CN110115645A - Multi path artificial nerve trachea and preparation method thereof - Google Patents

Abstract

The invention relates to a multi-channel artificial nerve conduit and a preparation method thereof, which can be used for customized bridging and repairing the distal and proximal ends of nerve defect sites. The multi-channel artificial nerve conduit that can be customized to imitate the natural anatomical structure of nerves disclosed in the present invention mainly includes a multi-channel stent (nerve conduit tube body) that imitates the natural anatomical structure of nerves, and a biological membrane structure ( nerve conduit wall). The multi-channel structure of the tube body can be customized to form a good match with the nerve bundles of different regions and sizes in the nerve to be repaired, and guide specific nerve bundles from the proximal part of the nerve defect to the distal end through different customized channel structures. , to reduce the divergent growth and intertwining phenomenon of nerve bundles during the growth process, which is beneficial to reduce the mismatch rate between the proximal new nerve bundle tissue and the distal target area.

Description

Multi-channel artificial nerve conduit and preparation method thereof

technical field

The invention relates to the field of nerve conduits, and relates to a customized biomimetic multi-channel artificial nerve conduit design and its preparation method imitating the natural anatomical structure of nerves.

Background technique

Peripheral nerve injury is a common clinical problem that is often caused by severe injury, resulting in neurological deficits. For defects larger than 4 mm, a simple direct suturing method cannot be used, but a graft bridge should be provided between the proximal and distal ends of the defect. Autologous nerve transplantation is currently the "gold standard" for repairing nerve defects, but it often results in damage to the donor site and incomplete functional recovery.

Nerve conduits have been developed as an alternative to autografting to repair nerve defects, and their structures are currently limited to simple single-channel or multi-channel structures with regular matrices. However, the natural anatomical structure of nerve bundles is not this simple single-channel lumen or regular matrix structure. The size and number of nerve bundles vary significantly between species and individuals, even if the same nerve is arranged in different cross-sections. Not all are the same.

Therefore, the current nerve conduit structure is limited to single-channel or regular matrix multi-channel nerve conduits, which cannot well simulate the arrangement and distribution of nerve bundles in the nerve, and cannot truly restore the three-dimensional space of multiple groups of nerve bundles in the primary nerve trunk. The above-mentioned nerve conduit structures are not the best choice for the restoration of peripheral nerve defects.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a multi-channel artificial nerve conduit, which is customized to imitate the natural anatomical structure of the nerve, and has multiple groups of anatomical channel structures similar to the target nerve to be repaired, which can axially guide the nerve bundle to reach the distal innervation area, bridging the nerve defect site with multiple well-matched isolated internal channels.

Another object of the present invention is to provide a method for preparing the above-mentioned multi-channel artificial nerve conduit.

The multi-channel artificial nerve conduit of the present invention mainly includes a nerve conduit tube body. The nerve conduit tube body is provided with a plurality of channels that imitate the natural anatomical structure of the nerve. The outer layer of the nerve conduit tube body wraps the nerve conduit tube wall, and the nerve conduit tube wall is Biofilm structures used to mimic epineurial tissue.

The nerve conduit tube body and the nerve conduit tube wall are composited by one or more materials, and have interconnected porous structures, which facilitate the exchange of nutrients and waste discharge between the inside and outside of the nerve conduit tube body. It can be understood that, the nerve conduit tube body is provided with a porous structure, and the nerve conduit tube wall is also provided with a porous structure. Since the nerve conduit tube wall wraps the nerve conduit tube body, the two are communicated with each other through the porous structure.

The number of channels is multiple, and each channel is designed with different contours and other parameters for different parts of the target nerve bundle. The diameter of the multi-channel lumen is usually 100-2000 μm.

The multi-channel structure set in the nerve conduit tube is based on the anatomical parameters of each nerve bundle in the target nerve to be repaired. In the multi-channel structure, a single channel preferentially matches a single nerve bundle. When a single channel needs to match multiple nerve bundles , and preferentially integrate multiple groups of nerve bundles with similar distance or target organ structure and function into one channel.

During the multi-channel artificial nerve catheter implantation process, the lumen of the multiple channels in the nerve catheter tube body can form a good matching relationship with the nerve bundle tissues at both ends of the nerve defect to be repaired.

Further, the multi-channel structure provides guidance consistent with the direction of the original nerve bundle, which helps the nerve tissue forming the bundle group at the proximal end to dock with the corresponding target area at the distal end, and promotes the recovery of nerve function by reducing the mismatch rate.

Both ends of the nerve conduit wall are longer than both ends of the nerve conduit tube body, and the length is 1-3 mm longer than the two ends of the tube body to provide bridging with the epineurium, and the thickness of the nerve conduit wall is 100-400 μm.

The nerve conduit wall provides protection for the repair process by:

When the nerve tissue is axially stretched by a certain strength, the nerve conduit wall stretches itself to disperse the tensile stress. When the axial stress exceeds the extension limit of the tube wall, the tube wall breaks to prevent the nerve tissue from being overloaded. pulled and further injured.

The outer tube wall and inner tube body have a synergistic mechanism in the process of nerve repair, and the tube wall and tube body have two degradation rates: fast and slow. The early degradation of the tube wall in the body can recruit more macrophages (cleaning effect) around the nerve regeneration chamber to provide a clean regeneration environment for the new nerve tissue. The slow degradation process of the tube body can provide a nerve regeneration channel with sufficient space size and supporting strength, which is a powerful guarantee for good blood circulation in the channel lumen, and can assist in the discharge of damaged cells and metabolic wastes in the nerve section.

The customized multi-channel artificial nerve conduit can drive the nerve bundles to form multiple channel (bundle group) structures similar to the original nerve trunk in the customized multi-channel. Consistent shape, the newly-born nerve tissue in the channel conforms to the shape and distribution of nerve bundles in natural nerves, and guides the newly-born nerve bundles at the proximal end to innervate the corresponding target areas at the distal end, thereby improving the accuracy of nerve repair. By matching the above-mentioned nerve regeneration channel with the nerve bundle tissue of the section of the nerve to be cut, it can reduce the intertwining and disorderly growth of the newly-born nerve bundles in the nerve graft, which is beneficial to reduce the relationship between the proximal newly-born nerve bundle tissue and the far-end nerve bundle tissue. The mismatch rate of the end target region.

The advantage of the present invention over the prior art is that the multi-channel structure of the nerve conduit tube has an engineered anatomical shape similar to the nerve bundle in the cross section of the nerve stump. Channels and nerve bundles can form a better matching relationship, so that one or more groups of nerve bundles with similar structures, functions and origins are confined to specific nerve regeneration channels, and nerve disorder is limited by the physical concentration of the nerve conduit wall. The divergent growth increases the number of axons per unit area and drives the newly formed nerves to form bundles within the channel. Further, the long axis of the multi-channel lumen of the catheter is consistent with the nerve bundles in the primary nerve tissue, which can guide the above-mentioned bundled nerves to the corresponding target area at the distal end for docking. Its beneficial effect is to improve the recovery of nerve function by reducing the incidence of nerve mismatch.

Description of drawings

In order to explain the embodiments of the present invention or the technical solutions in the prior art more clearly, the following briefly introduces the accompanying drawings that need to be used in the description of the embodiments or the prior art. Obviously, the accompanying drawings in the following description are only It is an embodiment of the present invention. For those of ordinary skill in the art, other drawings can also be obtained according to the provided drawings without creative efforts.

Figure 1 is a bionic New Zealand white rabbit sciatic nerve cross-sectional tissue section.

Fig. 2 is the sectional view of the lumen of the artificial nerve conduit with 4 channels designed by the present invention, and the matching relationship between the channel and the nerve bundle in the target nerve.

Figure 3 shows the process of preparing CANC by the mold method, and the illustration shows: 1. catheter forming mold, 2. catheter socket mold, 3. thimble, 4.15% (w/v) gelatin solution, 5. pipe body (multi-channel stent) , 6. Tube wall (PLGA biofilm), 7. CANC catheter.

Figure 4 shows the CANC structure observation under the scanning electron microscope, A. The PLGA biofilm on the outer layer of the catheter was observed under the scanning electron microscope (140 times magnification), and the rectangular box magnification (corresponding to B, 500 times magnification) shows the PLGA fiber diameter and pore structure , C is the multi-channel structure represented by the 4-channel conduit observed under the scanning electron microscope (magnification 20 times), and the enlarged rectangular box (corresponding to D, magnification 200 times) shows the pore structure of gelatin.

Figure 5 is a retrograde fluorescent tracer detection, A. New Zealand white rabbit sciatic nerve trunk (S) and its tibial nerve branch (T), common peroneal nerve branch (P), B. Retrograde fluorescent tracer detection mode diagram. (a, b). Respectively, the tracer retrograde transneural tract in the single-channel catheter (1-CANC) or multi-channel (4-CANC) simulation diagram, C.1-CANC and 4-CANC repair group The number of neurons labeled by NY, FB, FB-NY, and the percentage of retrograde fluorescently labeled neurons of FB-NY in D.1-CANC and 4-CANC repair groups.

Figure 6 shows the results and analysis of nerve tissue sections repaired by 1-CANC and 4-CANC, A. Toluidine blue staining and transmission electron microscope observation of nerve sections repaired by 1-CANC and 4-CANC catheters, B. The differences in the number, density, diameter and thickness of the myelin sheath of the regenerated nerve fibers between the above two kinds of nerve conduits were analyzed and compared.

Figure 7 shows the inner diameter parameters of each catheter lumen. The data in the table shows that the cross-sectional areas of all catheter lumens are basically the same. The matching index (MI) indicates the matching degree of nerve catheter channels and bundles. The matching between 4-CANC and the target nerve is higher than 1- CANC.

Detailed ways

Example 1

According to the biomimetic New Zealand white rabbit sciatic nerve histological section shown in Figure 1, a multi-channel artificial nerve conduit was custom-designed. The multi-channel artificial nerve conduit is a 4-channel nerve conduit (4-CANC), and the cross-section of the conduit is designed using computer-aided design software, as shown in the figure on the right in Figure 2 .

The nerve conduit tube body is an oval structure, the length of the nerve conduit tube body is 10 mm, the long axis of the nerve conduit tube body is 4 mm, and the short axis of the nerve conduit tube body is 2.5 mm.

There are 4 channels in the inner body of the nerve catheter, which are respectively defined as the 1# lumen channel, the 2# lumen channel, the 3# lumen channel, and the 4# lumen channel described in the figure.

The diameter of the 1# lumen channel is 1.2mm, the diameter of the 2# lumen channel is 0.7mm, the diameter of the 3# lumen channel is 0.7mm, and the diameter of the 4# lumen channel is 0.6mm.

The lumen circumference was 10.05 mm, the total lumen area was 2.18 mm ² , and the match index (MI) was 84.4% (Table 1).

Compared with the 4-channel nerve guide (4-CANC), the figure on the left in Figure 2 provides a 1-channel nerve guide (1-CANC), the 1# lumen channel is 1.7mm in diameter, and the matching index ( MI) 75.5% (Table 1).

The multi-channel artificial nerve conduit (CANC) with the above-mentioned structure is prepared by means of the mould shown in Fig. 3, and the above-mentioned data file is imported into the printer to prepare the mould, specifically as follows:

1. Preparation of nerve conduit body (multi-channel stent)

1. Dissolve the gelatin particles in deionized water to prepare a 15% (w/v) gelatin solution.

2. The gelatin solution is poured into the socket mold (2) and the corresponding forming mold (1), according to the channel diameter requirements, insert the corresponding diameter thimble (3) in turn, and freeze and solidify in a -80°C refrigerator.

As shown in Fig. 3, the sleeve die (2) has openings at both ends, and the openings at both ends are respectively embedded with a forming die (1), and a cavity for accommodating the gelatin solution is provided between the upper and lower forming die (1), and then respectively pass through A plurality of thimbles (3) are inserted into the two forming molds (1), and the purpose of inserting the thimbles (3) is to create a channel in the inner body of the nerve catheter, and the solidification is performed by freezing in a -80°C refrigerator.

3. After quickly removing the fixation at both ends of the mold, remove the ejector pin (3), push out the gelatin catheter, and place it in a pre-cooled freeze dryer for 48 hours to freeze dry.

After freeze-fixing, remove the thimble (3), remove the forming molds (1) at both ends of the sleeve mold (2), and then push out the frozen-solid gelatin catheter, and place it in a pre-cooled freeze dryer for lyophilization for 48 hours.

4. Freeze-drying and forming treatment: The parameters set for freeze-drying in the freeze-drying machine are: -30°C, 0.05mPa for continuous freeze-drying for 12 hours, heating to 0°C for 6 hours under vacuum, heating to 22°C and holding for 30-60min, release The vacuum was raised to room temperature to obtain an uncrosslinked nerve conduit tube.

5. Cross-linking process: placed in a mixed solution of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide (EDC, solubility 0.12mol/L) and N-hydroxysuccinimide (NHS, solubility 0.06mol/L) at 4 The cross-linking reaction was carried out at °C for 12 hours to obtain a multi-channel nerve conduit tube (5).

2. Preparation of nerve conduit wall (PLGA biofilm):

1. Dissolve PLGA particles (LA:GA=75:25, molecular weight 100000) in dichloromethane solution to prepare a PLGA solution with a concentration of 10% (w/v).

2. Transfer the PLGA solution into a 10 ml glass syringe of the electrospinning machine and connect to a Teflon tube.

3. Set the distance between the spinning needle of the electrospinning agent and the receiving plate to 15 cm, the horizontal movement speed of the receiving plate to be 0.00147 mm/s, adjust the injection rate of the syringe to 3 mL/h, and adjust the output voltage of the electrospinning machine to be 13-15kV, continuous spinning for 1 hour, to obtain PLGA biofilm (6), which is the nerve conduit wall.

The method for synthesizing the nerve conduit tube body and the nerve conduit tube wall is as follows: drying the obtained PLGA biofilm (6) in a vacuum oven at 40° C. and then fixing the obtained PLGA biofilm (6) on a small amount of gelatin solution. The surface of the nerve conduit body (5), CANC (7) was obtained and observed under a scanning electron microscope (Fig. 3).

The above CANC (7) was subjected to animal surgery and repair effects evaluation. Specifically, the obtained CANC (7) was sterilized by Co ⁶⁰ irradiation and then implanted into a New Zealand white rabbit sciatic nerve defect (10mm) model. Under the 8X special microscope for surgery, the nerve bundles at the distal and proximal ends of the nerve defect are arranged according to the structure (Figure 2). After matching the conduit channels with the nerve bundles, the PLGA biofilm (6) is fixed on both ends of the nerve defect with microscopic sutures. Epineurium structure. After routine feeding, the accuracy of nerve regeneration was assessed by retrograde fluorescence detection at a given time point (Figure 5).

Comparative Example 1

A 1-channel nerve conduit (1-CANC) was custom-designed according to the results of histological sections of the sciatic nerve of New Zealand white rabbits (Fig. 1), and the cross-section of the conduit was designed by computer-aided design software (Fig. 2). The long axis of the catheter was 4 mm, the short axis was 2.5 mm, the diameter of the 1# lumen channel was 1.7 mm, the lumen circumference was 5.34 mm, the total lumen area was 2.27 mm ² , and the matching index (MI) was 77.1%. The remaining steps are the same as those in Example 1.

experiment result shows:

Assessment of regeneration accuracy: 4-CANC and 1-CANC had the same total number of fluorescently labeled neurons (FB-, NY-, FB-NY), while mismatched neurons in 4-CANC (FB-NY) were relatively 1-CANC was less abundant (Fig. 5). Quantity and quality assessment of regenerated nerve fibers: 4-CANC and 1-CANC were the same in the total number and density of new myelinated nerve fibers, while 4-CANC was superior to the diameter of myelinated nerve fibers and the thickness of their myelin sheaths. 1-CANC (Figure 6).

To sum up, the embodiment limits the divergent growth of nerve bundles in the lumen compared to the comparative example, reduces the nerve mismatch rate, and shows the advantage of promoting the development and maturation of myelinated nerve fibers.

The above embodiments are only used to illustrate the technical solutions of the present invention, but not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that: The recorded technical solutions are modified, or some technical features thereof are equivalently replaced; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims (9)

1. a kind of multi path artificial nerve trachea, which is characterized in that including nerve trachea tube body, outside the nerve trachea tube body Layer package nerve trachea tube wall；Multiple channels are equipped in nerve trachea tube body；The nerve trachea tube body and the nerve trachea Tube wall is formed by one or more Material claddings, and has interconnected porous structure.

2. multi path artificial nerve trachea according to claim 1, which is characterized in that nerve trachea tube wall both ends Length is longer than the nerve trachea tube body.

3. a kind of preparation method of multi path artificial nerve trachea, which is characterized in that multi path artificial nerve trachea includes nerve Catheter tube, the outer layer covers nerve trachea tube wall of the nerve trachea tube body；Nerve trachea tube body the preparation method comprises the following steps: step One, gelatin particle is dissolved in deionized water, the gelatin solution that configuration concentration is 15%；Step 2 pours gelatin solution to mould Tool, freezing solid is carried out in -80 DEG C of refrigerators, obtains gelatin conduit；Gelatin conduit is lyophilized and shapes, do not handed over by step 3 The nerve trachea tube body of connection；Uncrosslinked nerve trachea tube body and mixed solution are carried out cross-linking reaction, obtain nerve by step 4 Catheter tube.

4. the preparation method of multi path artificial nerve trachea according to claim 3, which is characterized in that mold includes both ends The socket mold of opening, both ends open are respectively embedded into molding die, have between upper and lower two molding dies and accommodate gelatin solution Cavity.

5. the preparation method of multi path artificial nerve trachea according to claim 4, which is characterized in that in step 2, When gelatin solution is poured to mold, multiple thimbles are inserted by two molding dies respectively, are carried out in -80 DEG C of refrigerators cold Freeze solid, obtains gelatin conduit.

6. the preparation method of multi path artificial nerve trachea according to claim 5, which is characterized in that in step 3, Gelatin conduit is placed in the freeze drier being pre-chilled and is lyophilized 48 hours, obtains uncrosslinked nerve trachea tube body.

7. the preparation method of multi path artificial nerve trachea according to claim 6, which is characterized in that, will in step 4 Uncrosslinked nerve trachea tube body is placed in N- (3-dimethylaminopropyl)-N'-ethylcarbodiimide and N- In hydroxysuccinimide mixed solution, cross-linking reaction 12 hours at 4 DEG C obtain nerve trachea tube body.

8. the preparation method of multi path artificial nerve trachea according to claim 3, which is characterized in that nerve trachea tube wall The preparation method comprises the following steps: step 5, by PLGA grain dissolution in dichloromethane solution, being made into concentration is 10%PLGA solution；Step 6, PLGA solution is transferred in the 10ml glass syringe of electrostatic spinning machine and is connected with Teflon pipe, continuous spinning 1h is obtained Nerve trachea tube wall.

9. the preparation method of multi path artificial nerve trachea according to claim 8, which is characterized in that nerve trachea tube body With the mode of nerve trachea tube wall synthesis are as follows: the nerve trachea tube wall of acquisition is placed in 40 DEG C of vacuum drying oven after drying, is used The nerve trachea tube wall of acquisition is fixed on nerve trachea tube surfaces by a small amount of gelatin solution.