A composite bioglass and its preparation method and application

Abstract

The present application relates to the field of biological glass, in particular to a kind of composite biological glass and its preparation method and application.The present application uses rhBMP-2, doped antibacterial ion 45S5 bioactive glass and polyethylene glycol-lactide-glycolide polymer compound preparation slow-release antibacterial biological glass, the rhBMP-2 can occur synergistic effect with doped antibacterial ion 45S5 bioactive glass, compared with homologous growth factor can play more optimal synergy with antibacterial ion, so as to have higher antibacterial property and osteogenesis, while having longer slow-release effect in polyethylene glycol-lactide-glycolide polymer.The composite biological glass provided by the present application overcomes the pain points of the prior art for the treatment of oral bone defect, improves the treatment effect, reduces the amount of antibiotic use, and reduces the risk of infection.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological glass, and particularly relates to a composite biological glass as well as a preparation method and application thereof. BACKGROUND

[0002] Hench invented biological glass (BG) in 1970, which has been used for more than 50 years. With the continuous development of technology and the deepening of research, biological glass also shows strong vitality. For example, antibacterial ions are added to increase antibacterial properties; various drugs are added to be used as drug sustained-release carriers; special treatment is carried out to prepare biological glass scaffolds with greater strength; and coatings are made to be attached to the surface of other devices, etc. so that biological glass has more application scenarios and expands the use range.

[0003] In the field of oral cavity, biological glass plays a more important role. Biological glass is used in dental implantation, root canal treatment, alveolar ridge repair, and even tooth desensitization treatment. The oral cavity contains a variety of bacterial flora. Daily cleaning can maintain the balance of bacterial flora and reduce oral problems such as ulcers and inflammation. However, patients who have undergone oral surgery treatment are limited in daily cleaning, and the oral bacterial flora is unbalanced, which is more prone to additional oral problems, especially at the surgical site. Therefore, it is often supplemented with antibiotic treatment. However, long-term use of antibiotics can cause drug resistance and other problems. Therefore, it is urgent to provide a new and efficient sustained-release antibacterial biological glass to reduce the use of antibiotics, accelerate wound healing, reduce the risk of infection at the wound site, and reduce the pain of patients. SUMMARY

[0004] Therefore, the technical problem to be solved by the present application is a composite biological glass as well as a preparation method and application thereof. The composite biological glass provided by the present application has excellent biological activity, antibacterial property and osteogenic activity, good sustained-release effect, and is suitable for oral environment.

[0005] The present application provides a composite biological glass, which comprises: a polyethylene glycol-lactide-glycolide polymer gel and rhBMP-2 and 45S5 bioactive glass doped with antibacterial ions doped in the polyethylene glycol-lactide-glycolide polymer gel.

[0006] The antibacterial ions are selected from one or more of Ag, Zn, Cu and Ce.

[0007] Specifically, the composite bio-glass is obtained by cross-linking rhBMP-2, polyethylene glycol-lactide-glycolide polymer gel and 45S5 bioactive glass doped with antibacterial ions, wherein the rhBMP-2 is a growth factor; the polyethylene glycol-lactide-glycolide polymer gel is a hydrogel component; and the 45S5 bioactive glass doped with antibacterial ions is a bio-glass containing antibacterial components and is synthesized by a polymer template method. The mass ratio of the rhBMP-2, the polyethylene glycol-lactide-glycolide polymer and the 45S5 bioactive glass doped with antibacterial ions is (0.001-0.003):(2-3):(0.02-0.03), preferably (0.001-0.002):(2.3-2.7):(0.023-0.027), and more preferably 0.0018:2.5:0.025.

[0008] The doping amount of the antibacterial ions in the 45S5 bioactive glass doped with antibacterial ions is (5-15) wt%, preferably 8 wt%. The antibacterial ions are preferably selected from Zn, and the 45S5 bioactive glass doped with Zn has better protein adsorption capacity.

[0009] The polyethylene glycol-lactide-glycolide polymer gel is obtained by polymerization of polyethylene glycol, lactide and glycolide at a mass ratio of (18-22):(34-38):(8-12), preferably at a mass ratio of (19-21):(35-37):(9-11). Specifically, the polyethylene glycol-lactide-glycolide polymer sol is obtained by polymerization of polyethylene glycol, lactide and glycolide, and then the polyethylene glycol-lactide-glycolide polymer gel is obtained by solidification. The polyethylene glycol-lactide-glycolide polymer gel as a hydrogel component makes the release curve of the composite bio-glass more in line with the recovery rule. The molecular weight of the polyethylene glycol is 2000-6000. In some embodiments of the present application, the polyethylene glycol is PEG3000.

[0010] In the composite bio-glass, the rhBMP-2 can have a synergistic effect with the 45S5 bioactive glass doped with antibacterial ions, and compared with homologous growth factors, the rhBMP-2 can have better synergistic effect with the antibacterial ions, thereby having higher antibacterial property and osteogenic property, and having longer sustained-release effect in the polyethylene glycol-lactide-glycolide polymer.

[0011] The present application also provides a preparation method of the composite bio-glass, comprising the following steps:

[0012] The 45S5 bioactive glass doped with antibacterial ions and rhBMP-2 are solidified in a polyethylene glycol-lactide-glycolide polymer sol to obtain the composite bio-glass.

[0013] The antibacterial ions are selected from one or more of Ag, Zn, Cu and Ce.

[0014] Specifically, the 45S5 bioactive glass doped with antibacterial ions and rhBMP-2 are placed in a polyethylene glycol-lactide-glycolide polymer sol at room temperature, vacuumized for 8-12 min, and left to stand at 35-40 ℃ to solidify the sol, thereby obtaining the composite bio-glass.

[0015] In some embodiments of the present application, the polyethylene glycol-lactide-glycolide polymer is dissolved in physiological saline to prepare a 20-30 wt% polyethylene glycol-lactide-glycolide polymer sol; then rhBMP-2 is dispersed in the polyethylene glycol-lactide-glycolide polymer sol at room temperature to obtain a polyethylene glycol-lactide-glycolide polymer sol containing rhBMP-2; the 45S5 bioactive glass doped with antibacterial ions is completely immersed in the polyethylene glycol-lactide-glycolide polymer sol containing rhBMP-2 at room temperature, vacuumized for 8-12 min, and left to stand at 35-40 ℃ to solidify the sol, thereby obtaining the composite bio-glass.

[0016] The mass ratio of rhBMP-2, the polyethylene glycol-lactide-glycolide polymer sol and the 45S5 bioactive glass doped with antibacterial ions is 1.8 mg:2.5 g:0.025 g. The doping amount of antibacterial ions in the 45S5 bioactive glass doped with antibacterial ions is 5-15 wt%, preferably 8 wt%. The antibacterial ions in the 45S5 bioactive glass doped with antibacterial ions are preferably selected from Zn. The room temperature in the present application refers to the temperature in the natural state of a room, which is not dependent on external heating or cooling devices, and is preferably 25 ℃.

[0017] The polyethylene glycol-lactide-glycolide polymer sol of the present application is obtained by polymerization of polyethylene glycol, lactide and glycolide in a mass ratio of (18-22):(34-38):(8-12). Specifically, the polyethylene glycol-lactide-glycolide polymer is prepared by polymerization of polyethylene glycol, lactide and glycolide in the presence of an organic tin catalyst. More specifically, the polyethylene glycol is placed in a vacuum atmosphere for (2-4) h and argon is replaced once per hour while heating under stirring at (120-130) ℃ at a speed of (60-80) r / min; after natural cooling, the polyethylene glycol is added with lactide and glycolide for dissolution, and then an organic tin catalyst solution is added, followed by removal of the solvent and polymerization to obtain the polyethylene glycol-lactide-glycolide polymer sol.

[0018] The molecular weight of the polyethylene glycol of the present application is (2000-6000). In some embodiments of the present application, the polyethylene glycol is PEG3000. The organic tin catalyst of the present application is selected from at least one of stannous octoate, dibutyltin dilaurate, tributyltin oxide and dibutyltin oxide. The temperature of the polymerization reaction is (140-160) ℃, and the time of the polymerization reaction is (10-14) h.

[0019] The present application also provides the use of the composite bio-glass according to any of the technical solutions described above or the composite glass prepared by the preparation method according to any of the technical solutions described above as an oral bone repair material. The composite bio-glass provided by the present application has higher antibacterial property and osteogenic property, and has a longer and more regular release curve, and is very suitable for application in an oral environment as an oral bone repair material.

[0020] The present application provides a composite bio-glass, a preparation method and application thereof. The present application uses rhBMP-2, 45S5 bioactive glass doped with antibacterial ions and polyethylene glycol-lactide-glycolide polymer to prepare a slow-release antibacterial bio-glass. The rhBMP-2 can have a synergistic effect with the 45S5 bioactive glass doped with antibacterial ions, and can have a better synergistic effect with the antibacterial ions than the homologous growth factors, thereby having higher antibacterial property and osteogenic property, and having a longer slow-release effect in the polyethylene glycol-lactide-glycolide polymer. The composite bio-glass provided by the present application overcomes the pain points of the prior art in the treatment of oral bone defects, improves the treatment effect, reduces the amount of antibiotics used, and reduces the risk of infection. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 Biological activity results of the bio-glass doped with silver ions;

[0022] Figure 2 Biological activity result graph of the biological glass doped with zinc ions;

[0023] Figure 3 Biological activity result graph of the biological glass doped with copper ions;

[0024] Figure 4 Biological activity result graph of the biological glass doped with cerium ions;

[0025] Figure 5 Biological activity result graph of the rhBMP-2 / BG described in Example 1 of the present application;

[0026] Figure 6 Biological activity result graph of the polymer described in Comparative Example 1 of the present application;

[0027] Figure 7 Biological activity result graph of the rhBMP-4 / BG described in Comparative Example 2 of the present application. DETAILED DESCRIPTION

[0028] The present application discloses a composite biological glass, a preparation method and application thereof. Those skilled in the art can refer to the content herein and appropriately improve the process parameters to realize. It is particularly pointed out that all similar substitutions and changes are obvious to those skilled in the art, and they are considered to be included in the present application. The method and application of the present application have been described by preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the method and application herein without departing from the content, spirit and scope of the present application, to realize and apply the present application technology.

[0029] The present application is further described below in combination with examples:

[0030] Example 1

[0031] The antibacterial biological glass is preferably:

[0032] Step 1: Silver (Ag), zinc (Zn), copper (Cu) and cerium (Ce) are selected as antibacterial ions for doping, and 45S5 biological glass doped with different antibacterial ions is synthesized by a polymer template method, and the ion doping amount is 8 wt%; wherein the biological glass doped with silver ions is recorded as sample 1, the biological glass doped with zinc ions is recorded as sample 2, the biological glass doped with copper ions is recorded as sample 3, and the biological glass doped with cerium ions is recorded as sample 4;

[0033] Step 2: The biological glass doped with different antibacterial ions obtained in step 1 is cultured in vitro, and then its biological activity and antibacterial property are tested to optimize an antibacterial biological glass. The biological activity results of the biological glass doped with different antibacterial ions are as shown in Figures 1-4 Figure 1 ​Figure of bioactivity results of the bioactive glass doped with silver ions, Figure 2 Figure of bioactivity results of the bioactive glass doped with zinc ions, Figure 3 Figure of bioactivity results of the bioactive glass doped with copper ions, Figure 4 Figure of bioactivity results of the bioactive glass doped with cerium ions. The antibacterial results of the bioactive glass doped with different antibacterial ions are shown in Table 1:

[0034] Table 1

[0035]

[0036] Example 2

[0037] Preparation of rhBMP-2 / BG antibacterial bioactive glass composite

[0038] (1) Preparation of hydrogel

[0039] A three-necked flask containing 20.25 g of polyethylene glycol (PEG3000) was placed in an oil bath, and the temperature was set to 125°C and the stirring speed was set to 70 r / min. Vacuum was drawn for 3 h, and argon was replaced every hour. After natural cooling, 36.8 g of lactide and 9.9 g of glycolide were added, respectively, and after complete dissolution, stannous octoate toluene solution was added. The toluene was removed by vacuum, the oil bath temperature was set to 150°C, and the reaction was carried out for 12 h. The residual raw materials and small molecule products were removed by vacuum, and the polymer was obtained after hot water washing and freeze-drying. The obtained polymer is the hydrogel.

[0040] (2) Preparation of rhBMP-2 / BG

[0041] 2.5 g of the polymer prepared in (1) above was dissolved in 7.5 mL of normal saline to prepare a 25 wt% sol. 1.8 mg of rhBMP-2 dry powder was added to a 10 mL test tube containing 25 wt% hydrogel at room temperature, and the rhBMP-2 was fully dispersed in the sol by shaking for 1 min. 0.025 g of the antibacterial bioactive glass doped with 8 wt% zinc ions obtained in Example 1 was completely immersed in the sol at room temperature, vacuum was drawn for 10 min, and the sol was placed in a 37°C incubator until it solidified to obtain rhBMP-2 / BG.

[0042] (3) Performance test of rhBMP-2 / BG

[0043] The rhBMP-2 / BG obtained in (2) above was subjected to antibacterial test, bioactivity test, sustained release effect test, and osteogenesis effect (cell expression), and the methods were as follows:

[0044] ① Antibacterial test: antibacterial ring;

[0045] 2. Biological activity: Refer to YY / T 0964-2014 Surgical implants - Bioactive glass and glass-ceramic materials;

[0046] 3. Sustained release effect: Soak the sample in SBF solution, replace the SBF solution at regular intervals, and test the ion content in the solution;

[0047] 4. Osteogenic effect (cell expression): Seed bone mesenchymal stem cells (BMSCs) at the bottom of a 24-well plate, and embed 5 mg of scaffold sample in the upper part of a Transwell system. Both the cells and the microspheres are soaked in osteogenic induction medium (50 μg / mL ascorbic acid, 10 nM dexamethasone, and 10 mM β-glycerophosphate), and the medium is replaced every other day. After 7 days of culture, the expression of osteogenic genes, including Runt-related transcription factor 2 (RUNX2), osteocalcin (OCN), ALP, and Osterix, is quantitatively evaluated. Cell lysis and total RNA extraction are performed using TRIzol reagent (Invitrogen, USA). The extracted RNA is then reverse transcribed into complementary DNA (cDNA) using a commercial kit (Takara, Japan) and subjected to RT-PCR quantitative analysis, and Table 2 lists the specific primer sequences used.

[0048] Table 2

[0049]

[0050] The antibacterial effect time of rhBMP-2 / BG obtained in the above (2) is 22 d, and the specific test results are shown in Table 3; the sustained release effect time is 22 d; the test results of the in vitro osteogenic effect are shown in Table 4; and the test results of the biological activity are shown in Figure 5 Figure 5 The figure of the biological activity results of rhBMP-2 / BG described in Example 1 of the present application.

[0051] Table 3

[0052]

[0053] Table 4

[0054]

[0055] Comparative Example 1

[0056] ​A three-neck flask containing 20.25 g of polyethylene glycol was placed in an oil bath, and the temperature was set to 125°C and the stirring speed was set to 70 r / min. The flask was vacuumed for 3 h and then replaced with argon once per hour. After natural cooling, 36.8 g of lactide and 9.9 g of glycolide were added, respectively. After complete dissolution, a toluene solution of stannous octoate was added, and the toluene was removed by vacuum. The oil bath temperature was set to 150°C, and the reaction was carried out for 12 h. The residual raw materials and small molecule products were removed by vacuum, and the polymer was obtained after hot water washing and freeze-drying. The obtained polymer was a hydrogel.

[0057] The obtained polymer was subjected to antibacterial test, biological activity test, sustained-release effect test, and osteogenesis effect (cell expression) test, and the methods and examples 1 were the same, and thus will not be described herein.

[0058] The antibacterial effect time of the obtained polymer was 18 d, and the specific test results are shown in Table 5; the sustained-release effect time was 18 d; the test results of the in-vitro osteogenesis effect are shown in Table 6; and the test results of the biological activity are shown in Table 7. Figure 6 Figure 6 The biological activity results of the polymer of Comparative Example 1 of the present application are shown in the figure.

[0059] Table 5

[0060]

[0061] Table 6

[0062]

[0063] Comparative Example 2

[0064] A three-neck flask containing 20.25 g of polyethylene glycol was placed in an oil bath, and the temperature was set to 125°C and the stirring speed was set to 70 r / min. The flask was vacuumed for 3 h and then replaced with argon once per hour. After natural cooling, 36.8 g of lactide and 9.9 g of glycolide were added, respectively. After complete dissolution, a toluene solution of stannous octoate was added, and the toluene was removed by vacuum. The oil bath temperature was set to 150°C, and the reaction was carried out for 12 h. The residual raw materials and small molecule products were removed by vacuum, and the polymer was obtained after hot water washing and freeze-drying. The obtained polymer was a hydrogel.

[0065] 2.5 g of the polymer prepared above was dissolved in 7.5 mL of normal saline to prepare a 25 wt% sol. 1.8 mg of rhBMP-4 dry powder was added to a 10 mL 25 wt% hydrogel test tube at room temperature, and the rhBMP-4 was fully dispersed in the sol by shaking for 1 min. The antibacterial bioactive glass was completely immersed in the sol at room temperature, and vacuumed for 10 min. The sol was solidified by placing the test tube in a 37°C incubator, and rhBMP-4 / BG was obtained. ​

[0066] The rhBMP-4 / BG obtained above was subjected to antibacterial test, biological activity test, sustained-release effect test, and osteogenesis effect (cell expression) test, which were the same as those in Method and Example 1, and thus the description thereof will not be repeated.

[0067] The antibacterial effect of the rhBMP-4 / BG obtained above lasted for 21 days, and the test results are shown in Table 7; the sustained-release effect lasted for 21 days; the in vitro osteogenesis effect test results are shown in Table 8; and the biological activity test results are shown in Table 9. Figure 7 Figure 7 The biological activity results of the rhBMP-4 / BG according to Comparative Example 2 of the present application are shown in the following figure.

[0068] Table 7

[0069]

[0070] Table 8

[0071]

[0072] The above description is merely preferred embodiments of the present application, but the protection scope of the present application is not limited thereto. Any person skilled in the art, based on the technical content disclosed in the present application and the inventive concept thereof, can make equivalent replacements or changes within the technical scope of the present application, and such should be encompassed within the protection scope of the present application.​

Claims

1. A composite bioglass, characterized in that, It includes: Polyethylene glycol-lactide-glycolic acid polymer gel and rhBMP-2 doped in the polyethylene glycol-lactide-glycolic acid polymer gel and 45S5 bioactive glass doped with antibacterial ions; The antibacterial ions are selected from one or more of Ag, Zn, Cu and Ce.

2. The composite bioglass according to claim 1, characterized in that, The mass ratio of rhBMP-2, polyethylene glycol-lactide-glycolic acid polymer gel, and 45S5 bioactive glass doped with antibacterial ions is (0.001~0.003):(2~3):(0.02~0.03).

3. The composite bioglass according to claim 1, characterized in that, The doping amount of the antibacterial ions is (5~15)wt%.

4. The composite bioglass according to claim 1, characterized in that, The polyethylene glycol-lactide-glycol polymer gel is obtained by polymerizing polyethylene glycol, lactide and glycolide in a mass ratio of (18~22):(34~38):(8~12).

5. A method for preparing composite bioglass, characterized in that, Includes the following steps: The composite bioglass was obtained by solidifying 45S5 bioactive glass doped with antibacterial ions and rhBMP-2 in a polyethylene glycol-lactide-glycolic acid polymer sol. The antibacterial ions are selected from one or more of Ag, Zn, Cu and Ce.

6. The preparation method according to claim 5, characterized in that, The mass ratio of rhBMP-2, polyethylene glycol-lactide-glycolic acid polymer sol, and 45S5 bioactive glass doped with antibacterial ions is (0.001~0.003):(2~3):(0.02~0.03).

7. The preparation method according to claim 5, characterized in that, The doping amount of the antibacterial ions is (5~15)wt%.

8. The preparation method according to claim 5, characterized in that, The polyethylene glycol-lactide-glycol polymer sol is obtained by polymerizing polyethylene glycol, lactide and glycolide in a mass ratio of (18~22):(34~38):(8~12).

9. The preparation method according to claim 5, characterized in that, The polyethylene glycol-lactide-glycolic acid polymer sol was prepared by the following steps: Polyethylene glycol, lactide, and glycolide are polymerized under an organotin catalyst to obtain the polyethylene glycol-lactide-glycol polymer sol.

10. The application of the composite bioglass according to any one of claims 1 to 4 or the composite glass obtained by any one of the preparation methods according to claims 5 to 9 in the preparation of oral bone repair materials.

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