhanced, and mineralized nodules stained with alizarin red (FIG. 4) are significantly increased. Therefore, compared with the existing titanium substrate and the traditional ALD sprayed titanium implant, the PES implant can obviously increase the proliferation and osteogenic differentiation capacity of BMSCs, and the osteogenic speed of the implant surface is increased.

In vivo experiments, the PES implant can effectively accelerate the bone regeneration around the titanium implant, thereby reducing the early failure rate of the implant. Compared with the existing pure titanium Implant and the traditional ALD sprayed titanium Implant, the method can obviously improve the Bone-to-Implant Contact ratio (BIC%) and the Removal Torque (RTQ) of the Implant.

The titanium-based implant prepared by the invention has the advantages that the performance of the implant is obviously improved by loading the bovine islet beta cell exosomes, and a new product is provided for the field of medical implanted human body materials. The invention is suitable for popularization and application in industry and has great potential commercial value.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings without creative efforts.

FIG. 1 is a schematic diagram of the preparation of a PES implant;

FIG. 2 is a schematic view of a PES implant finished product;

FIG. 3 is a graph of in vitro biological activity of different implants; in the figure A, the proliferation of BMSCs in different implants; b is ALP concentration in different implants; c is the concentration of OCN in different implants;

FIG. 4 is a graph of alizarin red staining for different implants; in the figure, A is a pure titanium implant; b, ALD spraying the titanium implant; c is a PES implant; d is a comparison graph of optical density of different implants;

FIG. 5 is a histological evaluation of peri-implant bone formation; in the figure, A is the BIC comparison of different implants at week 4; b is an RTQ difference graph among different implants implanted in four weeks; c is a staining pattern of new bone formation around different implants at week 4.

Detailed Description

Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but as a more detailed description of certain aspects, features and embodiments of the invention.

It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention.

The materials, instruments and reagents used in the present invention are commercially available unless otherwise specified; the experimental methods used are all routine experimental methods in the field unless otherwise specified.

A schematic diagram of the preparation of the PES implant is shown in FIG. 1, and a schematic diagram of the finished PES implant is shown in FIG. 2.

EXAMPLE 1 preparation and Performance of titanium-based implants

1.1 preparation of bovine pancreatic beta cell exosomes

Isolation and culture of beta cells of bovine pancreas

a. Taking ox pancreas in a slaughterhouse, storing the ox pancreas in an ice box at a low temperature, and sending the ox pancreas to a biological safety laboratory within 6 hours.

b. After bovine pancreas was washed three times with 75% ethanol under sterile conditions, 10g of bovine pancreas was excised and soaked in 20mL of a standard phosphate-balanced salt buffer solution (pH 7.4).

c. And (2) placing the cleaned pancreas in a 6cm culture dish, shearing, adding 5mL of IV-type collagenase, digesting for 45min at 37 ℃, blowing a sample for several times by using a 1mL gun head during digestion, placing the sample in a constant-temperature incubation shaking table, and shaking for 10min at the temperature of 37 ℃ and 30rpm to ensure that the enzyme is fully contacted with the sample to achieve the optimal digestion effect.

d. 5mL of medium was added to stop digestion, the cell suspension was blown off with a pipette and filtered through a 200 mesh screen.

e. The filtrate was collected and centrifuged at 1000rpm for 10 min.

f. The supernatant was discarded, 4mL of fresh 10% DMEM medium (containing 10% serum and 90% DMEM medium) was added, and the mixture was vortexed. Inoculating into 6cm culture dish, and placing in culture dishIn the incubator, 5% CO₂And after culturing at 37 ℃ for 24h, replacing the culture medium in a full amount, and then replacing the culture medium every 2 days, and carrying out passage when the cells reach 80-90% confluence.

② extraction and purification of bovine pancreatic beta cell exosome

Culturing P2-5 generation ox pancreas beta cell to 70% fusion, removing culture medium supernatant, washing with PBS for 3 times, changing special serum-free culture medium for exosome, and continuously culturing at 37 deg.C with CO₂Culturing in an incubator for 48h, and collecting cell supernatant.

b. The collected supernatant was centrifuged at 300 g.times.10 min and 2000 g.times.10 min to remove the pellet (to remove floating cells and cell debris), and the supernatant was collected.

c. The collected supernatant was centrifuged at 4 ℃ for 10000 g.times.30 min to remove the pellet (remove cell debris), and the supernatant was collected.

d. Centrifuging the collected supernatant at 100000g at 4 deg.C for 70min with ultra-high speed centrifuge to collect precipitate. Resuspend the pellet with PBS.

e. Carefully lay the precipitate from step d on top of a 30% (w/v) sucrose pad of heavy water to form a clear interfacial layer. Centrifuging at 150000g for 70min at 4 ℃, discarding the PBS layer in the centrifuge tube, reserving the sucrose heavy water layer, adding PBS with at least 5 times volume, centrifuging at 100000g for 70min, and resuspending and precipitating with PBS to obtain purified exosome.

f. The purified exosomes were filter sterilized using 0.22 μm filter membranes and packed into sterile Ependorf tubes and frozen at-80 ℃.

Identification of bovine pancreas beta cell exosomes

a. A drop of the exosome suspension was taken and loaded on a 200 mesh copper mesh, left at room temperature for 10min, and the liquid was gently aspirated from the edge of the copper mesh with filter paper. And (3) dripping the phosphotungstic acid solution on a copper net, and carrying out negative dyeing for 5min in a room temperature environment. After washing twice with sterile deionized water, the mixture was dried under an incandescent lamp for 2 min. The exosome is placed under a transmission electron microscope to observe the size and the shape of the exosome, and an electron microscope picture is taken.

b. The exosomes were labeled with anti-TSG 101 and anti-CD 63 antibodies for immunowestern blotting to characterize the exosome surface-specific proteins.

c. The particle size of the exosomes was determined using a NanoSight analytical instrument.

1.2 fixing the bovine pancreas beta cell exosome on the titanium-based surface of the implant

Firstly, carrying out acid etching on the pure titanium implant to obtain an acid-etched implant.

And secondly, placing the acid-etched implant and the CD63 aptamer into an acetic acid buffer solution with the pH value of 5.0 at 37 ℃, rotating for 14 hours under the condition of keeping out of the sun by using tris (2-carboxyethyl) phosphine hydrochloride as a reducing agent, and cleaning for 3-4 times to obtain the implant loaded with the CD63 aptamer.

③ diluting the exosome of the purified bovine pancreatic beta cell to 4 multiplied by 10 by DMEM culture medium¹⁰particles/mL.

(iv) adding 50 microliters of exosome solution to the CD63 aptamer-loaded implants (1 mm diameter, 2mm length) placed in a 96-well plate.

Fifthly, 5 percent CO at 4 ℃ and pH7.4₂96-well plates were placed on a shaker at 100rpm under 95% relative humidity and the implants were incubated overnight in exosome solution.

Sixthly, repeating the step five to obtain the implant loaded with the bovine pancreas beta cell exosomes.

1.3 preparation of TiO by ALD₂Thin film fixed Exo-Ti structure

Placing the Exo-Ti implant on a glass sheet with the diameter of 13 mm.

② washing the Exo-Ti implant in isopropanol at the constant temperature of 37 ℃, and then washing twice in deionized water, each time for 5 min. Then N with a purity of 5.0% is used₂The carrier is dried.

③TiO₂The process of thin film deposition of the Exo-Ti surface is carried out in an atomic layer deposition system reactor.

And fourthly, alternately introducing the tetra (dimethylamino) titanium and the deionized water into the reaction chamber. Tetrakis (dimethylamino) titanium is used as the metal precursor and deionized water is the precursor of oxygen. After each use of the precursor, the reaction mixture was purified by passing N with a purity of 5.0%₂The reaction chamber is purged. After 0.2s of introduction of the metal precursor, wait for 3s, N₂Purified for 15s and then O is introduced₂Precursor 0.04s, wait 3s, N₂And a purification body 15 s.

Fifthly, the process is repeated for 1220 times and is carried out at the constant temperature of 40 ℃ and the atmospheric pressure of 66Pa, and meanwhile, the ambient temperature of the metal precursor is preheated to 70 ℃. The vacuum degree of the reaction chamber is 20mTorr when N is₂The reactor outlet pressure was stabilized at 364mTorr when injected into the reaction chamber.

Sixthly, the reaction chamber and the heater of the precursor are turned on one hour before the process starts to ensure the correct temperature is reached and to ensure that the TiO is not in contact with the process₂The film remains stable during the anchoring on the titanium-based surface.

⑦TiO₂And (5) analyzing the physical and chemical properties of the film.

a. Analysis of TiO by X-ray diffraction₂Crystallinity of the film.

b. Determination of the Final TiO Using Panalytical software according to the theory of Parlat₂Thickness of the coating, electron density and roughness.

c. The surface morphology and internal structure of the coating were observed and analyzed using a field emission Scanning Electron Microscope (SEM), an Atomic Force Microscope (AFM), and a Transmission Electron Microscope (TEM).

d. Evaluation of TiO by measuring the Water contact Angle deposited on the surface₂Hydrophilic/hydrophobic properties of the film. The TiO deposited on the cover plate was obtained using an OCA 25 goniometer from Dataphysik₂The contact angle of the fluid on the surface, the test liquid being water, was measured under normal conditions (temperature: 25 ℃; air humidity: 50%). All tests were repeated three times at different locations on the sample.

After atomic layer deposition, the titanium-based implant carrying the bovine pancreatic beta cell exosomes is manufactured (fig. 1A), and is packaged and transferred in vacuum.

1.4 detection of the Performance of the Ti-based planter of the bovine pancreas beta cell exosome

1.4.1 in vitro Performance testing

The experimental steps are as follows:

a. bone Marrow Mesenchymal Stem Cells (BMSCs) on PES implant surfaces were cultured in minimal medium, including minimal alpha medium with 15% fetal bovine serum.

b. Fresh medium was replaced twice weekly.

c. After 3, 7 and 14 days, respectively, the supernatants were collected and replaced with an equal amount of fresh minimal medium.

d. The medium was assayed for osteogenic key gene alkaline phosphatase (ALP) activity using the Roche Diagnostics ALP kit on the Cobas e602 platform.

e. Osteocalcin (OCN) activity was determined using an N-MID osteocalcin kit using an electrochemiluminescence immunoassay technique.

f. A total of 3 tests were performed and all data were analyzed and measured on the Cobas 8000 platform. Likewise, 1X 10⁵BMSCs were cultured in minimal medium.

g. On day 14, cells were fixed with 2.5% glutaraldehyde and stained with alizarin red. The precipitate was extracted with 10% solution of cetylpyridinium chloride (w/v) at room temperature (pH 7.0) for 2 hours. The content of alizarin red in the extraction buffer was determined by the optical density of the solution at 560 nm.

h. The BMSCs proliferation condition, ALP expression and OCN expression in pure titanium implant, traditional ALD sprayed titanium implant and PES implant are measured by adopting MTS method.

② experimental results: the results are shown in FIG. 1: BMSCs cultured on PES implant surface have increased proliferation capacity (FIG. 3A), and the expression of the osteogenic key genes ALP (FIG. 3B) and OCN (FIG. 3C) is enhanced.

The formation of the pure titanium implant group is checked by alizarin red staining and mineralization, and the result is shown in figure 4, the optical density of the ALD sprayed implant group is lower than that of the PES implant group, and the osteogenic differentiation of BMSCs on the surface of the PES implant is obviously higher than that of the titanium implant and the traditional ALD sprayed titanium implant.

1.4.2 in vivo Performance testing

The experimental steps are as follows:

a. new Zealand white rabbits (6 months) with a body weight of 2.90 + -0.32 kg were selected for the mandibular implant model. All rabbits were divided into three groups according to different implants.

b.2% sodium pentobarbital (30mg/kg) for intravenous anesthesia. A 10mm incision was made at the lower edge of the right mandible and the implant was implanted perpendicular to the bone surface from buccal side to lingual side using the Astra implant system.

c. At week 4 post-surgery, all rabbits were sacrificed and mandibular samples were excised, fixed, dehydrated and embedded in methyl methacrylate. Along the long axis of the implant, the tissue sections were ground to a thickness of 15 μm and stained with methylene blue/acid fuchsin.

d. Ten serial sections were observed in each implant and evaluated histologically using a Leica microscope. The central cross section of the Implant was selected for analysis and the amount of Bone around the Implant that had formed in each area was determined by the Bone-to-Implant Contact ratio (BIC%).

e. At

weeks

1, 2, 3 and 4 after surgery, mandibular bone specimens containing implants were tested using an AG-IS universal tester. The sampled peak torque value is recorded until the implant is rotated 90 degrees.

② experimental results:

by releasing the exosomes of bovine pancreatic beta cells in vivo, the PES implant can effectively accelerate bone regeneration around the titanium implant, thereby reducing the early failure rate of the implant. In vivo metrics that significantly improved over existing pure titanium implants and conventional ALD spray coated titanium implants include Bone-to-Implant Contact ratio (BIC%) (fig. 5A), Implant Removal Torque (Removal Torque, RTQ) (fig. 5B), and peri-Implant Bone formation (fig. 5C).

Comparative example 1 preparation of titanium-based implant

The preparation method of the titanium-based implant is the same as that of example 1, and the only difference is that the exosome is bone marrow mesenchymal stem cell (BMSC) exosome.

Comparative example 2 preparation of titanium-based implant

The titanium-based implant was prepared by the same method as in example 1, except that the pure titanium implant was not subjected to acid etching.

Comparative example 3 preparation of titanium-based implant

The titanium-based implant was prepared as in example 1, except that the CD63 aptamer was not added.

Comparative example 4 preparation of titanium-based implant

The titanium-based implant was prepared as in example 1, except that the temperature during the atomic deposition was 50 ℃.

The titanium-based implants prepared in comparative examples 1-4 were subjected to in vivo and in vitro performance measurements, the results of which are given in the following table:

to quantify bone formation, the percentage of bone regeneration around the PES implant at week 8 and the control implant was expressed as Bone Mineral Density (BMD) and bone volume/total volume (BV/TV).

Table 1: comparison of regeneration of new bone around five implants

The above-described embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solutions of the present invention can be made by those skilled in the art without departing from the spirit of the present invention, and the technical solutions of the present invention are within the scope of the present invention defined by the claims.

Claims

1. A titanium-based implant for promoting osseointegration, wherein bovine pancreatic beta cell exosomes are loaded on the titanium-based implant.

2. The titanium-based implant of claim 1, wherein the titanium-based implant is combined with bovine pancreatic beta cell exosomes to obtain Exo-Ti, and then TiO is prepared on the Exo-Ti surface₂A film.

3. The titanium-based implant of claim 2, wherein said titanium dioxide film is formed by atomic layer deposition.

4. A method for preparing the titanium-based implant of claim 1, comprising the steps of:

5. The method for preparing a titanium-based implant according to claim 4, wherein the fixing method in the step (1) is a chemical fixing method; the deposition method in the step (2) is an atomic layer deposition technology.

6. The method for preparing the titanium-based implant according to claim 5, wherein the chemical fixation method comprises the following specific steps:

7. The method for preparing a titanium-based implant according to claim 6, wherein the incubation is specifically: at 4 ℃ pH7.4, 5% CO₂And mixing the bovine pancreatic beta cell exosomes and the CD63 aptamer-loaded implant under the condition of 95% relative humidity, placing the mixture in a shaker at 100rpm, and incubating overnight to obtain the bovine pancreatic beta cell exosome-loaded implant.

8. The method for preparing a titanium-based implant according to claim 5, wherein the metal precursor of the atomic layer deposition technique is tetrakis (dimethylamino) titanium and the precursor of oxygen is deionized water.

9. Use of a titanium-based implant according to any one of claims 1 to 3 for the preparation of a medical implant body material.

10. The use of the titanium-based implant as defined in claim 9, wherein said medical implant material is an artificial implant.