Method for preparing biomimetic biomaterial and use thereof

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

Preparation method of biomimetic biomaterial and its application Technical Field

[0001] The present invention belongs to the field of tissue engineering and regenerative medicine, and in particular relates to a preparation method of a biomimetic biomaterial and application thereof. Background Art

[0002] Corneal blindness is the fourth leading cause of blindness worldwide, affecting over 10 million people. Due to a shortage of donor corneas, only approximately 185,000 corneal transplants are performed annually, leaving patients facing a lifelong risk of immune rejection. The corneal stroma, one of the five layers of the cornea, accounts for over 90% of its thickness and is crucial for corneal transparency and refractive function. Severe damage to the corneal stroma is the leading cause of corneal blindness. The anterior third of the corneal stroma is composed of interwoven unidirectional collagen fiber lamellae, while the posterior third consists of non-interwoven collagen fiber lamellae, with lamellae offset by 45° or 90°. This highly specialized structure is crucial for corneal transparency and mechanical strength. The complex structure of the corneal stroma makes it difficult to mimic through bioengineering methods. Various approaches are under investigation to identify optimal stroma substitutes. Advances in materials science have led to the development of a variety of biological tissue replacement materials, primarily collagen-based and acellular 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.

[0003] Summary of the Invention

[0004] In order to solve the problem that 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, including the following steps: immersing the mimicked biological tissue in a monomer polymer solution to allow the monomer polymer to fully penetrate into the mimicked biological tissue; forming a polymer network inside the mimicked biological tissue through a polymerization reaction; digesting and rinsing the mimicked 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.

[0005] 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 porcine cornea or decellularized porcine cornea; when the target biomimetic biomaterial is human skin, the mimetic biological tissue can be selected from porcine skin or decellularized porcine skin.

[0006] In a preferred embodiment, the biomimetic 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 .

[0007] In the present invention, the monomer polymer is selected from one or more of methylpropyl acylated polysaccharide, polyethylene glycol diacrylate or polyethylene glycol methyl ether methacrylate; wherein the methylpropyl acylated polysaccharide is selected from one or more of methacrylated hyaluronic acid, methacrylated chondroitin sulfate, and methacrylated dextran; and the concentration range of the monomer polymer solution is 5%-30% (w / v).

[0008] Furthermore, the monomer-polymer solution contains a photoinitiator or a thermal initiator. The concentration of the photoinitiator is 0.1%-0.5%, and the concentration of the thermal initiator is 0.1-0.2 mol / L. The photoinitiator is a blue light or ultraviolet light initiator, including 2,4,6-trimethylbenzoyl phosphate lithium salt (LAP) and photoinitiator 2959; the thermal initiator includes azobisisobutyronitrile (AIBN).

[0009] In a preferred embodiment, the monomer-polymer solution is a methyl propyl acylated polysaccharide containing 0.1-0.5% of a photoinitiator, with a concentration of 5-20% (w / v).

[0010] In a preferred embodiment, the monomer-polymer solution includes at least methacryloyl chondroitin sulfate.

[0011] In a more preferred embodiment, the monomer-polymer solution is a methacrylated chondroitin sulfate solution containing 0.2-0.3% photoinitiator, with a concentration of 10-15% (w / v).

[0012] In the present invention, the polymer network is formed by interlacing monomer polymers between simulated biological tissues through polymerization reaction.

[0013] In the present invention, the polymer skeleton is composed of a polymer network. Since the polymer network is formed inside the biomimetic 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 biomimetic biomaterial has a structure that is the same as or similar to that of the pseudo-bionic biomaterial.

[0014] 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.

[0015] 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.

[0016] The preparation method of the biomaterial solution takes the preparation of 20% collagen solution as an example. The method is as follows: 2 g of soluble atelocollagen type I protein is dissolved in 10 ml of phosphate buffer, and the mixture is shaken on a shaker at 120 rpm for 10 minutes.

[0017] During the soaking process, ultrasound, vacuuming or stirring can be used to accelerate the soaking or immersion process.

[0018] 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.

[0019] In the present invention, the polymerization reaction includes light activation and temperature activation. When light activation is used, irradiation is performed at a wavelength of 300nm-600nm for 2-3 minutes. When temperature activation is used, the reaction is performed at 60-70°C for 12-24 hours.

[0020] In a preferred embodiment, the photoinitiator is a 0.1%-0.3% LAP solution; and the activation time is 60-120 seconds.

[0021] In a more preferred embodiment, the polymerization reaction method is to polymerize the biomimetic tissue under 365nm ultraviolet light for 2 minutes.

[0022] In the present invention, the digestion includes but is not limited to acid digestion and enzyme digestion, and conventional methods can be used.

[0023] 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.

[0024] In the present invention, conventional rinsing methods can be used. 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 agitate it at room temperature for 15 minutes. Fresh phosphate buffer is then added for rinsing, and this step is repeated at least six times until the pH of the phosphate buffer reaches neutral (6.8-7.6).

[0025] In the present invention, the method of filling the biomaterial includes but is not limited to soaking, ultrasound, and vacuuming, and conventional methods can be used.

[0026] 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.

[0027] During the filling process, ultrasound and vacuum can be used to improve the efficiency of immersion. Preferably, the ultrasound frequency is 20-40 Hz and the vacuum degree is 100 mTorr.

[0028] In the present invention, the fixing method is cross-linking with a cross-linking agent to fix the monomer polymer within the polymer backbone. The types of cross-linking agents include but are not limited to EDC / NHS, CMC / NHS, genipin, proanthocyanidins, etc.

[0029] 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.

[0030] In a preferred embodiment, the fixation is cross-linked using EDC / NHS solution, comprising the following steps: taking out the biomimetic material soaked in the biomaterial, removing excess surface solution 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 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 0.01 mol / L phosphate buffer, placing it on a shaker at 120 rpm and shaking at room temperature for 15 minutes, then replacing it with new phosphate buffer for rinsing, and repeating this step at least 5 times.

[0031] In a preferred embodiment, the fixation adopts CMC / NHS cross-linking, comprising the following steps: taking out the biomimetic material soaked in 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 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 and shaking at room temperature for 15 minutes, then replacing it with new phosphate buffer for rinsing, and repeating this step at least 5 times.

[0032] 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.

[0033] Another aspect of the present invention provides the use of the biomimetic 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

[0034] The present invention uses a heterologous or homologous pseudo-bionic biomaterial (corneal stroma) as a template, injecting derivatives such as chondroitin sulfate into the gaps within the pseudo-bionic biomaterial (corneal stroma). After solidification, the original pseudo-bionic biomaterial (corneal stroma) is digested to produce a polymer skeleton that replicates the structural characteristics of the natural pseudo-bionic biomaterial (corneal stroma). Refilling with collagen yields a biomimetic biomaterial (corneal stroma) with the structural characteristics of the natural pseudo-bionic biomaterial (corneal stroma). The preparation method provided by the present invention removes heterologous proteins from natural biological tissues, reduces immunogenicity, and simultaneously retains the structural characteristics of natural tissues, achieving biomimetic imitation of natural biological tissue structures by artificial biomaterials. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 Ultrastructural characteristics of bionic corneal stroma and natural corneal stroma.

[0036] Figure 2 Ultrastructural characteristics of biomimetic corneal stroma, collagen-based artificial cornea, and cyanoacrylate gelatin-based cornea.

[0037] Figure 3 Comparison of light transmittance of biomimetic corneal stroma, natural cornea and collagen-based cornea.

[0038] Figure 4 Comparison of degradation rates of biomimetic corneal stroma, natural cornea, and collagen-based cornea.

[0039] Figure 5 Comparison of water absorption rates of biomimetic corneal stroma, natural cornea, and collagen-based cornea.

[0040] 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.

[0041] Figure 7 Comparison of the biomimetic corneal stroma repair effect using slit lamp, fluorescein staining, optical coherence tomography, and thickness scanning results in in vivo animal experiments. DETAILED DESCRIPTION

[0042] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative work shall fall within the scope of protection of the present invention.

[0043] Although steps, materials or substances, and reaction conditions similar or equivalent to those described herein can be used in the practice of the present invention, the preferred steps, materials or substances, and reaction conditions are described herein.

[0044] When a numerical range is described herein, unless otherwise indicated, the range is intended to include its endpoints and all integers and fractions within the range, and all values ​​within the range can achieve the effects of the present invention.

[0045] 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 art in the field of the invention or in the field to which the terms are applied.

[0046] As used herein, words in the singular include the plural and vice versa. Thus, "a," "an," and "the" generally include the plural form of the respective terms. As used herein, "one embodiment" or "an embodiment" refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The appearance of "in one embodiment" in various places in this specification does not necessarily refer to the same embodiment, nor does it refer to separate or selective embodiments that are mutually exclusive with other embodiments.

[0047] As used herein, "comprising," "having," "including," or "containing" is inclusive or open-ended and does not exclude additional, unrecited materials or method steps.

[0048] Decellularized cornea: The corneas of 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, ultra-static pressure treatment, nuclease treatment, and phospholipase treatment) to cause the stromal cells and endothelial cells to fall off, thus obtaining decellularized corneas.

[0049] Acellular porcine cornea: prepared by virus inactivation and decellularization process, including the anterior Descemet's layer and corneal stroma.

[0050] If the specific conditions are not specified in the examples, the experiments were carried out under conventional conditions or those recommended by the manufacturer. All reagents or instruments used, if the manufacturer is not specified, are commercially available conventional products.

[0051] Some of the materials in the examples of the present invention are from:

[0052] Fresh porcine cornea: fresh porcine cornea without denaturation purchased from the market.

[0053] SDS solution: sodium dodecyl sulfate solution, purchased from Beijing Solebow Technology Co., Ltd., product number S8010.

[0054] Nuclease: Deoxyribonuclease was purchased from Beijing Sino Biological Science and Technology Co., Ltd., catalog number SSNP01.

[0055] The photoinitiator LAP phenyl (2,4,6-trimethylbenzoyl) phosphate lithium salt was purchased from Shanghai Yuanye Biotechnology Co., Ltd., product number Y43995.

[0056] Methacrylated chondroitin sulfate: purchased from Suzhou Yongqinquan Intelligent Equipment Co., Ltd., product number EFL-ChsMA-001

[0057] PEGDA solution: polyethylene glycol diacrylate solution, purchased from Sigma, product number 455008.

[0058] PBS buffer: phosphate buffered saline, purchased from Beijing Solebow Technology Co., Ltd., catalog number P1020.

[0059] Rabbits were purchased from Xilingjiao Breeding and Propagation Center in Jinan.

[0060] 1-Ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) was purchased from Aladdin, product number E106172.

[0061] N-Hydroxysuccinimide (NHS): purchased from Aladdin, product number H109330.

[0062] 1-Cyclohexyl-2-morpholinoethylcarbodiimide p-toluenesulfonate (CMC): purchased from Maclean, product number M812751.

[0063] Morpholineethanesulfonic acid (MES): purchased from Maclean, product number M813436.

[0064] Example 1 Preparation of biomimetic biological cornea material

[0065] (1) Preparation of decellularized porcine cornea: fresh porcine cornea was scraped to remove the epithelium and endothelium, immersed in 0.5% SDS solution, treated with 500 U / ml nuclease for 2 hours, and then washed with sufficient PBS buffer for 6-8 times.

[0066] (2) Preparation of monomer and polymer solution: 10% methacrylated chondroitin sulfate solution was prepared with 0.3% LAP.

[0067] (3) Immersing in decellularized porcine cornea: Immerse the decellularized porcine cornea in at least 10 times the volume of methacryloyl chondroitin sulfate solution. Place the cornea in a vacuum oven and evacuate for 6 hours. Then, place the cornea in a shaker at 120 rpm and shake at room temperature for 18 hours.

[0068] (4) Polymerization reaction: The decellularized porcine cornea soaked with methacryloyl chondroitin sulfate was placed on a curvature-curved curvature plate and polymerized under 365 nm ultraviolet light for 2 minutes.

[0069] (5) Digestion of decellularized porcine cornea: The polymerized decellularized porcine cornea was placed in 30 mg / ml pepsin digestion solution and treated at 37°C and 120 rpm for 24 hours, and then rinsed with a large amount of physiological saline.

[0070] (6) Biomaterial refilling: The methacryloyl-modified chondroitin sulfate skeleton, after removal of acellular porcine cornea, was immersed in a 10% type I collagen solution at 120 rpm for 24 hours at room temperature. The collagen was then solidified at 4°C and cross-linked with a 1% CMC / NHS solution.

[0071] Example 2 Preparation of biomimetic biological cornea material

[0072] (1) Preparation of decellularized porcine cornea: fresh porcine cornea was scraped to remove the epithelium and endothelium, immersed in 0.5% SDS solution, treated with 500 U / ml nuclease for 2 hours, and then washed with sufficient PBS buffer for 6-8 times.

[0073] (2) Preparation of monomer and polymer solution: 10% methacrylated dextran solution was prepared with 0.25% LAP.

[0074] (3) Immersing the decellularized porcine cornea: Immerse the decellularized porcine cornea in at least 10 times the volume of methacrylated dextran solution. Place the cornea in a vacuum oven and evacuate for 6 hours. Then, place the cornea in a shaker at 120 rpm and shake at room temperature for 18 hours.

[0075] (4) Polymerization reaction: The decellularized porcine cornea soaked in methacrylated dextran solution was placed on a curing table with corneal curvature and polymerized under 365 nm ultraviolet light for 2 minutes.

[0076] (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.

[0077] (6) Biomaterial refilling: The methacrylated dextran skeleton from the decellularized porcine cornea was immersed in a 10% type I collagen solution at 120 rpm for 24 hours at room temperature. The collagen was then solidified at 4°C and cross-linked with a 5% CMC / NHS solution.

[0078] Example 3 Preparation of biomimetic biological cornea material

[0079] (1) Preparation of decellularized porcine cornea: fresh porcine cornea was scraped to remove the epithelium and endothelium, immersed in 0.5% SDS solution, treated with 500 U / ml nuclease for 2 hours, and then washed with sufficient PBS buffer for 6-8 times.

[0080] (2) Preparation of monomer and polymer solution: 0.2 g / ml PEGDA solution was prepared with 0.3% LAP.

[0081] (3) Immersing in decellularized porcine cornea: Immerse the decellularized porcine cornea in at least 10 times the volume of PEGDA solution. Place the cornea in a vacuum oven and evacuate for 6 hours. Then, place the cornea in a shaker at 120 rpm and shake at room temperature for 18 hours.

[0082] (4) Polymerization reaction: The decellularized porcine cornea soaked in PEGDA solution was placed on a curvature-curved curvature plate and polymerized under 365 nm ultraviolet light for 2 minutes.

[0083] (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.

[0084] (6) Biomaterial refilling: The PEGDA skeleton from the decellularized porcine cornea was immersed in a 10% type I collagen solution at room temperature and 120 rpm for 24 hours. The collagen was then solidified at 4°C and cross-linked with a 0.5% EDC / NHS solution.

[0085] Example 4 Preparation of bionic skin material

[0086] (1) Preparation of decellularized pig skin: Fresh pig skin was shaken with 0.25% trypsin for 6 hours, then immersed in 5% Triton X-100 solution and shaken for 6 hours, and finally washed with balanced salt solution 6 times.

[0087] (2) Preparation of monomer and polymer solution: 10% methacrylated hyaluronic acid solution was prepared with 0.3% LAP solution.

[0088] (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 the solution in a vacuum oven for 6 hours, then shake it in a shaker at 120 rpm for 18 hours.

[0089] (4) Polymerization reaction: Decellularized porcine skin soaked with methacrylated hyaluronic acid was spread out and polymerized under 365 nm ultraviolet light for 2 minutes.

[0090] (5) Digestion of decellularized pig skin: The polymerized decellularized pig skin was placed in a 30 mg / ml pepsin digestion solution and treated at 37°C and 120 rpm for 48 hours, followed by rinsing with a large amount of physiological saline.

[0091] (6) Biomaterial Refilling: A methacrylated hyaluronic acid scaffold, free of decellularized porcine skin, was immersed in a 10% type I collagen solution (Beijing Paisheng Biological) at 120 rpm for 24 hours at room temperature. The collagen-soaked polymer scaffold was then cross-linked with EDC / NHS to obtain a biomimetic skin material.

[0092] Comparative Example 1 Ultrastructural comparison between bionic cornea and human cornea

[0093] The biomimetic cornea and human cornea were fixed with glutaraldehyde, ultramicrotomed, and observed under transmission electron microscopy. The results showed that the ultrastructural similarity between the biomimetic corneal stroma and the human cornea was extremely high. Low-magnification observation revealed a lamellar structure similar to that of the natural cornea, while high-magnification observation revealed parallel arrangement of collagen fibers (Figure 1).

[0094] Comparative Example 2: Ultrastructure comparison between biomimetic corneal stroma and common artificial corneal repair materials

[0095] The biomimetic corneal stroma, along with methacrylated gelatin-based corneas and collagen-based corneas, was fixed with glutaraldehyde, ultramicrotomed, and observed under transmission electron microscopy. The results showed that, while the biomimetic corneal stroma had a precise ultrastructure similar to that of the natural cornea, the collagen-based corneas and methacrylated gelatin-based corneas had a homogeneous ultrastructure and lacked the histological structural characteristics of the corneal stroma (Figure 2).

[0096] Comparative Example 3 Comparison of physical properties of biomimetic corneal stroma with natural cornea and collagen-based cornea

[0097] The biomimetic corneal stroma, collagen-based cornea, and natural cornea were placed in a spectrophotometer cuvette and tested for transmittance between 300nm and 800nm. The results showed that the biomimetic corneal stroma had good transmittance and was more transparent than the natural cornea (Figure 3).

[0098] The biomimetic corneal stroma, collagen-based cornea, and natural cornea were placed in a 10U / ml collagenase solution. The weight changes of the samples were measured after 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 degraded the fastest, with 5.7% of the weight remaining on day 7. The natural cornea also retained 12.6% of its weight on day 7. The biomimetic corneal stroma degraded the slowest under the action of collagenase, with 38.4% of the weight remaining on day 7. This is related to the presence of a chondroitin sulfate biomimetic scaffold within the biomimetic corneal stroma (Figure 4).

[0099] The biomimetic corneal stroma, collagen-based cornea, and natural cornea were placed in PBS buffer 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 biomimetic corneal stroma remained stable in the 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)

[0100] Example 5 In vitro experiment - repairing corneal defects

[0101] (1) To achieve seamless repair, the biomimetic cornea prepared in Example 1 was immersed in at least 10 times the volume of a 20% methacrylated gelatin solution. The solution was placed in a 37°C constant temperature shaker at 120 rpm for 24 hours. The methacrylated gelatin on the surface of the biomimetic corneal stroma was then removed.

[0102] (2) A circular corneal stromal defect with a diameter of 6 mm and a depth of 500 μm was created on the surface of the pig cornea using a trephine drill and a lamellar knife. A biomimetic corneal stroma of the same size was placed over the corneal defect, heated to 37°C, and then irradiated with 405 nm visible light for 60 seconds.

[0103] (3) Comparing the repaired pig eyeball with the preoperative one, slit lamp observation revealed that the stromal defect was successfully closed. Anterior segment optical coherence tomography and thickness scanning confirmed that the stromal defect was filled and the corneal curvature was restored (Figure 6).

[0104] It is proven that bionic corneal materials can repair corneal defects and restore corneal thickness and curvature.

[0105] Example 6 In vitro experiment - repairing corneal stroma defects

[0106] A circular corneal stromal defect with a diameter of 3 mm and a depth of 200 μm was created on the rabbit cornea using a trephine drill and a lamellar knife. A biomimetic corneal stromal defect of equal size and thickness was placed over the defect and then irradiated with 405 nm visible light for 60 seconds. A group without biomimetic corneal stromal repair served as a control.

[0107] Four weeks after surgery, the biomimetic corneal stromal transplant group had clear corneas and complete epithelial regeneration. Optical coherence tomography showed that the biomimetic corneal stromal graft was stable, without displacement or detachment, and the stromal thickness had recovered, demonstrating good biocompatibility, no inflammatory response, and prevention of corneal stromal fibrosis, achieving excellent visual outcomes. The non-transplant group developed corneal scarring, loss of central corneal transparency, and a central corneal epithelial defect. Optical coherence tomography revealed corneal stromal fibrosis, a rough corneal surface, and a loss of central corneal thickness (Figure 7).

[0108] 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 intended to help understand the method of the present invention and its core concept. At the same time, changes or modifications made by those skilled in the art based on the concept of the present invention, the specific implementation methods of the present invention, and the scope of application are all within the scope of protection of the present invention. In summary, the contents of this specification should not be understood as limiting the present invention.

Claims

1. A method for preparing a biomimetic biomaterial, characterized in that: The method comprises the following steps: immersing the simulated biological tissue into 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 the 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.

2. The preparation method according to claim 1, 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.

3. The preparation method according to claim 2, characterized in that: The size of the bionic skin is 0.1-5cm 2 .

4. The preparation method according to claim 1, characterized in that: The monomer polymer solution also contains a photoinitiator or a temperature initiator.

5. The preparation method according to claim 4, 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.

6. 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.

7. The preparation method according to claim 1, 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.

8. 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% of a photoinitiator, and the concentration is 5-20% (w / v).

9. 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).

10. 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.

11. 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.

12. 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 it, and then place it on a shaker at 120 rpm at room temperature for 12-24 hours.

13. 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, and the activation time is 2-3 minutes; the temperature activation condition is 60-70°C, and the reaction time is 12-24 hours.

14. The preparation method according to claim 13, characterized in that: The polymerization reaction method is to polymerize the biomimetic tissue under 365nm ultraviolet light for 2 minutes.

15. 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 uses a hydrochloric acid solution with a concentration of 10%-25%; the biological enzyme digestion uses 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.

16. 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; soak the pre-fixed biomimetic material in the EDC / NHS solution 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 and shake at room temperature for 15 minutes, 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.

17. The biomimetic biomaterial prepared according to the preparation method according to any one of claims 1 to 16.

18. 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 such as tissue substitutes, medical dressings or drug carriers.

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