CN113730658B - Bionic bone cartilage integrated repair support and preparation method thereof - Google Patents
Bionic bone cartilage integrated repair support and preparation method thereof Download PDFInfo
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Abstract
The invention relates to the field of biomedical engineering osteochondral repair supports, in particular to a bionic osteochondral integrated repair support which consists of a surface cartilage repair layer, a middle transition layer and a subchondral bone repair layer, wherein the surface cartilage repair layer is a polycaprolactone nanofiber membrane with a core-shell structure, the inner core is sodium hyaluronate loaded with glucosamine, and the outer shell is polycaprolactone with good biocompatibility; the subchondral bone repair layer is a mineralized polycaprolactone crystal-stringy nanofiber membrane; the intermediate transition layer is a polycaprolactone crystal-tandem nanofiber membrane with a core-shell structure, so that support is provided for effective combination of the polycaprolactone crystal-tandem nanofiber membrane and the polycaprolactone crystal-tandem nanofiber membrane, and integrated repair of osteochondral is realized. The invention also relates to a preparation method of the bionic bone cartilage integrated repair bracket.
Description
Technical Field
The invention relates to a bionic osteochondral integrated repair bracket and a preparation method thereof, belongs to the field of biomedical engineering osteochondral repair brackets, and has wide application value in the osteochondral repair aspect.
Background
Osteochondral injury is a clinically common joint disease. Osteochondral tissues include articular cartilage and subchondral bone: articular cartilage, responsible for reducing friction and shock in motion; and subchondral bone, responsible for providing mechanical support. Osteochondral defects typically involve lesions of articular cartilage and subchondral bone that result from trauma, disease or aging. The clinical treatment is mainly carried out by means of autografting, allograft, microarthrosis, mosaic plasty and the like, but the application of the technologies is limited by the problems of limited donor sources, complicated operation, immunological rejection and the like. Due to the complexity of the osteochondral interface, the clinical treatment for osteochondral injury has great limitation, and tissue engineering provides a new idea for solving the problem.
In order to achieve simultaneous regeneration of cartilage and subchondral bone, the design of biological scaffolds is generally as follows: (1) a single-phase support; (2) a two-phase scaffold; (3) a multi-phase scaffold. Monophasic scaffolds do not meet the microenvironment required for osteoblast and chondrocyte growth. The biphase scaffold has the problems of poor interface connection and the like. The multi-phase scaffold is formed by adding an intermediate transition layer between a surface cartilage repair layer and a subchondral bone repair layer to ensure that the intermediate transition layer is tightly combined, and the multi-phase scaffold meets the requirements of osteochondral scaffolds. The electrostatic spinning technology is a commonly used technology for preparing tissue engineering scaffolds, and the produced fibers can effectively simulate the form of the natural extracellular matrix of bone or cartilage tissues.
Although the electrostatic spinning technology has a wide application prospect in tissue engineering, materials for preparing electrostatic spinning still need to be carefully selected. The stent constructed by different materials has advantages and disadvantages in the aspects of microstructure, surface appearance, mechanical property and the like. Polycaprolactone has good biocompatibility and mechanical properties, and a product generated after degradation is nontoxic. However, the further application of polycaprolactone is limited by the defects of strong hydrophobicity, poor cell adhesion and the like. Glucosamine can promote synthesis of mucopolysaccharide, increase viscosity of synovial fluid, improve metabolism of articular cartilage, and promote regeneration of cartilage.
Disclosure of Invention
The invention aims to provide a bionic osteochondral integrated repair scaffold and a preparation method thereof, and solves the problems of poor hydrophilicity of polycaprolactone and interface combination of the osteochondral repair scaffold in the prior art. The stent takes polycaprolactone as a base material, has good biocompatibility and mechanical property, and has the advantages of low cost, simple operation and the like. Preparing a polycaprolactone nanofiber membrane with a crystal structure as a subchondral bone repair layer by using an electrostatic spinning technology and a biomimetic mineralization method; fixing the nanofiber membrane on an electrostatic spinning receiver, and preparing the polycaprolactone string crystal nanofiber membrane with the core-shell structure as an intermediate transition layer by using a slightly soluble glue electrostatic spinning and self-induced crystallization technology; finally, preparing a polycaprolactone nanofiber membrane with a core-shell structure as a surface cartilage repair layer by using a slightly-soluble glue electrostatic spinning technology; mineralized polycaprolactone string crystals, polycaprolactone string crystals with a core-shell structure and polycaprolactone with a core-shell structure form a three-layer nanofiber membrane to biomimetically construct the osteochondral integrated repair scaffold.
The technical scheme adopted by the invention is as follows: the bionic osteochondral integrated repair bracket consists of a surface cartilage repair layer, a middle transition layer and a subchondral bone repair layer, wherein the surface cartilage repair layer is a polycaprolactone nanofiber membrane with a core-shell structure, the inner core is sodium hyaluronate loaded with glucosamine, and the outer shell is polycaprolactone with good biocompatibility; the subchondral bone repair layer is a mineralized polycaprolactone crystal-stringy nanofiber membrane; the intermediate transition layer is a polycaprolactone crystal-tandem nanofiber membrane with a core-shell structure, so that support is provided for effective combination of the polycaprolactone crystal-tandem nanofiber membrane and the polycaprolactone crystal-tandem nanofiber membrane, and integrated repair of osteochondral is realized.
The preparation method of the osteochondral integrated repair bracket comprises the following steps: preparing a mineralized polycaprolactone string crystal nanofiber membrane as a subchondral bone repair layer by using an electrostatic spinning technology and a biomimetic mineralization method; fixing the mineralized polycaprolactone string crystal nanofiber membrane on an electrostatic spinning receiver, and preparing the polycaprolactone string crystal nanofiber membrane with a core-shell structure as an intermediate transition layer by using a slightly soluble glue electrostatic spinning technology and an auto-induced crystallization method; finally, preparing a polycaprolactone nanofiber membrane with a core-shell structure as a surface cartilage repair layer by using a slightly-soluble glue electrostatic spinning technology; the mineralized polycaprolactone string crystal, the polycaprolactone string crystal with the core-shell structure and the polycaprolactone with the core-shell structure form a three-layer nanofiber membrane to biomimetically construct the osteochondral integrated repair scaffold.
The technological parameters of the slightly soluble glue electrostatic spinning are as follows: the voltage was 20kV, the distance between the spinneret and the receiving plate was 20cm, and the forwarding speed of the forwarding pump was 0.001 mm/s.
The self-induced crystallization method is that polycaprolactone dilute solution is dripped on the surface of a polycaprolactone nanofiber membrane with a core-shell structure to carry out self-induced crystallization, wherein the mass fraction of polycaprolactone in the polycaprolactone dilute solution is 0.1wt%, 0.2wt% and 0.5wt%, the volume ratio of glacial acetic acid to deionized water is 3:1, and the molecular weight of polycaprolactone is 80000.
Soaking the nanofiber membrane in a mineralization liquid for biomimetic mineralization, wherein the mineralization liquid is supersaturated calcium-phosphorus mixed liquid, and the mineralization time is 4 days.
The preparation method of the surface cartilage repair layer specifically comprises the following steps: mixing 1.2wt% of sodium hyaluronate hydrosol (loaded with glucosamine (1 mg/ml), Span-80 and dichloromethane at a high speed to obtain water-in-oil type emulsion, dissolving polycaprolactone and N, N-dimethylformamide in the emulsion to prepare spinning solution with the concentration of 9 wt%, and obtaining the polycaprolactone nanofiber membrane with the core-shell structure by utilizing an electrostatic spinning technology; the preparation method of the intermediate transition layer specifically comprises the following steps: dripping a polycaprolactone dilute solution on the surface of the nanofiber membrane, and obtaining a polycaprolactone string crystal nanofiber membrane with a core-shell structure after the solvent is volatilized; the preparation method of the subchondral bone repair layer specifically comprises the following steps: dissolving polycaprolactone in a mixed solution of dichloromethane and N, N-dimethylformamide (V: V =7: 3) to prepare a spinning solution with the concentration of 15wt%, and obtaining the polycaprolactone nanofiber membrane by an electrostatic spinning technology. And dropwise adding the diluted polycaprolactone solution onto the polycaprolactone nanofiber membrane, and incubating the fiber membrane in the saturated calcium-phosphorus mixed solution for 4 days after the solvent is completely volatilized to obtain the polycaprolactone nanofiber membrane with the crystal structure.
Further, in the preparation method of the surface cartilage repair layer, the mass fraction of the sodium hyaluronate hydrosol is 1.2wt%, and the volume ratio of the ammonia sugar aqueous solution to the sodium hyaluronate hydrosol is 1: 4; the volume ratio of the sodium hyaluronate hydrosol to the dichloromethane is 60.6, and the mass ratio of Span-80 to the dichloromethane is 1: 400; the volume ratio of dichloromethane to N, N-dimethylformamide was 1.43.
Further, the diluted polycaprolactone solution is diluted by glacial acetic acid and deionized water, wherein the volume ratio of the glacial acetic acid to the deionized water is 3:1, and the mass fractions of the polycaprolactone are 0.1wt%, 0.2wt% and 0.5 wt%.
The invention has the beneficial effects that: the invention designs the osteochondral integrated repair bracket from the bionic osteochondral natural structure according to the hierarchical structure of the osteochondral, the bracket takes polycaprolactone as a base material, has good biocompatibility and mechanical property, and has the advantages of low cost, simple operation and the like. Promoting the proliferation of chondrocytes by loading glucosamine into nanofiber cores in the surface cartilage repair layer; calcium phosphate is introduced into the subchondral bone repair layer to repair complex large-area bone defects and support the repair of the surface cartilage layer; the structure of the cross-crystal structure of the intermediate transition layer simulates the microstructure of collagen fibers, the hydrophilicity and the mechanical property of the polycaprolactone nanofiber membrane are improved, and support is provided for the effective combination of the surface cartilage repair layer and the subchondral bone repair layer.
Drawings
FIG. 1 is an SEM image of polycaprolactone nanofibers in core-shell structure;
FIG. 2 is a diameter distribution diagram of polycaprolactone nanofibers in a core-shell structure;
FIG. 3 is an SEM image of polycaprolactone nanofibers;
FIG. 4 is a diameter distribution plot of polycaprolactone nanofibers;
FIG. 5 is an SEM image of a polycaprolactone tandem crystal (SKMGPCL 02) nanofiber membrane with a core-shell structure;
FIG. 6 is an SEM image of a mineralized polycaprolactone string crystal (MSKPCL 02) nanofiber membrane;
FIG. 7 is the proliferation of MC3T3-E1 cells on different nanofiber materials;
PCL: a polycaprolactone nanofiber membrane; SKPCL 01: the polycaprolactone string crystal nanofiber membrane of example 1; SKPCL 02: the polycaprolactone string crystal nanofiber membrane of example 2; SKPCL05 polycaprolactone string crystal nanofiber membrane in example 3; MPCL: mineralizing the polycaprolactone nanofiber membrane; MSKPCL 01: mineralized polycaprolactone string crystal nanofiber membrane in example 1; MSKPCL 02: in example 2, a mineralized polycaprolactone string crystal nanofiber membrane; MSKPCL05 mineralized polycaprolactone string crystal nanofiber membrane in example 3.
Detailed Description
In order to make the technical scheme and advantages of the present invention clearer, the osteochondral integrated scaffold provided by the present invention will be further explained with reference to the accompanying drawings and specific embodiments. The embodiments described are only a few embodiments of the present invention, not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
1. Preparing a surface cartilage repair layer: and dissolving sodium hyaluronate in deionized water to obtain 1.2wt% sodium hyaluronate hydrosol. Mu.l of an aqueous ammonia sugar solution (1 mg/ml) was mixed into 200. mu.l of hyaluronic acid hydrosol, followed by addition of a mixture of 12.12ml of dichloromethane and 0.04g of Span-80 and high-speed stirring to obtain a water-in-oil emulsion. 2g of polycaprolactone and 8.44ml of N, N-dimethylformamide were added thereto, and the mixture was sufficiently stirred at room temperature to obtain a spinning dope. The electrostatic spinning process is carried out under the conditions of spinning voltage of 20kv, distance between a needle head and a collector of 20cm and advancing speed of 0.001mm/s, and the polycaprolactone nanofiber membrane with the core-shell structure is obtained after spinning is finished. FIG. 1 is a scanning electron microscope picture of polycaprolactone nanofiber membrane with core-shell structure, which shows a smooth and continuous nanofiber structure, and the diameter of nanofiber is 140.94 + -36.35 nm (FIG. 2) analyzed by ImageJ software.
2. Preparing an intermediate transition layer: dissolving polycaprolactone in a mixed solvent of glacial acetic acid and deionized water (V: V =3:1), and magnetically stirring at 70 ℃ to obtain a dilute solution with the mass fraction of 0.1 wt%. And (3) dripping the polycaprolactone dilute solution on the surface of the nanofiber membrane to perform self-induced crystallization, then performing vacuum drying for 24h, and removing the residual solvent to obtain the polycaprolactone crystal-string (SKMGPCL 01) nanofiber membrane with the core-shell structure.
3. Preparing a subchondral bone repair layer: dissolving polycaprolactone in a mixed solution (V: V =7: 3) of dichloromethane and N, N-dimethylformamide to prepare an electrostatic spinning solution with the concentration of 15wt%, sucking the spinning solution into an injector for electrostatic spinning, wherein the spinning voltage is 20kv, the distance between a spinning nozzle and a receiving plate is 20cm, the propelling speed of a propelling pump is 0.003mm/s, and the polycaprolactone nanofiber membrane is prepared. And (3) dripping a polycaprolactone dilute solution (0.1wt%) on the surface of the polycaprolactone nanofiber membrane for self-induced crystallization, then carrying out vacuum drying for 24h, and removing the residual solvent to obtain the polycaprolactone tandem crystal (SKPCL 01) nanofiber membrane. Soaking the nanofiber membrane in a saturated calcium hydroxide solution for pre-mineralization for 1h, rinsing with deionized water, and soaking in a supersaturated calcium-phosphorus mixed solution for 4d at a constant temperature of 37 ℃. And after mineralization, rinsing the sample by using deionized water, and drying in vacuum to obtain the mineralized polycaprolactone crystal string (MSKPCL 01) nanofiber membrane. FIG. 3 is a scanning electron microscope picture of polycaprolactone nanofiber membrane, which shows a smooth nanofiber structure, and the diameter of the nanofiber is 199.3 + -43.8 nm (FIG. 4) by ImageJ software analysis.
4. Preparing an osteochondral integrated repair bracket: and performing biomimetic construction on the surface cartilage repair layer, the intermediate transition layer and the subchondral bone repair layer by a layer-by-layer spinning method to form the osteochondral integrated repair scaffold.
5. The nanofiber membranes of the surface cartilage repair layer and the subchondral bone repair layer in the above examples are prepared into a disc shape with the diameter of 1cm and placed in a 48-well plate, and chondrocytes and MC3T3-E1 cells are prepared into cell suspension. Each sample well was inoculated with 400 μ l of cell suspension, and the cells were co-cultured with the material for 1, 4, and 7 days, with no nanofiber membrane as a blank control.
Example 2
1. Preparing a surface cartilage repair layer: same as example 1
2. Preparing an intermediate transition layer: dissolving polycaprolactone in a mixed solvent of glacial acetic acid and deionized water (V: V =3:1), and magnetically stirring at 70 ℃ to obtain a dilute solution with the mass fraction of 0.2 wt%. And (3) dripping a polycaprolactone dilute solution on the surface of the nanofiber membrane to perform self-induced crystallization, then performing vacuum drying for 24h, and removing residual solvent to obtain a polycaprolactone tandem crystal (SKMGPCL 02) nanofiber membrane with a core-shell structure, wherein FIG. 5 is a scanning electron microscope image of the SKMGPCL02 nanofiber membrane, and the lamellar crystal structure on the surface of the fiber can be obviously seen and is periodically arranged perpendicular to a fiber axis. Wherein, the crystal structure of the fiber with smaller diameter is more regular.
3. Preparing a subchondral bone repair layer: dissolving polycaprolactone in a mixed solution (V: V =7: 3) of dichloromethane and N, N-dimethylformamide to prepare an electrostatic spinning solution with the concentration of 15wt%, sucking the spinning solution into an injector for electrostatic spinning, wherein the spinning voltage is 20kv, the distance between a spinning nozzle and a receiving plate is 20cm, the propelling speed of a propelling pump is 0.003mm/s, and the polycaprolactone nanofiber membrane is prepared. And (3) dripping a polycaprolactone dilute solution (0.2wt%) on the surface of the nanofiber membrane for self-induced crystallization, then carrying out vacuum drying for 24h, and removing residual solvent to obtain the polycaprolactone crystal-string (SKPCL02) nanofiber membrane. Soaking the nanofiber membrane in a saturated calcium hydroxide solution for pre-mineralization for 1h, rinsing with deionized water, and soaking in a supersaturated calcium-phosphorus mixed solution for 4d at a constant temperature of 37 ℃. And after mineralization, rinsing the sample by using deionized water, and drying in vacuum to obtain the mineralized polycaprolactone crystal string (MSKPCL 02) nanofiber membrane. Fig. 6 is a scanning electron microscope image of MSKPCL02, which shows that the calcium phosphate coating uniformly covers the surface of MSKPCL02 nanofiber.
4. Preparing an osteochondral integrated repair bracket: the osteochondral integrated repair scaffold is prepared by compounding the surface cartilage repair layer, the intermediate transition layer and the subchondral bone repair layer by a layer-by-layer spinning method.
5. The nanofiber membranes of the surface cartilage repair layer and the subchondral bone repair layer in the above examples are prepared into a disc shape with the diameter of 1cm and placed in a 48-well plate, and chondrocytes and MC3T3-E1 cells are prepared into cell suspension. Each sample well was inoculated with 400 μ l of cell suspension, and the cells were co-cultured with the material for 1, 4, and 7 days, with no nanofiber membrane as a blank control.
Example 3
1. Preparing a surface cartilage repair layer: same as example 1
2. Preparing an intermediate transition layer: dissolving polycaprolactone in a mixed solvent of glacial acetic acid and deionized water (V: V =3:1), and magnetically stirring at 70 ℃ to obtain a dilute solution with the mass fraction of 0.5 wt%. And (3) dripping the polycaprolactone dilute solution on the surface of the nanofiber membrane to perform self-induced crystallization, then performing vacuum drying for 24h, and removing the residual solvent to obtain the polycaprolactone crystal-string (SKMGPCL 05) nanofiber membrane with the core-shell structure.
3. Preparing a subchondral bone repair layer: dissolving polycaprolactone in a mixed solution (V: V =7: 3) of dichloromethane and N, N-dimethylformamide to prepare an electrostatic spinning solution with the concentration of 15wt%, sucking the spinning solution into an injector for electrostatic spinning, wherein the spinning voltage is 20kv, the distance between a spinning nozzle and a receiving plate is 20cm, the propelling speed of a propelling pump is 0.003mm/s, and the polycaprolactone nanofiber membrane is prepared. And (3) dripping a polycaprolactone dilute solution (0.5wt%) on the surface of the nanofiber membrane for self-induced crystallization, then carrying out vacuum drying for 24h, and removing residual solvent to obtain the polycaprolactone crystal-string (SKPCL05) nanofiber membrane. Soaking the nanofiber membrane in a saturated calcium hydroxide solution for pre-mineralization for 1h, rinsing with deionized water, and soaking in a supersaturated calcium-phosphorus mixed solution for 4d at a constant temperature of 37 ℃. And after mineralization, rinsing the sample by using deionized water, and drying in vacuum to obtain the mineralized polycaprolactone crystal string (MSKPCL 05) nanofiber membrane.
4. Preparing an osteochondral integrated repair bracket: the osteochondral integrated repair scaffold is prepared by compounding a surface cartilage repair layer, an intermediate transition layer and a subchondral bone repair layer by a layer-by-layer spinning method.
5. The nanofiber membranes of the surface cartilage repair layer and the subchondral bone repair layer in the above examples are prepared into a disc shape with the diameter of 1cm and placed in a 48-well plate, and chondrocytes and MC3T3-E1 cells are prepared into cell suspension. Each sample well was inoculated with 400 μ l of cell suspension, and the cells were co-cultured with the material for 1, 4, and 7 days, with no nanofiber membrane as a blank control. As shown in FIG. 7, the test results showed that the sample of example 2 had a more significant effect on the proliferation of MC3T3-E1 cells.
Claims (5)
1. The utility model provides a support is restoreed in bionical bone cartilage integration which characterized in that: the cartilage repairing layer is a polycaprolactone nanofiber membrane with a core-shell structure, the inner core is sodium hyaluronate loaded with glucosamine, and the outer shell is polycaprolactone with good biocompatibility; the subchondral bone repair layer is a mineralized polycaprolactone crystal-stringy nanofiber membrane; the intermediate transition layer is a polycaprolactone crystal-tandem nanofiber membrane with a core-shell structure, so that support is provided for effective combination of the polycaprolactone crystal-tandem nanofiber membrane and the core-shell structure, and the surface cartilage repair layer, the intermediate transition layer and the subchondral bone repair layer realize integrated repair of osteochondral.
2. The method for preparing the osteochondral integrated repair scaffold according to claim 1, wherein the method comprises the following steps: preparing a mineralized polycaprolactone string crystal nanofiber membrane as a subchondral bone repair layer by using an electrostatic spinning technology and a biomimetic mineralization method; fixing the mineralized polycaprolactone string crystal nanofiber membrane on an electrostatic spinning receiver, and preparing the polycaprolactone string crystal nanofiber membrane with a core-shell structure as an intermediate transition layer by using a slightly soluble glue electrostatic spinning technology and an auto-induced crystallization method; finally, preparing a polycaprolactone nanofiber membrane with a core-shell structure as a surface cartilage repair layer by using a slightly-soluble glue electrostatic spinning technology; the mineralized polycaprolactone string crystal, the polycaprolactone string crystal with the core-shell structure and the polycaprolactone with the core-shell structure form a three-layer nanofiber membrane to biomimetically construct the osteochondral integrated repair scaffold.
3. The preparation method of the osteochondral integrated repair scaffold according to claim 2, wherein the technological parameters of the slightly soluble gel electrostatic spinning are as follows: the voltage was 20kv, the distance between the spinneret and the receiving plate was 20cm, and the advancing speed of the advancing pump was 0.001 mm/s.
4. The method for preparing the osteochondral integrated repair scaffold according to claim 2, wherein the method comprises the following steps: the self-induced crystallization method is that polycaprolactone dilute solution is dripped on the surface of a polycaprolactone nanofiber membrane with a core-shell structure to carry out self-induced crystallization, wherein the mass fraction of polycaprolactone in the polycaprolactone dilute solution is 0.1wt%, 0.2wt% and 0.5wt%, the volume ratio of glacial acetic acid to deionized water is 3:1, and the molecular weight of polycaprolactone is 80000.
5. The method for preparing the osteochondral integrated repair scaffold according to claim 2, wherein the method comprises the following steps: soaking the nanofiber membrane in a mineralization liquid for biomimetic mineralization, wherein the mineralization liquid is supersaturated calcium-phosphorus mixed liquid, and the mineralization time is 4 days.
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