A collagen-bioactive glass anti-inflammatory corneal repair material and its preparation method

Abstract

The present invention discloses a preparation method of a collagen-bioactive glass anti-inflammatory corneal repair material, comprising the following steps: (1) blending and grinding bioactive glass with lecithin, and then drying in vacuum to obtain modified bioactive glass; (2) adding the modified bioactive glass prepared in step (1) into ultrapure water and ultrasonically mixing to obtain a uniform suspension; (3) blending the suspension obtained in step (2) with a collagen solution to obtain a uniform and bubble-free mixed solution at room temperature; (4) air-drying the mixed solution obtained in step (3) to obtain a collagen-bioactive glass anti-inflammatory corneal repair material. The present invention also discloses a collagen-bioactive glass anti-inflammatory corneal repair material obtained by the above preparation method. On the premise of meeting the requirements of mechanical properties, moisture content, light transmittance, etc. required for corneal regeneration and repair materials, the corneal repair material of the present invention can regulate the polarization of macrophages, thereby achieving the effect of regulating inflammation.

Description

Technical Field

[0001] The present invention relates to the technical field of corneal repair materials, and particularly to a collagen-bioactive glass anti-inflammatory corneal repair material and a preparation method thereof. Background Art

[0002] Corneal diseases are the main cause of blindness. Currently, corneal transplantation is the only widely accepted treatment method. However, less than 5% of patients worldwide can undergo transplantation. The shortage of corneal donors, inflammation caused by rejection reactions, and infections are all important challenges faced by corneal transplantation. Therefore, biomaterials for corneal regeneration have become a research hotspot.

[0003] A suitable corneal replacement and repair material should have properties similar to those of a healthy human cornea, including water content, light transmittance, and permeability, to meet the transparency of the cornea and the supply of nutrients. The corneal repair material for corneal transplantation also needs to have a certain mechanical strength to resist the pulling of sutures during surgery. After the material is implanted into the body, it needs to exist stably and not cause an immune rejection reaction, and it must be non-toxic, so it also needs to have good biocompatibility. The long-term inflammatory phenomenon caused by the postoperative immune rejection reaction is one of the common complications after corneal transplantation. Inflammation is a basic process of corneal wound healing, but long-term exposure to an inflammatory microenvironment will also have adverse effects. Therefore, a repair material with anti-inflammatory function can effectively reduce the long-term inflammatory phenomenon after surgery and ensure the repair effect of the material. Currently, several feasible alternative solutions mainly include acellular corneal matrix, amniotic membrane, acrylate artificial cornea, and collagen-based cornea, etc., to replace the donor cornea or promote the regeneration of the natural cornea. However, these materials also have certain limitations, such as immunogenicity, poor transparency, easy induction of complications, and poor mechanical strength. An ideal corneal repair material should meet the requirements in terms of physical and chemical properties, achieve good epithelial repair effects in actual repair, and be less likely to cause postoperative inflammation to ensure the repair effect. How to prepare a corneal repair material that can meet the required mechanical strength for surgical suture, has good biocompatibility, and can effectively reduce postoperative inflammation has become a technical problem that still needs to be solved urgently in this field. Summary of the Invention

[0004] In order to overcome the above-mentioned disadvantages and deficiencies of the prior art, the purpose of the present invention is to provide a preparation method of a collagen-bioactive glass anti-inflammatory corneal repair material. The prepared collagen-bioactive glass anti-inflammatory corneal repair material can regulate the polarization of macrophages on the premise of meeting the requirements of mechanical properties, water content, light transmittance, etc. required for corneal regeneration and repair materials, so as to achieve the effect of regulating inflammation.

[0005] Another purpose of the present invention is to provide a collagen-bioactive glass anti-inflammatory corneal repair material prepared by the preparation method of the above-mentioned collagen-bioactive glass anti-inflammatory corneal repair material.

[0006] The object of the present invention is achieved by the following technical solutions:

[0007] A preparation method of a collagen-bioactive glass anti-inflammatory corneal repair material, comprising the following steps:

[0008] (1) Blend and grind bioactive glass and lecithin at 20-25 °C, and then vacuum dry to obtain modified bioactive glass; the mass ratio of the bioactive glass to lecithin is 22-28:1;

[0009] (2) Add the modified bioactive glass prepared in step (1) to ultrapure water and ultrasonically mix to obtain a uniform suspension;

[0010] (3) Blend the suspension obtained in step (2) with a collagen solution and react at room temperature for 20-25 hours to obtain a uniform and bubble-free mixed solution;

[0011] The mass ratio of collagen to modified bioactive glass in the mixed solution is 100:(1-7);

[0012] (4) Air-dry the mixed solution obtained in step (3) to obtain a collagen-bioactive glass anti-inflammatory corneal repair material.

[0013] Preferably, the composition of the bioactive glass is 24.5% Na2O - 24.5% CaO - 6% P2O5 - 45% SiO2.

[0014] Preferably, the preparation of the collagen solution is as follows: collagen is prepared into a collagen solution with a hydrochloric acid solution, and the molar concentration of the hydrochloric acid solution used is 0.001-0.01 mol / L.

[0015] Preferably, the collagen is type I collagen extracted from bovine tendon.

[0016] Preferably, the blending and grinding in step (1) is specifically:

[0017] Add absolute ethanol and blend and grind for 1.5-2.5 hours.

[0018] Preferably, the vacuum drying in step (1) is specifically:

[0019] Vacuum dry at 35-40 °C.

[0020] Preferably, the ultrasonic mixing in step (2) is specifically: ultrasonic mixing for 30-60 minutes.

[0021] Preferably, the air-drying of the mixed solution obtained in step (3) to obtain a collagen-bioactive glass anti-inflammatory corneal repair material in step (4) is specifically:

[0022] Cast the mixed solution described in step (3) in a mold, air-dry it at room temperature to form a composite film, wash it with deionized water multiple times, and air-dry it naturally at room temperature to obtain the collagen-bioactive glass anti-inflammatory corneal repair material.

[0023] Preferably, the mass ratio of collagen to modified bioactive glass in the mixed solution is 100:(6 - 7).

[0024] Preferably, the concentration of the collagen solution is 5.8 - 6.5 mg / mL.

[0025] More preferably, the concentration of the collagen solution is 5.8 - 6.3 mg / mL.

[0026] The collagen-bioactive glass anti-inflammatory corneal repair material prepared by the preparation method of the collagen-bioactive glass anti-inflammatory corneal repair material has a contact angle of 60° - 70°.

[0027] Preferably, the contact angle of the collagen-bioactive glass anti-inflammatory corneal repair material is 65° - 70°.

[0028] The collagen-bioactive glass anti-inflammatory corneal repair material has a layered structure, and bioactive glass is embedded between the layers of collagen.

[0029] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0030] (1) For the collagen-bioactive glass anti-inflammatory corneal repair material of the present invention, corneal epithelial cells can adhere and proliferate on the collagen-bioactive glass anti-inflammatory corneal repair material of the present invention. Under in vitro inflammatory conditions, the expression of anti-inflammatory cytokines such as IL-10 and ARG-1 can be up-regulated, thereby effectively reducing the inflammatory reaction caused by postoperative immune rejection.

[0031] (2) For the collagen-bioactive glass anti-inflammatory corneal repair material of the present invention, by compounding bioactive glass modified with lecithin and collagen, the obtained anti-inflammatory corneal repair material has a layered structure, good optical transparency, good water content, and good mechanical properties, and will not cause tearing when sutured with a surgical thread.

[0032] (3) For the collagen-bioactive glass anti-inflammatory corneal repair material of the present invention, the preparation raw materials used, namely collagen, lecithin, and bioactive glass, are all non-toxic and harmless, and have good biocompatibility and biodegradability. Description of the Drawings

[0033] Figure 1 is the cross-section of the scanning electron microscope (SEM) of the Col-BG composite film in Example 1 of the present invention

[0034] Figure 2 These are the test results of the light transmittance performance of the Col-BG composite film of Example 1 of the present invention.

[0035] Figure 3 These are the test results of the water contact angle of the Col-BG composite film of Example 1 of the present invention. Among them, (a) in the figure is the water contact angle of the Col film; (b) in the figure is the water contact angle of the Col-BG composite film.

[0036] Figure 4 These are the test results of the mechanical properties of the Col-BG composite film of Example 1 of the present invention.

[0037] Figure 5 These are the test results of the cytotoxicity of the Col-BG composite film of Example 1 of the present invention.

[0038] Figure 6a These are the in vitro anti-inflammatory effect diagrams (pro-inflammatory gene expression) of the corneal repair materials of Example 1 and Comparative Example 1 of the present invention, that is, the test results of the regulation of the expression level of related gene mRNA of macrophages RAW264.7 under inflammatory conditions by the composite film.

[0039] Figure 6b These are the in vitro anti-inflammatory effect diagrams (anti-inflammatory gene expression) of the corneal repair materials of Example 1 and Comparative Example 1 of the present invention, that is, the test results of the regulation of the expression level of related gene mRNA of macrophages RAW264.7 under inflammatory conditions. Detailed implementation manners

[0040] The present invention will be further described in detail below in conjunction with the embodiments, but the implementation manners of the present invention are not limited thereto.

[0041] Example 1

[0042] A collagen-bioactive glass anti-inflammatory corneal repair material (Col-BG composite film), the preparation method includes the following steps:

[0043] (1) Prepare modified bioactive glass by co-milling with a grinding medium for 2 hours at 20-25 °C according to the ratio of bioactive glass to lecithin of 25:1, and dry it in a vacuum drying oven at 37 °C;

[0044] (2) Weigh the prepared modified bioactive glass according to the ratio of 5:100 to the mass of collagen, and add it to ultrapure water and sonicate for 30 minutes to obtain a uniformly mixed suspension;

[0045] (3) Blend the mixed solution prepared in step (2) with the collagen solution, and react at room temperature for 24 hours to obtain a uniform and bubble-free mixed solution;

[0046] (4) Cast the mixed solution prepared in step (3) in a petri dish and let it air-dry naturally at room temperature to obtain a Col-BG composite film. Remove the mold, take out the composite film, wash it with deionized water multiple times, and let it air-dry naturally at room temperature to obtain the corneal repair material.

[0047] The bioactive glass was configured into a bioactive glass suspension with a concentration of 2 mg / mL using ultrapure water; type I collagen extracted from bovine Achilles tendon was configured into a collagen solution with a concentration of 6.0 mg / mL using a 0.01 mol / L hydrochloric acid solution.

[0048] By characterizing the physical and chemical properties of the Col-BG composite film material in Example 1, the following conclusions can be obtained:

[0049] Figure 1 It is a cross-sectional view of a scanning electron microscope (SEM) of the corneal repair material with anti-inflammatory effect obtained in this example. It can be seen that the bioactive glass is embedded between the layers of the collagen material, which is direct evidence of the successful preparation of this corneal repair material.

[0050] Figure 2 It is the test result of the light transmittance performance of the Col-BG composite film in Example 1. From Figure 2 it can be known that the Col-BG composite film material has good light transmittance performance. The addition of bioactive glass has little effect on the light transmittance performance, and the transmittance can reach more than 70% at most wavelengths;

[0051] Figure 3 It is the test result of the water contact angle of the Col-BG composite film in Example 1. From Figure 3 it can be known that compared with the Col film, the contact angle of the Col-BG composite film increases, indicating that the surface roughness increases slightly, which is beneficial to cell adhesion. The contact angle range of both is between 60° and 70°, which meets the contact angle range (45° - 75°) with better adhesion.

[0052] Figure 4 It is the test result of the mechanical properties of the Col-BG composite film in Example 1. From Figure 4 it can be known that compared with the Col material, the tensile strength of the Col-BG composite film material has increased.

[0053] Figure 5 It is the test result of the cytotoxicity of the Col-BG composite film in Example 1. From Figure 5 it can be known that the Col-BG composite film material has no cytotoxicity. The absorbance value of the Col-BG composite film material on the 5th day is slightly higher than that of the Col material, indicating that the composite film is more conducive to cell proliferation.

[0054] Figures 6a - 6b It is the in vitro anti-inflammatory effect diagram of the corneal repair materials in Example 1 and Comparative Example 1. FromFigure 6a , Figure 6b It can be seen that compared with the Col material, the Col-BG composite membrane material can significantly down-regulate the expression of INOS at 3 days, while up-regulating the expression of IL-10 and ARG-1, and there are significant differences. This indicates that RAW264.7 macrophages cultured on the Col-BG membrane polarize into M2 macrophages, thereby expressing anti-inflammatory cytokines. This shows that the addition of bioactive glass can endow the material with anti-inflammatory properties.

[0055] Example 2

[0056] A collagen-bioactive glass anti-inflammatory corneal repair material (Col-BG composite membrane), the preparation method includes the following steps:

[0057] (1) Prepare modified bioactive glass by co-milling with a grinding medium for 2 hours at a ratio of bioactive glass to lecithin of 25:1 under the condition of 20-25 °C, and dry it in a vacuum drying oven at 40 °C;

[0058] (2) Weigh the prepared modified bioactive glass at a ratio of 1:100 to the mass of collagen, add it to ultrapure water and ultrasonicate for 45 minutes to obtain a uniformly mixed suspension;

[0059] (3) Blend the mixed solution obtained in step (2) with the collagen solution and react at room temperature for 22 hours to obtain a uniform and bubble-free mixed solution;

[0060] (4) Pour the mixed solution obtained in step (3) into a petri dish, naturally air-dry it into a film at room temperature to obtain the Col-BG composite membrane, remove the mold, take out the composite membrane, wash it with deionized water multiple times, and naturally air-dry it at room temperature to obtain the corneal repair material.

[0061] The bioactive glass is configured into a bioactive glass suspension with a concentration of 1 mg / mL with ultrapure water; type I collagen extracted from bovine tendon is configured into a collagen solution with a concentration of 6.3 mg / mL with a 0.005 mol / L hydrochloric acid solution.

[0062] Example 3

[0063] A collagen-bioactive glass anti-inflammatory corneal repair material (Col-BG composite membrane), the preparation method includes the following steps:

[0064] (1) Prepare modified bioactive glass by co-milling with a grinding medium for 2 hours at a ratio of bioactive glass to lecithin of 25:1 under the condition of 20-25 °C, and dry it in a vacuum drying oven at 50 °C;

[0065] (2) Weigh the prepared modified bioactive glass according to the ratio of 7:100 to the mass of collagen, add it to ultrapure water, and ultrasonicate for 60 minutes to obtain a uniformly mixed suspension;

[0066] (3) Blend the mixed solution obtained in step (2) with the collagen solution and react at room temperature for 20 hours to obtain a uniformly mixed solution without bubbles;

[0067] (4) Pour the mixed solution obtained in step (3) into a petri dish, air-dry it naturally at room temperature to form a film, then remove the mold, take out the composite film, wash it several times with deionized water, and air-dry it naturally at room temperature to obtain the corneal repair material.

[0068] The bioactive glass is configured into a bioactive glass suspension with a concentration of 4 mg / mL using ultrapure water; type I collagen extracted from bovine Achilles tendon is configured into a collagen solution with a concentration of 6.5 mg / mL using a 0.001 mol / L hydrochloric acid solution.

[0069] Comparative Example 1

[0070] A collagen corneal repair material (Col film, Col), the preparation method includes the following steps:

[0071] (1) Stir the prepared collagen solution at room temperature for 24 hours to remove bubbles and obtain a uniformly mixed collagen solution without bubbles;

[0072] (2) Pour the collagen solution prepared in step (1) into a petri dish, air-dry it naturally at room temperature to form a film, then remove the mold, take out the Col film, wash it several times with deionized water, and air-dry it naturally at room temperature to obtain the corneal repair material.

[0073] Type I collagen extracted from bovine Achilles tendon is configured into a collagen solution with a concentration of 6.5 mg / mL using a 0.001 mol / L hydrochloric acid solution.

[0074] Result Detection

[0075] The specific detection results of the examples and comparative examples are shown in Table 1 and Table 2 below.

[0076] Table 1

[0077] Serial number Transmittance (%) Water contact angle (°) Tensile strength (MPa) Example 1 77.4±2.4 68.53±1.65 0.51±0.01 Example 2 79.07±2.7 63.17±2.49 0.53±0.04 Example 3 75.57±2.57 75.51±1.43 0.45±0.05 Comparative example 1 82.87±2.35 62.1±4.85 0.44±0.02

[0078] Table 2

[0079]

[0080]

[0081] The light transmittance (at 800 nm), contact angle, and tensile strength of each group of examples and comparative examples were compared. It was found that the addition of bioactive glass in the examples of the present invention had little effect on transparency, and the surface contact angle of the Col-BG composite film increased, indicating an increase in surface roughness which was beneficial to cell growth; moreover, the tensile strength also increased with the addition of bioactive glass, which might be related to the increase in the number of nucleation sites. As can be seen from Table 3, the composite films of each example could up-regulate the expression of anti-inflammatory factors such as ARG-1 and down-regulate the expression of pro-inflammatory factors such as INOS, and the regulatory effect of Example 1 was relatively the best, and there was no negative inhibitory effect on corneal materials in terms of light transmittance and tensile strength.

[0082] The above examples are the preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the above examples. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent substitution methods and are all included in the protection scope of the present invention.

Claims

1. A preparation method of a collagen-bioactive glass anti-inflammatory corneal repair material, characterized in that, It includes the following steps: (1) Blend and grind bioactive glass and lecithin at 20-25 °C, and then vacuum dry to obtain modified bioactive glass; the mass ratio of the bioactive glass to lecithin is 25:1; The composition of the bioactive glass is 24.5% Na2O - 24.5% CaO - 6% P2O5 - 45% SiO2; The blend and grind is specifically: Add absolute ethanol and blend and grind for 1.5 - 2.5 hours; The vacuum drying is specifically: Vacuum dry at 35 - 40 °C; (2) Add the modified bioactive glass prepared in step (1) into ultrapure water and ultrasonically mix to obtain a uniform suspension; The ultrasonic mixing is specifically: ultrasonically mix for 30 - 60 minutes; (3) Blend the suspension obtained in step (2) with the collagen solution and react at room temperature for 20 - 25 hours to obtain a uniform and bubble-free mixed solution; the mass ratio of the modified bioactive glass to collagen is 5:100; (4) Cast the mixed solution described in step (3) into a mold, air dry at room temperature to form a composite film, wash it with deionized water multiple times, and air dry naturally at room temperature to obtain the collagen-bioactive glass anti-inflammatory corneal repair material.

2. The preparation method of the collagen-bioactive glass anti-inflammatory corneal repair material according to claim 1, characterized in that, The preparation of the collagen solution is: prepare the collagen solution by dissolving collagen in a hydrochloric acid solution, and the molar concentration of the hydrochloric acid solution used is 0.001 - 0.01 mol / L.

3. The preparation method of the collagen-bioactive glass anti-inflammatory corneal repair material according to claim 1 or 2, characterized in that, The collagen is type I collagen extracted from bovine tendon.

4. The collagen-bioactive glass anti-inflammatory corneal repair material prepared by the preparation method of the collagen-bioactive glass anti-inflammatory corneal repair material according to any one of claims 1 to 3, characterized in that, Its contact angle is 60° - 70°.

5. The collagen-bioactive glass anti-inflammatory corneal repair material according to claim 4, wherein It has a layered structure, and the bioactive glass is embedded between the layers of collagen.

Images