Nerve repair scaffold and preparation method thereof

Abstract

The present application belongs to the field of biomaterial technology, and specifically relates to a nerve repair scaffold and a preparation method thereof. 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, and the PCL-dECM fiber filaments are PCL fiber filaments loaded with dECM. The PCL-dECM fiber filaments of the inner layer are arranged along the axial direction of the double-shell fiber structure. After implantation, the nerve repair scaffold can improve the recovery of nerve function and improve biocompatibility.

Description

Technical Field

[0001] The present application relates to the field of biomaterial technology, 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 current "gold standard" in 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; the frequent need for secondary surgery and possible complications at the donor site, which have limited its promotion in clinical practice.

[0003] Therefore, the use of functional biomaterials to replace autologous transplantation has been widely developed, including many commercial nerve guidance 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 a similar tissue microenvironment in the catheter can be achieved by constructing an extracellular matrix (ECM) mimic substrate or a cell-laden substrate. However, due to problems such as incomplete adhesion, lack of cell-matrix signal transduction 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 address the above-mentioned deficiencies in the prior art. A neural repair scaffold and a preparation method thereof are needed, which can improve the recovery of neural function and enhance 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, the present application provides a method for preparing a nerve repair scaffold as described in any embodiment of the present application, 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 ordered PCL-dECM fiber filaments by redissolving the PCL fiber filaments loaded with dECM using an electrohydrodynamic jet three-dimensional printing method; winding the PCL-dECM fiber filaments parallel to their 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 its preparation method 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 repair effect in the body; 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 components in different views. The same reference numerals with letter suffixes or different letter suffixes may represent different instances of similar components. The accompanying drawings generally illustrate various embodiments by way of example and not limitation, and together with the description and claims, serve to illustrate the claimed embodiments. Where 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 of the present apparatus or method.

[0009] Figure 1 A diagram showing a simulated structure of a nerve repair stent according to an embodiment of the present application;

[0010] Figure 2 Shows an SEM scan of a longitudinal section of a nerve repair scaffold according to an embodiment of the present application;

[0011] Figure 3 Shows a cross-sectional SEM scan of a nerve repair scaffold according to an embodiment of the present application;

[0012] Figure 4Images of rats with sciatic nerve injury after transplantation of a nerve repair scaffold according to an embodiment of the present application are shown, wherein the left image is an image taken 0 weeks after transplantation, and the right image is an image taken 12 weeks after transplantation;

[0013] Figure 5 A comparison diagram showing the recovery of the gastrocnemius muscle connected to the sciatic nerve after the nerve repair scaffold according to an embodiment of the present application was transplanted into the injured sciatic nerve of rats. DETAILED DESCRIPTION

[0014] 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 embodiments. The embodiments of the present application are further described in detail below in conjunction with the accompanying drawings and specific embodiments, but are not intended to limit the present application.

[0015] The terms "first," "second," and similar terms used in this application do not denote any order, quantity, or importance, but are used only to distinguish. The terms "include," "comprise," and similar terms mean that the elements preceding the term include the elements listed after the term, and do not exclude the possibility of also including other elements.

[0016] like Figure 1 As shown, according to an embodiment of the present application, a nerve repair stent is provided, which is a hollow double-shell fiber structure, the outer layer of the double-shell fiber structure is composed of PCL (polycaprolactone) nanofiber filaments, and 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.

[0017] The high strength and toughness of the outer layer of PCL fibers effectively protects the ordered structure of the inner layer, adapting to the complex environment of in vivo implantation. Once implanted, they maintain the original shape of the nerve conduit for extended periods, providing a strong physical environment for axonal extension and myelination while preventing immune rejection. PCL's slow degradation rate prevents rapid loss of dECM and prolongs its duration of action.

[0018] The inner layer of PCL-dECM fibers is axially arranged, forming a threaded inner layer that effectively mimics the topological cues of natural neurofibromin cables. This creates a guiding configuration within the lumen, which can better guide and promote nerve regeneration. Adjacent PCL-dECM fibers are spaced apart, forming microchannels that effectively guide and significantly promote neurite extension and Schwann cell migration. Furthermore, the dECM components on the fiber surface can significantly induce axonal extension and myelination.

[0019] 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 bioactivity, and significantly promote axon extension and myelin formation, and achieve a considerable degree of functional recovery.

[0020] The PCL fibers and PCL-dECM fibers included in the nerve repair scaffold have good biocompatibility and can avoid the immune rejection reaction caused by interventional surgery.

[0021] In some embodiments, the PCL-dECM filaments have a diameter of 5-10 μm, and the spacing between adjacent PCL-dECM filaments is 20-40 μm. These ultrafine PCL-dECM filaments, with spacing of approximately 30 μm between each filament, are not only optimal for protecting mature axons, but also have a diameter similar to that of a single neuronal axon, further facilitating axonal extension and myelination.

[0022] 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 fibers.

[0023] Fibers 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.

[0024] 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.

[0025] In some embodiments, the inner layer of the nerve repair scaffold is loaded with trophic 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 site and the stimulation of the trophic factors.

[0026] According to an embodiment of the present application, a method for preparing a nerve repair scaffold as described in any embodiment of the present application is also provided, 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 solution of PCL fiber filaments loaded with dECM; (4) preparing ordered PCL-dECM fiber filaments by redissolving the PCL fiber filaments loaded with dECM using an electrohydrodynamic jet three-dimensional printing method; (5) winding the PCL-dECM fiber filaments parallel to the extending direction on a metal collecting rod to obtain an inner layer of a double-layer fiber structure; and (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.

[0027] Dorsal root ganglia cultured on the surface of PCL nanofibers can form a well-developed extracellular matrix by utilizing the biocompatibility with PCL nanofibers. The morphology and structure contained in the extracellular matrix can regulate cell adhesion, gene expression, and differentiation. The decellularized matrix retains some biochemical complexity and is superior to the bioactivity of a simple PCL scaffold. The PCL scaffold containing ECM was decellularized and dissolved and then re-prepared into a new scaffold. The test results showed that some decellularized matrix components were not lost, which demonstrates the feasibility of this method. Compared with traditional surface adhesion or hydrogel perfusion methods, this method greatly increases the effective time of nerve repair and overcomes the shortcomings of surface adhesion, such as the easy loss of effective components and the poor biomechanical properties of perfused hydrogels.

[0028] 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.

[0029] Culturing cells on PCL fibers to deposit and then decellularizing the culture can maximize the retention of the effective components of dECM.

[0030] In some embodiments, the ganglion may include primary rat dorsal root ganglion, etc.

[0031] In some embodiments, the ganglia are seeded onto the PCL fibers and cultured for 20-35 days.

[0032] In some embodiments, the PCL fiber filaments with precipitated ECM are decellularized, comprising: 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 comprises PBS, NaCl and sodium 3-(decyldimethylamino)propanesulfonate, and the mixed solution B comprises 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 with ethanol and dried to obtain PCL fiber filaments loaded with dECM after decellularization.

[0033] 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 4-6 mM (mmol / L) PBS and 65-70 mM (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 for mixed solution E and mixed solution F can be water.

[0034] 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.

[0035] The initial treatment process of mixed solution A and mixed solution B can be performed only once or repeated twice.

[0036] In some embodiments, the mass fraction of ethanol used in gradient elution is 70%-100%.

[0037] In some embodiments, ordered PCL-dECM fiber filaments are prepared by redissolving the dECM-loaded PCL fiber filaments and utilizing an electrohydrodynamic jet 3D printing method, comprising dissolving the dECM-loaded PCL fiber filaments in a mixed solution C comprising HFIP, formic acid, and DMSO to obtain a mixed solution D; and then, based on the mixed solution D, printing out ordered PCL-dECM fiber filaments using a 3D printing method.

[0038] 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.

[0039] In some embodiments, the volume ratio of HFIP, formic acid, and DMSO in the mixed solution C is 6-9:1:1. Using the mixed solution C to dissolve the dECM-loaded PCL fibers is extremely gentle and reduces the damage of the organic solvent to the active ingredients.

[0040] 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.

[0041] In some embodiments, the method for preparing a PCL nanofiber outer layer 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.

[0042] 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.

[0043] 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%.

[0044] Example 1

[0045] 1) Scaffold Raw Material Preparation: To prepare PCL electrospun scaffolds, PCL powder was dissolved in a 6:4 volume ratio of DMF and DCM. Stirring dissolved the solution yielded a 7% PCL solution by weight. The solution was then drawn up through a syringe and placed in an electrospinning machine. The voltage was adjusted to 12 kV, the extrusion flow rate was 1 mL / h, and the collection distance was controlled at 15 cm. After 1 hour of collection, a planar PCL electrospun scaffold was obtained. Testing revealed that the diameter of the PCL nanofibers was approximately 200 nm. The scaffolds were disinfected with 70% ethanol and washed with PBS. Cultured primary rat dorsal root ganglia were inoculated onto the PCL filaments and cultured for 28 days to observe ECM secretion. After 28 days of culture, the ECM-precipitated PCL filaments were decellularized. The procedure was as follows: the scaffolds were washed three times with PBS and soaked in a mixture of 5 mM PBS and 68.5 mM NaCl. A 1.25 M sodium 3-(decyldimethylamino)propanesulfonate solution was added with gentle stirring at 4°C overnight. The PCL fibers, which had been precipitated with ECM, were then washed three times with PBS and placed in a mixture containing 0.14% sodium deoxycholate and 0.6 mM SB-16 with gentle stirring at 4°C overnight. This step was repeated twice and the scaffolds were stored at 4°C overnight with gentle stirring. The PCL fibers were collected from approximately 50 coverslips and dehydrated using a gradient of 70%-100% ethanol in a desiccator overnight. The PCL-dECM fibers were weighed before and after cell culture. The dried PCL-dECM fibers were dissolved in a mixture of HFIP:formic acid:DMSO (8:1:1) to yield a solution containing 16% PCL (w / v) and 10% dECM (w / w).

[0046] 2) Stent inner layer fabrication method: The solution obtained in the above steps was electrohydrodynamically three-dimensionally printed, with an extrusion flow rate of 0.2 mL / h, a local voltage of 3.0 kV at the jet needle, and a collection distance of 2 mm. Computer programming was used to print fibers with consistent direction, a diameter of 5-10 μm, and a spacing of 30 μm on a collection plate. The residual solvent was removed in a drying oven overnight. The collected PCL-dECM fibers were then wound parallel to the extension direction of the fibers around a metal collection rod with a diameter of 1.5 mm, ensuring that the direction of the PCL-dECM fibers was consistent with that of the metal collection rod, resulting in axially arranged PCL-dECM fibers.

[0047] 3) Fabrication of the stent outer layer: A PCL nanofiber outer layer was prepared using a pure PCL solution via electrospinning. The specific procedure was as follows: PCL powder was dissolved in a 6:4 volume ratio of DMF (N,N-dimethylformamide) and DCM (dichloromethane). The solution was stirred to obtain a 7% PCL solution by weight. The solution was then drawn up through a syringe and placed in an electrospinning machine. The voltage was adjusted to 12 kV, the extrusion flow rate was 1 mL / h, and the collection distance was controlled at 15 cm. A metal rod loaded with PCL-dECM fibers was fixed to a rotating collection device at 500 rpm to form the stent outer layer. This outer layer maintains the inner layer's thread structure while providing stable mechanical support for the nerve repair conduit. By controlling the electrospinning time, an outer layer with a thickness of approximately 300 μm was produced, resulting in a nerve repair scaffold with an ordered internal structure and a disordered external structure, uniformly and stably covered with dECM.

[0048] 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 found that electrohydrodynamic jet 3D printing technology effectively prepared ordered guided fiber filaments with a gap of 30μm. Subsequently, electrospinning technology was used to successfully prepare nerve repair conduits with an ordered inner layer and a uniform outer layer structure.

[0049] Experimental example

[0050] The procedure was as follows: the skin and muscle layers of the rat's right leg were incised, the sciatic nerve was exposed and transected, a 10 mm defect was created, the prepared nerve repair conduit was implanted into the nerve defect, and the skin and muscle layers were sutured using 3-0 and 6-0 nylon sutures. Nerve regeneration was assessed 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 in rats.

[0051] Twelve weeks after surgery, gastrocnemius muscle tissue was obtained from the surgical and contralateral normal sides for analysis. After euthanasia, the muscles were removed, weighed, and analyzed to determine the wet weight ratio and morphological comparison between the surgical and normal sides. Figure 5 The left side shows the gastrocnemius muscle after sciatic nerve injury repair, and the right side shows the gastrocnemius muscle in the normal position. Figure 5It has been shown that after losing innervation from the sciatic nerve, the gastrocnemius muscle gradually atrophies. If the sciatic nerve is repaired, the atrophy is reduced and gradually recovers. The recovery of the sciatic nerve can be assessed by observing the degree of atrophy of the gastrocnemius muscle. Experimental results have shown that implanting a nerve conduit in an injured sciatic nerve can improve the recovery of the gastrocnemius muscle.

[0052] Furthermore, 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., solutions that intersect various embodiments), adaptations, or changes. The elements of the claims are to be interpreted broadly based on the language employed in the claims and are not limited to the examples described in this specification or during the prosecution of the application, which examples are to be construed as non-exclusive. Therefore, it is intended that this specification and examples be considered merely as examples, with the true scope and spirit being indicated by the following claims and their full scope of equivalents.

[0053] The above description is intended to be illustrative and not restrictive. For example, the above examples (or one or more of their embodiments) 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 detailed description, various features may be grouped together to simplify the application. This should not be interpreted as an intention that a feature of an application that is not claimed for protection is essential to any claim. On the contrary, the subject matter of the present application may have less than all the features of an embodiment of a particular application. Therefore, the following claims are incorporated into the detailed description as examples or embodiments, with each claim independently serving as a separate embodiment, and it is contemplated that these embodiments may be combined with each other in various combinations or arrangements. The scope of the invention should be determined with reference to the appended claims and the full scope of equivalents to which such claims are entitled.

[0054] The above embodiments are merely exemplary embodiments of the present application and are not intended to limit the present invention. The scope of protection 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 spirit and scope of protection of the present application, and such modifications or equivalent substitutions shall also be deemed to fall within the scope of protection of the present invention.

Claims

1. A nerve repair scaffold, 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, and the PCL-dECM fiber filaments are PCL fiber filaments loaded with dECM. The PCL-dECM fiber filaments of the inner layer are arranged along the axial direction of the double-shell fiber structure; The preparation method of the nerve repair scaffold includes: preparing PCL nanofiber filaments by electrospinning; 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; 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, printing orderly arranged PCL-dECM fiber filaments by electrohydrodynamic jet three-dimensional printing; 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 electrospinning to obtain a nerve repair scaffold; The PCL fiber filaments with precipitated ECM are subjected to a decellularization treatment, comprising: sequentially immersing the PCL fiber filaments with precipitated ECM in a mixed solution A and a mixed solution B for initial treatment, wherein the mixed solution A comprises PBS, NaCl, and sodium 3-(decyldimethylamino)propanesulfonate, and the mixed solution B comprises sodium deoxycholate and SB-16, to obtain PCL-dECM fiber filaments after initial treatment; and gradiently dehydrating the PCL-dECM fiber filaments after initial treatment using ethanol, and drying them to obtain PCL fiber filaments loaded with dECM after decellularization treatment; The volume ratio of HFIP, formic acid, and DMSO in mixed solution C was 6-9:1:1; 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%.

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 nerve repair scaffold according to claim 1, characterized in that: Ganglia were seeded onto PCL fibers and cultured for 20-35 days.

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