WO2025044637A1 - Method for preparing biomimetic biomaterial and use thereof - Google Patents

Abstract

Disclosed in the present invention is a method for preparing a biomimetic biomaterial, which method comprises using heterologous or homologous corneal stroma or skin as a template, perfusing a chondroitin sulfate derivative into the interspace of the corneal stroma or skin, allowing the chondroitin sulfate derivative to cure and then digesting the original corneal stroma or skin, and thereby obtaining a polymer backbone having copied structural characteristics of a natural stroma. A biomimetic corneal stroma having the structural characteristics of a natural cornea or a biomimetic skin is obtained by means of refilling with collagen. The preparation method provided in the present invention removes heterologous proteins from a natural biological tissue and reduces the immunogenicity while maintaining the structural characteristics of the natural tissue, thereby realizing the biomimetics of an artificial biomaterial on a natural biological tissue structure.

Description

A preparation method of biomimetic biomaterial and its application Technical Field

The invention belongs to the field of tissue engineering and regenerative medicine, and specifically relates to a preparation method of a bionic biomaterial and application thereof.

Background Art

Corneal blindness is the fourth leading cause of blindness in the world, with more than 10 million people suffering from bilateral corneal blindness. Due to the shortage of donor corneas, there are only about 185,000 corneal transplants each year, and patients face the risk of immune rejection for life after transplantation. The corneal stroma is one of the five layers of the cornea, accounting for more than 90% of the thickness of the cornea, and is the most important layer for corneal transparency and refractive function. Severe damage to the corneal stroma is the main cause of corneal blindness. The front 1/3 of the corneal stroma is composed of unidirectionally arranged collagen fiber lamellae, and the back 3/2 layers are composed of non-interwoven collagen fiber lamellae, with a deviation of 45° or 90° between each lamella. This highly specialized structure is particularly important for corneal tissue transparency and mechanical strength. The complex stroma structure makes it difficult to simulate by bioengineering methods. In order to find the best stroma substitute, people are studying a variety of methods. With the development of materials science, a variety of biological tissue substitute materials have been developed in recent years, mainly including collagen-based and decellularized biomaterials. Collagen-based materials are prepared by cross-linking a single collagen protein and lack the ultrastructural characteristics of natural biological tissues. Decellularized materials are extracellular matrix scaffolds obtained by decellularizing xenogeneic animal tissues. They have the structural characteristics and mechanical properties of natural biological tissues, but there are still residual cell fragments, antigen exposure, and virus transmission during the decellularization process, and batch differences are difficult to control. Due to the complexity of the structure of natural biological tissues, there are few reports on the study of biomimetic ultrastructures of natural biological tissues. A Chinese invention patent (application number 202210102037.X) discloses a method for preparing an artificial cornea. The invention uses electrochemical deposition technology to prepare an artificial cornea composed of ordered collagen microfibers, but the invention still cannot simulate the orthogonally arranged lamellar structure characteristics of the natural cornea.

Summary of the invention

In order to solve the problem that the current biomaterials have a single structure and are difficult to simulate the ultrastructure of natural biological tissues, the present invention provides a method for preparing biomimetic biomaterials, comprising the following steps: immersing the simulated biological tissue in a monomer polymer solution to allow the monomer polymer to fully penetrate into the simulated biological tissue; forming a polymer network inside the simulated biological tissue through a polymerization reaction; digesting and flushing the simulated biological tissue to obtain a polymer skeleton composed of the polymer network; filling and fixing the biomaterial into the polymer skeleton to obtain the biomimetic biomaterial.

In the present invention, the mimetic biological tissue is selected from connective tissue having the same or similar structure as the target biomimetic biomaterial, and is derived from natural biological tissue or decellularized biological tissue of the same or different species. For example, when the target biomimetic biomaterial is human cornea, the mimetic biological tissue can be selected from pig cornea or decellularized pig cornea; when the target biomimetic biomaterial is human skin, the mimetic biological tissue can be selected from pig skin or decellularized pig skin.

In a preferred embodiment, the simulated biological tissue is selected from connective tissue mainly composed of extracellular matrix, such as cornea or skin, and preferably has a size of 0.1-5 cm ² .

In the present invention, the monomer polymer is selected from one or more of methyl propyl acylated polysaccharide, polyethylene glycol diacrylate or polyethylene glycol methyl ether methacrylate; wherein the methyl propyl acylated polysaccharide is selected from one of methacrylylated hyaluronic acid, methacrylylated chondroitin sulfate, methacrylylated dextran or more; the monomer polymer solution concentration range is 5%-30% (w/v).

Furthermore, the monomer polymer solution contains a photoinitiator or a temperature initiator. The concentration of the photoinitiator is 0.1%-0.5%; the concentration of the temperature initiator is 0.1-0.2 mol/L. The photoinitiator is a blue light or ultraviolet light initiator, including 2,4,6-trimethylbenzoyl lithium phosphate (LAP) and photoinitiator 2959; the temperature initiator includes azobisisobutyronitrile (AIBN).

In a preferred embodiment, the monomer polymer solution is a methyl propyl acylated polysaccharide containing 0.1-0.5% of a photoinitiator, and the concentration is 5-20% (w/v).

In a preferred embodiment, the monomer polymer solution includes at least methacryloyl chondroitin sulfate.

In a more preferred embodiment, the monomer polymer solution is a methacryloyl chondroitin sulfate solution containing 0.2-0.3% photoinitiator, with a concentration of 10-15% (w/v).

In the present invention, the polymer network is formed by interlacing monomer polymers between simulated biological tissues through polymerization reactions.

In the present invention, the polymer skeleton is composed of a polymer network. Since the polymer network is formed inside the bionic biomaterial, it has a structure that is the same as or similar to that of the pseudo-bionic biomaterial. After the biomaterial is filled and fixed to the polymer skeleton, the obtained bionic biomaterial has a structure that is the same as or similar to that of the pseudo-bionic biomaterial.

In the present invention, the biomaterial is selected from natural collagen, recombinant collagen and modified derivatives thereof, including but not limited to type I collagen, type III collagen, gelatin, recombinant human collagen or atelocollagen.

When the biomaterial is collagen, a solution with a concentration of 2%-20% is prepared; when the biomaterial is gelatin, a solution with a concentration of 10%-20% is prepared.

The preparation method of the biomaterial solution takes the preparation of 20% collagen solution as an example. The method is as follows: dissolve 2g of soluble atelocollagen type I protein in 10ml of phosphate buffer and shake on a shaker at 120rpm for 10 minutes.

During the above soaking process, ultrasound, vacuum or stirring can be used simultaneously to accelerate the soaking or immersion process.

In a preferred embodiment, the immersion is to completely immerse the biomimetic tissue in at least 10 times the volume of the monomer and polymer solution, place it on a shaker at 120 rpm, and shake at room temperature for 12-36 hours.

In the present invention, the polymerization reaction includes light activation and temperature activation. When light activation is used, the wavelength is 300nm-600nm and the irradiation time is 2-3 minutes. When temperature activation is used, the reaction time is 12-24 hours at 60-70°C.

In a preferred embodiment, the photoinitiator is a 0.1%-0.3% LAP solution; and the activation time is 60-120 seconds.

In a more preferred embodiment, the polymerization reaction method is to polymerize the biomimetic tissue under 365nm ultraviolet light for 2 minutes.

In the present invention, the digestion includes but is not limited to acid digestion and enzyme digestion, and conventional methods can be used.

In a preferred embodiment, acid digestion uses a hydrochloric acid solution with a concentration of 10%-25%; enzyme digestion uses a pepsin or collagenase solution with a concentration of 1%-5%; and the digestion time is 18-48 hours.

In the present invention, the conventional method can be used for the rinsing. In a preferred embodiment, the rinsing method is to immerse the digested biomimetic material in at least 20 volumes of 0.01 mol/L phosphate buffer, place it on a shaker at 120 rpm and shake at room temperature for 15 minutes. Then replace it with a new phosphate buffer and rinse it. This step is repeated at least 6 times until the pH value of the phosphate buffer is neutral. (6.8-7.6).

In the present invention, the method of filling the biomaterial includes but is not limited to immersion, ultrasound, and vacuuming, and conventional methods may be used.

In a preferred embodiment, the filling method is to immerse the washed biomimetic material in at least 10 times the volume of the biomaterial solution to completely immerse it, and then place it on a shaker at 120 rpm at room temperature for 12-24 hours.

During the filling process, the efficiency of immersion can be improved by ultrasound and vacuuming. Preferably, the ultrasound frequency is 20-40 Hz and the vacuum degree is 100 mTorr.

In the present invention, the fixing method is cross-linking with a cross-linking agent to fix the monomer polymer in the polymer skeleton. The types of the cross-linking agent include but are not limited to EDC/NHS, CMC/NHS, genipin, proanthocyanidin, etc.

In a preferred embodiment, the concentration of the cross-linking agent is 0.1%-5% CMC/NHS solution, and the solution for preparing the cross-linking agent is 50 mM MES solution.

In a preferred embodiment, the fixation adopts EDC/NHS solution cross-linking, comprising the following steps: taking out the biomimetic material soaked with the biomaterial, removing the excess solution on the surface with dust-free absorbent paper, and then placing it in a 4°C environment for pre-fixation for 15-30 minutes; then using a MES solution with a pH value of 4.7 and a concentration of 50mM to prepare a 0.5% EDC/NHS solution, and pre-cooling it in a 4°C environment; soaking the pre-fixed biomimetic material in the EDC/NHS solution for 8-12 hours; placing the fixed biomimetic material in at least 20 times the volume of 0.01mol/L phosphate buffer, placing it on a shaker at 120 rpm at room temperature for 15 minutes, and then replacing it with a new phosphate buffer for rinsing, and repeating this step at least 5 times.

In a preferred embodiment, the fixation adopts CMC/NHS cross-linking, which includes the following steps: taking out the biomimetic material soaked in biomaterial, removing the excess solution on the surface with dust-free absorbent paper, and then placing it in a 4°C environment for pre-fixation for 15-30 minutes; then using a MES solution with a pH value of 4.7 and a concentration of 50mM to prepare a 1% CMC/NHS solution, and pre-cooling it in a 4°C environment; soaking the pre-fixed biomimetic material in the CMS/NHS solution for 12 hours; placing the fixed biomimetic material in at least 20 times the volume of 0.01mol/L phosphate buffer, placing it on a shaker at 120 rpm at room temperature for 15 minutes, and then replacing it with a new phosphate buffer for rinsing, and repeating this step at least 5 times.

Another aspect of the present invention provides a biomimetic biomaterial prepared according to the above method, wherein the biomimetic biomaterial has the same or similar structure, transparency and mechanical properties as those of the simulated biological tissue.

Another aspect of the present invention provides the use of the bionic biomaterial prepared according to the above method and obtained in the preparation of medical devices for tissue substitutes, medical dressings or drug carriers, such as corneal stroma substitutes, skin substitutes, and ocular medical dressings.

Beneficial Effects

The present invention uses a heterogeneous or homogeneous pseudo-bionic biomaterial (corneal stroma) as a template, and injects derivatives such as chondroitin sulfate into the gap of the pseudo-bionic biomaterial (corneal stroma). After solidification, the original pseudo-bionic biomaterial (corneal stroma) is digested to obtain a polymer skeleton that copies the structural characteristics of the natural pseudo-bionic biomaterial (corneal stroma). After refilling with collagen, a biomimetic biomaterial (corneal stroma) with the structural characteristics of the natural pseudo-bionic biomaterial (corneal stroma) is obtained. The preparation method provided by the present invention removes heterogeneous proteins in natural biological tissues, reduces immunogenicity, and retains the structural characteristics of natural tissues, thereby realizing the biomimetic effect of artificial biomaterials on natural biological tissue structures.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 Ultrastructural characteristics of bionic corneal stroma and natural corneal stroma.

Figure 2 Ultrastructure of biomimetic corneal stroma with collagen-based artificial cornea and cyanoacrylate gelatin-based cornea Structural features.

Figure 3 Comparison of light transmittance between bionic corneal stroma, natural cornea and collagen-based cornea.

Fig. 4 Comparison of degradation rates of bionic corneal stroma, natural cornea and collagen-based cornea.

Figure 5 Comparison of water absorption between bionic corneal stroma, natural cornea and collagen-based cornea.

Figure 6 Gross photographs before and after bionic corneal stroma repair of corneal stromal defects, and comparison of optical coherence tomography and thickness scanning results.

Figure 7 Comparison of the bionic corneal stroma repair effect by slit lamp, fluorescein staining, optical coherence tomography and thickness scanning results in in vivo animal experiments.

DETAILED DESCRIPTION

The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative work are within the scope of protection of the present invention.

Although steps, materials or substances, and reaction conditions similar or equivalent to those disclosed herein can be used in the practice of the present invention, the preferred steps, materials or substances, and reaction conditions are described herein.

When a numerical range is described herein, unless otherwise specified, the range is intended to include its endpoints and all integers and fractions within the range, and all numerical values within the range can achieve the effects of the present invention.

Unless defined otherwise, all technical and scientific terms and abbreviations used herein have the same meaning as commonly understood by one of ordinary skill in the field of the invention or in the field to which the terms are applied.

As used herein, the singular form of a word includes the plural form and vice versa. Thus, "a", "an", and "the" generally include the plural form of the corresponding term. As used herein, "one embodiment" or "embodiment" refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The "in one embodiment" that appears in different places in this specification does not refer to the same embodiment, nor is it a separate or selective embodiment that is mutually exclusive with other embodiments.

As used herein, "comprising," "having," "including," or "containing" is inclusive or open-ended and does not exclude additional, unrecited materials or method steps.

Decellularized cornea: The corneas from mammals such as humans, pigs, horses, and cows are treated with conventional decellularization methods (including repeated freezing and thawing, high and low osmotic pressure treatment, surfactant treatment, ultrastatic pressure treatment, nuclease treatment, and phospholipase treatment) to cause the stromal cells and endothelial cells to fall off, thus obtaining decellularized corneas.

Acellular porcine cornea: prepared by virus inactivation and decellularization process, including the anterior Descemet's layer and corneal stroma.

If the specific conditions are not specified in the examples, the experiments were carried out under conventional conditions or conditions recommended by the manufacturer. If the manufacturers of reagents or instruments are not specified, they are all conventional products that can be obtained commercially.

Some of the materials in the embodiments of the present invention come from:

Fresh porcine cornea: fresh porcine cornea without denaturation purchased from the market.

SDS solution: Sodium dodecyl sulfate solution, purchased from Beijing Solebow Technology Co., Ltd., catalog number S8010.

Nuclease: Deoxyribonuclease, purchased from Beijing Sino Biological Technology Co., Ltd., catalog number SSNP01.

The photoinitiator LAP phenyl (2,4,6-trimethylbenzoyl) phosphate lithium salt was purchased from Shanghai Yuanye Biotechnology Co., Ltd., product number Y43995.

Methacrylated chondroitin sulfate: purchased from Suzhou Yongqinquan Intelligent Equipment Co., Ltd., Cat. No. EFL-ChsMA-001

PEGDA solution: polyethylene glycol diacrylate solution, purchased from Sigma, product number 455008.

PBS buffer: Phosphate buffered saline, purchased from Beijing Solebow Technology Co., Ltd., catalog number P1020.

Rabbits: purchased from Xilingjiao Breeding and Propagation Center, Jinan.

1-Ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) was purchased from Aladdin, product number E106172.

N-Hydroxysuccinimide (NHS): purchased from Aladdin, product number H109330.

1-Cyclohexyl-2-morpholinoethylcarbodiimide p-toluenesulfonate (CMC): purchased from McLean, product number M812751.

Morpholineethanesulfonic acid (MES): purchased from Maclean, product number M813436.

Example 1 Preparation of biomimetic corneal material

(1) Preparation of decellularized porcine cornea: The epithelium and endothelium of fresh porcine cornea were scraped off, and the cornea was immersed in 0.5% SDS solution and treated with 500 U/ml nuclease for 2 hours, followed by washing with sufficient PBS buffer for 6-8 times.

(2) Prepare monomer and polymer solution: Use 0.3% LAP to prepare 10% methacrylated chondroitin sulfate solution.

(3) Immersing in decellularized porcine cornea: Immerse the decellularized porcine cornea in at least 10 times the volume of methacrylylated chondroitin sulfate solution, place it in a vacuum oven, evacuate for 6 hours, and then place it in a shaker at 120 rpm and shake at room temperature for 18 hours.

(4) Polymerization reaction: The decellularized porcine cornea infiltrated with methacrylylated chondroitin sulfate was placed on a curvature-curved solidification table and polymerized under 365 nm ultraviolet light for 2 minutes.

(5) Digestion of decellularized porcine cornea: The polymerized decellularized porcine cornea was placed in a 30 mg/ml pepsin digestion solution and treated at 37°C and 120 rpm for 24 hours, followed by rinsing with a large amount of physiological saline.

(6) Biomaterial refilling: The methacryloyl-modified chondroitin sulfate skeleton with decellularized porcine cornea was immersed in a 10% type I collagen solution at room temperature at 120 rpm for 24 hours. The collagen was then solidified at 4°C and cross-linked with a 1% CMC/NHS solution.

Example 2 Preparation of biomimetic corneal material

(1) Preparation of decellularized porcine cornea: The epithelium and endothelium of fresh porcine cornea were scraped off, and the cornea was immersed in 0.5% SDS solution and treated with 500 U/ml nuclease for 2 hours, followed by washing with sufficient PBS buffer for 6-8 times.

(2) Preparation of monomer and polymer solution: Use 0.25% LAP to prepare 10% methacrylated dextran solution.

(3) Immersing in decellularized porcine cornea: Immerse the decellularized porcine cornea in at least 10 times the volume of methacrylated dextran solution, place it in a vacuum oven, evacuate for 6 hours, and then place it in a shaker at 120 rpm and shake at room temperature for 18 hours.

(4) Polymerization reaction: The decellularized porcine cornea soaked in methacrylated dextran solution was placed on a curvature-curved solidification table and polymerized under 365 nm ultraviolet light for 2 minutes.

(5) Digestion of decellularized porcine cornea: Digest the cornea in 30 mg/ml pepsin solution at 37°C and 120 rpm for 24 hours, then rinse with plenty of saline.

(6) Biomaterial refilling: The methacrylated dextran skeleton from which the decellularized porcine cornea was removed was immersed in a 10% type I collagen solution at room temperature at 120 rpm for 24 hours. The collagen was then solidified at 4°C and cross-linked with a 5% CMC/NHS solution.

Example 3 Preparation of biomimetic corneal material

(1) Preparation of decellularized porcine cornea: The epithelium and endothelium of fresh porcine cornea were scraped off, and the cornea was immersed in 0.5% SDS solution and treated with 500 U/ml nuclease for 2 hours, followed by washing with sufficient PBS buffer for 6-8 times.

(2) Preparation of monomer and polymer solution: 0.2 g/ml PEGDA solution was prepared with 0.3% LAP.

(3) Immersing in decellularized porcine cornea: Immerse the decellularized porcine cornea in at least 10 times the volume of the PEGDA solution, place it in a vacuum oven, evacuate for 6 hours, and then place it in a shaker at 120 rpm and shake at room temperature for 18 hours.

(4) Polymerization reaction: The decellularized porcine cornea soaked in PEGDA solution was placed on a curvature-curved curvature curvature plate and polymerized under 365 nm ultraviolet light for 2 minutes.

(5) Digestion of decellularized porcine cornea: The polymerized decellularized porcine cornea was placed in a 25% hydrochloric acid solution at 120 rpm for 48 hours, and then rinsed with a large amount of physiological saline.

(6) Biomaterial refilling: The PEGDA skeleton with decellularized porcine cornea removed was immersed in a 10% type I collagen solution and treated at room temperature at 120 rpm for 24 hours. The collagen was then solidified at 4°C and cross-linked with a 0.5% EDC/NHS solution.

Example 4 Preparation of bionic skin material

(1) Preparation of decellularized pig skin: Fresh pig skin was shaken with 0.25% trypsin for 6 hours, then immersed in 5% TritonX-100 solution and shaken for 6 hours, and finally washed 6 times with balanced salt solution.

(2) Preparation of monomer and polymer solution: 10% methacrylated hyaluronic acid solution was prepared with 0.3% LAP solution.

(3) Immersing in decellularized porcine skin: Immerse the decellularized porcine skin in at least 10 times the volume of the methacrylated hyaluronic acid solution, place it in a vacuum oven for 6 hours, and then place it in a shaker at 120 rpm for 18 hours.

(4) Polymerization reaction: Spread the decellularized porcine skin soaked with methacrylated hyaluronic acid and polymerize it under 365 nm ultraviolet light for 2 minutes.

(5) Digestion of decellularized porcine skin: The polymerized decellularized porcine skin was placed in a 30 mg/ml pepsin digestion solution at 37° C. and 120 rpm for 48 hours, and then rinsed with a large amount of physiological saline.

(6) Biomaterial refilling: The methacrylated hyaluronic acid skeleton with decellularized porcine skin removed was immersed in a 10% type I collagen solution (Beijing Paisheng Biological) at room temperature and 120 rpm for 24 hours. The polymer skeleton soaked with collagen was then cross-linked with EDC/NHS to obtain a biomimetic bioskin material.

Comparative Example 1 Ultrastructural comparison between bionic cornea and human cornea

The bionic cornea and human cornea were fixed with glutaraldehyde, ultramicrotomed, and observed under a transmission electron microscope. The results showed that the ultrastructure of the bionic corneal stroma was very similar to that of the human cornea. Low-magnification observation showed that the bionic corneal stroma had a lamellar structure similar to that of the natural cornea, and high-magnification observation showed that the bionic corneal stroma had parallel-arranged collagen fibers (Figure 1).

Comparative Example 2 Ultrastructure comparison between bionic corneal stroma and common artificial corneal repair materials

The biomimetic corneal stroma, methacrylated gelatin-based cornea, and collagen-based cornea were fixed with glutaraldehyde, ultramicrotomed, and observed under a transmission electron microscope. The results showed that, except for the biomimetic corneal stroma, which had a precise ultrastructure similar to that of the natural cornea, the ultrastructure of the collagen-based cornea and the methacrylated gelatin-based cornea was uniform and did not have the histological structural characteristics of the corneal stroma (Figure 2).

Comparative Example 3 Comparison of physical properties of bionic corneal stroma, natural cornea and collagen-based cornea

The bionic corneal stroma, collagen-based cornea and natural cornea were placed in the cuvette of the spectrophotometer and the transmittance between 300nm and 800nm was tested respectively. The results showed that the bionic corneal stroma had good transmittance and better transparency than the natural cornea. (Figure 3)

The bionic corneal matrix, collagen-based cornea and natural cornea were placed in 10U/ml collagenase solution, and the mass changes of the samples were detected at 3 hours, 6 hours, 12 hours, 1 day, 2 days, 3 days, 5 days and 7 days. The results showed that the collagen-based cornea had the fastest degradation rate, with 5.7% of the mass remaining on the 7th day. The natural cornea had 12.6% of the mass remaining on the 7th day. The bionic corneal matrix had the slowest degradation rate under the action of collagenase. The remaining mass on the 7th day was 38.4%. This is related to the presence of chondroitin sulfate bionic scaffold in the bionic corneal stroma. (Figure 4)

The bionic corneal matrix, collagen-based cornea and natural cornea were placed in PBS buffer respectively, and the quality of each sample was tested at 3, 6, 12, 24, 36 and 48 hours. The results showed that the collagen-based cornea and bionic corneal matrix remained stable in PBS buffer and did not swell significantly, with swelling rates of 100.2% and 101.8% respectively. The natural cornea swelled significantly, with a swelling rate of 489.1%. (Figure 5)

Example 5 In vitro experiment - repairing corneal defects

(1) To achieve seamless repair, the biomimetic cornea prepared in Example 1 is first immersed in a 20% methacrylic gelatin solution of at least 10 times the volume. The solution is placed in a 37°C constant temperature shaker at 120 rpm for 24 hours. The methacrylic gelatin on the surface of the biomimetic corneal stroma is then removed.

(2) A circular corneal stromal defect with a diameter of 6 mm and a depth of 500 μm was made on the surface of the cornea of the pig eye using a trephine drill and a lamellar knife. A biomimetic corneal stroma of the same size was placed on the corneal defect, heated to 37°C, and then irradiated with 405 nm visible light for 60 seconds.

(3) Comparing the repaired pig eyeball with the preoperative one, observation under the slit lamp showed that the stromal defect was successfully closed. Anterior segment optical coherence tomography and thickness scans proved that the stromal defect was filled and the corneal curvature was restored (Figure 6).

It is proven that bionic corneal materials can repair corneal defects and restore corneal thickness and curvature.

Example 6 In vitro experiment - repairing corneal stroma defects

A circular corneal stromal defect with a diameter of 3 mm and a depth of 200 μm was made on the surface of the rabbit cornea using a trephine and a lamellar knife. A biomimetic corneal stroma of the same size and thickness was placed at the corneal defect, and then irradiated with 405 nm visible light for 60 seconds. The group without biomimetic corneal stroma repair served as the control.

Four weeks after surgery, the cornea of the bionic corneal stromal transplantation group was transparent, the epithelium was completely regenerated, and optical coherence tomography showed that the bionic corneal stromal graft was stable, without displacement and peeling, and the thickness of the stromal graft was restored, proving that it had good biocompatibility, did not stimulate inflammatory response, and prevented corneal stromal fibrosis, achieving good visual effects. In the non-transplantation group, corneal scarring, central corneal transparency loss, and central corneal epithelial defect were formed. Optical coherence tomography showed corneal stromal fibrosis, rough corneal surface, and central thickness loss (Figure 7).

The embodiments of the present invention are described in detail above. Specific examples are used herein to illustrate the principles and implementation methods of the present invention. The description of the above embodiments is only used to help understand the method of the present invention and its core idea. At the same time, changes or deformations made by those skilled in the art based on the ideas of the present invention, the specific implementation methods and application scope of the present invention, all belong to the scope of protection of the present invention. In summary, the content of this specification should not be understood as limiting the present invention.

Claims (18)

A method for preparing a biomimetic biomaterial, characterized in that it comprises the following steps: immersing a simulated biological tissue in a monomer polymer solution, so that the monomer polymer is fully immersed in the simulated biological tissue; forming a polymer network in the simulated biological tissue through a polymerization reaction; digesting and washing the simulated biological tissue to obtain a polymer skeleton composed of the polymer network; filling and fixing a biomaterial into the polymer skeleton to obtain a biomimetic biomaterial; The mass volume concentration of the monomer polymer is 5%-30%, and is selected from one or more of methyl propyl acylated polysaccharide, polyethylene glycol diacrylate or polyethylene glycol methyl ether methacrylate; wherein the methyl propyl acylated polysaccharide is selected from one or more of methacryloyl hyaluronic acid, methacryloyl chondroitin sulfate and methacryloyl dextran; The biomaterial is selected from natural collagen, recombinant collagen and modified derivatives thereof, including type I collagen, type III collagen, gelatin, recombinant human collagen or atelocollagen. The preparation method according to claim 1 is characterized in that: the biomimetic biomaterial is a biomimetic corneal stroma, and the simulated biological tissue is a heterogeneous or homogeneous corneal stroma; or the biomimetic biomaterial is a biomimetic biological skin, and the simulated biological tissue is a heterogeneous or homogeneous skin. The preparation method according to claim 2, characterized in that the size of the bionic biological skin is 0.1-5 cm ² . The preparation method according to claim 1 is characterized in that the monomer polymer solution also contains a photoinitiator or a temperature initiator. The preparation method according to claim 4 is characterized in that: the concentration of the photoinitiator is 0.1%-0.5%; the concentration of the temperature initiator is 0.1-0.2 mol/L. The preparation method according to claim 4, characterized in that: the photoinitiator is a blue light or ultraviolet light initiator, including 2,4,6-trimethylbenzoyl lithium phosphate or photoinitiator 2959; the temperature initiator includes azobisisobutyronitrile. The preparation method according to claim 1 is characterized in that: when the biomaterial is collagen, a solution with a concentration of 2%-20% is prepared; when the biomaterial is gelatin, a solution with a concentration of 10%-20% is prepared. The preparation method according to claim 1, characterized in that the monomer polymer solution is a methyl propyl acylated polysaccharide solution containing 0.1-0.5% photoinitiator, and the concentration is 5-20% (w/v). The preparation method according to claim 8, characterized in that the monomer polymer solution is a methacryloyl chondroitin sulfate solution containing 0.2-0.3% phenyl-2,4,6-trimethylbenzoyl lithium phosphite, with a concentration of 10-15% (w/v). The preparation method according to claim 1, characterized in that: the fixing method is cross-linking with a cross-linking agent; the cross-linking agent is selected from EDC/NHS, CMC/NHS, genipin or proanthocyanidin. The preparation method according to claim 10, characterized in that: the cross-linking agent is 0.1%-5% CMC/NHS, and the solution for preparing the cross-linking agent is 50mM MES solution. The preparation method according to any one of claims 1 to 11, characterized in that: The immersion is to completely immerse the biomimetic tissue in at least 10 times the volume of the monomer polymer solution, place it on a shaker at 120 rpm, and shake at room temperature for 12-36 hours; The filling step is to immerse the washed biomimetic material in at least 10 times the volume of the biomaterial solution to completely immerse the biomimetic material, and then place the biomimetic material on a shaker at 120 rpm and room temperature for 12-24 hours. The preparation method according to any one of claims 1 to 11, characterized in that: the polymerization reaction includes light activation and temperature activation; the light activation condition is 300nm-600nm wavelength, the activation time is The temperature activation condition is 60-70°C and the reaction time is 12-24 hours. The preparation method as described in claim 13 is characterized in that: the polymerization reaction method is to polymerize the bionic tissue under 365nm ultraviolet light for 2 minutes. The preparation method according to any one of claims 1 to 11, characterized in that: the digestion is acid digestion or biological enzyme digestion; the acid digestion is to use a hydrochloric acid solution with a concentration of 10%-25%; the biological enzyme digestion is to use a pepsin or collagenase solution with a concentration of 1%-5%; the digestion time is 18-48 hours; The flushing comprises immersing the digested biomimetic material in at least 20 volumes of 0.01 mol/L phosphate buffer, placing it on a shaker at 120 rpm and shaking at room temperature for 10-30 minutes; repeating until the pH value of the phosphate buffer is 6.8-7.6. The preparation method according to any one of claims 1 to 11, characterized in that: When the fixation adopts EDC/NHS cross-linking, the following steps are included: after taking out the biomimetic material soaked with the biomaterial, use dust-free absorbent paper to remove the excess solution on the surface, and then place it in a 4°C environment for pre-fixation for 15-30 minutes; then use a MES solution with a pH value of 4.7 and a concentration of 50mM to prepare a 0.5% EDC/NHS solution, and place it in a 4°C environment for pre-cooling; place the pre-fixed biomimetic material in the EDC/NHS solution and soak it for 8-12 hours; place the fixed biomimetic material in at least 20 times the volume of 0.01mol/L phosphate buffer, place it on a shaker at 120 rpm at room temperature for 15 minutes, and then replace it with a new phosphate buffer for rinsing, and repeat this step at least 5 times; When CMC/NHS is used for fixation, the method comprises the following steps: taking out the biomimetic material soaked with the biomaterial, removing the excess solution on the surface with dust-free absorbent paper, and then placing it in a 4°C environment for pre-fixation for 15-30 minutes; then using a MES solution with a pH value of 4.7 and a concentration of 50 mM to prepare a 1% CMC/NHS solution, and pre-cooling it in a 4°C environment; soaking the pre-fixed biomimetic material in the CMS/NHS solution for 12 hours; placing the fixed biomimetic material in at least 20 times the volume of 0.01 mol/L phosphate buffer, placing it on a shaker at 120 rpm and shaking at room temperature for 15 minutes, then replacing it with a new phosphate buffer for rinsing, and repeating this step at least 5 times. A biomimetic biomaterial prepared according to the preparation method described in any one of claims 1 to 16. Use of the preparation method according to any one of claims 1 to 16 and the biomimetic biomaterial according to claim 17 in the preparation of medical devices for tissue substitutes, medical dressings or drug carriers.