Method for improving mechanical properties of fish scale-based biofilm and application thereof

Abstract

The application discloses a method for improving the mechanical property of fish scale-based biomembrane and application thereof, and relates to the field of medical materials in medicine and bioengineering. The fish scale-based biomembrane is soaked in a certain solution for 8-15 hours, and then is placed in a reaction kettle to react with new certain solution, the reaction temperature is 4-200 DEG C, the reaction pressure is 0.1-20 MPa, and the reaction time is 5-24 hours, and the fish scale-based biomembrane with improved mechanical property is obtained after the reaction. The fish scale-based biomembrane prepared by the method has good compliance, the biomechanical property is close to that of human cornea, the optical property is excellent, the biological activity is good, and the cytotoxicity is zero, so the fish scale-based biomembrane can be directly applied to clinical transplantation and treatment of various corneal diseases and has excellent application potential in the field of ophthalmic materials. The method has the advantages of simple operation, low toxicity, low production cost and suitability for industrial production.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of medical materials in medicine and bioengineering, in particular to a method for improving the mechanical properties of fish scale-based biomembrane and application. BACKGROUND

[0002] Cornea is an important structure on the anterior surface of the eyeball, which protects the eyeball and maintains vision. So far, the most thorough way to solve corneal blindness in clinic is to perform corneal transplantation. Due to cultural traditions, ethics and other reasons, the supply of donor corneal materials is severely insufficient, and the artificial corneal materials made of glass or polymer substrates have many complications, and it is difficult to achieve permanent replacement of the cornea. With the rise of tissue engineering, scholars turn their attention to the research of tissue-engineered corneal tissue, hoping to make up for the shortage of human corneal donors and the clinical defects of artificial cornea, and induce corneal tissue regeneration.

[0003] Tissue-engineered cornea is composed of corneal scaffold material, corneal stem cells and signal factors. The tissue-engineered corneal scaffold material is mainly made of biological macromolecules or synthetic polymers. So far, several corneal scaffold materials have been applied in clinic, and the typical ones are acellular porcine cornea and amniotic membrane, but they all have some performance defects: acellular porcine cornea can spread zoonotic disease pathogens, and may cause water swelling after transplantation, poor transparency recovery, even dissolution, and is only suitable for anterior lamellar corneal transplantation; amniotic membrane has poor mechanical strength, is difficult to suture during transplantation, is prone to wrinkles, and is prone to early degradation after transplantation, and is only suitable for ocular surface reconstruction.

[0004] Fish scale-based biological corneal material has low immunogenicity, similar structure and composition to cornea, and has attracted the attention of researchers. Unfortunately, in addition to good biological activity and optical performance, good biomechanical matching between the ideal tissue-engineered corneal scaffold material and the eyeball is also the key to the success of tissue-engineered corneal transplantation. In the past few years, many corneal grafts have failed to pass clinical trials due to poor tissue integration and adverse reactions in clinical practice, partly due to the mismatch of the biomechanical properties of the materials. Fish scale-based collagen materials have the same problem. It has been confirmed that fish scale-based collagen materials have certain biological activity, but the biomechanical compatibility of fish scale-based corneal materials with the eyeball tissue is poor, especially the compliance of the decalcified fish scale-based material is too poor, which leads to difficulty in suturing during implantation, easy stress cracking, severe mechanical stimulation after implantation, scar proliferation and implantation failure. SUMMARY

[0005] The purpose of the present application is to overcome the shortcomings of the prior art and provide a method for improving the mechanical properties of fish scale-based biomembrane and application.

[0006] The application is achieved by the following technical scheme: a method for improving the mechanical property of fish scale-based biomembrane, the fish scale-based biomembrane is soaked in a certain solution for 8-15 hours, then the fish scale-based biomembrane is placed in a reaction kettle and new certain solution is added for reaction, the reaction temperature is 4-200 DEG C, the reaction pressure is 0.1-20 MPa, and the reaction time is 5-24 hours, and the fish scale-based biomembrane with improved mechanical property is obtained after reaction; wherein the certain solution is a mixed solution of acid liquid and A liquid, and the A liquid is at least one of stearate, sodium dodecyl sulfonate, sodium dodecyl benzene sulfonate, sodium dodecyl sulfate, sodium glycocholate or glycerol.

[0007] Further, the acid liquid is at least one of formic acid, oxalic acid, nitric acid, sulfuric acid, hydrochloric acid, acetic acid, cane acid, malic acid, citric acid, boric acid, EDTA or soft acid.

[0008] Further, the mass-volume ratio of the fish scale-based biomembrane and the certain solution is 0.01-0.5 g:1 mL.

[0009] Further, the concentration of the acid liquid is 0.1-2 mol / L, the concentration of the A liquid is 0.1-5 mol / L, and the volume ratio of the acid liquid to the A liquid is 0.1-10:1.

[0010] Further, the preparation method of the fish scale-based biomembrane is as follows: after fresh fish scales are rinsed clean, defatting liquid, desugarizing liquid and decalcifying liquid are sequentially added for desugarizing, defatting and decalcifying treatment, wherein the defatting liquid is a mixed solution of alcohol and methanolic chloroform, and the volume ratio is 0.1-8:1; the desugarizing liquid is a sodium chloride solution with a mass fraction of 0.1-8 %; and the decalcifying liquid is a mixed solution of EDTA and nitric acid, and the mass fraction of EDTA in the mixed solution is 1-30 %, and the mass fraction of nitric acid is 1-20 %.

[0011] Further, the fish scales are from hypophthalmichthys molitrix, salmo salar, carassius auratus, grass carp, black carp, cyprinus carpio, oreochromis niloticus, sphyraena, cirrhinus molitorella or esox.

[0012] Further, the specific operation of defatting is as follows: after fresh fish scales are rinsed clean, the defatting liquid is added, a shaker is placed, the speed of the shaker is set to 60-160 r / min, and the defatting time is 1-3 hours; and the alcohol is any one of methanol, ethanol, propanol, isopropyl alcohol or glycerol.

[0013] Further, the specific operation of desugarizing is as follows: after the defatted fish scales are rinsed clean, the desugarizing liquid is added, a shaker is placed, the speed of the shaker is set to 60-160 r / min, and the desugarizing time is 1-3 hours.

[0014] Further, the specific operation of the decalcification treatment is that the desugared fish scales are rinsed clean and then put into a decalcification solution for 1-12 hours.

[0015] The fish scale-based biomembrane with improved mechanical properties prepared by the method is applied to an artificial cornea.

[0016] The present application has the following advantages:

[0017] (1) The method successfully improves the compliance of the fish scales, and the compressive elastic modulus is changed from 231.09 Mpa to 45.05 Kpa, which is closer to the compressive elastic modulus of the human cornea (about 20 Kpa).

[0018] (2) The tensile strength of the fish scale-based biomembrane material treated by the method is reduced from 20.8 Mpa to 3 Mpa, which is higher than the tensile strength of the human cornea (2.6 Mpa) and far higher than that of the gel corneal material; and the fish scale-based biomembrane treated by the method can be easily pierced, and the suturing strength can reach 29 gf, which is close to the suturing strength of the human cornea (35.6 gf) and can meet the suturing requirement.

[0019] (3) The method can not only improve the biomechanical properties of the fish scale-based biomembrane, but also weaken the ridge on the surface of the fish scale, greatly improving the optical performance of the material, and the light transmittance of the fish scale-based biomembrane prepared by the method can reach 92.8%, meeting the optical performance requirement of the cornea.

[0020] (4) The method does not destroy the ordered structure of the fish scale fiber arrangement, and can still simulate the fiber arrangement of the human cornea.

[0021] (5) The fish scale-based biomembrane prepared by the method has good compliance, biomechanical properties close to the human cornea, excellent optical performance, good biological activity, and no cytotoxicity, and can be directly applied to the clinical transplantation and treatment of various corneal diseases, and has excellent application potential in the field of ophthalmic materials.

[0022] (6) The method is simple in operation, low in toxicity, and low in production cost, and is suitable for industrialized production, and the method also provides a new solution for improving the mechanical properties of other decellularized materials. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 The figure is a nanoindentation test curve of the fish scale-based biomembrane before and after improvement by the method.

[0024] Figure 2 The figure is a tensile test curve of the fish scale-based biomembrane before and after improvement by the method.

[0025] Figure 3The fish scale-based biomembrane before and after the improvement of the method of the present application.

[0026] Figure 4 The light transmittance contrast diagram of the fish scale-based biomembrane before and after the improvement of the method of the present application.

[0027] Figure 5 The digital photo of the fish scale-based biomembrane before and after the improvement of the method of the present application, wherein A is a fresh fish scale, B is a decalcified fish scale, and C is a fish scale-based biomembrane with improved mechanical properties.

[0028] Figure 6 The electron microscope scanning contrast diagram of the surface ridge of the fish scale-based biomembrane before and after the improvement of the method of the present application, wherein A is a fresh fish scale, B is a decalcified fish scale, and C is a fish scale-based biomembrane with improved mechanical properties.

[0029] Figure 7 The electron microscope scanning contrast diagram of the cross section of the fish scale-based biomembrane before and after the improvement of the method of the present application, wherein A is a fresh fish scale, B is a decalcified fish scale, and C is a fish scale-based biomembrane with improved mechanical properties.

[0030] Figure 8 The FDA fluorescence diagram of L929 cultured by the fish scale-based biomembrane after the improvement of the method of the present application. DETAILED DESCRIPTION

[0031] The present application is further described below in conjunction with the accompanying drawings and examples, and the protection scope of the present application is not limited to the following description:

[0032] Example 1: a method for improving the mechanical properties of a fish scale-based biomembrane

[0033] S1. After the fresh silver carp fish scales are rinsed clean, they are added into a degreasing solution and placed on a shaker for degreasing, and the shaker is set at a speed of 60 r / min, and the degreasing time is 1 h; the degreasing solution is a mixed solution of methanol and methyl chloride, and the volume ratio is 0.1:1; the degreased fish scales are rinsed clean, added into a 0.1 % sodium chloride solution, and placed on a shaker for desugaring, and the shaker is set at a speed of 60 r / min, and the desugaring time is 3 h; the desugared fish scales are rinsed clean and placed in a decalcifying solution for 1 h of reaction, and the decalcifying solution is a mixed solution of EDTA and nitric acid, and the mass fraction of EDTA in the mixed solution is 30 %, and the mass fraction of nitric acid is 20 %;

[0034] S2. The fish scale-based biomembrane is soaked in a certain solution for 8 h, and after soaking, it is placed in a reaction kettle to add a new certain solution for reaction, the reaction temperature is 4 ℃, the reaction pressure is 0.1 MPa, and the reaction time is 5 h, and the fish scale-based biomembrane with improved mechanical properties is obtained after reaction; wherein the certain solution is a mixed solution of formic acid and sodium dodecyl sulfonate, the mass-volume ratio of the fish scale-based biomembrane to the certain solution for reaction is 0.01 g:1 mL; the concentration of the formic acid is 0.1 mol / L, the concentration of the sodium dodecyl sulfonate solution is 0.1 mol / L, and the volume ratio of the formic acid to the sodium dodecyl sulfonate solution is 0.1:1.

[0035] Example 2: A method for improving the mechanical properties of a fish scale-based biomembrane

[0036] S1. Fresh fish scales are rinsed clean and then added to a degreasing solution, and placed on a shaker for degreasing, with the shaker rate set to 160 r / min and the degreasing time set to 3 h; the degreasing solution is a mixed solution of propanol and dichloromethane with a volume ratio of 8:1; the degreased fish scales are rinsed clean and then added to a 8% mass fraction sodium chloride solution, and placed on a shaker for desugaring, with the shaker rate set to 160 r / min and the desugaring time set to 1 h; the desugared fish scales are rinsed clean and then placed in a decalcification solution for reaction for 12 h, and the decalcification solution is a mixed solution of EDTA and nitric acid with a mass fraction of EDTA of 1% and a mass fraction of nitric acid of 1%.

[0037] S2. The fish scale-based biomembrane is soaked in a certain solution for 15 h, and after soaking, it is placed in a reaction kettle to add a new certain solution for reaction, the reaction temperature is 200 ℃, the reaction pressure is 20 MPa, and the reaction time is 24 h, and the fish scale-based biomembrane with improved mechanical properties is obtained after reaction; wherein the certain solution is a mixed solution of an acid solution and A liquid, the A liquid is a mixed solution of sodium dodecyl benzene sulfonate and glycerol; the acid solution is a mixed solution of oxalic acid, nitric acid and sulfuric acid; the mass-volume ratio of the fish scale-based biomembrane to the certain solution for reaction is 0.5 g:1 mL; the concentration of the acid solution is 2 mol / L, the concentration of the A liquid is 5 mol / L, and the volume ratio of the acid solution to the A liquid is 10:1.

[0038] Example 3: A method for improving the mechanical properties of a fish scale-based biomembrane

[0039] S1. The fresh tilapia fish scales are rinsed clean and added to a degreasing solution, placed on a shaker for degreasing, the shaker speed is set to 90 r / min, and the degreasing time is 2.5 h; the degreasing solution is a mixed solution of isopropyl alcohol and chloroform, the volume ratio is 3:1; the degreased fish scales are rinsed clean, added to a 2% mass fraction of sodium chloride desugar solution, placed on a shaker for desugar, the shaker speed is set to 90 r / min, and the desugar time is 2.5 h; the desugared fish scales are rinsed clean and placed in a decalcification solution for 8 h, the decalcification solution is a mixed solution of EDTA and nitric acid, the mass fraction of EDTA in the mixed solution is 12%, and the mass fraction of nitric acid is 5%;

[0040] S2. The fish scale-based biofilm is soaked in a certain solution for 10 h, then placed in a reaction kettle and added with new certain solution for reaction, the reaction temperature is 90 ℃, the reaction pressure is 6 MPa, and the reaction time is 10 h, and the fish scale-based biofilm with improved mechanical properties is obtained after reaction; wherein the certain solution is a mixed solution of acid solution and A solution, the A solution is a mixed solution of sodium dodecyl benzene sulfonate, sodium dodecyl sulfate, sodium glycocholate and glycerol; the acid solution is a mixed solution of citric acid, boric acid, EDTA and soft acid; the mass-volume ratio of the fish scale-based biofilm to the certain solution for reaction is 0.1 g:1 mL; the concentration of the acid solution is 0.5 mol / L, the concentration of the A solution is 1 mol / L, and the volume ratio of the acid solution to the A solution is 3:1.

[0041] Example 4: A method for improving the mechanical properties of fish scale-based biofilm

[0042] S1. The fresh fish scales of bream are rinsed clean and added to a degreasing solution, placed on a shaker for degreasing, the shaker speed is set to 120 r / min, and the degreasing time is 2 h, the degreasing solution is a mixed solution of glycerol and tetrachloromethane, the volume ratio is 6:1; the degreased fish scales are rinsed clean, added to a 5% mass fraction of sodium chloride solution, placed on a shaker for desugar, the shaker speed is set to 145 r / min, and the desugar time is 2 h; the desugared fish scales are rinsed clean and placed in a decalcification solution for 4 h, the decalcification solution is a mixed solution of EDTA and nitric acid, the mass fraction of EDTA in the mixed solution is 23%, and the mass fraction of nitric acid is 16%;

[0043] S2. The fish scale-based biomembrane is soaked in a certain solution for 12 h, and after soaking, new certain solution is added to the reaction kettle for reaction, the temperature of the reaction is 155 DEG C, the pressure of the reaction is 16 MPa, and the reaction time is 20 h, and the fish scale-based biomembrane with improved mechanical properties is obtained after reaction; wherein the certain solution is a mixed solution of acid liquid and A liquid, the A liquid is a mixed solution of stearate, sodium dodecyl sulfonate and sodium glycocholate; the acid liquid is a mixed solution of nitric acid, sulfuric acid, hydrochloric acid, acetic acid, sugarcane acid, malic acid and citric acid; the mass-volume ratio of the fish scale-based biomembrane to the certain solution for reaction is 0.3 g:1 mL; the concentration of the acid liquid is 1.5 mol / L, the concentration of the A liquid is 3 mol / L, and the volume ratio of the acid liquid to the A liquid is 5:1.

[0044] Example 5: Compliance experiment

[0045] The fish scale-based biomembrane with improved mechanical properties prepared after treatment in Example 3 is subjected to nanoindentation test, and the surface elastic modulus is measured, a probe with a probe of 60.8 N / m and a tip radius of 45 um is used, and a curve comparison graph is shown in Figure 1 The Hertz formula in the software Piuma Dataviewer is used to fit the curve to obtain the elastic modulus of the material before and after treatment, and it can be seen from Figure 1 that the compliance of the fish scale-based biomembrane prepared by the application is indeed obviously improved, and the elastic modulus can be improved to 45.05 KPa, close to the elastic modulus of human cornea.

[0046] Example 6: Tensile strength experiment

[0047] The fish scale-based biomembrane with improved mechanical properties prepared in Example 3 is cut into strips, and stretched at a rate of 500 um / min, and a tensile curve graph is shown in Figure 2 , and the tensile strength is 3 MPa, close to the tensile strength of human cornea (about 2.6 MPa), and much higher than the tensile strength of gel corneal materials.

[0048] Example 7: Suture strength experiment

[0049] The fish scale-based biomembrane with improved mechanical properties prepared in Example 3 is threaded with ophthalmic special 10-0 nylon suture, and stretched at a rate of 6 mm / min, and a suture strength test is performed, and a curve graph is shown in Figure 3 , and the suture strength is 29 gf, close to the suture strength of human cornea (35.6 gf), and can meet the suture requirement.

[0050] Example 8: Light transmittance experiment

[0051] The fresh fish scales, decalcified fish scales and fish scale-based biomembrane with improved mechanical properties of Example 3 are subjected to digital photography, and a digital photograph comparison graph is shown inFigure 5 The transmittance of the fish scales treated by the method of the application was measured in the visible light wavelength range (400-800nm) by using a UV-visible spectrophotometer, and the transmittance curve was plotted, as shown in Figure 4 The changes of the transmittance of the fish scales treated by the method of the application were observed, and it was found that the transmittance of the fish scale-based biomembrane after treatment could reach 92.8% in the visible light wavelength range, so the fish scales improved by the method of the application could greatly improve the transmittance.

[0052] Example 9: Transparency experiment

[0053] The surfaces of the fresh fish scales, the decalcified fish scales and the fish scale-based biomembrane with improved mechanical properties of Example 3 were treated by gold spraying after drying, and then observed by scanning electron microscope, and the scanning electron microscope comparison chart is as shown in Figure 6 The results show that the ridges on the surface of the fish scale-based biomembrane after treatment are obviously weakened, which reduces the scattering and diffuse reflection of light, and greatly improves the transparency of the fish scale-based biomembrane material.

[0054] Example 10: Microstructure experiment

[0055] The cross sections of the fresh fish scales, the decalcified fish scales and the fish scale-based biomembrane with improved mechanical properties of Example 3 were treated by gold spraying after drying, and then observed by scanning electron microscope, and the scanning electron microscope comparison chart is as shown in Figure 7 The results show that the ordered structure of the fish scale fiber arrangement is not damaged in the treatment process of the application, and the fish scale-based biomembrane material can still simulate the fiber arrangement structure of human cornea.

[0056] Example 11: Biocompatibility experiment

[0057] After the fish scale-based biomembrane with improved mechanical properties of Example 3 was soaked by 75% alcohol and sterilized by ultraviolet, L929 cells were inoculated on the material and cultured for two days, the inoculation density was 20000 per square centimeter, FDA dye was added, and then photographed under an inverted fluorescence microscope, as shown in Figure 8 It can be observed from the figure that the cells grow well and spread well, indicating that the fish scale-based biomembrane material has good biocompatibility.

[0058] The above is only the preferred specific embodiment of the application, but the protection scope of the application is not limited thereto, any person skilled in the art can make equivalent replacement or change according to the technical scheme and the inventive concept of the application within the technical range disclosed by the application, which is covered by the protection scope of the application.

Claims

1. A method for improving the mechanical properties of fish scale-based biofilms, characterized in that, The fish scale-based biomembrane is soaked in a certain solution for 8-15 hours, and then is placed in a reaction kettle to react with new certain solution, the reaction temperature is 4-200 DEG C, the reaction pressure is 0.1-20 MPa, the reaction time is 5-24 hours, and the fish scale-based biomembrane with improved mechanical properties is obtained after reaction; wherein the certain solution is a mixed solution of acid liquid and A liquid, the A liquid is at least one of stearate, sodium dodecyl sulfonate, sodium dodecyl benzene sulfonate, sodium dodecyl sulfate or sodium glycocholate, and the acid liquid is at least one of formic acid, oxalic acid, nitric acid, sulfuric acid, hydrochloric acid, acetic acid, sugarcane acid, malic acid, citric acid, boric acid, EDTA or soft acid. The preparation method of the fish scale-based biomembrane comprises the following steps: rinsing fresh fish scales, and then adding degreasing liquid, desugaring liquid and decalcification liquid to carry out desugaring, degreasing and decalcification treatment.

2. The method for improving the mechanical properties of fish scale-based biofilms according to claim 1, characterized in that, The mass-volume ratio of the fish scale-based biomembrane and the certain solution is 0.01-0.5 g:1 mL.

3. The method of claim 1, wherein the fish scale-based biomembrane has improved mechanical properties. The concentration of the acid liquid is 0.1-2 mol / L, the concentration of the A liquid is 0.1-5 mol / L, and the volume ratio of the acid liquid to the A liquid is 0.1-10:

1.

4. The method of claim 1, wherein the fish scale-based biomembrane has improved mechanical properties. The degreasing liquid is a mixed solution of alcohol and methane chloro compound, and the volume ratio is 0.1-8:1; the desugaring liquid is a sodium chloride solution with a mass fraction of 0.1-8 %; and the decalcification liquid is a mixed solution of EDTA and nitric acid, wherein the mass fraction of EDTA in the mixed solution is 1-30 %, and the mass fraction of nitric acid is 1-20 %.

5. The method of claim 1, wherein the method is characterized by, The fish scales are from hypophthalmichthys molitrix, salmo salar, carassius auratus, grass carp, black carp, cyprinus carpio, tilapia, spearfish, cirrhinus molitorella or esox.

6. The method of claim 4, wherein the fish scale-based biomembrane is improved in mechanical properties. The specific operation of degreasing comprises the following steps: rinsing fresh fish scales, adding degreasing liquid, placing on a shaking table, setting the speed of the shaking table to 60-160 r / min, and degreasing for 1-3 hours; and the alcohol is any one of methanol, ethanol, propanol, isopropanol or glycerol.

7. The method of claim 4, wherein the fish scale-based biomembrane is improved in mechanical properties. The specific operation of desugaring comprises the following steps: rinsing the degreased fish scales, adding desugaring liquid, placing on a shaking table, setting the speed of the shaking table to 60-160 r / min, and desugaring for 1-3 hours.

8. The method of claim 4, wherein the fish scale-based biomembrane is improved in mechanical properties. The specific operation of decalcification treatment comprises the following steps: rinsing the desugared fish scales, adding decalcification liquid, and reacting for 1-12 hours.

9. The fish scale-based biomembrane with improved mechanical properties prepared by the method according to any one of claims 1-8 is applied to an artificial cornea.

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