Injectable nucleus pulposus repair product and preparation method thereof

Abstract

The present invention discloses an injectable nucleus pulposus repair product and its preparation method. The injectable nucleus pulposus repair product includes a cross-linking agent and a hydrogel precursor solution that are independently packaged. After the cross-linking agent is added to the hydrogel precursor solution, a hydrogel can be quickly formed under mild conditions. Nanofibers have a shell layer and a core layer, wherein the shell layer is insoluble in water and the core layer contains a nucleus pulposus repair substance. The nanofibers are independently packaged relative to the cross-linking agent and the hydrogel precursor solution, or are dispersed in the hydrogel precursor solution. The repair product can be instantly formed in the body during injection, avoiding gel leakage and compression of nerves. The nanofibers with a shell layer and a core layer can load various nucleus pulposus repair substances and bioactive factors, which are slowly released from both ends of the nanofibers. The dispersed nanofibers can also improve the mechanical properties of the hydrogel to twice that of native nucleus pulposus tissue while retaining the toughness of the hydrogel, thereby achieving dual mechanical and biological repair.

Description

Technical Field

[0001] The present invention relates to the technical field of preparation of artificial intervertebral disc nucleus pulposus scaffolds, and in particular to an injectable nucleus pulposus repair product and a preparation method thereof. Background Art

[0002] Intervertebral disc degeneration is a common disease worldwide, affecting over 500 million people. Currently, surgical procedures such as nucleus pulposus removal, disc fusion, and artificial disc replacement are commonly used to treat intervertebral disc degeneration. However, these procedures invariably cause new trauma and are prone to complications. Repairing the degenerated nucleus pulposus remains an unresolved challenge in the treatment of intervertebral disc degeneration, and there is currently no effective method for repairing degenerated and fragmented nucleus pulposus and restoring the biomechanical function of the intervertebral disc.

[0003] Although the development of tissue engineering regeneration technology has provided new technical means and prospects for the regeneration and repair of the intervertebral disc nucleus pulposus, due to the special anatomical position of the intervertebral disc, while it bears 80% of the human body's pressure, there is also the spinal nerve running behind it. The current products for repairing nucleus pulposus tissue have different effects. For example, platelet-rich plasma injections and high-strength hydrogel replacements only focus on biological repair or mechanical repair, and the effect on the regeneration and repair of intervertebral disc is not ideal. Summary of the Invention

[0004] In order to solve the shortcomings of existing nucleus pulposus cartilage repair products, the present invention provides an injectable nucleus pulposus repair product with both mechanical and biological repair capabilities and a preparation method thereof.

[0005] The technical solutions provided by the present invention are as follows:

[0006] In a first aspect, the present invention provides an injectable nucleus pulposus repair product, comprising:

[0007] A cross-linking agent and a hydrogel precursor solution are packaged independently of each other; after the cross-linking agent is added to the hydrogel precursor solution, a hydrogel can be quickly formed under mild conditions;

[0008] The nanofiber has a shell layer and a core layer, wherein the shell layer is insoluble in water and the core layer contains a nucleus pulposus repair material; the nanofiber is independently packaged relative to a crosslinking agent and a hydrogel precursor solution, or is dispersed in the hydrogel precursor solution.

[0009] In some embodiments provided by the present invention, when the nanofibers are independently packaged relative to the crosslinking agent and the hydrogel precursor solution, the nanofibers exist in the form of nanofiber membranes or short fibers. The nanofiber membranes are broken into short fibers before injection, and the core layer is exposed at both ends of the short fibers.

[0010] In some embodiments provided by the present invention, when the nanofibers are dispersed in the hydrogel precursor solution, the nanofibers exist in the form of nanofiber membranes, and the nanofiber membranes in the hydrogel precursor solution are broken into short fibers before injection.

[0011] In some embodiments provided by the present invention, the weight ratio of the hydrogel precursor to the nanofibers is 1:1 to 6:1.

[0012] In some embodiments provided by the present invention, the length of the nanofiber is 500 nm to 2000 nm; and the weight ratio of the shell layer to the core layer of the nanofiber is 1:1 to 10:1.

[0013] In some embodiments provided by the present invention, the shell layer of the nanofiber is at least one of polycaprolactone, chitosan, collagen, polyacrylonitrile, polyaniline, polyvinyl pyrrolidone, polyethylene oxide, silk fibroin, and polylactic acid, and the core layer of the nanofiber contains one or more of glucosamine, insulin-like growth factor, vascular endothelial growth factor, exosomes, transforming growth factor, and platelet-rich plasma.

[0014] In some embodiments provided by the present invention, the core layer further comprises at least one of chitosan, collagen, polyethylene glycol, polyvinyl alcohol, silk fibroin, and polylactic acid as a dispersant for the nucleus pulposus repair material.

[0015] In some embodiments provided by the present invention, the hydrogel precursor solution is a sodium alginate solution, and the cross-linking agent is a powder or solution containing calcium ions.

[0016] In some embodiments provided by the present invention, the mass concentration ratio of the sodium alginate solution to the cross-linking agent is 3:5-10.

[0017] In a second aspect, the present invention provides a method for preparing the above-mentioned injectable nucleus pulposus repair product, comprising:

[0018] The nanofiber membrane is prepared by coaxial electrospinning; the nanofiber membrane is broken into short fibers.

[0019] In some embodiments provided herein, preparing a nanofiber membrane by coaxial electrospinning comprises:

[0020] Silk fibroin and polycaprolactone in a weight ratio of 4 to 9:1 are dissolved in a solvent to prepare an electrospinning solution; coaxial electrospinning is performed to form a nanofiber membrane with silk fibroin and polycaprolactone as shell layers; and the nanofiber membrane is subjected to ethanol gradient cross-linking.

[0021] In some embodiments provided by the present invention, the solvent is an organic solvent that is harmless to the human body.

[0022] In a third aspect, the present invention provides a bionic scaffold made using the above-mentioned injectable nucleus pulposus repair product.

[0023] The injectable nucleus pulposus repair product provided by the present invention can reach the site to be repaired by injection and quickly form a hydrogel under mild conditions under the action of a cross-linking agent, making it easy and quick to use; the nanofibers evenly dispersed in the hydrogel can enhance the compressive strength of the hydrogel, and at the same time, the water-insoluble shell layer of the nanofibers can prevent the premature leakage of the nucleus pulposus repair material in the core layer. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 These are field emission electron micrographs of the nanofibers and hydrogels prepared in Examples 2 and 3. Figure A is a field emission electron micrograph of the nanofibers prepared in Example 2, Figure B is a field emission electron micrograph of the hydrogel obtained in Example 3, Figure C is a transmission electron micrograph of the nanofibers prepared in Example 2, and Figure D is a fluorescence confocal micrograph of the nanofibers prepared in Example 2.

[0025] Figure 2 Figure A is a field emission electron micrograph of the hydrogel obtained in accordance with the present invention. Figure B is a field emission electron micrograph of the hydrogel obtained in Example 2. Figure C is a field emission electron micrograph of the hydrogel obtained in Example 3. Figure D is a field emission electron micrograph of the hydrogel obtained in Example 4.

[0026] Figure 3 The mechanical properties and biocompatibility of the hydrogel obtained in the present invention are demonstrated. Figures A and B are the mechanical compression curves and calculated elastic moduli of the hydrogels obtained in Examples 1, 2, 3, and 4. Figures C and D are the live and dead cell detection of the hydrogel obtained in Example 3 and the fluorescence staining results of the cells after 1, 3, and 5 days of culture on the scaffold, with Calcein-AM (green) staining of live cells and PI (red) staining of dead cells. Scale bar = 500 μm. Figure E is the CCK-8 kit test of the hydrogel obtained in Example 3, and Figure F is the in vitro growth factor sustained release test of the hydrogel obtained in Example 3.

[0027] Figure 4 This is the use of the injectable nucleus pulposus repair product provided in Example 3 in a rat model of caudal disc degeneration. After establishing a rat model of caudal disc degeneration, different products were injected into the degenerated disc. X-rays, MRIs, and histological staining were performed 4 and 8 weeks after surgery. A control group was maintained without treatment. A shows X-ray images of the rat caudal vertebrae at different times, B shows MRI scans of the rat caudal vertebrae at different times, and C shows histological sections stained with Safranin Fast Green from the rat caudal vertebrae at different times. DETAILED DESCRIPTION

[0028] The present invention is further described below with reference to the following examples, which are intended to better illustrate the technical solutions of the present invention and are not intended to limit the claims. The present invention is not limited to the specific embodiments and implementations described herein. Further improvements and perfections can be readily made by those skilled in the art without departing from the spirit and scope of the present invention, and all such improvements and perfections fall within the scope of protection of the present invention.

[0029] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although any methods similar or equivalent to those described herein can be used in the practice or testing of the present invention, preferred methods will be described herein.

[0030] The numerical ranges used herein are intended to include every value and subgroup within the range, whether or not specifically disclosed. Further, these numerical ranges should be interpreted as providing support for claims directed to any value or subgroup of values ​​within the range.

[0031] Unless otherwise stated, the numerical values ​​provided herein should be taken up to and including the given endpoints.

[0032] The features and advantages of the present invention can be further understood by the following detailed description in conjunction with the accompanying drawings. However, these specific embodiments do not limit the scope of protection of the present invention in any way. The raw materials used in this embodiment are known compounds and can be purchased on the market.

[0033] The injectable nucleus pulposus repair product provided by the present invention comprises: a cross-linking agent and a hydrogel precursor solution that are packaged independently of each other, and nanofibers that are packaged independently relative to the cross-linking agent and the hydrogel precursor solution or dispersed in the hydrogel precursor solution; the nanofibers have a shell layer and a core layer, the shell layer is insoluble in water, and the core layer contains a nucleus pulposus repair substance. When the nanofibers are packaged independently relative to the cross-linking agent and the hydrogel precursor solution, the nanofibers are first dispersed into the hydrogel precursor solution before use, and then the cross-linking agent is added. After the cross-linking agent is added to the hydrogel precursor solution, a hydrogel can be quickly formed under mild conditions. The injectable nucleus pulposus repair product provided by the present invention can be instantly formed in the body during injection, avoiding gel leakage and compressing nerves; the nanofibers with a shell layer and a core layer can load various nucleus pulposus repair substances and bioactive factors, which are slowly released from both ends of the nanofibers; the dispersed nanofibers can also improve the mechanical properties of the hydrogel to twice that of the native nucleus pulposus tissue and retain the toughness of the hydrogel, thereby achieving dual mechanical and biological repair.

[0034] This injectable repair product is suitable for minimally invasive treatment of intervertebral discs. Its material is widely available, biocompatible, and its composition and structure are similar to natural cartilage. The release of the nucleus pulposus repair material in the body repairs and regenerates degenerated disc cells, resulting in minimal trauma and improved patient recovery, suggesting promising clinical application prospects.

[0035] In some embodiments provided by the present invention, when the nanofibers are independently packaged relative to the cross-linking agent and the hydrogel precursor solution, the nanofibers exist in the form of nanofiber membranes or short fibers. The nanofiber membranes are broken into short fibers before injection, and the core layers are exposed at both ends of the short fibers. The nanofibers are independently packaged relative to the cross-linking agent and the hydrogel precursor solution to avoid premature leakage of the nucleus pulposus repair material. Before use, the nanofibers in the form of short fibers are directly dispersed in the hydrogel precursor solution, or the nanofibers in the form of nanofiber membranes are broken into short fibers and then dispersed in the hydrogel precursor solution, and then injected together with the cross-linking agent to form a hydrogel. The core layer exposed at both ends of the short fibers slowly releases the nucleus pulposus repair material contained therein.

[0036] In some embodiments provided herein, when the nanofibers are dispersed in a hydrogel precursor solution, the nanofibers exist in the form of a nanofiber membrane. Prior to injection, the nanofiber membrane in the hydrogel precursor solution is broken into short fibers. The nanofibers exist in the hydrogel precursor solution in the form of a nanofiber membrane, resulting in a small exposed core area and minimal leakage of the nucleus pulposus repair material. Breaking the nanofiber membrane in the hydrogel precursor solution into short fibers prior to injection exposes a greater amount of the core layer, thereby improving the release of the nucleus pulposus repair material.

[0037] In some embodiments provided herein, the weight ratio of the hydrogel precursor to the nanofibers is 1:1 to 6:1. If the nanofiber ratio is too low, the elastic modulus of the resulting hydrogel is too low to achieve sufficient strength for use; if the nanofiber ratio is too high, it becomes difficult to evenly disperse the hydrogel precursor solution.

[0038] In some embodiments provided herein, the nanofibers have a shell-to-core weight ratio of 1:1 to 10:1, and the short fibers are 500 nm to 2000 nm in length. If the shell ratio is too low, the nanofibers are prone to damage, causing the nucleus pulposus repair material to be released explosively from the damaged shell. If the shell ratio is too high, the shell degradation time is prolonged, preventing the nucleus pulposus repair material from being released later through the shell.

[0039] In some embodiments provided herein, the nanofiber shell comprises at least one of polycaprolactone, chitosan, collagen, polyacrylonitrile, polyaniline, polyvinylpyrrolidone, polyethylene oxide, silk fibroin, and polylactic acid, and the nanofiber core comprises one or more of glucosamine, insulin-like growth factor, vascular endothelial growth factor, exosomes, transforming growth factor, and platelet-rich plasma. Preferably, the nanofiber shell is a blend of silk fibroin and polycaprolactone in a weight ratio of 8:2 to 9:1, and the blend is modified by gradient cross-linking with ethanol.

[0040] When the shell contains silk fibroin, the short fibers begin to slowly release nucleus pulposus repair substances from both ends. After a period of time, the silk fibroin in the shell degrades, and the nucleus pulposus repair substances increase the pathway for release from the gaps in the shell, compensating for the slower release rate caused by the decrease in the concentration of nucleus pulposus repair substances.

[0041] In some embodiments provided by the present invention, the core layer also contains at least one of chitosan, collagen, polyethylene glycol, polyvinyl alcohol, silk fibroin, and polylactic acid as a dispersant and fixative for the nucleus pulposus repair material. These substances can fix and disperse the nucleus pulposus repair material in the inner layer spinning solution, facilitating coaxial spinning.

[0042] In some embodiments provided herein, the hydrogel precursor solution is a sodium alginate solution, and the crosslinking agent is a powder or solution containing calcium ions. Preferably, the sodium alginate aqueous solution has a concentration of 1% to 5% (w / v), and the crosslinking agent is a calcium chloride aqueous solution having a concentration of 1% to 10% (w / v).

[0043] In some embodiments provided by the present invention, the injectable nucleus pulposus repair product is injected through a coaxial needle, the inner layer is a hydrogel precursor solution dispersed with short fibers, and the outer layer is a cross-linking agent.

[0044] In some embodiments provided by the present invention, the mass concentration ratio of the sodium alginate solution to the cross-linking agent is 3:5 to 10. When the ratio of the cross-linking agent is too low, the hydrogel is prone to leakage during extrusion.

[0045] In a second aspect, the present invention provides a method for preparing the above-mentioned injectable nucleus pulposus repair product, comprising:

[0046] The nanofiber membrane is prepared by coaxial electrospinning; the nanofiber membrane is broken into short fibers.

[0047] In some embodiments provided herein, preparing a nanofiber membrane by coaxial electrospinning comprises:

[0048] Silk fibroin and polycaprolactone in a weight ratio of 4 to 9:1 are dissolved in a solvent to prepare an electrospinning solution; coaxial electrospinning is performed to form a nanofiber membrane with silk fibroin and polycaprolactone as shell layers; and the nanofiber membrane is subjected to ethanol gradient cross-linking.

[0049] In some embodiments provided by the present invention, the coaxial electrospinning voltage is set to 15-20 kV, the core solution concentration is 5%-20%, the spinning is carried out at a speed of 0.1-0.9 mL / h, the shell solution is carried out at a speed of 0.8-1.6 mL / h, the distance from the receiver is set to 12-15 cm, the spinning temperature is 25-30°C, and the relative humidity is 30%-60%.

[0050] In some embodiments provided by the present invention, the nanofiber membrane is broken into short fibers by ultrasonic homogenization or mechanical ball milling.

[0051] In some embodiments provided by the present invention, the conditions for mechanical ball milling are 200-2000 rpm and 10-20 min; the conditions for ultrasonic homogenization are 8000-12000 rpm and 5-30 min.

[0052] In a third aspect, the present invention provides a bionic scaffold made from the above-mentioned injectable nucleus pulposus repair product, the compressive strength of which is 170% of that of ordinary sodium alginate hydrogel. The short fiber-hydrogel dual structure enables long-term sustained release of nucleus pulposus repair substances.

[0053] Unless otherwise specified, the regenerated silk fibroin in the following examples was prepared according to the method disclosed in CN109999227A.

[0054] Example 1

[0055] Weigh 0.3g of sodium alginate, add water to a volume of 10mL, and mechanically stir to mix to obtain a sodium alginate solution with a concentration of 3% (w / v). Add the sodium alginate solution to syringe 1, and add a 10% (w / v) calcium chloride aqueous solution to syringe 2. Syringes 1 and 2 are injected together through coaxial needles, with the inner layer being the sodium alginate solution and the outer layer being the calcium chloride aqueous solution. The injection speed of the inner and outer layers is 0.2mL / s. The field emission electron microscopy image of the resulting hydrogel is shown below. Figure 2 As shown in A.

[0056] Example 2

[0057] (1) Regenerated silk fibroin and polycaprolactone were weighed according to the ratio of silk fibroin to polycaprolactone = 8:2 (w / w), and both were dissolved in the organic reagent hexafluoroisopropanol (HFIP) and stirred until clear and transparent. This was used as the spinning outer layer solution, and the final concentration of silk fibroin was maintained at 8% (w / w).

[0058] Blood was collected from rats and centrifuged twice to obtain platelet-rich plasma. Calcein powder was added to the platelet-rich plasma to obtain calcein-labeled platelet-rich plasma. The calcein-labeled platelet-rich plasma was mixed with a 5% polyvinyl alcohol aqueous solution in a ratio of 7:3 (v / v) to serve as the spinning inner layer solution.

[0059] (2) The outer layer solution and the inner layer solution were prepared into nanofiber membranes by coaxial electrospinning. The electrospinning conditions were set as follows: voltage 20 kV, outer layer flow rate 0.8 mL / h, inner layer flow rate 0.2 mL / h, needle distance 15 cm from aluminum foil flat plate receiver, relative humidity 30°C, and relative humidity 30%. After the electrospinning was completed, the obtained nanofiber membrane was dried in a vacuum drying oven for 3 days to remove the residual HFIP. At this time, the field emission scanning electron microscopy image of the nanofiber membrane was as follows: Figure 1 As shown in Figure A. The dried nanofiber membrane was cross-linked with ethanol gradient to increase its water insolubility. The conditions for ethanol gradient cross-linking were: first soaking in 100% ethanol for 10 minutes, then soaking in 90% ethanol for 10 minutes, then soaking in 70% ethanol for 10 minutes, and finally washing with pure water three times to remove the ethanol, thus obtaining a water-insoluble nanofiber membrane. At this time, the structure of the nanofiber membrane was observed by transmission electron microscopy ( Figure 1 C), a relatively uniform distribution of the inner and outer layers can be observed. Further observation by confocal microscopy shows that calcein-labeled platelet-rich plasma is evenly distributed in the inner layer of the nanofibers ( Figure 1 D).

[0060] (3) The water-insoluble nanofiber membrane was broken up and dispersed into short fibers using a homogenizer at a speed of 12,000 rpm for 15 min. The short fibers were first frozen at -40°C and then freeze-dried at -20°C in a vacuum environment for 24 h before use.

[0061] (4) Sodium alginate and short fibers were weighed in a weight ratio of sodium alginate to short fibers = 6:1, that is, 0.3 g of sodium alginate and 0.05 g of short fibers were weighed, water was added to the solution volume to 10 mL, and mechanical stirring was performed to obtain a sodium alginate solution with short fibers dispersed therein. The sodium alginate solution with short fibers dispersed therein was added to syringe 1, and a 10% (w / v) calcium chloride aqueous solution was added to syringe 2. Syringes 1 and 2 were injected together through coaxial needles. The inner layer was the sodium alginate solution with short fibers dispersed therein, and the outer layer was the calcium chloride aqueous solution. The injection speed of the inner and outer layers was 0.2 mL / s. The field emission electron microscopy image of the obtained hydrogel is shown in FIG. Figure 2 As shown in B.

[0062] Example 3

[0063] The short fibers were prepared using the same method and parameters as in Example 2.

[0064] Sodium alginate and short fibers were weighed in a ratio of sodium alginate to short fibers = 3:1, that is, 0.3 g of sodium alginate and 0.1 g of short fibers were weighed, water was added to the solution volume to 10 mL, and mechanical stirring was performed to obtain a sodium alginate solution with short fibers dispersed therein. The sodium alginate solution with short fibers dispersed therein was added to syringe 1, and a 10% (w / v) calcium chloride aqueous solution was added to syringe 2. Syringes 1 and 2 were injected together through coaxial needles, with the inner layer being the sodium alginate solution with short fibers dispersed therein and the outer layer being the calcium chloride aqueous solution. The injection speed of both the inner and outer layers was 0.2 mL / s. The field emission electron microscopy image of the resulting hydrogel is shown below. Figure 1 As shown in B, the gel is porous, the pore walls between the pores are connected to each other, the short fibers cross each other, and a double-layer fiber structure can be seen. The gel has good compression resistance ( Figure 3 A and 3B).

[0065] Cell experiments:

[0066] After the hydrogel obtained in this example was co-cultured with nucleus pulposus cells for 1, 3, and 5 days, live and dead cell staining tests were performed. From the fluorescence confocal microscopy images ( Figure 3 C) The distribution of living and dead cells on the hydrogels can be seen. Living cells grow on all hydrogels. The hydrogels have the ability to support cell adhesion and growth. The survival rate of cells on the hydrogels is not less than 94% ( Figure 3 D). The results of CCK-8 test kit showed that the hydrogel had no cytotoxicity ( Figure 3 E). In addition, with the increase of co-culture time, cell proliferation was obvious. The ability of hydrogel to release growth factors in vitro was tested using Elisa kit, and the results showed that ( Figure 3 F), TGF-β1, PDGF-BB, IGF-1 and VEGF released by platelet-rich plasma can be released long-term for up to 40 days.

[0067] Animal experiments:

[0068] A rat model of coccygeal disc degeneration was prepared. The control group (Control) was not treated with the nucleus pulposus repair product. The nucleus pulposus repair product prepared according to this example (ExpA), the nucleus pulposus repair product prepared according to this example without PRP in the core layer (ExpB), and a simple sodium alginate hydrogel (ExpC) were injected into the rats with coccygeal disc degeneration model. Radiological X-ray and MRI examinations were performed at 4 weeks and 8 weeks, respectively, and tissue samples were obtained for histological staining. Figure 4 A and Figure 4As shown in Figure B, at 4 and 8 weeks, the control group showed a gradual decrease in disc signal intensity, loss of disc height, collapse of the intervertebral space, and decomposition and loss of the extracellular matrix. In contrast, at 8 weeks after surgery, the ExpA group showed recovery of disc signal intensity, gradual recovery of disc height, maintenance of the intervertebral space at a basic level, and restoration of the extracellular matrix. While degeneration improved in the ExpB and ExpC groups at 4 weeks, this improvement was not significant at 8 weeks.

[0069] Example 4

[0070] The short fibers were prepared using the same method and parameters as in Example 2.

[0071] Sodium alginate and short fibers were weighed in a ratio of sodium alginate to short fibers = 1:1, that is, 0.3 g of sodium alginate and 0.3 g of short fibers were weighed, water was added to the solution volume to 10 mL, and mechanical stirring was performed to obtain a sodium alginate solution with short fibers dispersed therein. The sodium alginate solution with short fibers dispersed therein was added to syringe 1, and a 10% (w / v) calcium chloride aqueous solution was added to syringe 2. Syringes 1 and 2 were injected together through coaxial needles, with the inner layer being the sodium alginate solution with short fibers dispersed therein and the outer layer being the calcium chloride aqueous solution. The injection speed of both the inner and outer layers was 0.2 mL / s. The field emission electron microscopy image of the resulting hydrogel is shown below. Figure 2 As shown in D.

[0072] Example 5

[0073] The short fibers were prepared using the same method and parameters as in Example 2.

[0074] Sodium alginate and short fibers were weighed in a ratio of sodium alginate to short fibers = 1:1, i.e., 0.3 g of sodium alginate and 0.6 g of short fibers were weighed, water was added to a solution volume of 10 mL, and mechanical stirring was performed to mix uniformly to obtain a sodium alginate solution with dispersed short fibers. The sodium alginate solution with dispersed short fibers was added to syringe 1, and a 10% (w / v) calcium chloride aqueous solution was added to syringe 2. Syringes 1 and 2 were injected together through coaxial needles, with the inner layer being the sodium alginate solution with dispersed short fibers and the outer layer being the calcium chloride aqueous solution. The injection speed of both the inner and outer layers was 0.2 mL / s to obtain a hydrogel.

[0075] Example 6

[0076] The short fibers were prepared using the same method and parameters as in Example 2.

[0077] Weigh sodium alginate and short fibers in a ratio of 3:1, that is, weigh 0.3g of sodium alginate and 0.1g of short fibers, add water to a solution volume of 10mL, and mechanically stir to mix to obtain a sodium alginate solution with dispersed short fibers. Add the sodium alginate solution with dispersed short fibers to syringe 1, and add a 1% (w / v) calcium chloride aqueous solution to syringe 2. Syringes 1 and 2 are injected together through coaxial needles, with the inner layer being the sodium alginate solution with dispersed short fibers and the outer layer being the calcium chloride aqueous solution. The injection speed of the inner and outer layers is 0.2mL / s to obtain a hydrogel. Take 0.5mL of the resulting hydrogel and immediately perform 30% deformation extrusion to observe whether the solution leaks under the extrusion.

[0078] Example 7

[0079] The short fibers were prepared using the same method and parameters as in Example 2.

[0080] Sodium alginate and short fibers were weighed in a ratio of sodium alginate to short fibers = 3:1, that is, 0.3g of sodium alginate and 0.1g of short fibers were weighed, water was added to the solution volume to 10mL, and mechanical stirring was performed to obtain a sodium alginate solution with dispersed short fibers. The sodium alginate solution with dispersed short fibers was added to syringe 1, and a 3% (w / v) calcium chloride aqueous solution was added to syringe 2. Syringes 1 and 2 were injected together through coaxial needles, with the inner layer being the sodium alginate solution with dispersed short fibers and the outer layer being the calcium chloride aqueous solution. The injection speed of the inner and outer layers was 0.2mL / s to obtain a hydrogel. 0.5mL of the obtained hydrogel was immediately subjected to 30% deformation extrusion to observe whether the solution leaked under the extrusion.

[0081] Example 8

[0082] The short fibers were prepared using the same method and parameters as in Example 2.

[0083] Sodium alginate and short fibers were weighed in a ratio of sodium alginate to short fibers = 3:1, that is, 0.3g of sodium alginate and 0.1g of short fibers were weighed, water was added to the solution volume to 10mL, and mechanical stirring was performed to obtain a sodium alginate solution with dispersed short fibers. The sodium alginate solution with dispersed short fibers was added to syringe 1, and a 5% (w / v) calcium chloride aqueous solution was added to syringe 2. Syringes 1 and 2 were injected together through coaxial needles, with the inner layer being the sodium alginate solution with dispersed short fibers and the outer layer being the calcium chloride aqueous solution. The injection speed of the inner and outer layers was 0.2mL / s to obtain a hydrogel. 0.5mL of the obtained hydrogel was immediately subjected to 30% deformation extrusion to observe whether the solution leaked under the extrusion.

[0084] Example 9

[0085] The short fibers were prepared using the same method and parameters as in Example 2.

[0086] Sodium alginate and short fibers were weighed in a ratio of 3:1, that is, 0.3g of sodium alginate and 0.1g of short fibers were weighed, water was added to a solution volume of 10mL, and mechanical stirring was performed to obtain a sodium alginate solution with dispersed short fibers. The sodium alginate solution with dispersed short fibers was added to syringe 1, and a 10% (w / v) calcium chloride aqueous solution was added to syringe 2. Syringes 1 and 2 were injected together through coaxial needles, with the inner layer being the sodium alginate solution with dispersed short fibers and the outer layer being the calcium chloride aqueous solution. The injection speed of the inner and outer layers was 0.2mL / s to obtain a hydrogel. 0.5mL of the resulting hydrogel was immediately subjected to 30% deformation extrusion to observe whether the solution leaked under the extrusion.

[0087] Example 10

[0088] (1) Weigh regenerated silk fibroin, dissolve it in hexafluoroisopropanol (HFIP), and stir until it becomes clear and transparent to serve as the spinning outer layer solution. Maintain the final concentration of silk fibroin at 8% (w / w).

[0089] Blood was collected from rats and centrifuged twice to obtain platelet-rich plasma. Calcein powder was added to the platelet-rich plasma to obtain calcein-labeled platelet-rich plasma. The calcein-labeled platelet-rich plasma was mixed with a 5% polyvinyl alcohol aqueous solution in a ratio of 7:3 (v / v) to serve as the spinning inner layer solution.

[0090] (2) The outer layer solution and the inner layer solution were prepared into nanofiber membranes by coaxial electrospinning. The electrospinning conditions were set as follows: voltage 20 kV, outer layer flow rate 0.8 mL / h, inner layer flow rate 0.2 mL / h, needle distance 15 cm from aluminum foil flat plate receiver, relative temperature 30°C, relative humidity 30%. After the electrospinning was completed, the obtained nanofiber membrane was dried in a vacuum drying oven for 3 days to remove residual HFIP. The dried nanofiber membrane was cross-linked with ethanol gradient to increase its water insolubility. The conditions of ethanol gradient cross-linking were: first soaking in 100% ethanol for 10 minutes, then soaking in 90% ethanol for 10 minutes, then soaking in 70% ethanol for 10 minutes, and finally washing with pure water 3 times to remove ethanol, thus obtaining a water-insoluble nanofiber membrane.

[0091] (3) The water-insoluble nanofiber membrane was broken up and dispersed into short fibers using a homogenizer at a speed of 12,000 rpm for 15 min. The short fibers were first frozen at -40°C and then freeze-dried at -20°C in a vacuum environment for 24 h.

[0092] Example 11

[0093] Preparation of staple fibers: Regenerated silk fibroin and polycaprolactone were weighed at a ratio of 9:1 (w / w) and dissolved in hexafluoroisopropanol (HFIP). Stirring was performed until the solution became clear and transparent, maintaining a final silk fibroin concentration of 8% (w / w). The remaining steps were the same as in Example 10.

[0094] Example 12

[0095] Preparation of staple fibers: Regenerated silk fibroin and polycaprolactone were weighed in a ratio of 8:2 (w / w) and dissolved in hexafluoroisopropanol (HFIP). Stirring was performed until clear and transparent, resulting in the outer spinning solution. The final concentration of silk fibroin was maintained at 8% (w / w). The remaining steps were the same as in Example 10.

[0096] Example 13

[0097] Preparation of staple fibers: Regenerated silk fibroin and polycaprolactone were weighed in a ratio of 7:3 (w / w) and dissolved in hexafluoroisopropanol (HFIP). Stirring was performed until the solution became clear and transparent, maintaining a final silk fibroin concentration of 8% (w / w). The remaining steps were the same as in Example 10.

[0098] Table 1: Parameters related to Examples 1-5

[0099]

[0100] “\” indicates no relevant data.

[0101] Table 2: Parameters related to Examples 6-9

[0102]

[0103] Table 3: Parameters related to Examples 10-13

[0104]

[0105]

[0106] The above description is only a preferred embodiment of the present invention, which certainly cannot be used to limit the scope of rights of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and changes can be made without departing from the principles of the present invention. These improvements and changes are also considered to be within the scope of protection of the present invention.

Claims

1. An injectable nucleus pulposus repair product, characterized in that: include: Cross-linking agent and hydrogel precursor solution packaged independently of each other; After adding a cross-linking agent to the hydrogel precursor solution, a hydrogel can be quickly formed under mild conditions; Nanofibers having a shell layer and a core layer, wherein the shell layer is insoluble in water and the core layer contains a nucleus pulposus repair material; the nanofibers are independently packaged relative to the crosslinking agent and the hydrogel precursor solution, or are dispersed in the hydrogel precursor solution; The weight ratio of the hydrogel precursor to the nanofiber is 1:1 to 6:1; the hydrogel precursor solution is a sodium alginate solution, and the crosslinking agent is a powder or solution containing calcium ions; the mass concentration ratio of the sodium alginate solution to the crosslinking agent is 3:5 to 10; the shell layer of the nanofiber is formed by blending silk fibroin and polycaprolactone in a weight ratio of 8:2 to 9:1, and the blend is modified by gradient cross-linking with ethanol; When the nanofibers are packaged independently from the crosslinking agent and the hydrogel precursor solution, the nanofibers exist in the form of nanofiber membranes or short fibers. The nanofiber membranes are broken into short fibers before injection, and the core layers are exposed at both ends of the short fibers. When the nanofibers are dispersed in the hydrogel precursor solution, the nanofibers exist in the form of nanofiber membranes. Before injection, the nanofiber membranes in the hydrogel precursor solution are broken into short fibers.

2. The injectable nucleus pulposus repair product according to claim 1, characterized in that: The weight ratio of the shell layer to the core layer of the nanofiber is 1:1 to 10:

1.

3. The injectable nucleus pulposus repair product according to claim 1, characterized in that: The nucleus pulposus repair material comprises one or more of glucosamine, insulin-like growth factor, vascular endothelial growth factor, exosomes, transforming growth factor, and platelet-rich plasma; the core layer further comprises at least one of chitosan, collagen, polyethylene glycol, polyvinyl alcohol, silk fibroin, and polylactic acid.

4. A method for preparing the injectable nucleus pulposus repair product according to any one of claims 1 to 3, characterized in that: include: Nanofibrous membranes were prepared by coaxial electrospinning; The nanofiber membrane is broken into pieces to obtain short fibers.

5. The method for preparing an injectable nucleus pulposus repair product according to claim 4, characterized in that: Preparation of nanofibrous membranes by coaxial electrospinning includes: Silk fibroin and polycaprolactone in a weight ratio of 4 to 9:1 are dissolved in a solvent to prepare an electrospinning solution; coaxial electrospinning is performed to form a nanofiber membrane with silk fibroin and polycaprolactone as shell layers; and the nanofiber membrane is subjected to ethanol gradient cross-linking.

6. A bionic scaffold made of the injectable nucleus pulposus repair product according to any one of claims 1 to 3.

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