Multifunctional posterior sclera reinforcement biomaterial with controllable mechanical and degradation properties and preparation method thereof

Abstract

The application discloses a multifunctional biomaterial for posterior sclera reinforcement, which is prepared by using layer-by-layer self-assembly (LbL) surface functionalization technology to coat silk fibroin (SF) on the surface of small intestinal submucosa (SIS), and preparing a light cross-linked SF (SFMA) microgroove coating with anisotropic characteristics on one side of the SIS for guiding cell arrangement and growth, promoting tissue regeneration and repair; and preparing a light cross-linked SF and sodium hyaluronate (SF-SH) mixed coating on the other side for improving biocompatibility and adhesion, so as to realize preparation of a multifunctional posterior sclera reinforcement biomaterial with controllable mechanical and degradation properties and capable of promoting surrounding tissue regeneration. The posterior sclera reinforcement biomaterial is cross-linked in the LbL mode to avoid toxic substance residues generated by cross-linking of SIS and traditional glutaraldehyde. The light cross-linked SFMA microgroove coating on one side can not only realize directional growth of fibroblasts, but also realize directional arrangement of collagen type I secreted by the cells, and effectively realize tissue integration. Meanwhile, the light cross-linked SF-SH mixed coating on the other side has excellent adhesion, which is helpful for positioning and attaching of the reinforcement material, and is of great significance to improving the effect of a posterior sclera reinforcement (PSR) operation and promoting long-term stable and effective treatment of pathological high myopia by using the biomaterial.

Description

Technical Field

[0001] This invention relates to the field of biomaterials technology, specifically to a multifunctional posterior scleral reinforcement biomaterial with controllable mechanical and degradation properties and its preparation method. Background Technology

[0002] Pathological high myopia often causes a series of degenerative changes in the fundus and a decline in visual function. Because the sclera of patients with pathological high myopia is thinner and weaker, the production of proteoglycans and collagen (especially type I collagen) decreases, and collagen fibers become thinner and more disorganized. These changes, under the influence of intraocular pressure, easily lead to posterior staphyloma, which in turn causes complications such as macular degeneration, macular holes, retinal schisis, or retinal detachment, ultimately resulting in irreversible vision loss and even blindness. Posterior scleral reinforcement (PSR) involves fixing a reinforcing material to the posterior sclera. By stimulating a series of beneficial inflammatory responses in the local tissues, the reinforcing material and the posterior sclera fuse together, forming a new sclera with increased thickness and enhanced biomechanics. This effectively shortens the axial length of the eye and reduces the incidence of intraocular complications, making it the most effective surgical method for treating pathological high myopia in clinical practice.

[0003] The choice of posterior scleral reinforcement material has a significant impact on the outcome of PSR surgery. Reinforcement materials include both biomaterials and synthetic materials. Biomaterials such as allogeneic sclera and bovine pericardium have good biocompatibility, but their availability is limited. Synthetic materials, such as artificial pericardial patches and polyester fiber meshes, are widely available and easily manufactured in various sizes, but often suffer from low biocompatibility and safety, difficulty in degradation, and challenges in biomechanical compatibility. Therefore, there is an urgent need to develop a multifunctional reinforcement material with good biocompatibility that simultaneously meets the requirements of mechanical properties, degradation performance, and tissue recovery.

[0004] Naturally derived extracellular matrix scaffolds, such as the submucosal layer of the small intestine (SIS), possess significant bioactivity, effectively promoting tissue regeneration and repair, and have been widely used in experiments on various tissues including the bladder, abdominal wall, skin, and dura mater. However, rapid in vivo degradation and structural and mechanical instability limit the further application of SIS. Silk fibroin (SF) from silkworm silk, as a natural cross-linking agent, can controllably regulate the mechanical properties and degradation rate of materials by altering the number of SF layers through layer-by-layer self-assembly (LbL). Sodium hyaluronate (SH), first discovered in the vitreous humor of the eye, exhibits high viscoelasticity, hydrophilicity, and biocompatibility, and is widely used in ophthalmic surgeries such as those for glaucoma and cataracts. Simultaneously leveraging the advantages of SF and SH, an SF coating with directional grooves is constructed on the outer side of the SIS membrane, while a high-viscosity SF-SH coating is constructed on the inner layer close to the sclera. This develops a novel multifunctional posterior scleral reinforcement biomaterial that simultaneously possesses high biocompatibility, controllable mechanical and degradation properties, and promotes the regeneration of surrounding tissues. This will be of great significance for the long-term, stable, and effective treatment of pathological high myopia. Summary of the Invention

[0005] To design biomaterials for posterior scleral reinforcement with controllable mechanical and degradation properties for effective reinforcement of the posterior sclera, we prepared an anisotropic photocrosslinked SF (SFMA) microgroove coating on one side of a layer-by-layer self-assembled (LbL) SIS to guide cell alignment and growth, promoting tissue regeneration and repair. On the other side, we prepared a hybrid coating of photocrosslinked SF and sodium hyaluronate (SF-SH) to improve biocompatibility and adhesion. This resulted in the preparation of a multifunctional posterior scleral reinforcement material (SFMA / LbL / SIS / SH) with controllable mechanical and degradation properties, advancing the development and application of biomaterials in ophthalmic surgery.

[0006] The technical solution of the present invention is as follows:

[0007] A multifunctional biomaterial for posterior scleral reinforcement is proposed. To avoid the problem of insufficient structural and mechanical retention caused by the rapid degradation of SIS during current clinical use, the multifunctional coated SIS biomaterial uses SF LbL technology to modify SIS, thus preparing a SIS membrane material with controllable degradation and mechanical retention.

[0008] The preparation method of the SFMA / LbL / SIS / SH multifunctional posterior scleral reinforcement biomaterial in this invention includes the following steps:

[0009] (1) Preparation of SIS membrane: SIS was immersed in a mixed solution of methanol and chloroform (1:1, V / V) for 12 h, then digested in 0.05% trypsin at 37℃ for 12 h, then treated with 0.5% sodium dodecyl sulfate (SDS), and then immersed in 0.1% peracetic acid, and freeze-dried to obtain SIS membrane.

[0010] (2) Preparation of SF solution: 5 g of silkworm cocoons were immersed in boiling 0.02 M Na2CO3 solution for 1 h, then removed, washed 5 times in ultrapure water, and dried in an oven at 45 °C for 24 h. Subsequently, the dried silk fibers were placed in 9.3 M lithium bromide solution at 60 °C for 3 h until completely dissolved. Next, the SF solution was dialyzed in a dialysis bag with a molecular weight cutoff of 3500 for 3 days. After 3 days, the SF solution was centrifuged at 12000 rpm for 15 min to obtain the SF solution.

[0011] (3) Preparation of SF LbL surface-functionalized SIS (denoted as SF / LbL / SIS): Place the SIS membrane in a petri dish and add an equal volume of 1 mg / mL SF solution and 1 mg / mL methacrylamide SF solution. Shake on a decolorizing shaker for 15 min. Then immerse the SIS membrane in 90% methanol solution and finally dry it with nitrogen to obtain photocurable group-modified SF / LbL / SIS.

[0012] (4) Preparation of photocurable SF microgroove coating SIS (denoted as SFMA / LbL / SIS): Weigh 30 g of polydimethylsiloxane (PDMS) and 3 g of curing agent, mix them evenly, pour the mixture onto a silicon plate with a 20 μm wide microgroove, vacuum dry for 1 h, and then transfer it to an 85℃ oven to dry for 4 h. Take 120 μL of SFMA solution and drop it into a PET frame, then place the pre-wetted LbL SIS on the solution, and irradiate with ultraviolet light for 35 s to cure the SFMA solution and form SFMA / LbL / SIS with a stable microgroove structure on the outside.

[0013] (5) Preparation of photocurable SH-SF adhesive coating SIS (denoted as SFMA / LbL / SIS / SH): A mixed solution of 15 mg / mL photocurable SH and 5 mg / mL SFMA was prepared using 0.25% photoinitiator (LAP). 50 μL of the mixed solution was evenly coated onto the inner surface of SFMA / LbL / SIS, irradiated with ultraviolet light for 8 s to cure, and then placed in a 37℃ constant temperature oven for slow drying to form a multifunctional SFMA / LbL / SIS / SH material.

[0014] The beneficial effects of this invention are as follows: The synthesized SFMA LbL SIS SH exhibits high stability, antibacterial properties, low toxicity, and good biocompatibility. This multifunctional SFMA LbL SIS SH posterior scleral reinforcement biomaterial possesses high biocompatibility, controllable mechanical properties and degradation rate, good tissue integration ability, and excellent adhesion, making it suitable for clinical applications. Furthermore, the multifunctional SFMA LbL SIS SH posterior scleral reinforcement biomaterial integrates well with the surrounding normal tissue, with type I collagen on one side of the SFMA microgroove exhibiting directional alignment. These results indicate that SFMA LbL SIS SH is a posterior scleral reinforcement biomaterial that simultaneously possesses high biocompatibility, controllable mechanical properties, and degradation characteristics to match the regeneration rate, showing great potential for improving postoperative PSR outcomes and promoting the application of biomaterials in ophthalmic surgery. Attached Figure Description

[0015] Figure 1. Surface morphology of SIS and SIS modified with different numbers of SF layers (a) Scanning electron microscope (b) Confocal microscope image

[0016] Figure 2. Morphological characterization of SIS, SF / LbL / SIS, and SFMA / LbL / SIS: (a) Scanning electron microscope image; (b) 3D optical imaging.

[0017] Figure 3. Mechanical retention analysis of SIS and SF / LbL / SIS during enzymatic degradation (ac). Stress-strain curves of different membranes at 1, 3, and 7 days of enzymatic degradation (df). Statistical analysis of stress, strain, and Young's modulus of different membranes at 1, 3, and 7 days of enzymatic degradation.

[0018] Figure 4. In vitro cell compatibility evaluation of biomaterials (a) Cytoskeleton staining (b) CCK-8 analysis

[0019] Figure 5. Cell behavior analysis on the surface of biomaterials (a) Surface morphology of the cytoskeleton (b) CCK-8 analysis (c) Immunofluorescence staining of type I collagen Detailed Implementation

[0020] The technical solution of the present invention will be further described below with reference to specific embodiments.

[0021] Example 1: Preparation of SIS membrane

[0022] The SIS membrane was immersed in a 1:1, V / V mixture of methanol and chloroform for 12 h, then digested in 0.05% trypsin at 37 °C for 12 h, treated with 0.5% sodium dodecyl sulfate (SDS), and then immersed in 0.1% peracetic acid. After freeze-drying and sterilization, the SIS membrane was obtained.

[0023] Example 2: Preparation of SF6 solution

[0024] Five g of silkworm cocoons were immersed in boiling 0.02M Na₂CO₃ solution for 1 hour, then removed, washed five times in ultrapure water, and dried in a 45℃ oven for 24 hours. The dried silk fibers were then placed in a 9.3M lithium bromide solution at 60℃ for 3 hours until completely dissolved. Next, the SF₂ solution was dialyzed for 3 days in a dialysis bag with a molecular weight cutoff of 3500. After 3 days, the SF₂ solution was centrifuged at 12000 rpm for 15 minutes to obtain the SF₂ solution.

[0025] Example 3: Preparation of SF LbL surface-functionalized SIS film (denoted as SF / LbL / SIS)

[0026] The SIS membrane was placed in a petri dish and an equal volume mixture of 1 mg / mL SF solution and 1 mg / mL methacrylamide SF solution was added. The mixture was shaken on a decolorizing shaker for 15 min. Then the SIS membrane was immersed in 90% methanol solution and finally dried with nitrogen gas to obtain photocurable group-modified SF / LbL / SIS.

[0027] Example 4: Preparation of photocurable SF microgroove coated SIS film (denoted as SFMA / LbL / SIS)

[0028] 30 g of polydimethylsiloxane (PDMS) and 3 g of curing agent were weighed and mixed evenly, then poured onto a silicon substrate with 20 μm wide microgrooves. The mixture was vacuum dried for 1 hour, followed by drying in an 85°C oven for 4 hours. 120 μL of SFMA solution was added dropwise into a PET frame, and then a pre-wetted LbL SIS was placed on the solution. The SFMA solution was cured by UV irradiation for 35 seconds, forming an SFMA / LbL / SIS film with a stable microgrooved structure. Example 5: Preparation of a photocurable SH-SF adhesive coating double-sided SIS film (denoted as SFMA / LbL / SIS / SH).

[0029] A mixed solution of 15 mg / mL photocurable SH and 5 mg / mL SFMA was prepared using 0.25% photoinitiator (LAP). 50 μL of the mixed solution was uniformly coated onto the surface of SFMA / LbL / SIS, irradiated with UV light for 8 seconds to cure, and then slowly dried in a 37°C oven to form a bilayer functionalized SFMA / LbL / SIS / SH biomaterial.

[0030] Example 6: Mechanical retention of SF / LbL / SIS membrane

[0031] The tensile mechanical properties of SIS and SF / LbL / SIS membranes under enzymatic degradation conditions were tested using a dynamic mechanical analyzer (DMA Q800). Samples were degraded in a collagenase (C0130, Sigma) solution. Prior to testing, samples were rinsed and air-dried, followed by tensile testing. All samples were cut into rectangles 1 cm long and 2 mm wide. All tensile tests were conducted at room temperature at a speed of 1.5 N / min. Stress-strain data were obtained directly from the testing machine based on the initial sample dimensions. Tensile strength is defined as the maximum stress at failure, and the fracture strain at failure is defined as the maximum strain value. The elastic modulus of the membrane was calculated between 1% and 3% of the strain.

[0032] Example 7: In vitro cell compatibility of SFMA / LbL / SIS membrane

[0033] Mouse embryonic fibroblasts (NIH3T3) were used for in vitro cell compatibility studies of the material. Cell proliferation on SIS, SF / LbL / SIS, and SFMA / LbL / SIS was measured using a cell counting kit (CCK-8). After co-culturing cells with the material for 1, 3, and 7 days, the culture medium was removed, and fresh medium containing 10% CCK-8 solution was added to each well. After reacting at 37°C for 1 h, the absorbance of the supernatant at 450 nm was measured using a spectrophotometer. The arrangement of collagen matrix secreted by fibroblasts cultured on the membrane was detected using Collagen I immunofluorescence staining. Cells were seeded on SIS, SF / LbL / SIS, and SFMA / LbL / SIS and cultured for 3 days. Cell samples were fixed with 4% paraformaldehyde for 30 min, washed three times with PBS, and then treated with 0.2% Triton X-100 at room temperature for 5 min. Subsequently, the slides were blocked with 2% bovine serum albumin for 2 h, then incubated overnight at 4°C with mouse anti-Collagen I (1:200) monoclonal antibody. After washing with PBS, the slides were incubated with fluorescent secondary antibody (goat anti-mouse IgG-Alexa Fluor 594, 1:300) at 37°C for 1 h, followed by PBS washing. Finally, the slides were mounted with anti-fluorescence quenching mounting medium (containing DAPI) and observed and photographed using CLSM.

Claims

1. A multifunctional biomaterial for posterior scleral reinforcement, namely: preparing an anisotropic photocrosslinked SF microgroove coating on one side of a layer-by-layer self-assembled SIS, and preparing a mixed coating of photocrosslinked SF and sodium hyaluronate on the other side, thereby constructing a multifunctional biomaterial with controllable mechanical and degradation properties and high biocompatibility.

2. The method for preparing the multifunctional biomaterial for posterior scleral reinforcement as described in claim 1 is as follows: (1) Preparation of SIS membrane: SIS was immersed in a 1:1, V / V mixed solution of methanol and chloroform for 12 h, then digested in 0.05% trypsin at 37℃ for 12 h, then treated with 0.5% sodium dodecyl sulfate, and then immersed in 0.1% peracetic acid, freeze-dried and sterilized to obtain SIS membrane; (2) Preparation of SF solution: 5 g of silkworm cocoons were put into boiling 0.02M Na2CO3 solution for 1 h and then taken out. They were washed 5 times in ultrapure water and dried in an oven at 45℃ for 24 h. Then the dried silk fibers were placed in 9.3M lithium bromide solution at 60℃ for 3 h until completely dissolved. Next, the SF solution was placed in a dialysis bag with a molecular weight cutoff of 3500 and dialyzed for 3 days. After 3 days, the SF solution was centrifuged at 12000 rpm for 15 min to obtain SF solution. (3) Preparation of SF LbL surface functionalized SIS: Place the SIS membrane in a petri dish and add an equal volume of 1 mg / mL SF solution and 1 mg / mL methacrylamide SF solution. Shake on a decolorizing shaker for 15 min. Then immerse the SIS membrane in 90% methanol solution and finally dry it with nitrogen to obtain photocurable group modified SF LbL surface functionalized SIS. (4) Preparation of photocurable SF microgroove coating SIS: Weigh 30 g of polydimethylsiloxane and 3 g of curing agent and mix them evenly. Pour the mixture onto a silicon plate with a 20 μm wide microgroove and vacuum dry for 1 h. Then transfer it to an 85℃ oven and dry for 4 h. Take 120 μL of SFMA solution and drop it into a PET frame. Then place the pre-wetted LbL SIS on the solution and irradiate it with ultraviolet light for 35 s to cure the SFMA solution and form a photocurable SF microgroove coating SIS with a stable microgroove structure on the outside. (5) Preparation of photocurable SH-SF adhesive coating SIS: A mixed solution of 15 mg / mL photocurable SH and 5 mg / mL SFMA was prepared using 0.25% photoinitiator LAP. 50 μL of the mixed solution was evenly coated onto the inner surface of the photocurable SF microgroove coating SIS. It was cured by UV irradiation for 8 s and then placed in a 37℃ constant temperature oven for slow drying to form a multifunctional photocurable SH-SF adhesive coating SIS material.

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