A reel type nerve conduit with collagen fibers aligned and a preparation method and application thereof

Abstract

The application discloses a collagen fiber directional arrangement reel type nerve conduit and a preparation method and application thereof, and belongs to the field of medical biomaterials. Collagen in the nerve conduit is arranged in the axial direction of the conduit, a friendly biomimetic environment is provided for nerve cells, and the directional arrangement of collagen fibers provides a guide clue for nerve cell migration and axon directional extension; the nerve conduit can be used for bridging a damaged nerve, guiding directional growth of nerve cells, and promoting nerve repair and reconstruction and functional recovery.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of medical biomaterials, in particular to a collagen fiber oriented arrangement of a reel type nerve conduit and its preparation method and application. BACKGROUND

[0002] Peripheral nerve injury is a common clinical injury, which usually causes motor and sensory dysfunction, and has a high disability rate for patients, causing heavy medical and economic burden to society. The injured peripheral nerve is difficult to reconstruct, especially the regeneration ability of large segment defect is very limited. The gold standard for repairing large segment nerve defect is autologous nerve transplantation, however, the donor source is limited, the function of the donation site is severely lacking, and the donor and recipient are not easy to match. The nerve conduit, also known as artificial nerve and artificial nerve graft, is a tubular structure made of natural and / or synthetic biopolymers, which serves as a bridge covering / filling the injured nerve or connecting the nerve stumps, providing structural support and nutritional guarantee for axon regeneration along the conduit. The nerve conduit overcomes many disadvantages of autologous and allogeneic nerve transplantation, and a number of nerve conduits have been used for clinical repair of nerve injury. More and more professionals believe that nerve conduits can gradually replace nerve transplantation and play a key role in peripheral nerve repair in the future.

[0003] At present, natural polymers, synthetic polymers and their composites are used to manufacture nerve conduits, among which collagen and polycaprolactone are widely used in the manufacture of natural biopolymers and synthetic polymers of nerve conduits. Natural biopolymers such as collagen have excellent biocompatibility and tissue regeneration inducing ability, and can provide a friendly living environment for cells, but the mechanical properties are weak. Synthetic polymers have good mechanical properties, but the biocompatibility and biological activity are poor. Therefore, natural biopolymers and synthetic polymers are usually used together, natural biopolymers provide a biomimetic environment for cells, and synthetic polymers provide appropriate structural / mechanical support.

[0004] There are various structural types of nerve conduits, such as hollow non-porous single wall, hollow porous single wall, hollow groove single wall, multi-channel, single wall filled with fibers or hydrogel. Nutrient substance and growth factor penetration are key requirements for nerve conduits, and non-porous single wall design cannot meet such requirements, and this structure design has been rarely used; groove and multi-channel design can guide the directional growth of axons well, but the manufacturing technology is high; nerve conduits with hydrogel fillers can prevent axons from growing into the conduit, therefore, porous single wall and fiber-filled structure are the most common in currently used nerve conduits in clinic, such as is a hollow porous single wall round tube made of type I collagen, 3D nerve conduit is based on the above, the inside is filled with a porous matrix composed of type I collagen and chondroitin sulfate-6.

[0005] An ideal nerve conduit requires a bionic structure, proper mechanical properties, sufficient permeability to provide nutrition, flexibility in use, and biodegradability, to provide a suitable microenvironment for axon regeneration, improve the efficiency of nerve regeneration, and quickly restore impaired motor and sensory functions. The currently used nerve conduits are far from ideal, and are generally used for nerve defect repair with a defect length of less than 3 cm, and the effect of longer nerve repair is unsatisfactory. In addition, compared with other treatment methods, autologous nerve transplantation, as the gold standard, has the best effect, but the success rate after transplantation is only 50%. The development of nerve conduits with excellent effects on the treatment of peripheral nerve injury has been the direction of efforts of clinicians and researchers. SUMMARY

[0006] The present application provides a collagen fiber oriented arrangement of the reel type nerve conduit and its preparation method and application, which can be used to bridge the damaged nerve and guide the directional growth of nerve cells, promote nerve repair and functional recovery.

[0007] The present application first provides a nerve conduit, which is curled from collagen protein slices;

[0008] The directional and ordered arrangement of collagen protein in the collagen protein slice is the natural and inherent directional and ordered arrangement of collagen protein in the raw material tissue.

[0009] The nerve conduit described above is made of collagen protein slices curled at least one turn;

[0010] Preferably, the collagen protein slices are curled for multiple turns to form a reel, ensuring that there is also directional arrangement of collagen protein inside the nerve conduit.

[0011] The arrangement direction of collagen protein in the collagen protein slice is the same as or similar to the long axis direction of the nerve conduit.

[0012] The nerve conduit described above, the raw material tissue is an animal tissue rich in collagen protein and arranged in a directional and ordered manner, including but not limited to tendon, ligament or nerve.

[0013] The preparation method of the collagen protein slice comprises the following steps:

[0014] The raw material tissue is cut into slices, cells and macromolecules in the tissue slices are removed, and the collagen protein slice is obtained by drying.

[0015] The raw material tissue is an animal tissue rich in collagen protein and arranged in a directional and ordered manner; including but not limited to at least one of tendon, ligament and nerve;

[0016] The thickness of the raw material tissue cut into slices is 5 microns to 500 microns, preferably 100 microns to 300 microns.

[0017] The method for removing cells in the tissue slice can adopt the method of repeated freeze-thawing or adopt chemical reagents;

[0018] Specifically, the chemical reagents adopted include but are not limited to formic acid, peracetic acid, sodium hydroxide, sodium dodecyl sulfonate, Triton X-100, sodium deoxycholate and a combination thereof.

[0019] The combination includes but is not limited to a combination of 0.5-2% (volume percentage) Triton X-100 in Tris-HCL buffer and 1-5% (weight percentage) sodium deoxycholate solution, a combination of 0.1-5% (volume percentage) peracetic acid / 5-40% (volume percentage) ethanol solution and 0.01-0.2 M sodium hydroxide / 0.05-0.2 M EDTA solution.

[0020] The macromolecules removed in the tissue slice include DNA and RNA, which can be removed by ribonuclease and deoxyribonuclease.

[0021] The slicing into thin pieces is to obtain a slice in a directionally ordered arrangement mode of collagen in the raw material tissue, rather than to completely destroy the tissue structure and then reassemble.

[0022] The slicing into thin pieces includes but is not limited to tissue frozen section or tissue paraffin-embedded section.

[0023] The collagen thin pieces can be modified first and then rolled into a roll shape to obtain the nerve conduit;

[0024] The modification is any one of the following:

[0025] (1) chemical coupling of the collagen thin pieces;

[0026] Specifically, the reagent for the chemical coupling is any one or a combination of at least two of aldehyde compounds, carbodiimide, genipin and proanthocyanidin; the aldehyde compound is preferably glutaraldehyde and / or formaldehyde;

[0027] (2) adsorption or chemical coupling of biological factors or polypeptides promoting nerve regeneration on the collagen thin pieces;

[0028] The biological factors can be at least one of nerve growth factor, brain-derived neurotrophic factor, basic fibroblast growth factor, vascular endothelial growth factor, insulin-like growth factor and platelet-derived growth factor;

[0029] The polypeptide can be a polypeptide YIGSR which can promote differentiation of nerve cells.

[0030] (3) directly culturing stem cells on the collagen lamella;

[0031] Specifically, the stem cells are at least one of neural stem cells, bone marrow mesenchymal stem cells, adipose tissue mesenchymal stem cells, umbilical cord mesenchymal stem cells and induced pluripotent stem cells.

[0032] After removing the main non-collagen components in the lamella, chemical coupling or biological factor modification, the collagen lamella is dried for storage and transportation. The drying method includes air drying, blow drying or freeze drying.

[0033] The dried collagen lamella can also be subjected to sterilization treatment, and the sterilization treatment methods include ethylene oxide sterilization, high-energy electron beam irradiation sterilization or gamma ray sterilization.

[0034] The application further provides the use of the above-mentioned nerve conduit in the preparation of nerve repair materials or nerve repair.

[0035] The preparation method of the above-mentioned nerve conduit comprises the following steps: flattening the collagen lamella, wetting it with normal saline, placing a medical silk braided wire parallel to the collagen fibers at one end of the collagen lamella, curling the collagen lamella to cover the silk braided wire, then straightening the silk braided wire with both hands, rotating to drive the collagen lamella to curl, and finally extracting the silk braided wire.

[0036] In clinical use, the dried collagen lamella is cut into a certain shape according to the length and diameter of the damaged nerve to be repaired, wetted and then curled into a nerve conduit with the required length and diameter.

[0037] The nerve conduit has different optimal degradation times in the body for different nerve defect sites, thicknesses, lengths and widths, and the degradation time of the collagen lamella in the body needs to be optimized; the degradation time of the collagen lamella in the body can be optimized by changing the thickness of the collagen lamella or by chemical coupling.

[0038] The application has the following beneficial effects:

[0039] (1) The collagen in the nerve conduit prepared by the application is derived from allogeneic tissues or xenogeneic tissues, and the special feature is that the collagen in the nerve conduit maintains its natural directional arrangement mode in the raw material tissues, is directionally arranged along the axis of the conduit, provides a friendly biomimetic environment for nerve cells, and the directionally arranged collagen fibers provide guidance clues for the migration of nerve cells and the directional extension of axons.

[0040] (2) The present application rolls the two-dimensionally arranged collagen sheet into a three-dimensionally rolled structure, and realizes a multi-layer filled nerve conduit, in which the collagen in the outer membrane is arranged in order, and the collagen in the inner part is also arranged in order, which is beneficial to the directional migration and growth of the internal and external cells of the nerve, and better promotes the regeneration of the axon;

[0041] (3) The collagen in the nerve conduit of the present application is arranged in order according to the arrangement of the collagen in the raw material tissue, and the manufacturing process is simple, and the product quality is easy to control, without directly manipulating the collagen molecules and fibers;

[0042] (4) The nerve conduit prepared by the present application can be cut into a certain shape according to the size of the nerve injury and the diameter / length of the nerve to be bridged, and then rolled into a nerve conduit with the required length and diameter, so that there is no need to prepare multiple types of products during the operation, and the personalized nerve conduit can be conveniently rolled out before repairing the nerve. BRIEF DESCRIPTION OF DRAWINGS

[0043] Figure 1 The collagen sheet prepared in Example 1 and the morphology of the collagen fibers on the surface thereof.

[0044] Figure 2 Bone marrow stem cells grown on the collagen sheet.

[0045] Figure 3 The method for manufacturing the nerve conduit.

[0046] Figure 4 The cross section of the nerve conduit.

[0047] Figure 5 Repairing the sciatic nerve defect of a rat by the nerve conduit.

[0048] Figure 6 The sciatic nerve of a rat repaired for 12 weeks. DETAILED DESCRIPTION

[0049] The present application will be further described in detail below in conjunction with the specific embodiments, and the examples given are only for illustrating the present application, but not for limiting the scope of the present application.

[0050] In the following examples, the experimental methods are conventional methods, unless otherwise specified.

[0051] In the following examples, the materials, reagents, etc. used, unless otherwise specified, can be obtained from commercial channels.

[0052] Ribonuclease in the following examples was purchased from Sigma-Aldrich, Cat. No. R5503; Deoxyribonuclease was purchased from Sigma-Aldrich, Cat. No. DN25;

[0053] Ethyl dimethylaminopropyl carbodiimide was purchased from Aldrich, Cat. No. E106172; N-hydroxysuccinimide was purchased from Aldrich, Cat. No. H109330;

[0054] Calcien was purchased from Biyun Tian, Cat. No. C2012.

[0055] Example 1, Preparation of Collagen Grafts

[0056] Fresh bovine tendons were obtained from slaughterhouse, and 50 mm long segments were cut from the tendon region, immersed in OCT freezing embedding medium, and sliced longitudinally with a freezing microtome at 100 μm thickness. The tendon slices were then immersed in physiological saline to remove the embedding medium. The tendon slices were repeatedly frozen and thawed for 5 cycles (-80°C for 10 min, 37°C for 10 min), and then treated with ribonuclease (150 IU / mL) and deoxyribonuclease (100 μg / mL) in phosphate buffer solution at 37°C for 4 hours, washed with distilled water, frozen in a refrigerator (-80°C for 10 min), and then freeze-dried in a freeze-drying machine (instrument limit vacuum degree ≤ 5 Pa). The obtained collagen grafts are shown in Fig. A of Figure 1 Finally, the collagen grafts were sealed in product packaging bags, sterilized by γ irradiation at a dose of 25 kGy, and stored at room temperature. The surface of the collagen grafts was observed by scanning electron microscope, and the collagen fibers were arranged in an oriented and ordered manner (see Fig. B of Figure 1

[0057] Using the same method, collagen grafts derived from bovine sciatic nerve can be prepared.

[0058] Example 2, Chemical Coupling of Collagen Grafts

[0059] 1) The collagen grafts (bovine tendon) prepared in Example 1 were immersed in a coupling buffer (0.1 M carbonate, 0.15 M NaCl, pH 8.5) containing 1% glutaraldehyde (volume percent), and reacted at room temperature for 6 hours, then rinsed with distilled water and freeze-dried (frozen in a refrigerator at -80°C for 10 min, and then freeze-dried in a freeze-drying machine).

[0060] ​2) The collagen sheet prepared in Example 1 (bovine Achilles tendon) was immersed in a coupling buffer (0.1 M MES (morpholinoethanesulfonic acid), pH 4.7) containing 50 mM ethyldimethylaminopropyl carbodiimide and 20 mM N-hydroxysuccinimide, and reacted at room temperature for 2 hours, after which it was rinsed with distilled water and freeze-dried (frozen for 10 minutes in a freezer at -80°C, and then freeze-dried in a freeze-dryer).

[0061] 3) The collagen sheet prepared in Example 1 (bovine Achilles tendon) was immersed in a physiological saline solution containing 1% genipin (mass percent), and reacted at room temperature for 24 hours, after which it was rinsed with distilled water and freeze-dried (frozen for 10 minutes in a freezer at -80°C, and then freeze-dried in a freeze-dryer).

[0062] Example 3, Biological factor-modified collagen sheet

[0063] The collagen sheet was chemically coupled with glutaraldehyde or ethyldimethylaminopropyl carbodiimide using the method 1) or 2) in Example 2, rinsed with distilled water, cooled to semi-dry at room temperature, and nerve growth factor (mouse nerve growth factor, a biologically active protein with a molecular weight of 26.5 KD, extracted from the submandibular gland of a mouse) was added dropwise onto the collagen sheet (50 μL / cm 2 ) in an aqueous solution (50 μg / mL) at 4°C, and left to stand for 12 hours, and then freeze-dried (frozen for 10 minutes in a freezer at -80°C, and then freeze-dried in a freeze-dryer).

[0064] Example 4, Collagen sheet loaded with stem cells

[0065] The collagen sheet prepared in Example 1 (bovine Achilles tendon) was trimmed to a size of 1.0 cm x 1.5 cm, and placed on the bottom of a bacterial culture dish. The collagen sheet was first infiltrated with a cell culture medium (DMEM added with 10% (v / v) fetal bovine serum), and then 200 μL of a suspension of rat bone marrow stem cells (2 x 10 5 cell / mL) was uniformly loaded onto the collagen sheet, and then the material was placed in a cell culture incubator and cultured at 37°C under 5% (v / v) CO2for 3 hours. After the cells had sufficiently adhered to the material, the culture medium was slowly added along the wall of the culture dish to completely immerse the collagen sheet, and the culture medium was changed every other day. After 7 days of culture, the sample was stained with calcein, and the cell morphology and arrangement were observed under a laser confocal scanning microscope. It was found that many cells were arranged in the direction of the collagen fibers (white arrows represent the direction of the collagen fibers). Figure 2

[0066] Example 5, Fabrication of nerve conduit

[0067] ​A rectangular collagen sheet (10) was taken out of the package, the collagen fiber arrangement direction was indicated by the broken and solid lines (10) Figure 3 ), and laid flat. A medical silk braided thread (11) parallel to the collagen fiber was placed at one end of the collagen sheet, and the collagen sheet was rolled to cover the silk braided thread. Then the silk braided thread was pulled straight with both hands, and rotated to drive the collagen sheet to roll, to form a roll (12) with multiple layers of collagen fibers arranged axially. Finally, the silk braided thread was pulled out of the nerve conduit. The collagen sheet can be cut into different shapes as needed, for example, cut into a shape with a wide upper part and a narrow lower part, and rolled according to the same method above to form a roll-type nerve conduit with a sleeve at both ends. In use, the nerve stumps are filled into the sleeves, and then sutured and fixed. The sleeves can limit the growth of foreign cells into the anastomosis gap, and better promote nerve repair. If a thick nerve conduit is needed, after rolling one collagen sheet, the rolled roll is used as the axis to continue rolling the second, third, and so on collagen sheets until the desired diameter is reached, and used for repairing nerves. The nerve conduit is embedded with OCT freezing section embedding agent, frozen, and transversely sectioned. The sections are stained with aniline blue, photographed, and the results are shown in Figure 4 .

[0068] Example 6, Repair of Rat Sciatic Nerves

[0069] Twelve SD rats with a body weight of about 300-350 g were randomly assigned to an injury group, an autograft group, a nerve conduit group, and a nerve growth factor modified nerve conduit group. The rats were anesthetized by intraperitoneal injection of 2% (wt%) sodium pentobarbital. After the rats were anesthetized, they were fixed on an operating table, the left posterolateral thigh was shaved, iodine was used for disinfection, the skin was incised 1 cm at the posterior median depression, the muscles were carefully separated along the intermuscular space, and the sciatic nerve (20) Figure 5 ) was exposed. The middle segment of the sciatic nerve was resected to form a 10 mm defect. In the injury group, no repair was performed after the 10 mm nerve defect was created. In the autograft group, the sciatic nerve was cut for 10 mm and sutured in situ. In the nerve conduit group, a collagen sheet (bovine Achilles tendon) prepared in Example 1 was used to make a nerve conduit with a diameter slightly larger than that of the sciatic nerve and a length of 10 mm, according to the method of Example 5. The nerve conduit was connected to the distal and proximal ends of the damaged nerve, and the joint was sutured with 9-0 surgical suture thread Figure 5The nerve conduit group modified with nerve growth factor was prepared using collagen sheets (conjugated with ethyldimethylaminopropylcarbodiimide) modified with nerve growth factor as described in Example 3. Following the method in Example 5, nerve conduits with a diameter slightly thicker than the sciatic nerve and a length of 10 mm were fabricated to connect the distal and proximal ends of the damaged nerve. The junction was sutured with 9-0 surgical sutures. After nerve suturing, the incision was closed layer by layer, and the muscle and epidermis were sutured with 4-0 surgical sutures. All rats were raised under standard experimental conditions. Twelve weeks post-surgery, the rats were euthanized. It was found that the sciatic nerve in the nerve conduit group was morphologically connected (…). Figure 6 The thinner central portion represents the regenerated nerve. Tibialis anterior muscle was harvested from the experimental side and the unoperated control side of each rat, and muscle weight was measured. The weight ratios of the surgical side to the unoperated side of the tibialis anterior muscle in the damaged group, autologous transplantation group, nerve conduit group, and nerve growth factor-modified nerve conduit group were 0.171, 0.429, 0.335, and 0.412, respectively. These results confirm that the nerve conduit can repair sciatic nerve defects in rats, significantly slowing tibialis anterior muscle atrophy, and that nerve growth factor modification helps the nerve conduit repair sciatic nerve defects in rats.

Claims

1. A neural conduit, characterized by: The nerve conduit is curled from the collagen sheet; the collagen sheet is curled for multiple turns to form a reel; The collagen in the collagen sheet is arranged in a directional order, which is the natural inherent directional order of collagen in the raw material tissue; The arrangement direction of the collagen in the collagen sheet is the same as or similar to the long axis direction of the nerve conduit; the two-dimensional directional arrangement of the collagen sheet is curled to form a three-dimensional reel structure, realizing a multi-layer filled nerve conduit, in which the collagen in the outer membrane is arranged in order and the collagen filled in the inner part is also arranged in order, which is beneficial to the directional migration and growth of the cells inside and outside the nerve and better promotes the regeneration of axons; The preparation method of the collagen sheet comprises the following steps: The raw material tissue is cut into a sheet, cells and macromolecules in the tissue slice are removed, and the collagen sheet is obtained by drying; the thickness of the sheet is 100-300 microns; The preparation method of the nerve conduit comprises the following steps: the collagen sheet is laid flat, wetted with normal saline, and a medical silk braided wire parallel to the collagen fiber is placed at one end of the collagen sheet, the collagen sheet is curled to cover the silk braided wire, then the silk braided wire is straightened with both hands, rotated to drive the collagen sheet to curl, and a reel with multiple layers of collagen fibers arranged in the axial direction is formed, and finally the silk braided wire is pulled out.

2. The neural conduit of claim 1, wherein: The raw material tissue is an animal tissue rich in collagen and arranged in a directional order; the method for removing cells in the tissue slice adopts a repeated freeze-thaw method or a chemical reagent; the macromolecules include DNA and RNA, and the DNA and RNA are removed by ribonuclease and deoxyribonuclease.

3. The nerve conduit of claim 2, wherein: The raw material tissue is at least one of a tendon, a ligament and a nerve.

4. The nerve conduit of any one of claims 1-3, wherein: The collagen sheet is subjected to chemical coupling; the reagent for the chemical coupling is any one or a combination of at least two of aldehyde compounds, carbodiimides, genipin and proanthocyanidins.

5. The nerve conduit according to any one of claims 1-3, wherein: A biological factor or a polypeptide promoting nerve regeneration is adsorbed or chemically coupled on the collagen sheet.

6. The nerve conduit of claim 5, wherein: The biological factor is at least one of a nerve growth factor, a brain-derived neurotrophic factor, a basic fibroblast growth factor, a vascular endothelial cell growth factor, an insulin-like growth factor and a platelet-derived growth factor; the polypeptide is a polypeptide YIGSR capable of promoting the differentiation of nerve cells.

7. The nerve conduit of any one of claims 1-3, wherein: Stem cells are directly cultured on the collagen sheet.

8. The nerve conduit of claim 7, wherein: The stem cells are at least one of neural stem cells, bone marrow mesenchymal stem cells, adipose tissue mesenchymal stem cells, umbilical cord mesenchymal stem cells and induced pluripotent stem cells.

9. Use of the nerve conduit in any one of claims 1-8 in the preparation of a nerve repair material.

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