阅读说明：本技术 一种具有生物活性表面的牙种植体及其制备方法 (Dental implant with bioactive surface and preparation method thereof ) 是由 曲哲 郭英 张久文 于 2021-01-21 设计创作，主要内容包括：本发明提供一种具有生物活性表面的牙种植体及其制备方法,牙种植体包括植体本体和覆盖在种植体本体外表面上的缓释层,缓释层具体为负载有EPO的壳聚糖纳米微球复合层。EPO负载于壳聚糖纳米微球中,然后涂布于牙种植体本体表面,在牙种植体本体表面形成含有EPO的壳聚糖纳米微球复合层结构。本发明所得牙种植体上的EPO可持续释放15d,具有持久、高效的促进血管内皮细胞生长,促进血管化的作用,进一步能够支持多种细胞(如成骨细胞、软骨细胞)的粘附、存活、生长与分化,具有广阔的应用前景。(The invention provides a dental implant with a bioactive surface and a preparation method thereof. EPO is loaded in the chitosan nano-microspheres and then coated on the surface of the dental implant body, and a composite layer structure of the chitosan nano-microspheres containing EPO is formed on the surface of the dental implant body. The EPO on the dental implant obtained by the invention can be sustainably released for 15d, has the effects of durably and efficiently promoting the growth of vascular endothelial cells and promoting vascularization, further can support the adhesion, survival, growth and differentiation of various cells (such as osteoblasts and chondrocytes), and has wide application prospect.)

1. A dental implant with a bioactive surface, characterized in that it comprises an implant body (6) and a slow-release layer (5); the surface of the implant body (6) is provided with threads, the two ends of the implant body (6) are respectively provided with a crown opening end (1) and an implanting end (2), the surface of one side, close to the implanting end (2), of the implant body (6) is provided with a self-tapping groove (3), the slow release layer (5) covers the outer surface of the implant body (6), and the outer surfaces of the threads, the crown opening end (1), the implanting end (2) and the self-tapping groove (3) are covered with the slow release layer (5); the slow release layer (5) is a chitosan nano microsphere composite layer coated with EPO.

2. A dental implant according to claim 1, characterised in that the implant body (6) is of an inverted cone shape, the end of the implantation end (2) remote from the coronal end (1) is of a circular arc surface (4), the coronal end (1) having a cross-sectional diameter greater than the cross-sectional diameter of the implantation end (2); the number of the self-tapping grooves (3) is 2-4, and the self-tapping grooves (3) are uniformly arranged on the outer surface of the implant body (6).

3. The dental implant of claim 1, wherein the chitosan is a water-soluble Hydroxypropyl Chitosan (HCS); the particle size of the chitosan nano-microsphere is 150-350 nm; the encapsulation efficiency of the EPO is 72-80%.

4. A method for preparing a dental implant according to any one of claims 1 to 3, comprising the steps of:

(a) pre-treating the surface of the implant body;

(b) immersing the implant body in the EPO-loaded chitosan nano-microspheres for 20-30 min;

(c) after washing with deionized water, sterilization treatment was performed.

5. The method of claim 4, wherein the step (a) comprises the steps of:

(1) cleaning and drying the implant body to be treated;

(2) carrying out sand blasting treatment;

(3) cleaning the implant body subjected to sand blasting, and drying;

(4) acid etching treatment;

(5) neutralizing the acid-etched implant body;

(6) after cleaning, soaking the fabric in pure acetone for 8 to 12 minutes, and then soaking the fabric in absolute ethyl alcohol for 8 to 12 minutes;

(7) and (3) putting the implant body into 150mM sodium chloride electrolyte for preservation, wherein the pH value of the electrolyte is 6-7.

6. The production method according to claim 5, wherein in the step (2), the blasting sand is alumina; the sand blasting pressure is 0.1-0.5 Mpa; the sand blasting time is 50-70 s; the distance between the nozzle and the implant body is 5-8 cm; the sandblasting angle is 15 ~ 20.

7. The preparation method according to claim 5, wherein the acid etching treatment is acid etching at 60-70 ℃ for 25-35 min; the acid etching liquid medicine contains 25-35% of sulfuric acid, 10-25% of hydrochloric acid and the balance of water by volume fraction.

8. The preparation method according to claim 5, wherein the neutralization treatment comprises incubating the implant body in 5mol/L NaOH solution at 55-75 ℃ for 24-48 h, and adjusting the pH value to 7.0.

9. The method according to claim 4, wherein in the step (b), the EPO-loaded chitosan nanospheres are prepared as follows: 1) dissolving HCS and EPO in double distilled water, stirring, standing for 1-1.5 h, filtering with 0.45-micron filter paper, and adjusting the pH value to 6 with a small amount of acetic acid; 2) dissolving sodium Tripolyphosphate (TPP) in double distilled water, and filtering with 0.22 micrometer filter paper; 3) taking the solution in the step 1), dropwise adding a TPP solution at a speed of 20-30 drops/minute, continuously stirring for 25-35 min by magnetic force, and storing at low temperature;

the mass ratio of the HCS to the EPO is 1: 1; the final concentration of HCS is 1.0-2.0 g/l, and the final concentration of TPP is 0.5-1.0 g/l.

10. The preparation method according to claim 4, wherein the step (c) comprises washing the dental implant with deionized water, adjusting the pH to 7.0, and sterilizing the dental implant with cobalt 60 radiation for 24-48 h.

Technical Field

The invention relates to the technical field of dental implants, in particular to a dental implant with an EPO (erythropoietin) -containing bioactive surface and a preparation method thereof.

Background

In order to improve the bioactivity of titanium and titanium alloy, shorten the planting period, accelerate bone healing and improve the binding force between bone and implant, the surface modification technology of the planting material is gradually developed and matured day by day. After the surface of the implant is modified, the physical and chemical properties of the surface are changed. However, the study on the implant surface by the broad scholars is still continuously explored, how to load the implant in the early stage even immediately, thereby improving the living standard and the repair quality of the patient. In order to shorten the load time of the implant, how to construct a novel implant with better early retention and make immediate load possible is the focus of advanced research.

Tresguerres IF et al carried out animal experiments to implant threaded implants in the femur of rabbits,local additive for implant implantation Adding recombinant human auxin freeze-dried powderThe bone-implant binding rate of a group of cortical bone areas locally using auxin is 30 percent higher than that of a control group, and the difference is obvious. This study demonstrated for the first time the promotion of osteointegration by topical application of auxin and demonstrated that topical application of auxin promotes osteointegration early in the implantation. Brama et al coated bone morphogenetic proteins on the implant surface and observed their osseointegration in animal experiments, followed by the application of this technique to coat human reconstructive bone morphogenetic protein-2 or TGF- β 1 on the implant surface, early improved peri-implant bone formation. However, the methods for coating the cytokine have the defects of quick release and degradation in the early stage of implant implantation, limited action of single cytokine, uneven coating on the surface of the implant, low coating concentration and the like.

Disclosure of Invention

According to the technical problems, the invention provides a dental implant, the surface of which is provided with a water-soluble hydroxypropyl chitosan-EPO nano microsphere composite layer, Erythropoietin (EPO) can be uniformly coated on the surface of the implant, and EPO is slowly released and secreted to promote early vascularization and bone formation in the early implantation period of the implant, so that an ideal biological material with biocompatibility and the implant surface which best meets the oral physiological requirements are obtained.

The technical means adopted by the invention are as follows:

a dental implant with a bioactive surface comprises an implant body and a slow release layer; the surface of the implant body is provided with threads, the two ends of the implant body are respectively a coronal end and an implanting end, the surface of the implant body close to one side of the implanting end is provided with a self-tapping groove, the slow release layer covers the outer surface of the implant body, and the outer surfaces of the threads, the coronal end, the implanting end and the self-tapping groove are all covered with the slow release layer; the slow release layer is a chitosan nano-microsphere composite layer coated with EPO.

Furthermore, the implant body is an inverted cone, one end of the implanted end, which is far away from the coronal end, is an arc surface, and the diameter of the cross section of the coronal end is larger than that of the implanted end.

Furthermore, the number of the self-tapping grooves is 2-4, and the self-tapping grooves are uniformly arranged on the outer surface of the implant body.

Further, the chitosan is water-soluble Hydroxypropyl Chitosan (HCS); the particle size of the chitosan nano-microspheres is 150-350 nm, and the encapsulation rate of the encapsulated EPO is 72-80%.

The invention also provides a preparation method of the dental implant with the bioactive surface, which comprises the following steps:

(a) pre-treating the surface of the implant body;

(b) immersing the implant body in the EPO-loaded chitosan nano-microspheres for 20-30 min;

(c) after washing with deionized water, sterilization treatment was performed.

Further, the step (a) includes the steps of:

(1) cleaning and drying the implant body to be treated;

(2) carrying out sand blasting treatment;

(3) cleaning the implant body subjected to sand blasting, and drying;

(4) acid etching treatment;

(5) neutralizing the acid-etched implant body;

(6) after cleaning, soaking the fabric in pure acetone for 8 to 12 minutes, and then soaking the fabric in absolute ethyl alcohol for 8 to 12 minutes;

(7) the implant body is placed in 150mM sodium chloride electrolyte, and the physiological saline is closer to the in-vivo environment, so that the implant can be effectively stored, and the external pollution is prevented; the pH value of the electrolyte is 6-7.

Further, in the step (2), the sand blasting material is aluminum oxide; the sand blasting pressure is 0.1-0.5 Mpa; the sand blasting time is 50-70 s; the distance between the nozzle and the implant body is 5-8 cm; the sandblasting angle is 15 ~ 20.

Further, the acid etching treatment is specifically acid etching for 25-35 min at 60-70 ℃; the acid etching liquid medicine contains 25-35% of sulfuric acid, 10-25% of hydrochloric acid and the balance of water by volume fraction.

Further, the neutralization treatment specifically comprises the steps of placing the implant body in a 5mol/L NaOH solution at the temperature of 55-75 ℃ for incubation for 24-48 h, and adjusting the pH value to 7.0.

Further, in the step (b), the preparation method of the EPO-loaded chitosan nanoparticle is as follows: 1) dissolving HCS and EPO in double distilled water, stirring, standing for 1-1.5 h to fully dissolve the HCS and EPO, filtering by 0.45-micron filter paper, and adjusting the pH value to 6 by using a small amount of acetic acid; 2) dissolving sodium Tripolyphosphate (TPP) in double distilled water, and filtering with 0.22 micrometer filter paper; 3) taking the solution in the step 1), dropwise adding a TPP solution at a speed of 20-30 drops/minute, continuously stirring for 25-35 min by magnetic force, and storing at low temperature;

the mass ratio of the HCS to the EPO is 1: 1; the final concentration of HCS is 1.0-2.0 g/l, and the final concentration of TPP is 0.5-1.0 g/l.

Further, the step (c) is specifically that the dental implant is washed by deionized water, the pH value is adjusted to 7.0, and the dental implant is sterilized by cobalt 60 radiation for 24-48 h.

Compared with the prior art, the invention has the following advantages:

aiming at the problems of low early osseointegration rate and long final repair time caused by low surface free energy and low surface activity of an implant, the invention selects Erythropoietin (EPO) which is researched more at present to promote bone tissue and blood vessel regeneration by screening according to the capacity of enhancing cell adhesion and improving osteoblast activity of extracellular matrix protein components and hydrophilic surfaces, constructs a water-soluble hydroxypropyl chitosan nano microsphere as a carrier through chitosan and derivatives thereof, and combines the EPO and the water-soluble hydroxypropyl chitosan nano microsphere on the surface of the implant to carry out slow-release coating to construct a novel EPO coated hydrophilic implant, thereby influencing the adhesion of organic and inorganic molecules around the implant and playing the roles of promoting bone regeneration around the implant and improving osseointegration.

After the implant is implanted into a body, EPO is gradually released into surrounding tissues along with the slow dissolution of the surface of the chitosan nanoparticle coating, can last for 10-15 days, has a slow release effect, can persistently and efficiently promote the growth of vascular endothelial cells and vascularization, further can support the adhesion, survival, growth and differentiation of various cells (such as osteoblasts, chondrocytes and the like), and has a wide application prospect.

Drawings

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

In fig. 1, (a) is a schematic structural view of a dental implant, and (b) is a schematic partial sectional view.

FIG. 2 is an EPO release rate of the dental implant obtained in example 2.

FIG. 3 shows the results of alkaline phosphatase activity assay.

FIG. 4 shows the results of measurement of the secretion of osteocalcin.

Detailed Description

The technical scheme of the invention is explained in detail by the following examples, and the equipment and raw materials related to the examples can be obtained by conventional methods if not specifically stated.

Example 1

The utility model provides a dental implant with bioactive surface, includes implant body 6 and slow-release layer 5, and 6 surfaces of implant body are provided with the screw thread, and the both ends of implant body 6 are respectively crown mouth end 1 and implantation end 2, and implant body 6 is the obconic body, and the one end that crown mouth end 1 was kept away from to implantation end 2 is arc surface 4, and 1 cross section diameter of crown mouth end is greater than implantation end 2 cross section diameter.

The outer surface of the implanting end 2 is provided with two self-tapping grooves 3, and the two self-tapping grooves 3 are uniformly distributed on the outer surface of the implanting end 2; the depth of the self-tapping groove 3 becomes gradually shallower from the end of the implanting end 2 far away from the coronal end 1 to the direction of the implanting end 2 close to the coronal end 1.

The slow release layer 5 covers the outer surface of the implant body 6, and the slow release layer 5 covers the outer surfaces of the thread, the coronal end 1, the implanting end 2 and the self-tapping groove 3; the slow release layer 5 is a chitosan nano microsphere composite layer coated with EPO; the chitosan nano-microsphere is a water-soluble Hydroxypropyl Chitosan (HCS) nano-microsphere with the particle size of 200-350 nm; the EPO encapsulation efficiency is about 75 percent.

Example 2

A preparation method of a dental implant with a bioactive surface containing EPO comprises the following specific preparation steps:

(1) preparation of water-soluble hydroxypropyl chitosan nano microsphere

Taking 2g of Chitosan (Chitosan, CS) in a three-neck flask, adding 15ml of NaOH solution with the mass fraction of 20% and 24ml of isopropanol, stirring for 1h at room temperature, adding 1ml of catalyst tetramethylammonium hydroxide, slowly dropwise adding 15ml of propylene oxide, shaking, stirring and uniformly mixing for 5h in a water bath at the temperature of 60 ℃, and cooling to room temperature after the reaction is finished. Adjusting pH to 7 with HCl, adding into acetone water solution, stirring, filtering, washing with anhydrous ethanol for several times, and vacuum drying at 60 deg.C.

(2) Preparation of water-soluble hydroxypropyl chitosan-EPO nano microsphere

Dissolving 0.2g of HCS and 0.2g of EPO in 50ml of double distilled water by adopting an ion gel method, stirring, standing for 1 hour to fully dissolve the HCS and the EPO, filtering by using 0.45-micron filter paper, and adjusting the pH value to 6 by using a small amount of acetic acid; dissolving 0.1g sodium Tripolyphosphate (TPP) in 50ml double distilled water, filtering with 0.22 micron filter paper; 20ml of HCS solution is taken, 20ml of TPP solution is dripped at the speed of 20 drops/minute, and the final concentration of HCS is 2.0g/l and the final concentration of TPP is 1.0g/l under the condition of magnetic force continuous stirring for 30 min; the diameter of the nano microspheres is 150-350 nm; storing at 4 deg.C. The encapsulation rate of EPO in the nano microspheres is about 75%, and the sustained release time of EPO from the nano microspheres can reach more than 10 days.

(3) Pretreatment of implant body surface

Cleaning the implant body to be treated by deionized water, drying and then carrying out sand blasting treatment; the sand blasting material is alumina, the sand blasting pressure is 0.5Mpa, the time is 70s, the distance between the nozzle and the implant body is 8cm, and the sand blasting angle is 20 degrees; cleaning the implant body subjected to sand blasting, drying, and carrying out acid etching at 70 ℃ for 30 min; the acid etching liquid medicine contains 35% of sulfuric acid, 25% of hydrochloric acid and the balance of water in volume fraction; after acid etching, placing the implant body in 5mol/L NaOH solution at 60 ℃ for incubation for 48h, and adjusting the pH value to 7.0; after being cleaned by deionized water, the mixture is firstly soaked in pure acetone for 10 minutes and then soaked in absolute ethyl alcohol for 10 minutes; and (3) putting the implant body into 150mM sodium chloride electrolyte with pH of 7 for preservation.

The surface of the implant body after pretreatment is detected by a laser confocal microscope, and Roughness parameters roughnessparameter (micrometer) are as follows:

sa, (arithmetic mean deviation of the surface): 1.3+0.02

Sq, (root mean square deviation root-mean-square deviation of the surface): 1.6+0.03

St, (maximum peak-to-valley height of the surface): 8.1+0.2

Sk, (amplitude distribution skew): 4.9+1.8

(4) Preparation of dental implant with water-soluble hydroxypropyl chitosan-EPO (erythropoietin) nano microsphere surface

And (3) placing the pretreated implant body with the acid etching and sand blasting surfaces into a water-soluble hydroxypropyl chitosan-EPO nano microsphere solution for soaking for 30min, adjusting the pH value to 7.0 when the nano microspheres are adsorbed on the surface of the implant body to reach a saturated state, and sterilizing for 24h under cobalt 60 radiation sterilization to obtain the dental implant with the EPO-containing bioactive surface.

Example 3

The dental implant prepared in example 2 is tested in a drug release test, which comprises the following specific steps:

(1) placing the treated dental implant on the surface of the water-soluble hydroxypropyl chitosan-EPO nano microsphere in 20ml of phosphate buffer (pH is 7.2), and carrying out water bath at 37 ℃;

(2) taking out 0.5ml of supernatant at intervals, measuring the content of free EPO in the supernatant by using a Coomassie brilliant blue protein measuring kit, and simultaneously keeping the volume constant by using an equivalent amount of phosphate buffer;

(3) each experiment was repeated 3 times, and the average of 3 determinations was taken.

The results are shown in FIG. 1, and good EPO delayed-release effect is still displayed after 200 h.

Example 4

Preparing different surface experimental groups and control groups to carry out bone formation capability test by detecting alkaline phosphatase and osteocalcin, and specifically comprising the following steps:

control 1 (Plastic): 24-well cell culture plates (15 mm diameter);

control 2(Plastic + EPO): processing a porous cell culture plate by using water-soluble hydroxypropyl chitosan-EPO nano microspheres;

experimental group 1 (SLA): carrying out surface acid etching and sand blasting on the titanium plate, wherein the diameter of the titanium plate is 15mm, and the height of the titanium plate is 1 mm;

experimental group 2(SLA + EPO): and (3) treating the acid-etched and sand-blasted titanium disc by using the water-soluble hydroxypropyl chitosan-EPO nano microspheres, wherein the diameter of the titanium disc is 15mm, and the height of the titanium disc is 1 mm.

(1) Alkaline phosphatase detection

Taking 3 rd generation human alveolar bone osteoblast with cell density of 9300cells/cm²The cell concentration of (2) is inoculated in a 24-well cell culture plate containing an experimental group and a control group, and the intervention is carried out according to experimental groups; at 37 ℃ with a volume fraction of 5% CO₂Culturing in a cell culture box, replacing the culture solution every 48h, and removing the culture solution after 7 days to harvest cells; centrifuging the cells, washing the precipitate, adding 200 μ L cell lysate (PBS containing 0.2% Triton X-100), and ultrasonically lysing the cells at 4 deg.C; the total cellular protein content was determined using the protein assay BCA kit.

The determination of alkaline phosphatase activity is carried out by converting colorless p-nitrophenyl phosphate into colored p-nitrophenol and completing the experiment according to the experimental flow of the alkaline phosphatase determination kit. The color change was measured by a spectrophotometer at 405nm, quantifying the enzyme released by the cells and comparing with the same standard curve. The alkaline phosphatase level was normalized to the relative total protein content, and the results are shown in FIG. 2, where the total amount of SLA + EPO-produced protein was the highest.

(2) Cellular osteocalcin secretion assay

Taking 3 rd generation human alveolar bone osteoblast with cell density of 9300cells/cm²The cell concentration of (2) is inoculated in a 24-well cell culture plate containing an experimental group and a control group, and the intervention is carried out according to experimental groups; at 37 ℃ with a volume fraction of 5% CO²Culturing in a cell culture box, replacing the culture solution every 48h, and removing the culture solution for later use after 8 days; the osteocalcin secretion in the culture solution is finished by a commercial osteocalcin detection kit, the experiment is measured by enzyme-linked immunoassay, and the measurement operation flow is strictly executed according to the kit instruction. The sensitivity (0.4. mu.g/L) commonly used for this measurement, and the results are shown in FIG. 3, where SLA + EPO exhibited good bone formation ability.

Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.