Neural repair stent and preparation method thereof

Abstract

The invention belongs to the technical field of biological materials, and particularly relates to a nerve repair scaffold and a preparation method thereof.The nerve repair scaffold is of a hollow double-shell fiber structure, the outer layer of the double-shell fiber structure is composed of PCL nanofiber filaments, the inner layer of the double-shell fiber structure comprises PCL-dECM fiber filaments, and the outer layer of the double-shell fiber structure is composed of PCL nanofiber filaments. The PCL-dECM cellosilk is a PCL cellosilk loaded with dECM, the PCL-dECM cellosilk of the inner layer is arranged in the axial direction of the double-shell fiber structure, and after the nerve repair stent is implanted, the nerve function recovery can be improved, and the biocompatibility is improved.

Description

Technical Field

[0001] The present application relates to the technical field of biomaterials, and more specifically, to a nerve repair scaffold and a preparation method thereof. Background Art

[0002] Peripheral nerve injury not only causes loss of motor function in the innervated area, but is also often accompanied by neuropathic pain, which seriously affects the patient's quality of life. It is a global problem in the field of nerve injury. For patients who cannot be repaired by tension-free suture, autologous transplantation can be used for nerve reconstruction, which is the "gold standard" in current nerve repair practice. However, there are many challenges in clinical practice, such as limited donor nerve resources and difficulty in obtaining nerve tissue of appropriate size; and secondary surgery is often required, as well as possible complications at the donor site, which limit its promotion in clinical practice.

[0003] Therefore, the use of functional biomaterials to replace autologous transplantation has been widely developed, including many commercialized nerve guide catheters. Although various synthetic polymer materials, natural materials and their composites have been developed, the restoration of neural function after implantation is still unsatisfactory due to the lack of bioactive molecules and the lack of topological guidance at the cellular level. Studies have shown that the construction of similar tissue microenvironments in catheters can be achieved by constructing extracellular matrix (ECM) mimicking substrates or cell-laden substrates. However, due to problems such as incomplete adhesion, lack of cell-matrix signaling and cell behavior defects, there are only a few successful examples of specific materials. Summary of the invention

[0004] The present application is provided to solve the above defects in the prior art. A nerve repair scaffold and a preparation method thereof are needed, which can improve the recovery of nerve function and improve biocompatibility after implantation.

[0005] In a first aspect of the present application, a nerve repair scaffold is provided, which is a hollow double-shell fiber structure, the outer layer of the double-shell fiber structure is composed of PCL nanofiber filaments, the inner layer of the double-shell fiber structure includes PCL-dECM fiber filaments, and the PCL-dECM fiber filaments are PCL fiber filaments loaded with dECM, and the PCL-dECM fiber filaments of the inner layer are arranged along the axial direction of the double-shell fiber structure.

[0006] In a second aspect of the present application, a method for preparing a nerve repair scaffold as described in any embodiment of the present application is provided, the preparation method comprising: preparing PCL nanofiber filaments by an electrospinning method; inoculating ganglia onto the PCL nanofiber filaments and culturing them to obtain PCL nanofiber filaments with precipitated ECM after culturing; decellularizing the PCL nanofiber filaments with precipitated ECM to obtain PCL fiber filaments loaded with dECM; preparing orderly arranged PCL-dECM fiber filaments by re-dissolving the PCL fiber filaments loaded with dECM using an electrohydrodynamic jet three-dimensional printing method; winding the PCL-dECM fiber filaments parallel to the extension direction on a metal collecting rod to obtain an inner layer of a double-layer fiber structure; preparing an outer layer of PCL nanofiber filaments on the outer surface of the inner layer by an electrospinning method to obtain a nerve repair scaffold.

[0007] The nerve repair scaffold and preparation method thereof provided in each embodiment of the present application have a double-layer structure, the outer layer is PCL nanofiber filaments, which have good biological stability, greatly prolonging the repair time of the nerve repair scaffold and ensuring its in vivo repair effect; the inner layer is axially arranged PCL-dECM fiber filaments, and the axially arranged microchannels promote the extension of neurites and the migration of Schwann cells, accelerating nerve repair; and the organic polymer matrix selected for the scaffold has high biocompatibility, avoiding the immune rejection reaction caused by interventional surgery, making it have a wide range of use value. BRIEF DESCRIPTION OF THE DRAWINGS

[0008] In the drawings, which are not necessarily drawn to scale, the same reference numerals may describe similar parts in different views. The same reference numerals with letter suffixes or different letter suffixes may represent different instances of similar parts. The accompanying drawings generally illustrate various embodiments by way of example and not limitation, and together with the specification and claims, are used to illustrate the embodiments applied for. When appropriate, the same reference numerals are used throughout the drawings to refer to the same or similar parts. Such embodiments are illustrative and are not intended to be exhaustive or exclusive embodiments of the present apparatus or method.

[0009] Figure 1 A simulated structural diagram of a nerve repair stent according to an embodiment of the present application is shown; Figure 2 A SEM scan of a longitudinal section of a nerve repair scaffold according to an embodiment of the present application is shown; Figure 3 A SEM scan of a cross section of a nerve repair scaffold according to an embodiment of the present application is shown; Figure 4 The images of the injured sciatic nerve of a rat transplanted with a nerve repair scaffold according to an embodiment of the present application are shown, wherein the left image is an image taken at 0 weeks after transplantation, and the right image is an image taken at 12 weeks after transplantation; Figure 5 A comparison diagram showing the recovery of the gastrocnemius muscle connected to the sciatic nerve after the sciatic nerve injury site in rats was transplanted with a nerve repair scaffold according to an embodiment of the present application. DETAILED DESCRIPTION

[0010] In order to enable those skilled in the art to better understand the technical solution of the present application, the present application is described in detail below in conjunction with the accompanying drawings and specific implementation examples. The embodiments of the present application are further described in detail below in conjunction with the accompanying drawings and specific implementation examples, but are not intended to limit the present application.

[0011] The words "first", "second" and similar words used in this application do not indicate any order, quantity or importance, but are only used to distinguish. The words "include" or "comprise" and similar words mean that the elements before the word include the elements listed after the word, and do not exclude the possibility of including other elements.

[0012] like Figure 1 As shown, according to an embodiment of the present application, a nerve repair stent is provided, wherein the nerve repair stent is a hollow double-shell fiber structure, the outer layer of the double-shell fiber structure is composed of PCL (polycaprolactone) nanofiber filaments, the inner layer of the double-shell fiber structure includes PCL-dECM fiber filaments, and the PCL-dECM fiber filaments are PCL fiber filaments loaded with dECM (decellularized matrix), and the PCL-dECM fiber filaments of the inner layer are arranged along the axial direction of the double-shell fiber structure.

[0013] The PCL fibers in the outer layer have high strength and toughness, which can effectively protect the ordered structure of the inner layer and adapt to the complex environment of the implantation process in the body. When implanted in the body, they can maintain the original form of the nerve conduit for a long time, provide a strong physical environment guarantee for axon extension and myelination, and avoid immune rejection in the body. The slow degradation rate of PCL can avoid the rapid loss of dECM and prolong the duration of action.

[0014] The inner layer of PCL-dECM fibers are arranged axially to form an inner layer with a clue structure, which can effectively simulate the topological clues of natural neurofibromin cables, and construct a guiding configuration in the cavity to better guide and promote nerve regeneration. Adjacent PCL-dECM fibers have spacing to form microchannels, which can effectively guide and significantly promote the extension of neurites and the migration of Schwann cells, and the dECM components on the surface of the fibers can significantly induce axon extension and myelination.

[0015] dECM is a more complex complex of bioactive components, including collagen, fibronectin, glycosaminoglycans and many cytokines from the natural extracellular matrix (ECM), which can provide a relaxed microenvironment for cell proliferation, migration and differentiation; it can also enhance the affinity of cells to synthetic materials, reduce the physical and mechanical limitations of dECM, retain its biological activity, and can significantly promote axon extension, myelin formation, and achieve a considerable degree of functional recovery.

[0016] The PCL fibers and PCL-dECM fibers included in the nerve repair scaffold have good biocompatibility and can avoid immune rejection reactions caused by interventional surgery.

[0017] In some embodiments, the diameter of the PCL-dECM fiber is 5-10 μm, and the spacing between adjacent PCL-dECM fiber is 20-40 μm. PCL-dECM fiber is ultra-fine fiber, and the spacing between each fiber is controlled at about 30 μm, which is not only the best condition for protecting mature axons, but also the diameter is close to the diameter of a single neuron axon, which is more conducive to axon extension and myelination.

[0018] In some embodiments, the outer diameter of the nerve repair scaffold is 1-3 mm, that is, the total outer diameter of the double-shell fiber structure formed by the PCL nanofibers covering the PCL-dECM fiber.

[0019] Fiber filaments with corresponding diameters and gaps can be designed according to different injury sites, different damage modes, and different cell growth environments to prepare nerve repair scaffolds with different pore sizes and lengths.

[0020] In some embodiments, the thickness of the outer layer of the nerve repair scaffold is 90-110 μm. This can improve the mechanical support effect of the nerve repair scaffold. In some embodiments, the inner layer of the nerve repair scaffold is loaded with nutrient factors, so as to achieve the effect of nerve repair through the dual effects of the nerve repair scaffold on the spatial structure of the repair position and the stimulation of nutrient factors.

[0021] According to an embodiment of the present application, there is also provided a method for preparing a nerve repair scaffold as described in any embodiment of the present application, the preparation method comprising: (1) preparing PCL nanofiber filaments by an electrospinning method; (2) inoculating ganglia onto the PCL nanofiber filaments and culturing them to obtain PCL nanofiber filaments with precipitated ECM after culturing; (3) decellularizing the PCL nanofiber filaments with precipitated ECM to obtain a preparation stock solution of PCL fiber filaments loaded with dECM; (4) redissolving the PCL fiber filaments loaded with dECM and preparing orderly arranged PCL-dECM fiber filaments by electrohydrodynamic jet three-dimensional printing; (5) winding the PCL-dECM fiber filaments parallel to the extension direction on a metal collecting rod to obtain an inner layer of a double-layer fiber structure; (6) preparing an outer layer of PCL nanofiber filaments on the outer surface of the inner layer by an electrospinning method to obtain a nerve repair scaffold.

[0022] Dorsal root ganglia cultured on the surface of PCL nanofibers can form a good extracellular matrix by utilizing the biocompatibility with PCL nanofibers. The morphology and structure contained in it can regulate cell adhesion, gene expression and differentiation, and the decellularized matrix retains some biochemical complexity, which is better than the biological activity of a simple PCL scaffold. The PCL scaffold containing ECM was decellularized and dissolved and re-prepared into a new scaffold. The test results showed that some decellularized matrix components had not been lost, which indicates the feasibility of this method. Compared with traditional surface adhesion or hydrogel perfusion, this method greatly increases the effective time of nerve repair and overcomes the shortcomings of easy loss of effective components of surface adhesion and poor biomechanical properties of perfusion hydrogel.

[0023] Using 3D printing, the PCL solution containing dECM components is printed into ultra-fine fiber filaments. The 3D printing preparation method is controllable, and fiber filaments with a certain directionality can be prepared according to requirements, which can further induce the migration of Schwann cells and thus accelerate nerve repair.

[0024] Cultivating cells on PCL fibers for deposition and then decellularizing the culture can maximize the retention of the active ingredients of dECM.

[0025] In some embodiments, the ganglia may include primary rat dorsal root ganglia, etc.

[0026] In some embodiments, the ganglia are seeded onto the PCL fibers and cultured for 20-35 days.

[0027] In some embodiments, the PCL fiber filaments with precipitated ECM are decellularized, including: the PCL fiber filaments with precipitated ECM are sequentially immersed in mixed solution A and mixed solution B for initial treatment, wherein the mixed solution A includes PBS, NaCl and sodium 3-(decyldimethylamino)propanesulfonate, and the mixed solution B includes sodium deoxycholate and SB-16 (sulfobetaine-16) to obtain PCL-dECM fiber filaments after initial treatment; the PCL-dECM fiber filaments after initial treatment are gradiently dehydrated and dried using ethanol to obtain PCL fiber filaments loaded with dECM after decellularization.

[0028] In some embodiments, mixed solution A includes mixed solution E and mixed solution F, mixed solution E and mixed solution F are mixed to obtain mixed solution A, mixed solution E includes 4-6mM (mmol / L) PBS and 65-70mM (mmol / L) NaCl; mixed solution F includes 1.2-1.3 M sodium 3-(decyldimethylamino)propanesulfonate. The volume ratio of mixed solution E to mixed solution F is 9:1. The solvent of mixed solution E and mixed solution F can be water.

[0029] In some embodiments, the mass fraction of sodium deoxycholate in the mixed solution B is 0.1-0.2%, and the concentration of SB-16 is 0.4-0.8 mM (mmol / L). The solvent in the mixed solution B can be water.

[0030] The initial treatment process of mixed solution A and mixed solution B can be performed only once or repeated twice.

[0031] In some embodiments, the mass fraction of ethanol used for gradient elution is 70%-100%.

[0032] In some embodiments, orderly arranged PCL-dECM fiber filaments are prepared by redissolving the dECM-loaded PCL fiber filaments using an electrohydrodynamic jet 3D printing method, including dissolving the dECM-loaded PCL fiber filaments in a mixed solution C, wherein the mixed solution C includes HFIP, formic acid and DMSO to obtain a mixed solution D; then, based on the mixed solution D, orderly arranged PCL-dECM fiber filaments are printed using a 3D printing method.

[0033] In some embodiments, the conditions for electrohydrodynamic jet 3D printing of PCL-dECM filaments include: an extrusion flow rate of 0.1-0.3 mL / h, a local voltage of the jet needle of 2-4 kV, and a collection distance controlled at 1-3 mm.

[0034] In some embodiments, the volume ratio of HFIP, formic acid and DMSO in the mixed solution C is 6-9:1:1. The mixed solution C is used to dissolve the PCL fibers loaded with dECM. This method is extremely gentle and reduces the damage of organic solvents to the active ingredients.

[0035] In some embodiments, the mass volume ratio of PCL in the mixed solution D is 12-18%, and the mass percentage of dECM in the mixed solution D is 5-15%. The solvent of the mixed solution D can be water.

[0036] In some embodiments, the method for preparing an outer layer of PCL nanofiber filaments on the outer surface of the inner layer by electrospinning includes: fixing a metal rod with PCL-dECM fiber filaments on a rotating collection device; dissolving PCL in a mixed solution of DMF and DCM, and extruding it using an electrospinning machine while the rotating collection device rotates.

[0037] In some embodiments, the extrusion conditions include: a voltage of 10-13 kV, an extrusion flow rate of 0.8-1.5 mL / h, and a collection distance controlled at 12-17 cm.

[0038] In some embodiments, PCL is dissolved in a mixed solution of DMF and DCM, wherein the volume ratio of DMF to DCM in the mixed solution of DMF and DCM is 5-7:4; after dissolution, the mass volume ratio of PCL to the mixed solution of DMF and DCM is 5-10%.

[0039] Example 1 1) Preparation of raw materials for the scaffold: Preparation of PCL electrospinning scaffold: Dissolve PCL powder in a mixed solution of DMF and DCM with a volume ratio of 6:4, stir and dissolve to obtain a PCL solution with a mass volume ratio of 7%, draw it out from the syringe and place it in the electrospinning machine, adjust the voltage to 12kV, the extrusion flow rate to 1mL / h, control the collection distance to 15cm, and obtain a planar PCL electrospinning scaffold after collecting for 1h. It was tested that the diameter of the PCL nanofibers was about 200nm. Disinfect with 70% ethanol and wash with PBS. The cultured dorsal root ganglia of primary rats were inoculated onto the PCL fibers and cultured for 28 days to observe the secretion of ECM. After 28 days of culture, the PCL fibers with precipitated ECM were decellularized. The steps are as follows: the scaffold was washed three times with PBS, immersed in a mixed solution of 5 mM PBS and 68.5 mM NaCl, and 1.25 M sodium 3-(decyldimethylamino)propanesulfonate solution was added under gentle stirring at 4 °C, and stirred overnight. Then, the PCL fibers with precipitated ECM were washed three times with PBS, placed in a mixed solution containing 0.14% sodium deoxycholate and 0.6 mM SB-16, and stirred gently at 4 °C overnight. The above steps were repeated twice and stored under gentle stirring at 4 °C overnight. The PCL fibers with precipitated ECM of about 50 coverslips were collected, dehydrated with 70%-100% ethanol gradient, and stored in a drying oven overnight. The weight of PCL-dECM fibers before and after cell culture was weighed. The dried PCL-dECM fibers were dissolved in a mixed solution of HFIP:formic acid:DMSO (8:1:1) to obtain a solution of 16% PCL (W / V) and 10% dECM (w / w).

[0040] 2) Method for making the inner layer of the stent: The solution obtained in the above steps was subjected to electrohydrodynamic three-dimensional printing, the extrusion flow rate was controlled to be 0.2 mL / h, the local voltage of the jet needle was 3.0 kV, the collection distance was controlled to be 2 mm, and the fiber filaments with consistent direction, diameter of 5-10 μm, and interval of 30 μm were printed on the collection plate through computer programming. The residual solvent was removed in a drying oven overnight, and the collected PCL-dECM fiber filaments were wound parallel to the extension direction on a metal collection rod with a diameter of 1.5 mm, so that the direction of the PCL-dECM fiber filaments was consistent with the direction of the metal collection rod, and PCL-dECM fiber filaments arranged in the axial direction were obtained.

[0041] 3) Preparation method of the outer layer of the stent: The outer layer of PCL nanofibers was prepared using pure PCL solution as raw material through electrospinning technology: The specific operation process is as follows: PCL powder was dissolved in a mixed solution of DMF (N,N-dimethylformamide) and DCM (dichloromethane) with a volume ratio of 6:4, and the solution was stirred and dissolved to obtain a PCL solution with a mass volume ratio of 7%. The solution was drawn into the electrospinning machine, the voltage was adjusted to 12kV, the extrusion flow rate was 1mL / h, the collection distance was controlled at 15cm, and the metal rod with PCL-dECM fiber was fixed on the rotating collection device, and the speed was set to 500rpm to prepare the outer layer of the stent. The outer layer of the stent maintains the thread structure of the inner layer of the stent and provides stable mechanical support for the nerve repair catheter. By controlling the time of electrospinning, an outer layer of the stent with a thickness of about 300 μm was prepared, so that a nerve repair stent with an ordered structure inside and a disordered structure outside and uniformly and stably covered with dECM was obtained.

[0042] The prepared nerve repair scaffold was observed using a SEM scanning electron microscope, and the following Figure 2 and Figure 3 The results are shown. Figure 2 and Figure 3 It can be concluded that electrohydrodynamic jet 3D printing technology effectively prepared ordered guided fiber filaments with a gap of 30 μm, and then electrospinning technology was used to successfully prepare nerve repair conduits with an ordered inner layer and a uniform outer layer structure.

[0043] Experimental example The operation process is as follows: the skin and muscle layers of the right leg of the rat were cut, the sciatic nerve was exposed and transected, a 10 mm defect was created, the prepared nerve repair catheter was implanted into the nerve defect, and the skin and muscle layers were sutured with 3-0 and 6-0 nylon sutures. The nerve regeneration was evaluated 12 weeks after surgery. Figure 4 As shown, through Figure 4 The obtained nerve repair conduit has good biocompatibility, does not cause obvious inflammatory response after transplantation, and has a good nerve repair effect on rats.

[0044] At 12 weeks post-surgery, gastrocnemius muscle tissues from the operated and contralateral normal sides were obtained for analysis. After euthanasia, the muscles were removed, weighed, and analyzed to determine the wet weight ratio and morphological comparison between the operated and normal sides. Figure 5 The left side shows the gastrocnemius muscle after the sciatic nerve injury was repaired, and the right side shows the gastrocnemius muscle in the normal position. Figure 5It can be seen that after losing the innervation of the sciatic nerve, the gastrocnemius muscle will gradually atrophy. If the sciatic nerve is repaired, the atrophy will be alleviated and gradually recovered. The recovery of the sciatic nerve can be judged by observing the atrophy of the gastrocnemius muscle. The experimental results show that the gastrocnemius muscle recovers well after the injured sciatic nerve is implanted with a nerve catheter.

[0045] In addition, although exemplary embodiments have been described herein, the scope includes any and all embodiments based on the present application with equivalent elements, modifications, omissions, combinations (e.g., various embodiments intersecting schemes), adaptations or changes. The elements in the claims are to be interpreted broadly based on the language adopted in the claims, and are not limited to the examples described in this specification or during the practice of this application, and the examples are to be interpreted as non-exclusive. Therefore, this specification and examples are intended to be considered as examples only, and the true scope and spirit are indicated by the following claims and the full scope of their equivalents.

[0046] The above description is intended to be illustrative rather than restrictive. For example, the above examples (or one or more schemes thereof) may be used in combination with each other. For example, a person of ordinary skill in the art may use other embodiments when reading the above description. In addition, in the above-mentioned specific embodiments, various features may be grouped together to simplify the present application. This should not be interpreted as an intention that a feature of an application that does not require protection is necessary for any claim. On the contrary, the subject matter of the present application may be less than all the features of an embodiment of a particular application. Thus, the following claims are incorporated herein in the specific embodiments as examples or embodiments, wherein each claim is independently a separate embodiment, and it is considered that these embodiments may be combined with each other in various combinations or arrangements. The scope of the present invention should be determined with reference to the appended claims and the full scope of equivalent forms granted by these claims.

[0047] The above embodiments are only exemplary embodiments of the present application and are not intended to limit the present invention. The protection scope of the present invention is defined by the claims. Those skilled in the art may make various modifications or equivalent substitutions to the present invention within the essence and protection scope of the present application, and such modifications or equivalent substitutions shall also be deemed to fall within the protection scope of the present invention.

Claims

1. A nerve repair stent, characterized in that: The nerve repair scaffold is a hollow double-shell fiber structure, the outer layer of the double-shell fiber structure is composed of PCL nanofiber filaments, the inner layer of the double-shell fiber structure includes PCL-dECM fiber filaments, the PCL-dECM fiber filaments are PCL fiber filaments loaded with dECM, and the PCL-dECM fiber filaments of the inner layer are arranged along the axial direction of the double-shell fiber structure.

2. The nerve repair scaffold according to claim 1, characterized in that: The diameter of the PCL-dECM fiber is 5-10 μm, and the distance between adjacent PCL-dECM fiber is 20-40 μm.

3. The nerve repair scaffold according to claim 1, characterized in that: The outer diameter of the nerve repair stent is 1-3 mm.

4. The nerve repair scaffold according to claim 1, characterized in that: The thickness of the outer layer of the nerve repair scaffold is 90-110 μm.

5. The method for preparing a nerve repair scaffold according to any one of claims 1 to 4, characterized in that: The preparation method comprises: preparing PCL nanofiber filaments by an electrostatic spinning method; inoculating ganglia onto the PCL nanofiber filaments and culturing them to obtain PCL nanofiber filaments with precipitated ECM after culturing; decellularizing the PCL nanofiber filaments with precipitated ECM to obtain PCL fiber filaments loaded with dECM; preparing orderly arranged PCL-dECM fiber filaments by re-dissolving the PCL fiber filaments loaded with dECM using an electrohydrodynamic jet three-dimensional printing method; winding the PCL-dECM fiber filaments on a metal collecting rod in parallel with their extending direction to obtain an inner layer of a double-layer fiber structure; preparing an outer layer of PCL nanofiber filaments on the outer surface of the inner layer by an electrostatic spinning method to obtain a nerve repair scaffold.

6. The preparation method according to claim 5, characterized in that: Ganglia were seeded onto PCL fibers and cultured for 20-35 days.

7. The preparation method according to claim 5, characterized in that: The PCL fiber filaments with precipitated ECM are subjected to a decellularization treatment, including: the PCL fiber filaments with precipitated ECM are sequentially immersed in a mixed solution A and a mixed solution B for initial treatment, wherein the mixed solution A includes PBS, NaCl and sodium 3-(decyldimethylamino)propanesulfonate, and the mixed solution B includes sodium deoxycholate and SB-16, to obtain PCL-dECM fiber filaments after initial treatment; and the PCL-dECM fiber filaments after initial treatment are subjected to gradient dehydration and drying using ethanol to obtain PCL fiber filaments loaded with dECM after decellularization treatment.

8. The preparation method according to claim 5, characterized in that: PCL-dECM fiber filaments are prepared by re-dissolving the PCL fiber filaments loaded with dECM and using an electrohydrodynamic jet 3D printing method, including dissolving the PCL fiber filaments loaded with dECM in a mixed solution C, wherein the mixed solution C includes HFIP, formic acid and DMSO to obtain a mixed solution D; then based on the mixed solution D, orderly arranged PCL-dECM fiber filaments are printed using an electrohydrodynamic jet 3D printing method.

9. The preparation method according to claim 8, characterized in that: The volume ratio of HFIP, formic acid and DMSO in the mixed solution C is 6-9:1:

1.

10. The preparation method according to claim 8, characterized in that: The mass volume ratio of PCL in the mixed solution D is 12-18%, and the mass percentage of dECM in the mixed solution D is 5-15%.

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