Double-sided self-mineralized guided bone regeneration collagen membrane as well as preparation method and application thereof

Abstract

The invention relates to the field of biomedicine, in particular to a double-sided self-mineralized guided bone regeneration collagen membrane as well as a preparation method and application thereof, and the preparation method of the membrane comprises the following steps: preparing a collagen solution, adding a sodium alginate solution, stirring, cross-linking, and freeze-drying to form a collagen membrane; preparing a calcium phosphate oligomer, coating the surface of the collagen membrane with the calcium phosphate oligomer for biomineralization, and finally performing radiation sterilization treatment. The GBR membrane prepared by the preparation method has the advantages of high tensile strength, good biocompatibility and the like, can be used in clinical operations, and is used as a dental implant guided bone regeneration barrier isolating membrane and a dental restoration membrane.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a double-sided self-mineralized collagen membrane for guiding bone regeneration, and a preparation method and application thereof. Background Art

[0002] Guided bone regeneration (GBR) membrane plays an irreplaceable role in dental implant surgery. Clinical studies have shown that absorbable collagen membranes often have better repair effects. However, pure collagen membranes have low strength, short duration in the body, and are easy to swell when absorbing water, which can easily cause compression to periodontal tissues. GBR membranes, which are filling materials, grow different cells on both sides: osteoblasts and fibroblasts. Under the support of the GBR membrane, osteoblasts have enough time to grow and develop, and can act as a barrier to prevent the penetration of fibroblasts. Therefore, double-sided collagen membranes with different molecular pore sizes are more suitable for clinical surgery.

[0003] Low-strength collagen membranes need to be modified to enhance tensile strength. Among the common collagen cross-linking modification methods, physical cross-linking (including radiation, heating, etc.) and chemical cross-linking (glutaraldehyde, etc.) are toxic to cells to a certain extent. Therefore, non-toxic cross-linking agents should be selected to ensure that GBR membranes do not cause secondary toxic damage to the human body. Summary of the invention

[0004] In order to solve the above technical problems existing in the prior art, the present invention proposes a double-sided self-mineralized guided bone regeneration collagen membrane and its preparation method and application, and its specific technical scheme is as follows: A method for preparing a double-sided self-mineralized guided bone regeneration collagen membrane comprises the following steps: Step 1, preparing a collagen solution: taking a collagen material and dissolving it in a glacial acetic acid solution, stirring evenly, to prepare an acidic collagen solution; Step 2, preparing a sodium alginate solution: dissolving powdered sodium alginate in deionized water, and stirring to obtain a sodium alginate solution; Step 3, preparing a sodium hydroxide solution: taking sodium hydroxide particles and dissolving them in sterile water to obtain a sodium hydroxide solution; Step 4, cross-linking: adding the sodium hydroxide solution prepared in step 3 dropwise to the acidic collagen solution in step 1 to neutralize the collagen solution to a pH of 7.2 to 7.4; Then, add the sodium alginate solution in step 2 dropwise into the neutralized collagen solution; Step 5, freeze-drying to form a film: pre-freeze the collagen solution obtained in step 4, and then freeze-dry it in a freeze dryer to obtain a collagen film with a smooth surface; Step 6, irradiation sterilization: subjecting the collagen membrane obtained in step 5 to ultraviolet sterilization; Step 7, preparing calcium phosphate oligomers: adding calcium salt as a calcium source, triethylamine as a stabilizer, and phosphoric acid as a phosphorus source to an organic solvent to prepare a calcium phosphate oligomer solution, and then removing excess organic solvent and triethylamine by dialysis; Step 8, mineralization: the calcium phosphate oligomer solution prepared in step 7 is evenly applied to one side of the surface of the collagen membrane sterilized in step 6, and then placed in a 37° C. air drying oven for mineralization, thereby obtaining a collagen membrane with one side surface mineralized as calcium phosphate crystals, thereby forming a collagen membrane with a double-sided structure; Step 9, irradiation sterilization: the collagen membrane obtained in step 8 is sterilized by ultraviolet irradiation.

[0005] Furthermore, during the process of dissolving the collagen in step 1, the ambient temperature is maintained at 4° C., and stirring is stopped until the collagen is completely dissolved.

[0006] Furthermore, the ambient temperature in step 4 is -4°C, and the quality ratio of sodium alginate to collagen is controlled between 1:5 and 1:3.

[0007] Furthermore, the pre-freezing temperature of step 5 is -20°C, the duration is 6 to 8 hours, the freezing temperature of the freeze dryer is between -45°C and -40°C, and the freezing time is not less than 12 hours.

[0008] Furthermore, in step 7, the calcium salt is calcium chloride, the molar ratio of the calcium salt to phosphoric acid is 1.2 to 1.68, and the concentration of triethylamine is 0.1 to 6 mol / L.

[0009] Furthermore, in step 8, the mineralization time is not less than 2 hours.

[0010] Furthermore, in step 9, both sides of the collagen membrane are irradiated for 6 to 8 hours.

[0011] A double-sided self-mineralized guided bone regeneration collagen membrane comprises 95wt% collagen and 5wt% calcium phosphate crystals.

[0012] A double-sided self-mineralized guided bone regeneration collagen membrane is used as a dental implant guided bone regeneration barrier isolation membrane and an oral repair membrane.

[0013] The double-sided biomineralized GBR membrane of the present invention solves the problems of low tensile strength, short in vivo retention time and high swelling rate of existing collagen membranes, has good biocompatibility, can be used in clinical surgery, and is used as a dental implant guided bone regeneration barrier isolation membrane and an oral repair membrane. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1Schematic diagram of a smooth-surfaced collagen membrane formed by cross-linking sodium alginate and collagen in a certain proportion and then freeze-drying according to an embodiment of the present invention; Figure 2a Schematic diagram of the mineralized surface of the double-sided self-mineralized guided bone regeneration collagen membrane of the embodiment of the present invention under an electron microscope; Figure 2b Schematic diagram of the mineralized cross-sectional structure of the collagen membrane for guiding bone regeneration according to an embodiment of the present invention; Figure 3 Schematic diagram of cell activity in an MC3T3-E1 cell proliferation detection experiment using the guided bone regeneration collagen membrane of an embodiment of the present invention; Figure 4 is a schematic diagram of the retention ratio of a degradation experiment using the guided bone regeneration collagen membrane of an embodiment of the present invention; Figure 5 It is a schematic diagram of the results of a swelling test using the guided bone regeneration collagen membrane according to an embodiment of the present invention. DETAILED DESCRIPTION

[0015] In order to make the purpose, technical scheme and technical effect of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments.

[0016] The double-sided self-mineralized guided bone regeneration collagen membrane of the present invention comprises 95wt% collagen and 5wt% calcium phosphate crystals. The preparation process of the membrane mainly comprises the following steps: Example 1: Preparation of collagen membrane: Step 1, prepare collagen solution: weigh 70 mg of bovine Achilles tendon collagen and dissolve it in 10 mL of 0.5 M glacial acetic acid solution, stir evenly, and prepare 10 mL of 7 mg / mL collagen solution, at which the solution pH = 3.7; keep the ambient temperature at 4°C during the dissolution of collagen until the collagen is completely dissolved, and stop stirring.

[0017] Step 2, preparing a sodium alginate solution: weigh 100 mg of sodium alginate powder and dissolve it in 10 mL of deionized water, stirring evenly for at least 1 hour to obtain a 10 mg / mL sodium alginate solution.

[0018] Step 3, prepare sodium hydroxide solution: weigh 1.6 g of sodium hydroxide particles and dissolve them in 10 mL of sterile water to prepare a 4 M sodium hydroxide solution.

[0019] Step 4, cross-linking: adding the 4M sodium hydroxide solution prepared in step 3 dropwise to the acidic collagen solution in step 1, so that the pH of the collagen solution reaches 7.2-7.4; Then, 2.3 mL of the sodium alginate solution in step 2 was added dropwise to the collagen solution, stirring while adding, and slowly stirred for two hours for cross-linking. The ambient temperature was controlled at -4°C, and the mass ratio of sodium alginate to collagen was controlled between 1:5 and 1:3.

[0020] Step 5, freeze-drying to form a membrane: Pour the collagen solution obtained in step 4 into a culture dish with a diameter of 10 cm, keep the solution height no higher than 3 mm, then pre-freeze in a -20°C refrigerator for 6 to 8 hours, and then freeze in a freeze dryer at -45°C to -40°C for no less than 12 hours to obtain a smooth collagen membrane. Figure 1 .

[0021] Step 6, irradiation sterilization: the collagen membrane obtained in step 5 is treated with ultraviolet sterilization for 6 hours.

[0022] Example 2: Preparation of mineralized collagen membrane: Step 7, preparation of calcium phosphate oligomers: weigh 5.0g of CaCl2·2H2O and dissolve it in 45mL of anhydrous ethanol to form a transparent solution. Measure 1.9mL of triethylamine solution and add it dropwise to the above ethanol solution, the concentration of triethylamine is 0.1-6mol / L. Then, add 175μL of phosphoric acid dropwise to 5mL of anhydrous ethanol and mix well to obtain a phosphoric acid ethanol solution, and add it dropwise to the ethanol solution containing triethylamine and calcium chloride, stir overnight, collect and obtain a calcium phosphate oligomer solution, the molar ratio of calcium chloride to phosphoric acid is 1.2-1.68, and then add it to a 500 Da dialysis bag, change the water in the morning and evening, dialyze for five days to remove excess anhydrous ethanol and triethylamine, and finally obtain a 0.25g / mL calcium phosphate oligomer solution.

[0023] Step 8, mineralization: The calcium phosphate oligomer solution prepared in step 7 is evenly applied to a unit area of ​​1 cm according to different masses, such as 0.2 g, 0.4 g, 0.8 g, and 1.6 g. 2 The surfaces of the collagen membrane prepared in step 6 are respectively denoted as CSaM-em0, CSaM-em1, CSaM-em2, and CSaM-em3, and placed in a 37°C air drying oven for no less than 2 hours. The resulting collagen membrane has a smooth unmineralized side and a rough mineralized calcium phosphate crystal side, i.e., a double-sided collagen membrane. Figure 2a The electron microscopy results show that the formed hydroxyapatite is evenly distributed on the surface of the collagen membrane. The mineralized cross section shows a relatively compact multilayer structure. Figure 2b .

[0024] Step 9, irradiation sterilization: irradiate both sides of the collagen membrane obtained in step 8 with ultraviolet rays for sterilization for 6 to 8 hours. The irradiation time should not be too long to avoid reducing the pores between protein molecules.

[0025] Cytocompatibility testing: The collagen membrane prepared in Example 2 was used to prepare sample extracts according to GB / T16886.5-2017 "Biological Evaluation of Medical Devices Part 5: In Vitro Cytotoxicity Test", and L929 cells and MC3T3-E1 cells were inoculated into 96-well plates and cultured for 24 hours. Cell activity was measured after 48 hours. The results showed that the cells in the experimental group were in good condition.

[0026] Cell proliferation assay: The collagen membrane prepared in Example 2 was cut into 10 mm diameter discs and placed in a 48-well plate for MC3T3-E1 cell proliferation experiment. The cell activity was measured by CCK-8 method on days 1, 3, 5, and 7. The results showed that compared with the collagen membrane in Example 1, the mineralized collagen membrane was more conducive to the growth of MC3T3-E1 cells. Figure 3 .

[0027] Degradation experiment: The collagen membrane prepared in Example 2 was cut into 10 mm diameter discs, placed in a 48-well plate, and added with physiological saline. The membrane was dried and weighed on days 0, 3, 7, 14, 21, and 28. The results showed that about 60% of the collagen membrane remained after 28 days, indicating that it can exist in the body for a long time. Figure 4 .

[0028] Swelling test: The collagen membrane in Example 2 was cut into 10 mm diameter discs, placed in a 48-well plate, and added with physiological saline. The volume of the collagen membrane was measured after 0 min, 15 min, 30 min, 60 min, 180 min, 240 min, 600 min, and 1440 min, respectively. The results showed that its water absorption swelling rate decreased by 20%, and the reference Figure 5 .

[0029] Mechanical tensile test: The collagen membrane in Example 2 was cut into 0.5 cm The size of the collagen membrane is 1 cm, and a tensile test is performed at a speed of 5 mm / min using a universal material testing machine, and the tensile strain is about 5%, and the Young's modulus is about 20.804±0.016 MPa. Compared with the Young's modulus of 10.75±0.05 MPa obtained by the collagen membrane test in Example 1, the tensile performance is improved by about 93%.

[0030] In summary, the introduction of the bionic mineralization method enables the collagen membrane to have some components similar to human bones, adapting to the human body environment while also strengthening the barrier capacity. Therefore, the double-sided mineralized collagen membrane of the present invention can be used as a barrier membrane for guided bone regeneration of dental implants and an oral repair membrane.

[0031] The above is only a preferred implementation case of the present invention and does not limit the present invention in any form. Although the implementation process of the present invention is described in detail above, for those familiar with the art, they can still modify the technical solutions recorded in the above examples, or replace some of the technical features therein with equivalents. All modifications, equivalent replacements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing a double-sided self-mineralized collagen membrane for guiding bone regeneration, characterized in that: The following steps are involved: Step 1, preparing a collagen solution: taking a collagen material and dissolving it in a glacial acetic acid solution, stirring evenly, to prepare an acidic collagen solution; Step 2, preparing a sodium alginate solution: dissolving powdered sodium alginate in deionized water, and stirring to obtain a sodium alginate solution; Step 3, preparing a sodium hydroxide solution: taking sodium hydroxide particles and dissolving them in sterile water to obtain a sodium hydroxide solution; Step 4, cross-linking: adding the sodium hydroxide solution prepared in step 3 dropwise to the acidic collagen solution in step 1 to neutralize the collagen solution to a pH of 7.2 to 7.4; Then, add the sodium alginate solution in step 2 dropwise into the neutralized collagen solution; Step 5, freeze-drying to form a film: pre-freeze the collagen solution obtained in step 4, and then freeze-dry it in a freeze dryer to obtain a collagen film with a smooth surface; Step 6, irradiation sterilization: subjecting the collagen membrane obtained in step 5 to ultraviolet sterilization; Step 7, preparing calcium phosphate oligomers: adding calcium salt as a calcium source, triethylamine as a stabilizer, and phosphoric acid as a phosphorus source to an organic solvent to prepare a calcium phosphate oligomer solution, and then removing excess organic solvent and triethylamine by dialysis; Step 8, mineralization: the calcium phosphate oligomer solution prepared in step 7 is evenly applied to one side of the surface of the collagen membrane sterilized in step 6, and then placed in a 37° C. air drying oven for mineralization, thereby obtaining a collagen membrane with one side surface mineralized as calcium phosphate crystals, thereby forming a collagen membrane with a double-sided structure; Step 9, irradiation sterilization: the collagen membrane obtained in step 8 is sterilized by ultraviolet irradiation.

2. The method for preparing a double-sided self-mineralized collagen membrane for guided bone regeneration according to claim 1, characterized in that: During the process of dissolving collagen in step 1, the ambient temperature is maintained at 4° C., and stirring is stopped until the collagen is completely dissolved.

3. The method for preparing a double-sided self-mineralized collagen membrane for guided bone regeneration according to claim 1, characterized in that: The ambient temperature in step 4 is -4°C, and the mass ratio of sodium alginate to collagen is controlled between 1:5 and 1:

3.

4. The method for preparing a double-sided self-mineralized collagen membrane for guided bone regeneration according to claim 1, characterized in that: The pre-freezing temperature of step 5 is -20°C, the duration is 6 to 8 hours, the freezing temperature of the freeze dryer is between -45°C and -40°C, and the freezing time is not less than 12 hours.

5. The method for preparing a double-sided self-mineralized collagen membrane for guided bone regeneration according to claim 1, characterized in that: In step 7, the calcium salt is calcium chloride, the molar ratio of the calcium salt to phosphoric acid is 1.2 to 1.68, and the concentration of triethylamine is 0.1 to 6 mol / L.

6. The method for preparing a double-sided self-mineralized collagen membrane for guided bone regeneration according to claim 1, characterized in that: In step 8, the mineralization time is not less than 2 hours.

7. The method for preparing a double-sided self-mineralized collagen membrane for guided bone regeneration according to claim 1, characterized in that: In the step 9, both surfaces of the collagen membrane are irradiated, and the irradiation time is 6 to 8 hours.

8. A collagen membrane prepared by the preparation method according to any one of claims 1 to 7, characterized in that: Comprises 95wt% collagen and 5wt% calcium phosphate crystals.

9. Use of the collagen membrane as claimed in claim 8 in dental implant guided bone regeneration barrier isolation membrane and oral repair membrane.

Images