CN116115824A - An injection type nucleus pulposus repair product and its preparation method - Google Patents

Abstract

The invention discloses an injection type nucleus pulposus repairing product and a preparation method thereof, wherein the injection type nucleus pulposus repairing product comprises a cross-linking agent and a hydrogel precursor solution which are packaged independently; the hydrogel precursor solution can quickly form hydrogel under mild conditions after the cross-linking agent is added into the hydrogel precursor solution; a nanofiber having a shell layer and a core layer, the shell layer being insoluble in water, the core layer comprising a nucleus pulposus repair substance; the nanofibers are packaged separately from the crosslinker and the hydrogel precursor solution or dispersed in the hydrogel precursor solution. The repair product can be formed in vivo immediately during injection, so that gel leakage is prevented from being pressed to nerves; the nanofiber with the shell layer and the core layer can be loaded with various nucleus pulposus repairing substances and bioactive factors, and slow release is carried out from two ends of the nanofiber; the dispersed nano fiber can also improve the mechanical property of the hydrogel to 2 times of the original nucleus pulposus tissue and retain the toughness of the hydrogel, thereby realizing the dual restoration of mechanics and biology.

Description

An injection type nucleus pulposus repair product and its preparation method

technical field

The invention relates to the technical field of preparation of an artificial intervertebral disc nucleus pulposus support, in particular to an injection-type nucleus pulposus repair product and a preparation method thereof.

Background technique

Intervertebral disc degeneration is a relatively common disease in the world, and more than 500 million people in the world are suffering from intervertebral disc degeneration. At present, surgical operations such as nucleus pulposus excision, intervertebral disc fusion, and artificial intervertebral disc replacement are generally used clinically to treat intervertebral disc degeneration. These surgical operations without exception will cause new trauma and easily lead to complications. It is an unsolved problem in the treatment of intervertebral disc degeneration. There is no effective means to repair the degenerated and fragmented nucleus pulposus and reconstruct the biomechanical function of the intervertebral disc.

Although the development of tissue engineering regeneration technology has provided new technical means and prospects for the regeneration and repair of the nucleus pulposus of the intervertebral disc, due to the particularity of the anatomical position of the intervertebral disc, while bearing 80% of the pressure of the human body, there is still a spinal nerve running behind it, while The current products for repairing nucleus pulposus tissue have different effects. For example, platelet-rich plasma injection and high-strength hydrogel replacement only focus on biological repair or mechanical repair, and the effect on regeneration and repair of intervertebral disc is not ideal.

Contents of the invention

In order to solve the shortcomings of existing nucleus pulposus cartilage repair products, the present invention provides an injection-type nucleus pulposus repair product with both mechanical and biological repairs and a preparation method thereof.

The technical scheme provided by the invention is as follows:

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

A cross-linking agent and a hydrogel precursor solution packaged independently of each other; the hydrogel precursor solution can quickly form a hydrogel under mild conditions after adding a cross-linking agent;

A nanofiber with a shell and a core, the shell is insoluble in water, and the core contains a nucleus pulposus repairing substance; the nanofiber is packaged independently of the cross-linking agent and the hydrogel precursor solution, or dispersed in front of the hydrogel in body solution.

In some embodiments provided by the present invention, when the nanofibers are packaged independently of the crosslinking agent and the hydrogel precursor solution, the nanofibers exist in the form of nanofiber films or short fibers, and the nanofiber films are broken into short pieces before injection. fiber, the ends of the staple fiber expose the core layer.

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 a nanofiber film, and the nanofiber film in the hydrogel precursor solution is broken into short fibre.

In some embodiments provided by the present invention, the weight ratio of the hydrogel precursor to the nanofiber is 1:1˜6:1.

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

In some embodiments provided by the present invention, the shell layer of the nanofiber is polycaprolactone, chitosan, collagen, polyacrylonitrile, polyaniline, polyvinylpyrrolidone, polyethylene oxide, silk fibroin, poly At least one of lactic acid, 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.

In some embodiments provided by the present invention, the core layer further includes at least one of chitosan, collagen, polyethylene glycol, polyvinyl alcohol, silk fibroin, and polylactic acid as a dispersant for the nucleus pulposus repairing substance.

In some embodiments provided by the present invention, the hydrogel precursor solution is a sodium alginate solution, and the crosslinking agent is a calcium ion-containing powder or solution.

In some embodiments provided by the present invention, the mass concentration ratio of the sodium alginate solution and the crosslinking agent is 3:5-10.

In a second aspect, the present invention provides a method for preparing the above-mentioned injection-type nucleus pulposus repair product, comprising:

The nanofibrous membrane is prepared by coaxial electrospinning; the nanofibrous membrane is broken to obtain short fibers.

In some embodiments provided by the invention, the preparation of nanofibrous membranes by coaxial electrospinning includes:

Dissolve silk fibroin and polycaprolactone in a weight ratio of 4-9:1 together in a solvent to make an electrospinning solution; carry out coaxial electrospinning to form a shell with silk fibroin and polycaprolactone layer of nanofibrous membrane; the nanofibrous membrane was cross-linked with ethanol gradient.

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

In the third aspect, the present invention provides a bionic stent made by using the above injection-type nucleus pulposus repair product.

The injection-type 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, which is convenient and quick to use; the uniformly dispersed nano The fiber can enhance the compressive strength of the hydrogel, and at the same time, the water-insoluble shell of the nanofiber can prevent the early leakage of the nucleus pulposus repair substance in the core layer.

Description of drawings

Fig. 1 is the field emission electron microscope figure of the nanofiber prepared by embodiment 2,3 and hydrogel, and figure A is the field emission electron microscope figure of the nanofiber prepared by embodiment 2, and figure B is the resultant hydrogel of embodiment 3 Field emission electron microscope image, Figure C is a transmission electron microscope image of the nanofiber prepared in Example 2, and Figure D is a fluorescence confocal microscope image of the nanofiber prepared in Example 2.

Fig. 2 is the field emission electron microscope figure of the obtained hydrogel of the present invention, figure A is the field emission electron microscope figure of the hydrogel gained in embodiment 1, and figure B is the field emission electron microscope figure of the hydrogel gained in embodiment 2, figure C is a field emission electron microscope image of the hydrogel obtained in Example 3, and Figure D is a field emission electron microscope image of the hydrogel obtained in Example 4.

Fig. 3 is a display of the mechanical properties and biocompatibility of the hydrogel obtained in the present invention. Figures A and B are the mechanical compression curves and calculated elastic modulus of the hydrogels obtained in Examples 1, 2, 3, and 4. Figures C and D are the detection of live and dead cells of the hydrogel obtained in Example 3 and the results of fluorescent staining of cells cultured on the scaffold for 1, 3, and 5 days, Calcein-AM (green) staining of live cells, PI (red) of dead cells Staining, scale bar = 500 μm. Figure E is the CCK-8 kit detection of the hydrogel obtained in Example 3, and Figure F is the in vitro growth factor slow-release detection of the hydrogel obtained in Example 3.

Fig. 4 is the application of the injection-type nucleus pulposus repair product provided in Example 3 in the rat tail intervertebral disc degeneration model. After the rat tail intervertebral disc degeneration model was prepared, different products were injected into the degenerated intervertebral disc. Imaging X-rays, MRI and histological staining were performed at 4 and 8 weeks after operation, and no treatment measures were taken with uninjected products no control group. A is the X-ray examination images of the rat tail vertebrae at different times, B is the MRI scanning images of the rat tail vertebrae at different times, and C is the histological section stained with Safranin fast green of the rat tail vertebrae at different times.

Detailed ways

The present invention will be further described below in conjunction with the examples, and the description is only for better illustrating the technical solution of the present invention rather than limiting the claims. The invention is not limited to the particular examples and implementations described herein. Anyone skilled in the art can easily make further improvements and perfections without departing from the spirit and scope of the present invention, and all fall within the protection scope of the present invention.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods similar or equivalent to those described herein can be used in the practice or testing of the invention, the preferred methods are now described.

As used herein, numerical ranges are intended to include every value and subgroup within that range, whether specifically disclosed or not. Further, these numerical ranges should be construed as providing support for claims directed to any value or subgroup of values within the range.

Unless otherwise specified, numerical values presented herein should be up to and including the given endpoints.

The features and advantages of the present invention can be further understood through the following detailed description in conjunction with the accompanying drawings. But these specific embodiments do not limit the protection scope of the present invention in any way. The raw materials used in this embodiment are known compounds, which are commercially available.

The injection-type nucleus pulposus repair product provided by the present invention includes: a cross-linking agent and a hydrogel precursor solution packaged independently of each other, and the cross-linking agent and the hydrogel precursor solution are independently packaged or dispersed in the hydrogel precursor solution The nanofiber in; this nanofiber has shell and core layer, and its shell layer is insoluble in water, and core layer contains nucleus pulposus repair substance, when nanofiber is packaged independently with respect to cross-linking agent and hydrogel precursor solution, before use The nanofibers are firstly dispersed into the hydrogel precursor solution, and then a cross-linking agent is added. After the cross-linking agent is added to the hydrogel precursor solution, a hydrogel can be rapidly formed under mild conditions. The injection-type nucleus pulposus repair product provided by the present invention can be molded instantly in the body during injection, avoiding gel leakage and oppressing nerves; nanofibers with a shell and a core layer can be loaded with various nucleus pulposus repair substances and biologically active factors, Slow release from both ends of the nanofibers; the dispersed nanofibers can also increase the mechanical properties of the hydrogel to twice that of the original nucleus pulposus tissue and retain the toughness of the hydrogel, thereby achieving a dual repair of mechanics and biology.

The injectable form of the repair product can be suitable for the minimally invasive treatment of the intervertebral disc, and the material has a wide range of sources and good biocompatibility, and its composition cost and structure are similar to natural cartilage tissue. The release of nucleus pulposus repair substances in the body repairs and regenerates the cells of the degenerated intervertebral disc, and the treatment trauma is small, which is more conducive to the recovery of patients, and has a good clinical application prospect.

In some embodiments provided by the present invention, when the nanofibers are packaged independently of the crosslinking agent and the hydrogel precursor solution, the nanofibers exist in the form of nanofiber films or short fibers, and the nanofiber films are broken into short pieces before injection. fiber, the ends of the staple fiber expose the core layer. The nanofibers are packaged independently of the crosslinking agent and the hydrogel precursor solution, which avoids the early leakage of the nucleus pulposus repair substance. Before use, the nanofibers in the form of short fibers are directly dispersed in the hydrogel precursor solution, or The nanofibers existing in the form of nanofibrous film are broken into short fibers and then dispersed in the hydrogel precursor solution, and then injected together with the cross-linking agent to form the hydrogel. The exposed core layer at both ends of the short fiber slowly releases the contained nucleus pulposus repairing substances.

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 a nanofiber film, and the nanofiber film in the hydrogel precursor solution is broken into short fibre. Nanofibers exist in the hydrogel precursor solution in the form of nanofiber membranes, the exposed area of the core layer is small, and the leakage of nucleus pulposus repair substances is small. Before injection, the nanofiber membranes in the hydrogel precursor solution are broken into short fibers , to expose more of the core layer and improve the release ability of the nucleus pulposus repairing substances.

In some embodiments provided by the present invention, the weight ratio of the hydrogel precursor to the nanofiber is 1:1˜6:1. When the proportion of nanofibers is too low, the elastic modulus of the formed hydrogel is too small to achieve the use strength; when the proportion of nanofibers is too high, it is difficult to disperse uniformly in the hydrogel precursor solution.

In some embodiments provided by the present invention, the weight ratio of the shell layer to the core layer of the nanofiber is 1:1-10:1, and the length of the short fiber is 500nm-2000nm. When the proportion of the shell is too low, it is easy to be damaged, causing the nucleus pulposus repairing substances to explode from the damaged shell; when the proportion of the shell is too high, the shell degrades for a long time, and the nucleus pulposus repairing substances cannot be released through the shell at a later stage.

In some embodiments provided by the present invention, the shell layer of the nanofiber is polycaprolactone, chitosan, collagen, polyacrylonitrile, polyaniline, polyvinylpyrrolidone, polyethylene oxide, silk fibroin, poly At least one of lactic acid, 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. Preferably, the shell layer of the nanofiber is formed by blending silk fibroin and polycaprolactone at a weight ratio of 8:2 to 9:1, and the blend is modified by ethanol gradient crosslinking.

When the shell contains silk fibroin, the short fibers start to slowly release the nucleus pulposus repair substance from both ends. The release rate slowed down due to the decrease in the concentration of the nucleus pulposus repair substance.

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 for the nucleus pulposus repair substance and Fixing agent, these substances can fix and disperse the nucleus pulposus repairing substance in the inner spinning solution, which is convenient for coaxial spinning.

In some embodiments provided by the present invention, the hydrogel precursor solution is a sodium alginate solution, and the crosslinking agent is a calcium ion-containing powder or solution. Preferably, the sodium alginate aqueous solution has a concentration of 1% to 5% (w/v), and the crosslinking agent is calcium chloride aqueous solution, and the concentration thereof is 1% to 10% (w/v).

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.

In some embodiments provided by the present invention, the mass concentration ratio of the sodium alginate solution and the crosslinking agent is 3:5-10. When the proportion of cross-linking agent is too low, the hydrogel is easy to squeeze and leak.

In a second aspect, the present invention provides a method for preparing the above-mentioned injection-type nucleus pulposus repair product, comprising:

The nanofibrous membrane is prepared by coaxial electrospinning; the nanofibrous membrane is broken to obtain short fibers.

In some embodiments provided by the invention, the preparation of nanofibrous membranes by coaxial electrospinning includes:

Dissolve silk fibroin and polycaprolactone in a weight ratio of 4-9:1 together in a solvent to make an electrospinning solution; carry out coaxial electrospinning to form a shell with silk fibroin and polycaprolactone layer of nanofibrous membrane; the nanofibrous membrane was cross-linked with ethanol gradient.

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

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

In some embodiments provided by the present invention, the conditions of mechanical ball milling are 200-2000 rpm, 10-20 minutes; the conditions of ultrasonic homogenization are 8000-12000 rpm, 5-30 minutes.

In the third aspect, the present invention provides a biomimetic stent made by using the above-mentioned injectable nucleus pulposus repair product, and the compressive strength of the biomimetic stent is 170% of that of ordinary sodium alginate hydrogel. And the dual structure of short fiber-hydrogel realizes long-term sustained release of nucleus pulposus repairing substances.

Unless otherwise specified, the regenerated silk fibroin in the following examples are all prepared according to the method disclosed in CN109999227A.

Example 1

Weigh 0.3 g of sodium alginate, add water to a volume of 10 mL, stir and mix mechanically to obtain a sodium alginate solution with a concentration of 3% (w/v). Add sodium alginate solution into syringe 1, add calcium chloride aqueous solution with a concentration of 10% (w/v) into syringe 2, inject syringe 1 and syringe 2 together through a coaxial needle, and the inner layer is sodium alginate Solution, the outer layer is calcium chloride aqueous solution, the injection speed of the inner layer and the outer layer is 0.2mL/s, and the field emission electron microscope image of the obtained hydrogel is shown in Figure 2A.

Example 2

(1) Weigh the regenerated silk fibroin and polycaprolactone according to silk fibroin:polycaprolactone=8:2 (w/w), and dissolve the two together in the organic reagent hexafluoroisopropanol (HFIP), Stir until clear and transparent, and keep the final concentration of silk fibroin at 8% (w/w) as the spinning outer layer solution.

Blood was collected from rats and centrifuged twice to obtain platelet-rich plasma, and calcein powder was added to platelet-rich plasma to obtain calcein-labeled platelet-rich plasma; : 3 (v/v) ratio mixed as spinning inner layer solution.

(2) Prepare the spinning outer layer solution and the spinning inner layer solution into nanofiber membranes by coaxial electrospinning. The electrospinning conditions are set as: voltage 20kV, outer layer flow rate 0.8mL/h, inner layer flow rate 0.2mL/h, the needle is 15cm away from the aluminum foil plate receiver, the relative temperature is 30°C, and the relative humidity is 30%. After the electrospinning was completed, the obtained nanofiber membrane was dried in a vacuum oven for 3 days to remove residual HFIP. At this time, the field emission scanning electron microscope image of the nanofiber membrane is shown in Figure 1A. The dry nanofibrous membrane is cross-linked with ethanol gradient to increase its water insolubility. The conditions of ethanol gradient cross-linking are: first soak in 100% ethanol for 10 minutes, then soak in 90% ethanol for 10 minutes, then soak in 70% ethanol for 10 minutes, Finally, it was washed with pure water for 3 times to remove ethanol, and a water-insoluble nanofibrous membrane was obtained. At this time, the structure of the nanofibrous membrane was observed through a transmission electron microscope (FIG. 1C), and a relatively uniform distribution of inner and outer layers could be observed. Further observation by confocal microscopy showed that the calcein-labeled platelet-rich plasma was uniformly distributed in the inner layer of the nanofibers (Fig. 1D).

(3) The water-insoluble nanofibrous membrane was crushed and dispersed into short fibers with a homogenizer, the homogenization speed was 12000 rpm, and the homogenization time was 15 minutes. The short fibers were first frozen at -40°C, and then continuously freeze-dried at -20°C for 24 hours in a vacuum environment for later use.

(4) Weigh sodium alginate and short fiber according to the weight ratio of sodium alginate:short fiber=6:1, that is, weigh 0.3g sodium alginate and 0.05g short fiber, add water until the solution volume is 10mL, mechanically stir and mix Homogenize to obtain a sodium alginate solution dispersed with short fibers. The sodium alginate solution dispersed with short fibers was added to syringe 1, and the calcium chloride aqueous solution with a concentration of 10% (w/v) was added to syringe 2, and syringe 1 and syringe 2 were injected together through a coaxial needle, and the inner The layer is a sodium alginate solution dispersed with short fibers, the outer layer is a calcium chloride aqueous solution, and the injection speed of both the inner and outer layers is 0.2mL/s. The field emission electron microscope image of the obtained hydrogel is shown in Figure 2B.

Example 3

Short fibers were prepared according to the same method and parameters as in Example 2.

Weigh sodium alginate and short fiber according to the ratio of sodium alginate:short fiber = 3:1, that is, weigh 0.3g sodium alginate and 0.1g short fiber, add water until the volume of the solution is 10mL, mechanically stir and mix to obtain a dispersion Sodium alginate solution with short fibers. The sodium alginate solution dispersed with short fibers was added to syringe 1, and the calcium chloride aqueous solution with a concentration of 10% (w/v) was added to syringe 2, and syringe 1 and syringe 2 were injected together through a coaxial needle, and the inner The layer is a sodium alginate solution dispersed with short fibers, the outer layer is a calcium chloride aqueous solution, and the injection speed of the inner and outer layers is both 0.2mL/s. The field emission electron microscope image of the obtained hydrogel is shown in Figure 1B. The gel was porous, and the pore walls between the pores were connected to each other, short fibers intersected among them, and a double-layer fiber structure could be seen, and the gel had good compression resistance (Figure 3A and 3B).

Cell experiments:

After the co-culture of the hydrogel obtained in this example and the nucleus pulposus cells for 1, 3, and 5 days, the living and dead cell staining test was carried out. From the fluorescent confocal observation image (Fig. 3C), it can be seen that the living cells and dead cells in the hydrogel Viable cells grew on all the hydrogels, and the hydrogels had the ability to support cell adhesion and growth, and the survival rate of cells on the hydrogels was not less than 94% (Fig. 3D). The test results of CCK-8 kit showed that the hydrogel had no cytotoxicity (Fig. 3E). In addition, with the increase of co-culture time, the cell proliferation was obvious. The Elisa kit was used to detect the slow-release ability of growth factors in vitro of the hydrogel. The results showed (Fig. 3F) that TGF-β1, PDGF-BB, IGF-1 and VEGF released from platelet-rich plasma could be released for a long time up to 40 days.

Animal experiment:

Prepare the rat tail intervertebral disc degeneration model, take the untransplanted nucleus pulposus repair product and do not take treatment as the control group (Control), the nucleus pulposus repair product (ExpA) prepared by this embodiment, the core layer prepared by this embodiment are not PRP-containing nucleus pulposus repair product (ExpB) and pure alginate hydrogel (ExpC) were injected into rats with caudal disc degeneration. Radiological X-ray and MRI examinations and tissue samples were obtained for histological staining at 4 and 8 weeks, respectively. As shown in Figure 4A and Figure 4B, the intervertebral disc signal gradually became lower in the Control group at 4 weeks and 8 weeks, the intervertebral disc height was lost, the gap collapsed, and the extracellular matrix was decomposed and lost. In the ExpA group, the signal of the intervertebral disc recovered, the height of the intervertebral disc gradually recovered, the gap maintained the basic level, and the extracellular matrix recovered at the 8th week after the operation and the 4th week after the operation. Although the degeneration of the ExpB group and ExpC group improved at the 4th week, the improvement was not obvious at the 8th week.

Example 4

Short fibers were prepared according to the same method and parameters as in Example 2.

Weigh sodium alginate and short fiber according to the ratio of sodium alginate:short fiber = 1:1, that is, weigh 0.3g sodium alginate and 0.3g short fiber, add water until the volume of the solution is 10mL, stir and mix mechanically to obtain a dispersion Sodium alginate solution with short fibers. The sodium alginate solution dispersed with short fibers was added to syringe 1, and the calcium chloride aqueous solution with a concentration of 10% (w/v) was added to syringe 2, and syringe 1 and syringe 2 were injected together through a coaxial needle, and the inner The layer is a sodium alginate solution dispersed with short fibers, the outer layer is a calcium chloride aqueous solution, and the injection speed of both the inner and outer layers is 0.2mL/s. The field emission electron microscope image of the obtained hydrogel is shown in Figure 2D.

Example 5

Short fibers were prepared according to the same method and parameters as in Example 2.

Weigh sodium alginate and short fiber according to the ratio of sodium alginate:short fiber = 1:1, that is, weigh 0.3g sodium alginate and 0.6g short fiber, add water until the volume of the solution is 10mL, mechanically stir and mix to obtain a dispersion Sodium alginate solution with short fibers. The sodium alginate solution dispersed with short fibers was added to syringe 1, and the calcium chloride aqueous solution with a concentration of 10% (w/v) was added to syringe 2, and syringe 1 and syringe 2 were injected together through a coaxial needle, and the inner The layer is sodium alginate solution dispersed with short fibers, the outer layer is calcium chloride aqueous solution, and the injection speed of both the inner layer and the outer layer is 0.2mL/s to obtain a hydrogel.

Example 6

Short fibers were prepared according to the same method and parameters as in Example 2.

Weigh sodium alginate and short fiber according to the ratio of sodium alginate:short fiber = 3:1, that is, weigh 0.3g sodium alginate and 0.1g short fiber, add water until the volume of the solution is 10mL, mechanically stir and mix to obtain a dispersion Sodium alginate solution with short fibers. The sodium alginate solution dispersed with short fibers is added to syringe 1, and the calcium chloride aqueous solution with a concentration of 1% (w/v) is added to syringe 2, and syringe 1 and syringe 2 are injected together through a coaxial needle, and the inner The layer is sodium alginate solution dispersed with short fibers, the outer layer is calcium chloride aqueous solution, and the injection speed of both the inner layer and the outer layer is 0.2mL/s to obtain a hydrogel. Take 0.5 mL of the obtained hydrogel and immediately perform 30% deformation extrusion, and observe whether the solution leaks under the extrusion.

Example 7

Short fibers were prepared according to the same method and parameters as in Example 2.

Weigh sodium alginate and short fiber according to the ratio of sodium alginate:short fiber = 3:1, that is, weigh 0.3g sodium alginate and 0.1g short fiber, add water until the volume of the solution is 10mL, mechanically stir and mix to obtain a dispersion Sodium alginate solution with short fibers. The sodium alginate solution dispersed with short fibers was added to syringe 1, and the calcium chloride aqueous solution with a concentration of 3% (w/v) was added to syringe 2, and syringe 1 and syringe 2 were injected together through a coaxial needle, and the inner The layer is sodium alginate solution dispersed with short fibers, the outer layer is calcium chloride aqueous solution, and the injection speed of both the inner layer and the outer layer is 0.2mL/s to obtain a hydrogel. Take 0.5 mL of the obtained hydrogel and immediately perform 30% deformation extrusion, and observe whether the solution leaks under the extrusion.

Example 8

Short fibers were prepared according to the same method and parameters as in Example 2.

Weigh sodium alginate and short fiber according to the ratio of sodium alginate:short fiber = 3:1, that is, weigh 0.3g sodium alginate and 0.1g short fiber, add water until the volume of the solution is 10mL, mechanically stir and mix to obtain a dispersion Sodium alginate solution with short fibers. The sodium alginate solution dispersed with short fibers is added to syringe 1, and the calcium chloride aqueous solution with a concentration of 5% (w/v) is added to syringe 2, and syringe 1 and syringe 2 are injected together through a coaxial needle, and the inner The layer is sodium alginate solution dispersed with short fibers, the outer layer is calcium chloride aqueous solution, and the injection speed of both the inner layer and the outer layer is 0.2mL/s to obtain a hydrogel. Take 0.5 mL of the obtained hydrogel and immediately perform 30% deformation extrusion, and observe whether the solution leaks under the extrusion.

Example 9

Short fibers were prepared according to the same method and parameters as in Example 2.

Weigh sodium alginate and short fiber according to the ratio of sodium alginate:short fiber = 3:1, that is, weigh 0.3g sodium alginate and 0.1g short fiber, add water until the volume of the solution is 10mL, mechanically stir and mix to obtain a dispersion Sodium alginate solution with short fibers. The sodium alginate solution dispersed with short fibers was added to syringe 1, and the calcium chloride aqueous solution with a concentration of 10% (w/v) was added to syringe 2, and syringe 1 and syringe 2 were injected together through a coaxial needle, and the inner The layer is sodium alginate solution dispersed with short fibers, the outer layer is calcium chloride aqueous solution, and the injection speed of both the inner layer and the outer layer is 0.2mL/s to obtain a hydrogel. Take 0.5 mL of the obtained hydrogel and immediately perform 30% deformation extrusion, and observe whether the solution leaks under the extrusion.

Example 10

(1) Weigh the regenerated silk fibroin, dissolve it in hexafluoroisopropanol (HFIP), stir until it is clear and transparent as the spinning outer layer solution, and keep the final concentration of silk fibroin at 8% (w/w).

Blood was collected from rats and centrifuged twice to obtain platelet-rich plasma, and calcein powder was added to platelet-rich plasma to obtain calcein-labeled platelet-rich plasma; : 3 (v/v) ratio mixed as spinning inner layer solution.

(2) Prepare the spinning outer layer solution and the spinning inner layer solution into nanofiber membranes by coaxial electrospinning. The electrospinning conditions are set as: voltage 20kV, outer layer flow rate 0.8mL/h, inner layer flow rate 0.2mL/h, the needle is 15cm away from the aluminum foil plate receiver, the relative temperature is 30°C, and the relative humidity is 30%. After the electrospinning was completed, the resulting nanofibrous membrane was dried in a vacuum oven for 3 days to remove residual HFIP. The dry nanofibrous membrane is cross-linked with ethanol gradient to increase its water insolubility. The conditions of ethanol gradient cross-linking are: first soak in 100% ethanol for 10 minutes, then soak in 90% ethanol for 10 minutes, then soak in 70% ethanol for 10 minutes, Finally, it was washed with pure water for 3 times to remove ethanol, and a water-insoluble nanofibrous membrane was obtained.

(3) The water-insoluble nanofibrous membrane was crushed and dispersed into short fibers with a homogenizer, the homogenization speed was 12000 rpm, and the homogenization time was 15 minutes. The short fibers were first frozen at -40°C, and then continuously freeze-dried at -20°C under vacuum for 24 hours.

Example 11

Prepare short fibers: take regenerated silk fibroin and polycaprolactone according to the ratio of silk fibroin:polycaprolactone=9:1 (w/w), dissolve the two in hexafluoroisopropanol (HFIP), Stir until clear and transparent as the spinning outer layer solution, keeping the final concentration of silk fibroin at 8% (w/w). Remaining steps are identical with embodiment 10.

Example 12

Preparation of short fibers: Take regenerated silk fibroin and polycaprolactone according to the ratio of silk fibroin:polycaprolactone=8:2 (w/w), dissolve the two in hexafluoroisopropanol (HFIP), Stir until clear and transparent as the spinning outer layer solution, keeping the final concentration of silk fibroin at 8% (w/w). Remaining steps are identical with embodiment 10.

Example 13

Preparation of short fibers: Take regenerated silk fibroin and polycaprolactone according to the ratio of silk fibroin:polycaprolactone=7:3 (w/w), dissolve the two in hexafluoroisopropanol (HFIP), Stir until clear and transparent as the spinning outer layer solution, keeping the final concentration of silk fibroin at 8% (w/w). Remaining steps are identical with embodiment 10.

Table 1: Relevant parameters of Examples 1-5

"\" means no relevant data.

Table 2: Relevant parameters of embodiments 6-9

Table 3: Relevant parameters of Examples 10-13

The above description is only a preferred embodiment of the present invention, and of course the scope of rights of the present invention cannot be limited by this. It should be pointed out that for those of ordinary skill in the art, they can also Several improvements and changes are made, and these improvements and changes are also regarded as the protection scope of the present invention.

Claims (10)

1. An injectable nucleus repair product comprising:

a cross-linking agent and a hydrogel precursor solution packaged independently of each other; the hydrogel precursor solution can quickly form hydrogel under mild conditions after the cross-linking agent is added into the hydrogel precursor solution;

a nanofiber having a shell layer and a core layer, the shell layer being insoluble in water, the core layer comprising a nucleus pulposus repair substance; the nanofibers are packaged separately from the crosslinker and the hydrogel precursor solution or dispersed in the hydrogel precursor solution.

2. The injectable nucleus repair product of claim 1, wherein: when the nanofiber is independently packaged relative to the cross-linking agent and the hydrogel precursor solution, the nanofiber exists in the form of a nanofiber membrane or a short fiber, the nanofiber membrane is broken into the short fiber before injection, and the two ends of the short fiber are exposed out of the core layer.

3. The injectable nucleus repair product of claim 1, wherein: 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.

4. The injectable nucleus repair product of claim 1, wherein: the weight ratio of the hydrogel precursor to the nanofiber is 1:1-6:1; the weight ratio of the shell layer to the core layer of the nanofiber is 1:1-10:1.

5. The injectable nucleus repair product of claim 1, wherein: the shell layer of the nanofiber is at least one of polycaprolactone, chitosan, collagen, polyacrylonitrile, polyaniline, polyvinylpyrrolidone, polyethylene oxide, silk fibroin and polylactic acid, and the nucleus pulposus repairing substance comprises one or more of glucosamine, insulin-like growth factors, vascular endothelial growth factors, exosomes, transforming growth factors and platelet-rich plasma; the core layer also comprises at least one of chitosan, collagen, polyethylene glycol, polyvinyl alcohol, silk fibroin and polylactic acid.

6. The injectable nucleus repair product of claim 1, wherein: the hydrogel precursor solution is sodium alginate solution, and the cross-linking agent is calcium ion-containing powder or solution.

7. The injectable nucleus repair product of claim 6, wherein: the mass concentration ratio of the sodium alginate solution to the cross-linking agent is 3:5-10.

8. A method of preparing the injectable nucleus repair product of any one of claims 1-7, comprising:

preparing a nanofiber membrane by coaxial electrostatic spinning; breaking the nanofiber membrane to obtain the short fibers.

9. The method of preparing an injectable nucleus repair product according to claim 8 wherein: the preparation of nanofiber membranes by in-line electrospinning comprises:

the silk fibroin and polycaprolactone with the weight ratio of 4-9:1 are dissolved in a solvent together to prepare an electrostatic spinning solution; carrying out coaxial electrostatic spinning to form a nanofiber membrane taking silk fibroin and polycaprolactone as shell layers; the nanofiber membrane was subjected to ethanol gradient crosslinking.

10. A biomimetic scaffold made using the injectable nucleus repair product of any one of claims 1-7.

Images