CN112315815B

CN112315815B - Preparation method and application of bioactive material containing nano calcium fluoride

Info

Publication number: CN112315815B
Application number: CN202011225435.8A
Authority: CN
Inventors: 刘瑾; 徐华焜; 李昂; 代泉
Original assignee: Xian Jiaotong University
Current assignee: Xian Jiaotong University
Priority date: 2020-11-05
Filing date: 2020-11-05
Publication date: 2022-08-26
Anticipated expiration: 2040-11-05
Also published as: CN112315815A

Abstract

The invention discloses a preparation method and application of a bioactive material containing nano calcium fluoride. Belongs to the technical field of dental biomaterials. The method comprises the following steps: mixing glycidyl methacrylate and triethylene glycol dimethacrylate at a mass ratio of 1:1 to obtain BT, and mixing the BT with camphorquinone and ethyl 4- (N, N-diethylamino) benzoate at a mass ratio of 100:0.2:0.8 to obtain a mixture A; mixing the boron aluminum silicate barium glass particles with 3- (methacryloyloxy) propyl trimethoxy silane and n-propylamine, stirring by using a magnetic stirrer, and standing overnight at normal temperature until the liquid is clear and transparent to obtain a mixture B; and mixing the nano calcium fluoride with the mixture A and the mixture B. Compared with the prior art, the invention has the following beneficial effects: the bioactive material added with the nano calcium fluoride has good mechanical property, can slowly release fluorine ions and calcium ions, and is beneficial to regeneration of periodontal tissues.

Description

Preparation method and application of bioactive material containing nano calcium fluoride

Technical Field

The invention relates to the technical field of dental biomaterials, in particular to a preparation method and application of a bioactive material containing nano calcium fluoride.

Background

Periodontitis and root-surface caries are both causal agents, the occurrence of root-surface caries is related to age and the amount of cementum exposed in the mouth, and periodontitis is also exacerbated by food and plaque retention caused by root-surface caries. Periodontitis can cause recession of the gums, exposure of the root surface, and increased incidence of root surface caries. Filling therapy remains the dominant approach at present.

Materials used for the filling are silver amalgam, composite resin and glass ion composite, with composite resin being the most common. The composite resin is composed of a resin matrix and an inorganic filler, has a large tensile strength and is less expanded, contracted and deformed during curing, and has the most prominent advantages of beauty and optimal color matching with adjacent teeth. However, the existing filling resin material has certain irritation to gingiva, which causes further progress of periodontal inflammation, and the common filling material cannot release biological ions which are beneficial to regeneration of periodontal tissues.

In view of the above, it is an urgent need to solve the problem of providing a filling material that is less irritating to the gums and can release bio-ions that are beneficial to regeneration of periodontal tissues for a long period of time.

Disclosure of Invention

In view of this, the invention provides a preparation method and application of a bioactive material containing nano calcium fluoride.

In order to achieve the purpose, the invention adopts the following technical scheme:

a preparation method of a bioactive material containing nano calcium fluoride comprises the following steps:

(1) mixing glycidyl methacrylate (BisGMA) and triethylene glycol dimethacrylate (TEGDMA) in a mass ratio of 1:1 to obtain BT, and then mixing and stirring BT, Camphorquinone (CQ) and 4- (N, N-diethylamino) ethyl benzoate at a speed of 200rpm in a dark place at a mass ratio of 100:0.2:0.8 to obtain a mixture A;

(2) mixing the barium boroaluminosilicate glass particles with 3- (methacryloyloxy) propyl trimethoxy silane and n-propylamine according to the mass ratio of 100:4:2, and then stirring overnight by using a magnetic stirrer at normal temperature and 200rpm until the liquid is clear and transparent to obtain a mixture B;

(3) mixing nanometer calcium fluoride (Nano-CaF) ₂ ) Mixing with the mixture A and the mixture B to obtain the bioactive material containing the nano calcium fluoride.

The beneficial effects are as follows: nanometer calcium fluoride biological material is added into the filling material, and when the carious cavity is filled, fluorine ions and calcium ions which are slowly released can enter periodontal tissues through gingival sulcus fluid, so that osteogenic differentiation and cementum differentiation of periodontal ligament stem cells are promoted, and regeneration of the periodontal tissues is facilitated.

Furthermore, the mass ratio of the nano calcium fluoride to the mixture A to the mixture B in the step (3) is (10-20) to (30) (50-60).

The beneficial effects are as follows: if the addition amount of the nano fluorine oxide is too small, the release amount of calcium ions and fluorine ions is small; if added too much, it will interfere with the proper curing of the resin.

Further, the mass ratio of the nano calcium fluoride to the mixture A to the mixture B in the step (3) is 10:30: 60.

Further, the mass ratio of the nano calcium fluoride to the mixture A to the mixture B in the step (3) is 15:30: 55.

Further, the mass ratio of the nano calcium fluoride to the mixture A to the mixture B in the step (3) is 20:30: 50.

Further, the median particle diameter of the barium boroaluminosilicate glass particles is 1.4 μm.

The bioactive material containing nano calcium fluoride prepared by the method is applied to the periodontitis filling material.

According to the technical scheme, compared with the prior art, the bioactive material added with the nano calcium fluoride has good mechanical properties of the filling material, can slowly release fluorine ions and calcium ions, is beneficial to periodontal tissue regeneration, and is suitable for periodontal patients.

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 embodiments or the prior art descriptions will be briefly described below, it is obvious that the drawings in the following description are only embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the provided drawings without creative efforts.

FIG. 1 is a graph showing the results of experiment 1 according to the present invention, wherein a is a graph showing the results of flexural strength, b is a graph showing the results of elastic modulus, and c is a graph showing the results of hardness;

FIG. 2 is a graph showing the results of measurement in experiment 2 of the present invention, wherein a is a graph showing the results of calcium ion release (in mmol/L day) and b is a graph showing the results of fluorine ion release (in mmol/L day);

FIG. 3 is a graph showing the results of cell proliferation assay in experiment 3 of the present invention, in which a is a graph showing the proliferation results of hPDLSCs, b is a graph showing the SEM results (bioactive material prepared in example 3), and c is an enlarged view of the dotted frame portion in FIG. 3 b;

FIG. 4 is a graph showing the result of the cell staining assay of experiment 3 of the present invention, in which a is 0% Nano-CaF ₂ FIG. 1 day results, b is 20% Nano-CaF ₂ Results of group 1 day, c is results of Heliomolar group 1 day, d is 0% Nano-CaF ₂ FIG. 7 day results, e is 20% Nano-CaF ₂ Results of group 7 days, f is results of Heliomolar group 7 days, g is 0% Nano-CaF ₂ Group 14 days results plot, h is 20% Nano-CaF ₂ The result chart of the group at 14 days, i is the result chart of the Heliomolar group at 14 days, j is the result chart of the density of the living cells, and k is the result chart of the percentage of the living cells;

FIG. 5 is a graph showing the results of osteoblast gene changes in experiment 4 of the present invention, in which a is the expression of ALP gene, b is the expression of RUNX2 gene, c is the expression of OPN gene, and d is the expression of COL1 gene;

FIG. 6 is a graph showing the results of the cementum-forming gene changes in experiment 4 of the present invention, wherein a is the expression of BSP gene, b is the expression of CEMP1 gene, and c is the expression of CAP gene;

FIG. 7 is a graph showing the results of measurement of the change in ALP activity in experiment 5 of the present invention;

FIG. 8 is a graph showing the results of ARS staining in experiment 6 of the present invention;

FIG. 9 is a graph showing the result of quantitative analysis of mineralized nodules in experiment 6 according to the present invention.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

The required medicament is a conventional experimental medicament, and is purchased from a market channel; the unrecited experimental method is a conventional experimental method, and is not described in detail herein.

Example 1

(1) mixing glycidyl methacrylate and triethylene glycol dimethacrylate at a mass ratio of 1:1 to obtain BT, and mixing BT with camphorquinone and ethyl 4- (N, N-diethylamino) benzoate at a mass ratio of 100:0.2:0.8 to obtain a mixture A.

(2) Mixing the boron aluminum silicate barium glass particles with 3- (methacryloyloxy) propyl trimethoxy silane and n-propylamine according to the mass ratio of 100:4:2, stirring by using a magnetic stirrer, and standing overnight at normal temperature until the liquid is clear and transparent to obtain a mixture B.

(3) And mixing the nano calcium fluoride with the mixture A and the mixture B to obtain the bioactive material containing the nano calcium fluoride, wherein the mass ratio of the nano calcium fluoride to the mixture A to the mixture B is 10:30: 60.

(4) Molding the prepared bioactive material: the cover of a sterile 96-well plate is used as a mold to form a circular sheet with the diameter of 8mm and the thickness of 1 mm. ② the prepared bioactive material wafer is photo-cured (irradiated by a photo-curing machine) for 1 minute, and then is immersed in distilled water of 37 ℃ and stirred for 1 day to eliminate the uncured monomer. And thirdly, finally sterilizing by using ethylene oxide, and then degassing for 3 days for reuse. The cells were soaked in DMEM medium for 24 hours before the experiments.

Example 2

The mass ratio of the nano calcium fluoride to the mixture A and the mixture B in the step (3) is 15:30:55, and the rest of the operation is the same as that in the example 1.

Example 3

The mass ratio of the nano calcium fluoride to the mixture A and the mixture B in the step (3) is 20:30:50, and the rest of the operation is the same as that in the embodiment 1.

Comparative example 1

(1) Mixing glycidyl methacrylate and triethylene glycol dimethacrylate in a mass ratio of 1:1 to obtain BT, and mixing BT with camphorquinone and ethyl 4- (N, N-diethylamino) benzoate in a mass ratio of 100:0.2:0.8 to obtain a mixture A.

(3) And mixing the mixture A and the mixture B according to the mass ratio of 30: 70.

(4) Molding the prepared bioactive material: the cover of a sterile 96-well plate is used as a mold to form a circular sheet with the diameter of 8mm and the thickness of 1 mm. ② the prepared bioactive material wafer is photo-cured (irradiated by a photo-curing machine) for 1 minute, and then immersed in distilled water at 37 ℃ and stirred for 1 day to eliminate all uncured monomer. And thirdly, finally sterilizing by using ethylene oxide, and then degassing for 3 days for reuse. The cells were soaked in DMEM medium for 24 hours before the experiments.

Experiment 1 measurement of flexural Strength, elastic modulus and hardness

(1) Measurement target: the bioactive material wafer prepared by the methods in examples 1-3, the bioactive material wafer prepared by the method in comparative example 1, and the composite resin Heliomolar.

(2) The determination method comprises the following steps:

the flexural strength and the modulus of elasticity were measured using a universal tester (5500R, MTS, Cary, NC, USA) at a loading rate of 1 mm/min.

Hardness values were evaluated using a Vickers diamond indenter durometer (HMV-g21dt, Shimadzu, Kyoto). Three impressions were made and measured at various points on each sample, with a load of 200g and a dwell time of 15 seconds.

(3) And (3) measuring results: the measurement results are shown in FIG. 1.

As can be seen from the results of FIG. 1, FIGS. 1a and 1c show four Nano-CaF ₂ There was no difference in flexural strength and hardness between the groups, but the flexural strength of Heliomolar was lower than that of the other four groups (p)<0.05). FIG. 1b shows 0% Nano-CaF ₂ The elastic modulus of the Himoomolar contrast is slightly less than that of other Nano-CaF ₂ Group (p)<0.05). The results show that the addition of Nano-CaF ₂ Compared with the Heliomolar group, the mechanical properties of flexural strength, hardness and elastic modulus are good, and the composite material can be used for filling a cavity.

Experiment 2 calcium fluoride ion Release experiment

(1) Measurement target: bioactive material wafers (2 mm. times.2 mm. times.12 mm) and composite resin Heliomolar (2 mm. times.2 mm. times.12 mm) prepared by the methods of examples 1-3.

(2) The measuring method comprises the following steps: the mass concentration of NaCl and HEPES in the soaking solution was 133mmol/L and 50mmol/L, respectively. The samples were immersed in 50mL of the immersion solution and the concentration of fluorine and calcium released from the samples on

days

1, 2, 4, 7, 14, 21, 28 and 56 of immersion were determined. At each time period, 2ml of the soak solution was taken for evaluation. The amount of fluorine ions was measured using a fluorine ion concentration measuring instrument. For calcium ions, the concentration of calcium ions in the soak solution was measured spectrophotometrically. Each sample was replicated 3 times.

(3) And (3) measuring results: the measurement results are shown in FIG. 2.

FIG. 2 illustrates the addition of Nano-CaF in different proportions ₂ Can release effective fluoride ions and calcium ions for 56 days, and 20 percent of Nano-CaF ₂ The effect of the group is optimal. The Heliomolar group released only a very low concentration of fluoride ions, and did not release calcium ions. 20% Nano-CaF ₂ The fluorine ion release amount of the group was about 30 times that of Heliomolar. The bioactive material prepared by the invention can release fluorine and calcium ions simultaneously, and is more beneficial to the differentiation of osteogenic bone and cementum.

Experiment 3 biocompatibility experiment

(1) Measurement target: the bioactive material wafer prepared by the methods in the embodiments 1 to 3, the bioactive material wafer prepared by the method in the comparative example 1 and the composite resin Heliomolar.

(2) The determination method comprises the following steps: to determine the Nano-CaF ₂ Whether the adhesion bioactivity of hPDLSCs is affected by mixing into the resin composite material or not, the cell viability on the bioactive material disc and Helimalor disc is measured by a cell counting kit 8 (CCK-8).

1ml of hPDLSCs was seeded into each well with discs at a cell density of 5000 cells/well. The medium was refreshed every 3 days. Cell proliferation was measured on

days

1, 4, 7, 14 and 21 using the CCK8 kit. The cell-bearing discs were washed with phosphate buffered saline and transferred to new 48-well plates(ii) a Then put in CO ₂ The incubator is 2 hours. Absorbance at optical density of 450nm was measured using a microplate reader.

(3) And (3) measuring results: the measurement results are shown in fig. 3 and 4.

FIG. 3a shows the addition of Nano-CaF ₂ Does not adversely affect cell proliferation. Growth of attached hPDLSCs (human periodontal ligament stem cells) on bioactive materials was similar to Heliomolar. Representative SEM images show hPDLSCs on the bioactive material prepared in example 3 at 14 days (fig. 3 b). An enlarged view of the red dashed box is shown in fig. 3 c. The hPDLSCs formed long cytoplasmic extensions (yellow arrows) on the bioactive material, indicating that the bioactive material was biocompatible and promoted adhesion of the hPDLSCs.

FIGS. 4a to 4i show fluorescence live/dead staining photographs of hPDLSCs on bioactive materials, which show that there are a large number of live cells (green staining) and few dead cells (red staining) on bioactive materials. The number of viable cells increased from 1 day to 14 days with the culture time. In FIG. 4j, 20% Nano-CaF ₂ The viable cell density (viable cell count per unit area) was slightly higher than 0% Nano-CaF ₂ And the viable cell density of the Hemolmolar control (approximately 1.3-fold and 1.5-fold at 7 days, respectively). In FIG. 4k, the percentage of viable cells was nearly constant (p) in all three groups>0.1). Fig. 4 shows that hPDLSCs can grow well in the experimental group with the addition of nano calcium fluoride material by dead-live staining of cells.

The results of fig. 3 and 4 show that the bioactive material prepared by the invention has good biocompatibility.

Experiment 4 Effect of bioactive materials on cell osteogenic and cementogenic differentiation

(1) Measurement target: bioactive material discs prepared in example 3, bioactive material discs prepared in comparative example 1, and composite resin Heliomolar.

(2) The determination method comprises the following steps:

the hPDLSCs are processed at 5 × 10 ⁴ Individual cells/well were seeded on the surface of different bioactive material discs in 24-well plates. Waiting for hPDLSCs to attach to the surface 24 of the bioactive material waferAfter hours, the medium was changed to osteogenic medium, which contained DMEME growth medium, 10% FBS +100nM dexamethasone, 10mM beta-glycerophosphate, 0.05mM ascorbic acid, and 10nM 1 alpha, 25-dihydroxyvitamin D3(Sigma-Aldrich) osteogenic medium. After culturing for 1, 7, 14 and 21d, the gene expression of osteoblasts and cementoblasts in hPDSCs was detected by quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR). RNA was reverse transcribed into cDNA using a high capacity cDNA reverse transcription kit. qPCR was performed by SYBR's PCR kit. Primer sequences are listed in table 1. qPCR was performed by Biosystems Prism 7000 detection system with 0% Nano-CaF ₂ Ct values at day 1 of the group were used as calibration values.

TABLE 1 PCR primer sequences

(3) And (3) measuring results: the measurement results are shown in fig. 5 and 6.

As can be seen from FIG. 5, the ALP gene expression peaked at 14 days, while RUNX2, OPN and COL1 peaked at 21 days.

As can be seen from FIG. 6, 20% of Nano-CaF was observed in the cementogenic gene at 14 days (CAP and CEMP1 genes) and 21 days (BSP gene) ₂ Group significantly elevated (p)<0.05)。

The results of fig. 5 and 6 show that the bioactive material prepared by the invention can promote the osteogenic differentiation of cells and the differentiation of cementum.

Experiment 5 ALP Activity

(2) The determination step comprises: hPDLSCs at 10 ⁴ The density of individual cells/well was seeded onto biologically active material discs in 48-well plates. On

days

1, 7, 14 and 21, detection by ALP kit (BioA)ssay Systems, Cambridge, MA, USA) to assess ALP activity: the adhered hPDLSCs were digested and washed, resuspended and stirred in 0.2% Triton-X100 lysis buffer for 30 minutes. Then centrifuged at 1500rpm for 5 minutes. The ALP activity of the supernatant was then determined using an ALP working solution containing 200. mu.L of assay buffer, 5. mu.L of magnesium acetate (final 5mM) and 2. mu.L of pNPP liquid matrix (10mM) at a ratio of 20. mu.L sample supernatant/180. mu.LALP working solution. After mixing, at OD ₄₀₅ The mixture was measured at nm with an absorbance meter and after 4 minutes the mixture was measured again using a microplate reader (SpectraMax M5). Finally, ALP activity was normalized using protein mass.

(3) And (3) measuring results: the measurement results are shown in FIG. 7.

The results in FIG. 7 show that ALP activity of hPDLSCs increases with time from 1 day to 14 days, and then gradually decreases again from 14 days to 21 days. On

days

7, 14 and 21, 20% Nano-CaF ₂ ALP activity of group hPDLSCs was 0% Nano-CaF ₂ 57-, 78-and 55-fold for the control group.

Experiment 6 alizarin red staining

(2) The determination step comprises: hPDLSCs at 1 × 10 ⁴ Individual cells/well were seeded onto bioactive material discs and cultured in osteogenic medium for 1, 7, 14 and 21 days. Then, bone mineral secreted from hPDLSCs on the bioactive material was examined by Alizarin Red Staining (ARS).

The calcium material secreted by the hPDLSCs can be stained deep red by fixing the hPDLSCs on the bioactive material discs with 4% paraformaldehyde for 30 minutes and then staining with 2% ARS solution for 30 minutes. The ARS solution was then removed and the bioactive material discs were washed with PBS to eliminate any loose alizarin red. The sample is then imaged.

For quantitative testing, ARS-stained hPDLSCs on composite material were destained for 15 min in 10% cetylpyridinium chloride. At OD ₆₅₂ The absorbance of the solution was evaluated at nm using a microplate reader.

(3) And (3) measuring results:

ARS pictures of mineralized nodules synthesized by hPDLSCs are shown in FIG. 8. Bone mineral is stained red. All groups had no mineral nodules on day 1. But for 20% Nano-CaF ₂ hPDLSC starts to synthesize bone mineral at 7 and 14 days. The bioactive material disc was covered by a new layer of mineralized bone matrix secreted by hPDLSCs, which became thicker and more abundant at 21 days. In contrast, at 14 and 21 days, 0% Nano-CaF ₂ And the Heliomolar disc had much fewer bone mineral nodules.

FIG. 9 plots the quantification. hPDSCs in 20% Nano-CaF ₂ Bone mineral secretion on bioactive materials increases significantly with prolonged culture time. 20% Nano-CaF ₂ The mineral secretion of hPDLSCs in the groups at day 14 and day 21 was almost 2-fold higher than that in the other groups (p)<0.05). Indicating 20% Nano-CaF ₂ The group has a good effect of promoting the formation of bone mineralization nodules.

The embodiments in the present description are described in a progressive manner, each embodiment focuses on differences from other embodiments, and the same and similar parts among the embodiments are referred to each other.

The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

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Claims

1. A preparation method of a bioactive material containing nano calcium fluoride is characterized by comprising the following steps:

(1) mixing glycidyl methacrylate and triethylene glycol dimethacrylate in a mass ratio of 1:1 to obtain BT, and mixing BT with camphorquinone and ethyl 4- (N, N-diethylamino) benzoate in a mass ratio of 100:0.2:0.8 to obtain a mixture A;

the median particle diameter of the boron aluminum silicate barium glass particles is 1.4 mu m;

(3) mixing the nano calcium fluoride with the mixture A and the mixture B to obtain a bioactive material containing the nano calcium fluoride;

the mass ratio of the nano calcium fluoride to the mixture A to the mixture B is (10-20) to (30) (50-60).

2. The method for preparing the bioactive material containing the nano-calcium fluoride according to claim 1, wherein the mass ratio of the nano-calcium fluoride to the mixture A to the mixture B in the step (3) is 10:30: 60.

3. The method for preparing the bioactive material containing the nano calcium fluoride according to claim 1, wherein the mass ratio of the nano calcium fluoride to the mixture A to the mixture B in the step (3) is 15:30: 55.

4. The method for preparing the bioactive material containing the nano calcium fluoride according to claim 1, wherein the mass ratio of the nano calcium fluoride to the mixture A to the mixture B in the step (3) is 20:30: 50.

5. The application of the bioactive material containing nano calcium fluoride prepared by the method of any one of claims 1 to 4 in preparation of a periodontitis filling material.