A composite scaffold for bone repair and its preparation method

Abstract

This invention discloses a composite scaffold for bone repair and its preparation method. The composite scaffold for bone repair consists of a porous polylactic acid (PLA) scaffold in the middle and an outer layer of small intestinal submucosal matrix sponge. The ratio of the diameter of the composite scaffold to the diameter of the PLA porous scaffold is 2-4. The preparation method includes the following steps: S1: preparing small intestinal submucosal matrix; S2: preparing a small intestinal submucosal matrix solution; S3: preparing a PLA porous scaffold; S4: preparing a composite scaffold of small intestinal submucosal matrix sponge and PLA porous scaffold; S5: cross-linking treatment, placing the composite scaffold of small intestinal submucosal matrix sponge and PLA porous scaffold in a cross-linking treatment solution, and finally freeze-drying to obtain the composite scaffold for bone repair. The composite scaffold for bone repair prepared by this invention has good mechanical properties and will be gradually degraded and replaced and absorbed by newly formed bone tissue.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a composite scaffold for bone repair and its preparation method. Background Technology

[0002] Bones are the largest tissues and organs in the human body. They not only provide mechanical support, protect internal organs, and maintain bodily movement, but also play a crucial role in hematopoiesis and regulating the balance of calcium and phosphorus ions in the body's environment. With societal development, increased life expectancy, and a rapidly aging population, bone defects caused by fractures, osteoporosis, bone tumors, osteomyelitis, and congenital factors have become one of the major diseases affecting human health.

[0003] When bone defects are small, the bone can heal itself following the process described above. However, when bone defects are large, the bone cannot heal normally through this process. To achieve a better repair effect for large bone defects, bone repair materials are needed. These materials are implanted into the defect site, acting as support and bridging agents. They can be considered part of the bone matrix in the normal bone healing process. They absorb proteins, activate receptor tissues and the coagulation system, and provide carriers for cells and growth factors, thus promoting the bone repair process.

[0004] Ideal bone repair materials not only need good biocompatibility and bioactivity (bone formation, osteoconduction, and osteoinductive properties) to stimulate rapid bone tissue remodeling, but also require suitable pore structure and certain biodegradability and mechanical properties. Furthermore, early vascularization of bone repair materials plays an indispensable role in new bone formation during the bone repair process. During bone growth, blood vessels not only provide essential nutrients for bone repair, but special cell populations within them also secrete cytokines that promote bone repair. Therefore, vascularization of bone repair materials is currently an important means to improve the effectiveness of bone defect repair. Summary of the Invention

[0005] The purpose of this invention is to provide a composite scaffold for bone repair and its preparation method to solve the problems in the prior art.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] A composite scaffold for bone repair comprises a polylactic acid porous scaffold in the middle and an outer layer of small intestinal submucosal matrix sponge, wherein the ratio of the diameter of the composite scaffold for bone repair to the diameter of the polylactic acid porous scaffold is 2-4.

[0008] Furthermore, the polylactic acid porous scaffold is perforated, with pinholes of 300-600 μm in diameter distributed around its perimeter. The spacing between the pinholes is 2-6 mm. The polylactic acid porous scaffold has a top-to-bottom channel structure inside, and the channel structure is interconnected with the pinholes. The pore size of the channel structure is 5-25 μm.

[0009] Furthermore, the submucosal matrix sponge of the small intestine has a porous structure with a pore size of 100-300 μm. The submucosal matrix sponge of the small intestine is immersed in the polylactic acid porous scaffold through the pinholes around the polylactic acid porous scaffold, thereby making the submucosal matrix sponge of the small intestine tightly bonded to the polylactic acid porous scaffold.

[0010] A method for preparing a composite scaffold for bone repair, comprising the following steps:

[0011] S1: Preparation of small intestinal submucosal matrix; the small intestinal mucosa, serosa, and muscularis tissue of the animal are removed to obtain the small intestinal submucosal layer; the small intestinal submucosal layer is defatted and decellularized, and then placed in sterile PBS and shaken on a shaker to obtain the small intestinal submucosal matrix;

[0012] S2: Prepare a small intestinal submucosal matrix solution; cut the small intestinal submucosal matrix into small pieces and place it in an acetic acid aqueous solution containing pepsin and stir for 24h-96h to obtain a fully dissolved small intestinal submucosal matrix solution;

[0013] S3: Preparation of polylactic acid porous scaffold; 4-8wt% polylactic acid solution is poured into a first hollow mold, a metal block is connected to the bottom of the first hollow mold, and the metal block is brought into contact with liquid nitrogen to solidify the polylactic acid solution. Then, it is immersed in cold water at 0-10℃ for 24-96 hours, and finally freeze-dried to obtain the polylactic acid scaffold. At the same time, holes are punched around the polylactic acid scaffold with a needle with a diameter of 300-600μm; thus, the polylactic acid porous scaffold is obtained.

[0014] S4: Prepare a composite scaffold of small intestinal submucosal matrix sponge and polylactic acid porous scaffold. Place the polylactic acid porous scaffold in the center of the second hollow mold, and then pour the small intestinal submucosal matrix solution around the polylactic acid porous scaffold. The small intestinal submucosal matrix solution will solidify to form a small intestinal submucosal matrix sponge. Then freeze-dry the second hollow mold to obtain the composite scaffold of small intestinal submucosal matrix sponge and polylactic acid porous scaffold.

[0015] S5: Crosslinking treatment: The small intestinal submucosal matrix sponge and polylactic acid porous scaffold composite scaffold are placed in a crosslinking treatment solution at a temperature of 15-25°C and shaken on a shaker for 1-5 days. Finally, they are freeze-dried to obtain a composite scaffold for bone repair.

[0016] Further, the defatting and decellularization steps in step S1 are as follows: the submucosa of the small intestine is soaked in a defatting solution for 6-12 hours, the submucosa of the small intestine is removed and placed in a decellularization solution, shaken on a shaker at room temperature for 20-24 hours, the submucosa of the small intestine is then removed and placed in sterile PBS and shaken on a shaker for 2-3 hours.

[0017] Further, the defatting solution is one or more of acetone, anhydrous ethanol, ethylene glycol ethyl ether, or isopropanol, and the decellularization solution is one or more of sodium hydroxide, Triton X-100 solution, SDS solution, or trypsin.

[0018] Further, in step S2, the pepsin mass fraction in the submucosal matrix solution of the small intestine is 0.1wt%-0.5wt%, the acetic acid mass fraction is 1wt%-5wt%, and the submucosal matrix mass fraction of the small intestine is 1wt%-3wt%.

[0019] Furthermore, the drilling distance in step S3 is 2-6mm.

[0020] Furthermore, the inner diameter of the first hollow mold is 3-6mm, and the metal block is a copper block.

[0021] Furthermore, the crosslinking agent in the crosslinking treatment solution in step S5 is one of genipin, glutaraldehyde, or EDC, and the concentration of the crosslinking agent is 0.2%-2%, and the pH is 5.0-7.0.

[0022] Compared to existing technologies, the advantages of this invention are as follows: In this invention, a needle with a diameter of 300-600 μm is used to perforate the periphery of the polylactic acid (PLA) scaffold, allowing the small intestinal submucosal matrix solution to fully penetrate the PLA scaffold. This enables the porous PLA scaffold to tightly bond with the small intestinal submucosal matrix sponge after freeze-drying, forming a composite scaffold with a porous PLA scaffold in the middle and a small intestinal submucosal sponge on the outer layer. The small intestinal submucosal matrix sponge on the outer layer of this composite scaffold is mainly composed of collagen, exhibiting good biocompatibility. Furthermore, the small intestinal submucosal matrix also contains growth factors such as transforming growth factor-β (TGF-β), fibroblast growth factor (bFGF), and vascular endothelial growth factor (VEGF), which are beneficial for osteoblast differentiation and angiogenesis. Simultaneously, the small intestinal submucosal matrix sponge has a relatively loose porous structure, allowing for blood vessel ingrowth. The polylactic acid (PLA) porous scaffold in the middle section has a top-to-bottom pore structure, which facilitates osteoblasts to crawl into the scaffold and to transport nutrients and exchange substances with surrounding tissues. Furthermore, the PLA porous scaffold possesses good mechanical properties, providing excellent support and connection for bone defects. Although the small pore size of the PLA porous scaffold hinders cell and blood vessel ingrowth, cells and blood vessels from the submucosal matrix sponge of the small intestine in the outer layer of the composite scaffold can ingrow into it along the 300–600 μm pores around the PLA porous scaffold. This enables early vascularization of bone defect repair, providing sufficient nutrients and material exchange for the formation of new bone tissue.

[0023] The diameter of the bone repair composite scaffold is (2-4):1 with that of the polylactic acid porous scaffold. In the early stages of bone defect repair, as the submucosal sponge of the small intestine gradually degrades, the composite scaffold fuses with the tissue at the bone defect site. At the same time, compared with the submucosal sponge of the small intestine, the polylactic acid porous scaffold degrades more slowly, which means it can provide better mechanical properties in the early stages of bone defect repair and will not cause secondary damage to the bone defect. Furthermore, as new bone tissue forms, the polylactic acid porous scaffold will also be gradually degraded. During the degradation process, the pore size of the internal channels of the polylactic acid porous scaffold increases, which is conducive to osteoblasts crawling into the scaffold and eventually being replaced and absorbed by the new bone tissue, thereby achieving the purpose of repairing bone defects.

[0024] The composite scaffold for bone repair prepared by this invention has good mechanical properties and will be gradually degraded and replaced and absorbed by new bone tissue.

[0025] The above description is merely an overview of the technical solution of the present invention. In order to better understand the technical means of the present invention and to implement it in accordance with the contents of the specification, the preferred embodiments of the present invention are described in detail below. Attached Figure Description

[0026] Figure 1 This is a schematic diagram of the composite scaffold for bone repair according to the present invention.

[0027] Figure 2 The images show the cross-sectional microstructures of the composite scaffolds used for bone repair in Examples 1-3 of the present invention; wherein a1 is a cross-sectional microstructure of the small intestinal submucosal matrix sponge of Example 1, a2 is a cross-sectional microstructure of the polylactic acid porous scaffold of Example 1, b1 is a cross-sectional microstructure of the small intestinal submucosal matrix sponge of Example 2, b2 is a cross-sectional microstructure of the polylactic acid porous scaffold of Example 2, c1 is a cross-sectional microstructure of the small intestinal submucosal matrix sponge of Example 3, and c2 is a cross-sectional microstructure of the polylactic acid porous scaffold of Example 3.

[0028] Figure 3 The images show the longitudinal cross-sectional microstructures of the composite scaffolds used for bone repair in Examples 1-3 of the present invention; wherein d1 is the longitudinal cross-sectional microstructure of the small intestinal submucosal matrix sponge of Example 1, d2 is the longitudinal cross-sectional microstructure of the polylactic acid porous scaffold of Example 1, e1 is the longitudinal cross-sectional microstructure of the small intestinal submucosal matrix sponge of Example 2, e2 is the longitudinal cross-sectional microstructure of the polylactic acid porous scaffold of Example 2, f1 is the longitudinal cross-sectional microstructure of the small intestinal submucosal matrix sponge of Example 3, and f2 is the longitudinal cross-sectional microstructure of the polylactic acid porous scaffold of Example 3.

[0029] Figure 4 These are mechanical performance test diagrams for the composite scaffolds used in bone repair in Examples 1-3. Detailed Implementation

[0030] The technical solution of the present invention will be further described below with reference to specific embodiments. However, the present invention should not be limited to these embodiments. Unless specifically stated otherwise, all features can be replaced by other equivalent or similar alternative features. Unless specifically stated otherwise, each feature is only one example of a series of equivalent or similar features. The terminology used in the present invention, unless otherwise stated, generally has the meaning commonly understood by those skilled in the art. In the following embodiments, unless otherwise stated, concentration % refers to mass percentage; all substances used are commercially available. The technical solution of the present invention will be further described below with reference to specific embodiments. However, the present invention should not be limited to these embodiments. Unless specifically stated otherwise, all features can be replaced by other equivalent or similar alternative features. Unless specifically stated otherwise, each feature is only one example of a series of equivalent or similar features. The terminology used in the present invention, unless otherwise stated otherwise, generally has the meaning commonly understood by those skilled in the art.

[0031] A composite scaffold for bone repair comprises a polylactic acid porous scaffold in the middle and an outer layer of small intestinal submucosal matrix sponge. The ratio of the diameter of the composite scaffold for bone repair to the diameter of the polylactic acid porous scaffold is 2-4, and the ratio can be 2, 3, 4, or any value within 2-4.

[0032] The polylactic acid (PLA) porous scaffold is perforated, with pinholes of 300-600 μm in diameter distributed around its perimeter. The pinhole size can be 300, 400, 500, or 600 μm, or any value within this range. The spacing between the pinholes is 2-6 mm, which can be 2 mm, 3 mm, or 6 mm, or any value within this range. The PLA porous scaffold has a top-to-bottom channel structure, which is interconnected with the pinholes. The pore size of the channel structure is 5-25 μm, which can be 5 μm, 10 μm, or 25 μm, or any value within this range.

[0033] The submucosal matrix sponge of the small intestine has a porous structure with a pore size of 100-300 μm, which can be 100 μm, 150 μm, 300 μm, or any value within the range of 100-300 μm. The submucosal matrix sponge of the small intestine is immersed in the polylactic acid porous scaffold through the pinholes around the scaffold, thereby making the submucosal matrix sponge of the small intestine tightly bonded to the polylactic acid porous scaffold.

[0034] A method for preparing a composite scaffold for bone repair, comprising the following steps:

[0035] S1: Preparation of small intestinal submucosal matrix; The small intestinal mucosa, serosa, and muscularis propria of the animal are removed to obtain the small intestinal submucosa; the small intestinal submucosa is immersed in a defatting solution for 6-12 hours, then removed and placed in a decellularized solution, shaken on a shaker at room temperature for 20-24 hours, then removed again and placed in sterile PBS, shaken on a shaker for 2-3 hours, to obtain the small intestinal submucosal matrix; the animal small intestine can be selected from any of pigs, cattle, and sheep. The defatting solution is one or more of acetone, anhydrous ethanol, ethylene glycol ethyl ether, or isopropanol, and the decellularized solution is one or more of sodium hydroxide, Triton X-100 solution, SDS solution, or trypsin.

[0036] S2: Preparation of a small intestinal submucosal matrix solution; the small intestinal submucosal matrix is ​​shredded and then placed in an aqueous acetic acid solution containing pepsin and stirred for 24-96 hours to obtain a fully dissolved small intestinal submucosal matrix solution; the stirring time can be 24 hours, 48 ​​hours, 96 hours, or any value within 24-96 hours. The pepsin mass fraction is 0.1 wt%-0.5 wt%, the acetic acid mass fraction is 1 wt%-5 wt%, and the small intestinal submucosal matrix mass fraction is 1 wt%-3 wt%.

[0037] S3: Preparation of a porous polylactic acid (PLA) scaffold; a 4-8 wt% PLA solution is poured into a first hollow mold, a metal block is connected to the bottom of the first hollow mold, and the metal block is brought into contact with liquid nitrogen to solidify the PLA solution. Then, the solution is immersed in cold water at 0-10°C for 24-96 hours, and finally freeze-dried to obtain the PLA scaffold. At the same time, holes are punched around the PLA scaffold using a needle with a diameter of 300-600 μm; thus, the porous PLA scaffold is obtained. The mass fraction of PLA can be any value among 4%, 6%, 8%, and 4%-8%.

[0038] S4: Prepare a composite scaffold of small intestinal submucosal matrix sponge and polylactic acid porous scaffold. Place the polylactic acid porous scaffold in the center of the second hollow mold, and then pour the small intestinal submucosal matrix solution around the polylactic acid porous scaffold. The small intestinal submucosal matrix solution will solidify to form a small intestinal submucosal matrix sponge. Then freeze-dry the second hollow mold to obtain the composite scaffold of small intestinal submucosal matrix sponge and polylactic acid porous scaffold.

[0039] S5: Crosslinking treatment. The composite scaffold of small intestinal submucosal matrix sponge and polylactic acid porous scaffold is placed in a crosslinking treatment solution at a temperature of 15-25℃ and shaken on a shaker for 1-5 days. Finally, it is freeze-dried to obtain a composite scaffold for bone repair. The temperature can be any temperature among 15℃, 20℃, 25℃, and 15-25℃, and the shaking days can be 1 day, 3 days, 5 days, or any number of days within 1-5 days. The crosslinking agent in the crosslinking treatment solution is one of genipin, glutaraldehyde, or EDC, and the concentration of the crosslinking agent is 0.2%-2%, and the pH is 5.0-7.0.

[0040] The following detailed description uses specific embodiments, in which there are only minor variations between the embodiments for the purpose of comparison, but it does not mean that the material parameters and reaction parameters in the embodiments of the present invention are limited to the parameters in the following embodiments.

[0041] Example 1

[0042] A method for preparing a composite scaffold for bone repair includes the following steps:

[0043] S1: Cut the pig's small intestine into 20cm segments and cut along the midline of the small intestine with scissors. After cleaning, remove the small intestinal mucosa, serosa, and muscularis tissue by mechanical scraping to obtain the small intestinal submucosa. Soak the small intestinal submucosa in a defatted acetone solution for 9 hours. Then, remove the small intestinal submucosa and place it in a decellularized sodium hydroxide solution. Shake it on a shaker at room temperature for 22 hours. Then, remove the small intestinal submucosa and place it in sterile PBS. Shake it on a shaker for 2.5 hours to obtain the small intestinal submucosa matrix.

[0044] S2: Preparation of small intestinal submucosal matrix solution; The small intestinal submucosal matrix was cut into small pieces and then placed in an acetic acid aqueous solution containing pepsin and stirred for 48 hours to obtain a fully dissolved small intestinal submucosal matrix solution; The pepsin mass fraction was 0.3 wt%, the acetic acid mass fraction was 3 wt%, and the small intestinal submucosal matrix mass fraction was 1 wt%.

[0045] S3: Preparation of polylactic acid porous scaffold; 4wt% polylactic acid solution (solvent is 1,4-dioxane) is poured into a first hollow mold with an inner diameter of 4mm. A copper block is connected to the bottom of the first hollow mold, and the copper block is brought into contact with liquid nitrogen to solidify the polylactic acid solution. Then, it is immersed in cold water at 5℃ for 48h, and finally freeze-dried to obtain the polylactic acid scaffold. At the same time, holes are punched around the polylactic acid scaffold with a diameter of 450μm, and the distance between the holes is 4mm; thus, the polylactic acid porous scaffold is obtained.

[0046] S4: Prepare a composite scaffold of small intestinal submucosal matrix sponge and polylactic acid porous scaffold. Place the polylactic acid porous scaffold in the center of a second hollow mold with an inner diameter of 10 mm, pour the small intestinal submucosal matrix solution around the polylactic acid porous scaffold, let it stand for 30 min, and then freeze-dry the second hollow mold to obtain the composite scaffold of small intestinal submucosal matrix sponge and polylactic acid porous scaffold.

[0047] S5: Crosslinking treatment. The composite scaffold of small intestinal submucosal matrix sponge and polylactic acid porous scaffold is placed in a crosslinking treatment solution at 20°C and shaken for 3 days. Finally, it is freeze-dried to obtain a composite scaffold for bone repair. The crosslinking agent in the crosslinking treatment solution is genipin; the concentration of the crosslinking agent is 1%, the pH is 6, and the solvent is 70% alcohol. The ratio of the small intestinal submucosal matrix sponge and polylactic acid porous scaffold composite scaffold to the crosslinking treatment solution is 0.1 g / mL.

[0048] A composite scaffold for bone repair is prepared using the above-described method.

[0049] Example 2

[0050] A method for preparing a composite scaffold for bone repair includes the following steps:

[0051] S1: Cut the pig's small intestine into 20cm segments and cut along the midline of the small intestine with scissors. After cleaning, remove the small intestinal mucosa, serosa, and muscularis tissue by mechanical scraping to obtain the small intestinal submucosa. Soak the small intestinal submucosa in a defatted acetone solution for 9 hours. Remove the small intestinal submucosa and place it in a decellularized sodium hydroxide solution. Shake it on a shaker at room temperature for 22 hours. Then remove the small intestinal submucosa and place it in sterile PBS. Shake it on a shaker for 2.5 hours to obtain the small intestinal submucosa matrix.

[0052] S2: Preparation of small intestinal submucosal matrix solution; The small intestinal submucosal matrix was cut into small pieces and then placed in an acetic acid aqueous solution containing pepsin and stirred for 48 hours to obtain a fully dissolved small intestinal submucosal matrix solution; The pepsin mass fraction was 0.3 wt%, the acetic acid mass fraction was 3 wt%, and the small intestinal submucosal matrix mass fraction was 2 wt%.

[0053] S3: Preparation of a porous polylactic acid scaffold; a 6wt% polylactic acid solution (solvent: 1,4-dioxane) was poured into a first hollow mold with an inner diameter of 4mm. A copper block was connected to the bottom of the first hollow mold, and the copper block was brought into contact with liquid nitrogen to solidify the polylactic acid solution. The solution was then immersed in cold water at 5°C for 48 hours, and finally freeze-dried to obtain the polylactic acid scaffold. At the same time, holes were punched around the polylactic acid scaffold with a diameter of 450μm, and the distance between the holes was 4mm; thus, the porous polylactic acid scaffold was obtained.

[0054] S4: To prepare a composite scaffold of small intestinal submucosal matrix sponge and polylactic acid porous scaffold, the polylactic acid porous scaffold is placed in the center of a second hollow mold with an inner diameter of 10 mm. The small intestinal submucosal matrix solution is then poured around the polylactic acid porous scaffold and allowed to stand for 45 min. The second hollow mold is then freeze-dried to obtain the composite scaffold of small intestinal submucosal matrix sponge and polylactic acid porous scaffold.

[0055] S5: Crosslinking treatment. The composite scaffold of small intestinal submucosal matrix sponge and polylactic acid porous scaffold is placed in a crosslinking treatment solution at 20°C and shaken for 3 days. Finally, it is freeze-dried to obtain a composite scaffold for bone repair. The crosslinking agent in the crosslinking treatment solution is genipin; the concentration of the crosslinking agent is 1%, the pH is 6, and the solvent is 70% alcohol. The ratio of the small intestinal submucosal matrix sponge and polylactic acid porous scaffold composite scaffold to the crosslinking treatment solution is 0.1 g / mL.

[0056] A composite scaffold for bone repair is prepared using the above-described method.

[0057] Example 3

[0058] A method for preparing a composite scaffold for bone repair includes the following steps:

[0059] S1: Cut the pig's small intestine into 20cm segments and cut along the midline of the small intestine with scissors. After cleaning, remove the small intestinal mucosa, serosa, and muscularis tissue by mechanical scraping to obtain the small intestinal submucosa. Soak the small intestinal submucosa in a defatted acetone solution for 9 hours. Remove the small intestinal submucosa and place it in a decellularized sodium hydroxide solution. Shake it on a shaker at room temperature for 22 hours. Then remove the small intestinal submucosa and place it in sterile PBS. Shake it on a shaker for 2.5 hours to obtain the small intestinal submucosa matrix.

[0060] S2: Preparation of small intestinal submucosal matrix solution; The small intestinal submucosal matrix was cut into small pieces and then placed in an acetic acid aqueous solution containing pepsin and stirred for 48 hours to obtain a fully dissolved small intestinal submucosal matrix solution; The pepsin mass fraction was 0.3 wt%, the acetic acid mass fraction was 3 wt%, and the small intestinal submucosal matrix mass fraction was 3 wt%.

[0061] S3: Preparation of polylactic acid porous scaffold; 8wt% polylactic acid solution (solvent is 1,4-dioxane) is poured into a first hollow mold with an inner diameter of 4mm. A copper block is connected to the bottom of the first hollow mold, and the copper block is brought into contact with liquid nitrogen to solidify the polylactic acid solution. Then, it is immersed in cold water at 5℃ for 48h, and finally freeze-dried to obtain the polylactic acid scaffold. At the same time, holes are punched around the polylactic acid scaffold with a diameter of 450μm, and the distance between the holes is 4mm; thus, the polylactic acid porous scaffold is obtained.

[0062] S4: To prepare a composite scaffold of small intestinal submucosal matrix sponge and polylactic acid porous scaffold, the polylactic acid porous scaffold is placed in the center of a second hollow mold with an inner diameter of 10 mm. The small intestinal submucosal matrix solution is then poured around the polylactic acid porous scaffold and allowed to stand for 60 min. The second hollow mold is then freeze-dried to obtain the composite scaffold of small intestinal submucosal matrix sponge and polylactic acid porous scaffold.

[0063] S5: Crosslinking treatment. The composite scaffold of small intestinal submucosal matrix sponge and polylactic acid porous scaffold is placed in a crosslinking treatment solution at 20°C and shaken for 3 days. Finally, it is freeze-dried to obtain a composite scaffold for bone repair. The crosslinking agent in the crosslinking treatment solution is genipin; the concentration of the crosslinking agent is 1%, the pH is 6, and the solvent is 70% alcohol. The ratio of the small intestinal submucosal matrix sponge and polylactic acid porous scaffold composite scaffold to the crosslinking treatment solution is 0.1 g / mL.

[0064] A composite scaffold for bone repair is prepared using the above-described method.

[0065] A schematic diagram of the composite scaffold for bone repair of the present invention is shown below. Figure 1 As shown.

[0066] SEM detection

[0067] The composite scaffolds for bone repair prepared in Examples 1-3 were immersed in liquid nitrogen and fractured to obtain cross-sectional and longitudinal sections of the samples. The cross-sectional and longitudinal sections of each sample were then fixed on a horizontal electron microscope stage. The stage was then placed in a gold sputtering machine for gold sputtering treatment. The microstructure of the cross-sectional and longitudinal sections of the composite scaffolds for bone repair in Examples 1, 2, and 3 was photographed using a scanning electron microscope at an accelerating voltage of 20 kV. The test results are as follows: Figure 2 He Ru Figure 3 As shown.

[0068] from Figure 2 The cross-sectional microstructure of the composite scaffold reveals that both the inner and outer layers possess porous structures. Furthermore, compared to the polylactic acid porous scaffold in the inner layer, the small intestinal submucosal matrix sponge in the outer layer exhibits a larger pore structure. The pore size is largest in Example 1, concentrated in the range of 200-300 μm, while that in Example 3 is smallest, concentrated in the range of 100-200 μm. A pore size of 100-300 μm facilitates sufficient cell and blood vessel ingrowth, thus the small intestinal submucosal matrix sponge of this composite scaffold can meet the requirements for cell and blood vessel ingrowth. In Example 1, the polylactic acid porous scaffold in the inner layer suffers from a lower polylactic acid concentration, resulting in uneven dispersion of polylactic acid during pore formation, thus failing to form a uniform pore structure. The polylactic acid porous scaffolds in Examples 2 and 3, however, possess a more uniform porous structure with pore sizes ranging from 5 to 25 μm, which is sufficient for nutrient exchange.

[0069] from Figure 3 The longitudinal section microstructure of the composite scaffold shows that the longitudinal section microstructure and cross-sectional microstructure of the small intestinal submucosal matrix sponge of the outer layer of the composite scaffold are the same. At the same time, the polylactic acid porous scaffold of the inner layer of the composite scaffold has a pore structure from top to bottom. In Example 1, the polylactic acid porous scaffold of the inner layer of the composite scaffold cannot form a uniformly distributed and interconnected pore structure due to the low concentration of polylactic acid. The polylactic acid porous scaffolds in Examples 2 and 3 both have a uniformly distributed and interconnected pore structure from top to bottom. This structure is conducive to the migration of osteoblasts into the interior of the scaffold.

[0070] Mechanical property testing

[0071] The composite scaffolds for bone repair prepared in Examples 1-3 were cut into samples with a height of 5 mm. The samples were then placed on an Instron for compression testing at a compression rate of 0.5 mm / min and a maximum compressive strain of 90%. The test results are as follows: Figure 4As shown, the composite scaffold for bone repair of the present invention has good mechanical properties. The composite scaffolds prepared in Examples 1-3 have a maximum compressive strength of 3-4 MPa when deformed to 90%, all of which have good compressive strength and can meet the mechanical performance requirements in the bone tissue repair process, providing good mechanical support at the bone defect site.

[0072] In vitro degradation test

[0073] The outer layer of small intestinal submucosal matrix sponge and the inner layer of polylactic acid porous scaffold in the composite scaffolds for bone repair prepared in Examples 1-3 were separated and weighed separately, recorded as M0. Then, each group of samples was placed in phosphate buffered saline (PBS, pH = 7.4) and tested in a temperature-controlled shaker. The parameters of the temperature-controlled shaker were set as follows: temperature 37°C, shaking speed 100 r / min. During the test, samples were removed after 30, 60, 90, 120, 150, 180, 240, 300, 360, 450, and 540 days of degradation, respectively. After lyophilization, the samples were weighed and recorded as M1. Fresh PBS buffer was then added, and the test was continued. The degradation rate was calculated using the following formula:

[0074]

[0075] The results are shown in Table 1 below.

[0076]

[0077] Table 1

[0078] As shown in Table 1, the spongy submucosa of the small intestine degrades first, followed by the polylactic acid porous scaffold. This property allows the composite scaffold to fuse with the tissue at the bone defect site, providing better mechanical properties in the early stages of bone defect repair without causing secondary damage to the bone defect. Furthermore, as new bone tissue forms, the polylactic acid porous scaffold is gradually degraded. During the degradation process, the pore size of the internal channels of the polylactic acid porous scaffold increases, which is conducive to osteoblasts crawling into the scaffold and eventually being replaced and absorbed by the new bone tissue, thereby achieving the purpose of repairing the bone defect.

[0079] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A composite scaffold for bone repair, characterized in that, The middle layer is a polylactic acid porous scaffold, and the outer layer is a small intestinal submucosal matrix sponge. The ratio of the diameter of the composite scaffold used for bone repair to the diameter of the polylactic acid porous scaffold is 2-4. The polylactic acid porous scaffold is perforated, with pinholes of 300-600 μm in diameter distributed around its perimeter. The spacing between the pinholes is 2-6 mm. The polylactic acid porous scaffold has a channel structure running from top to bottom inside. The channel structure is interconnected with the pinholes, and the pore size of the channel structure is 5-25 μm. The submucosal matrix sponge of the small intestine has a porous structure with a pore size of 100-300μm. The submucosal matrix sponge of the small intestine is immersed in the polylactic acid porous scaffold through the pinholes around the polylactic acid porous scaffold, thereby making the submucosal matrix sponge of the small intestine tightly bonded to the polylactic acid porous scaffold. The polylactic acid porous scaffold is obtained through the following steps: 6-8 wt% polylactic acid solution is poured into a first hollow mold, a metal block is connected to the bottom of the first hollow mold, and the metal block is brought into contact with liquid nitrogen to solidify the polylactic acid solution. Then, it is immersed in cold water at 0-10℃ for 24-96 hours, and finally freeze-dried to obtain the polylactic acid scaffold. At the same time, holes are punched around the polylactic acid scaffold using a needle with a diameter of 300-600μm; thus, the polylactic acid porous scaffold is obtained.

2. A method for preparing a composite scaffold for bone repair, used to prepare the composite scaffold for bone repair as described in claim 1, characterized in that, Includes the following steps: S1: Preparation of small intestinal submucosal matrix; removing the animal's small intestinal mucosa, serosa, and muscularis tissue to obtain the small intestinal submucosal layer; defatting and decellularizing the small intestinal submucosal layer, and then placing it in sterile PBS and shaking it on a shaker to obtain the small intestinal submucosal matrix; S2: Prepare a small intestinal submucosal matrix solution; cut the small intestinal submucosal matrix into small pieces and place it in an acetic acid aqueous solution containing pepsin and stir for 24h-96h to obtain a fully dissolved small intestinal submucosal matrix solution; S3: Preparation of polylactic acid porous scaffold; 6-8 wt% polylactic acid solution is poured into a first hollow mold, a metal block is connected to the bottom of the first hollow mold, and the metal block is brought into contact with liquid nitrogen to solidify the polylactic acid solution. Then, it is immersed in cold water at 0-10℃ for 24-96 hours, and finally freeze-dried to obtain the polylactic acid scaffold. At the same time, holes are punched around the polylactic acid scaffold with a needle with a diameter of 300-600μm; thus, the polylactic acid porous scaffold is obtained. S4: Prepare a composite scaffold of small intestinal submucosal matrix sponge and polylactic acid porous scaffold. Place the polylactic acid porous scaffold in the center of a second hollow mold, and then pour the small intestinal submucosal matrix solution around the polylactic acid porous scaffold. The small intestinal submucosal matrix solution will solidify to form a small intestinal submucosal matrix sponge. Then freeze-dry the second hollow mold to obtain the composite scaffold of small intestinal submucosal matrix sponge and polylactic acid porous scaffold. S5: Crosslinking treatment: The small intestinal submucosal matrix sponge and polylactic acid porous scaffold composite scaffold are placed in a crosslinking treatment solution at a temperature of 15-25°C and shaken on a shaker for 1-5 days. Finally, they are freeze-dried to obtain a composite scaffold for bone repair.

3. The method for preparing a composite scaffold for bone repair according to claim 2, characterized in that, The defatting and decellularization steps in step S1 are as follows: the submucosal layer of the small intestine is soaked in a defatting solution for 6-12 hours, the submucosal layer of the small intestine is removed and placed in a decellularization solution, shaken on a shaker at room temperature for 20-24 hours, the submucosal layer of the small intestine is then removed and placed in sterile PBS and shaken on a shaker for 2-3 hours.

4. The method for preparing a composite scaffold for bone repair according to claim 3, characterized in that, The defatting solution is one or more of acetone, anhydrous ethanol, ethylene glycol ethyl ether, or isopropanol, and the decellularization solution is one or more of sodium hydroxide, Triton X-100 solution, SDS solution, or trypsin.

5. The method for preparing a composite scaffold for bone repair according to claim 2, characterized in that, In step S2, the submucosal matrix solution of the small intestine contains pepsin at a mass fraction of 0.1 wt%-0.5 wt%, acetic acid at a mass fraction of 1 wt%-5 wt%, and submucosal matrix of the small intestine at a mass fraction of 1 wt%-3 wt%.

6. The method for preparing a composite scaffold for bone repair according to claim 2, characterized in that, In step S3, the drilling distance is 2-6mm.

7. The method for preparing a composite scaffold for bone repair according to claim 2, characterized in that, The inner diameter of the first hollow mold is 3-6mm, and the metal block is a copper block.

8. The method for preparing a composite scaffold for bone repair according to claim 2, characterized in that, The crosslinking agent in the crosslinking treatment solution in step S5 is one of genipin, glutaraldehyde, or EDC, with a concentration of 0.2%-2% and a pH of 5.0-7.0.

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