CN113559332A - Preparation method of multi-dimensional multi-branch microporous polymer nerve graft catheter - Google Patents

Abstract

The invention discloses a preparation method of a multi-dimensional multi-branch microporous polymer nerve graft catheter. The multi-dimensional multi-branch nerve conduit can be integrally processed, and is prepared by utilizing the processes of natural macromolecule freezing glass state forming, hot melting wax method demoulding and freeze drying. The method comprises the following specific steps: (1) designing and manufacturing wax cores with different bifurcation structures; (2) preparing a biological macromolecule solution, and precooling; (3) uniformly doping the frozen paraffin particles in the biomacromolecule solution, and cooling to a frozen glass state; (4) coating a wax inner core by utilizing the characteristic that the natural macromolecular frozen glass state has solid and liquid states, controlling the thickness and performing denaturation forming; (5) heating to remove the waxy inner core, and washing with water to remove the wax; (6) drying to obtain the natural polymer nerve graft conduit with multi-dimensional and multi-branch micropores. The catheter of the invention can be used for clinical repair of bifurcated nerve lesions, supporting regeneration of transected axons in their respective independent distal directions.

Description

Preparation method of multi-dimensional multi-branch microporous polymer nerve graft catheter

Technical Field

The invention relates to a preparation method of a multi-dimensional multi-branch microporous natural polymer nerve graft catheter for nerve repair, belonging to the field of biomedical materials and implantable medical devices.

Background

Because the nerve injuries caused by accident traffic accidents, natural disasters and war conflicts are rare, the current gold standard for treating various nerve injuries is still autologous transplantation, but the autologous nerve transplantation is still limited by the source of donor grafts, insufficient length of injured nerves, postoperative dysfunction, poor regeneration capacity and the like, so that the artificial graft made of various biological materials is considered to be an effective alternative treatment method and has achieved better treatment effect. However, many nerves do not grow in a single direction under physiological conditions, but rather need to branch at specific sites for better substance transport or information transfer. Therefore, the construction of the tissue engineering artificial nerve graft with different bifurcation structures has important clinical significance and application value for better realizing nerve injury repair. However, to date, there have been few reports of constructing artificial nerve grafts having different branch structures for nerve damage repair.

The most mature method for preparing the nerve graft developed at present is a casting molding method, but the method is difficult to realize the demoulding of the catheter with a complex structure, so that the nerve graft with a bifurcation structure cannot be prepared. Some researches prepare the 'Y' catheter by a 3D printing technology, an electrostatic spinning technology and the like, but the preparation method is more complicated, and the bifurcations in the same plane can only be realized. Under physiological conditions, the bifurcation of nerves is a more complex three-dimensional structure, and the preparation difficulty can be further increased and the feasibility is greatly reduced when the condition is met.

How to construct hollow biomaterial catheters with different branch structures in vitro is a problem which needs to be solved urgently at present.

Disclosure of Invention

The purpose of the invention is as follows: aiming at the problem that the existing catheter with a multi-dimensional multi-bifurcation structure is single in structure, all of which are straight tube type and can not meet the requirement of repairing and regenerating bifurcation nerves, the invention provides a preparation method of a multi-dimensional multi-bifurcation micropore polymer nerve graft catheter. The microporous natural polymer nerve graft conduit with the multidimensional and multi-bifurcation structure can be used for repairing bifurcation nerve injury, supports the regeneration of transverse axons along the independent distal directions of the axons, and has important clinical application significance.

The technical scheme is as follows: in order to achieve the purpose, the invention adopts the following technical scheme:

a multi-dimensional multi-branch microporous high-molecular nerve graft catheter is prepared through preparing the inner core of multi-dimensional multi-branch catheter with wax, freezing the natural high-molecular material in glass state, demoulding by hot-smelting wax, and drying.

The natural macromolecule frozen glassy state is an amorphous solid state, namely a frozen glassy state, which can be presented by a high-concentration biological macromolecule solution under a low-temperature condition. The polymer is natural biological material, such as chitosan, fibrin or collagen.

The high-concentration biomacromolecule solution is obtained by dissolving chitosan in acetic acid water solution or dissolving silk fibroin and collagen in water. The concentration of the chitosan acetic acid aqueous solution is 4-6%, the concentration of the silk fibroin aqueous solution is 10-20%, and the concentration of the collagen solution is preferably that the solution concentration of the collagen solution presents a frozen glass state under a low temperature condition (the temperature is 0 +/-4 ℃).

The biomacromolecule solution can be added with frozen wax particles, and in the subsequent process, the frozen wax particles are removed by heating to obtain micropores, the pore size of the micropores can be controlled and can be changed along with the size of the wax particles and the processing process, and is generally controlled to be 10-40 μm, and is optimally 20-30 μm.

The specific operation steps are as follows:

step 1), designing and manufacturing a multi-dimensional multi-bifurcation catheter inner core with different bifurcation structures;

melting wax blocks, pouring the melted wax blocks into metal molds of different specifications, standing the melted wax blocks at high temperature to eliminate bubbles, cooling the melted wax blocks until the melted wax blocks are completely solidified, demolding the melted wax blocks to obtain wax cylinders of different diameters, and assembling the wax cylinders into the multidimensional multi-forked inner core.

The inner diameter of the metal die has different specifications of 2mm, 3mm and 4mm so as to prepare wax inner cores with corresponding different inner diameters.

The assembly is to bond and assemble the single wax inner core into the multi-dimensional multi-branched wax inner core by a local heating method.

Step 2), sufficiently dissolving biological macromolecules, standing at a low temperature, and then cooling the solution to a frozen glassy state solution; adding the frozen wax particles, and mixing uniformly.

Step 3) utilizing the characteristic that the natural macromolecular frozen glass state has solid and liquid states, uniformly coating the biological macromolecular solution on the prepared multi-dimensional multi-branched wax inner core, and performing the whole operation process at a low temperature to ensure that the solution keeps the frozen glass state and has poor liquidity, so that the solution is conveniently coated to form uniform thickness and the thickness is controlled;

step 4), quickly setting;

step 5), heating to remove the wax inner core, and fully washing with water until the wax is completely removed;

heating in water bath to 80 ℃ after complete molding, and removing the wax inner core by hot melting; transferring the hollow catheter without the waxy inner core to new 80 ℃ hot water for cleaning; the washing with hot water was repeated until the residual wax was completely removed.

And 6) drying to obtain the multi-dimensional multi-branch microporous natural polymer nerve graft conduit. The drying comprises vacuum drying or freeze drying technology, wherein the vacuum drying is carried out for 48 hours at room temperature, and the freeze drying technology is to adopt a freeze dryer to carry out freeze drying treatment for 24 hours.

Has the advantages that:

(1) the inventor adopts wax to make a casting mould, coats a molding material to form an integral conduit casting mould, and heats the casting mould to remove the wax to form a conduit cavity. The wax has excellent plasticity and is beneficial to the manufacture of complex structures. In addition, the hot melting property of the mould can easily realize the demoulding of a complex structure.

(2) High concentration biomacromolecule solutions can be in an amorphous solid state, i.e., a frozen glassy state, at low temperatures. Under the solid state with the glass-like characteristic, the wax core has plasticity and low fluidity, can be uniformly coated on the wax core, and can be rapidly shaped. Meanwhile, frozen wax particles can be added into the frozen glassy state biomacromolecule solution to be uniformly dispersed. And then in the subsequent hot water washing process, the wax particles are melted and removed to form uniform micropores.

(3) The multi-dimensional multi-branch microporous natural polymer nerve graft catheter is prepared by adopting the multi-dimensional multi-branch inner core made of wax and combining the technologies of natural macromolecule freezing glass state forming, hot melting wax method demoulding and the like, the catheter is made of biological macromolecules, provides a guiding effect for the growth of tissues and cells, can accelerate the repair of nerve injury, and has certain mechanical property, no toxicity and good tissue compatibility, and is beneficial to the proliferation and growth of the cells. Meanwhile, the pore diameter of the micropores of the catheter can be adjusted, thereby being beneficial to exchanging nutrient substances and promoting nerve regeneration. The method has the advantages of simple process, good controllability, high efficiency and the like.

Drawings

FIG. 1 is a schematic view of a multi-dimensional multi-pronged microporous chitosan nerve graft catheter; (a) macroscopic view, (b) external surface electron microscope view, and (c) internal surface electron microscope view.

FIG. 2 is the target muscle loss ratio of example 3: carrying out statistics on wet-weight ratio of 12w gastrocnemius muscles and tibialis anterior muscles after operation;

FIG. 3 is a 8w post-operative axon and remyelination immunofluorescence plot of example 3 (green: NF; red: S100; blue: DAPI);

figure 4 is a schematic view of a multi-dimensional, multi-pronged microporous silk fibroin nerve graft conduit;

FIG. 5 is a diagram of the effect of multi-dimensional multi-branch microporous silk fibroin nerve graft catheter on sciatic nerve repair (remyelination: sciatic nerve distal end fluoroscopy at 3 months after operation, bar is 100 μm);

figure 6 is a graph of the effect of multi-dimensional multi-branched microporous silk fibroin nerve graft catheter on sciatic nerve repair (target muscle loss-to-weight ratio: 12w post-operative calf muscle and tibialis anterior muscle wet-to-weight ratio statistics, representing the group compared to the defect group, p < 0.05 indicating significant difference).

FIG. 7 is a multi-dimensional bifurcated nerve conduit obtained in example 1 applied to FIG. 7 is a facial trigeminal anatomical diagram of a rat.

FIG. 8 is a diagram of the multi-dimensional bifurcated nerve conduit obtained in example 1 applied to the sciatic nerve of New Zealand rabbit.

Detailed Description

In order to make the present invention more fully understood by those skilled in the art, the following technical solutions of the present invention are described with reference to examples and drawings, but the present invention is not limited thereto in any way.

Example 1: preparation of multidimensional multi-branch microporous chitosan nerve graft catheter

1) Preparation of multi-furcation structure waxy inner core:

melting the wax block, pouring the wax block into metal molds with different specifications, cooling and demolding to obtain wax cylinders with different diameters, wherein the inner diameters of the used metal molds have different specifications of 2mm, 3mm and 4mm so as to prepare wax inner cores with corresponding different inner diameters; and (3) bonding and assembling the wax columns with different diameters into the three-dimensional multi-branched wax inner core by a local heating method.

2) Preparation of a frozen glassy chitosan solution containing wax particles:

6g of chitosan powder was weighed and dissolved in 100mL of 3% acetic acid to prepare a 6% chitosan solution. And adding 1-5% of frozen wax particles after fully dissolving, fully mixing uniformly, standing at a low temperature, and keeping the temperature of the solution at-4-4 ℃ to obtain a glassy chitosan solution.

3) Casting and shaping of the guide pipe:

uniformly coating the frozen glassy state chitosan solution containing wax particles on a wax inner core, wherein the wall thickness is 1-2 mm; then the mixture is put into a sodium hydroxide solution for forming.

4) Demolding:

fully soaking the chitosan conduit in an alkali solution until the chitosan conduit is completely formed, heating the alkali solution to 80 ℃, and melting and hydrolyzing the waxy inner core; transferring the hollow chitosan catheter without the waxy inner core to new hot water at 80 ℃ for cleaning; the washing with hot water was repeated until the residual wax was completely removed.

5) And (3) drying:

vacuum drying at room temperature for 48h, or freeze-drying for 24h with a freeze dryer to obtain the multidimensional multi-branched microporous chitosan nerve graft conduit (see figure 1, it can be seen that the forming effect of the multidimensional multi-branched structure of the chitosan conduit is good, and the inner and outer surfaces have uniform pores).

Example 2: preparation of three-dimensional multi-branch silk fibroin conduit with nano pores

1) Preparation of multi-furcation structure waxy inner core: the same as in example 1.

2) Preparation of regenerated silk fibroin

50g of raw silkworm silk is put into 2000mL of 0.5% sodium carbonate solution, treated for 3 times at the temperature of 98-100 ℃ for 30min each time, cleaned, drawn and dried to obtain refined silk for later use. Preparing 50mL of 9.3M lithium bromide aqueous solution, dissolving 10g of refined silk for 0.5h at the temperature of 60 ℃ to obtain a mixed solution, filling the mixed solution into a dialysis bag, changing water every two hours, dialyzing for 72h to obtain a pure silk fibroin solution, and then putting the pure silk fibroin solution into a freeze dryer for freeze drying for 48h to obtain regenerated silk fibroin.

3) Preparation of frozen glassy silk fibroin solution containing wax particles:

10g of regenerated silk fibroin is weighed and dissolved in 100mL of water to prepare a 10% silk fibroin solution. And adding 1-5% of frozen wax particles after fully dissolving, fully and uniformly mixing, standing at a low temperature, and cooling the solution to obtain the silk fibroin solution in a frozen glass state.

4) Casting and shaping of the guide pipe:

the frozen glassy state silk fibroin solution containing wax particles is uniformly coated on a wax inner core, the wall thickness is 1-2mm, and then the mixture is processed by ethanol for forming.

5) Demolding:

fully soaking the fibroin catheter in an ethanol solution until the fibroin catheter is completely formed, and heating in a water bath until the waxy inner core is melted; and transferring the hollow silk fibroin catheter without the waxy inner core into new hot water for cleaning, and repeatedly using the hot water for cleaning until residual wax is completely removed.

6) And (3) drying:

vacuum drying at room temperature for 48h, or freeze drying with a freeze dryer for 24 h. The obtained multi-dimensional multi-branch microporous silk fibroin nerve graft conduit (see fig. 4, the molding effect of the multi-dimensional multi-branch structure of the silk fibroin conduit is good, and the inner surface and the outer surface of the silk fibroin conduit are provided with uniform pores).

Example 3:

SD rat is used as experimental subject, multidimensional bifurcation nerve conduit is applied to regeneration and repair of facial trigeminal nerve (see figure 7), a customized multidimensional bifurcation nerve conduit graft is sutured at the facial nerve defect of the rat, and 8-0 absorbable suture is used for connecting the nerve adventitia with the conduit. Evaluating the functional recovery condition at 2 weeks, 4 weeks, 8 weeks and 16 weeks after operation, wherein the functional evaluation index is beard movement and morphological analysis of nucleus connected with each bifurcation nerve. The result shows that the catheter obtained by the application can accelerate the repair of nerve injury, and the pore diameter of the micropores of the catheter is beneficial to nutrient exchange and promotes nerve regeneration.

Example 4:

the multidimensional bifurcated nerve conduit is applied to regeneration and repair of sciatic nerve by taking a new zealand rabbit as an experimental object (see figure 8). A custom-made multi-dimensional bifurcated nerve conduit graft is sutured to the sciatic nerve defect and an 8-0 absorbable suture connects the adventitia to the conduit. The recovery of function was evaluated at 2, 4, 8, and 16 weeks after the operation. Function evaluation indexes: electromyography of each bifurcation, wet-weight ratio of muscles innervated by each bifurcation, and analysis of muscle morphology. The result shows that the catheter obtained by the application can accelerate the repair of nerve injury, and the pore diameter of the micropores of the catheter is beneficial to nutrient exchange and promotes nerve regeneration.

Example 5: experimental results of sciatic nerve repair Using the Multi-dimensional branched Chitosan nerve graft catheter obtained in example 1

1. Wet weight ratio of target muscle

In addition to causing a loss of sensation and function to the patient after the neurological deficit, it can also lead to atrophy of the surrounding muscles. The experiment is mainly used for observing whether the atrophy degrees of the tibialis anterior muscle and gastrocnemius muscle around the sciatic nerve change after 3 months. The gastrocnemius and tibialis anterior muscles on the healthy and operative sides were perfused and harvested 3 months after the catheter bridging operation, and the wet-to-weight ratio was calculated. As can be seen from the results, at 12w, there was no significant difference in the wet-weight ratio of the target muscles between the chitosan catheter group and the autologous group, indicating that the repair effect of the chitosan catheter group can approach the gold standard, i.e., autologous suture, and the results are shown in FIG. 2.

2. Axon and remyelination

The experiment mainly comprises the steps of crosscutting and freezing a catheter, and observing the regeneration condition of sciatic nerves in the catheter through tissue immunostaining. Axons and myelin sheaths of regenerated sciatic nerves were labeled with both NF and S100 antibodies. Immunofluorescent staining results showed that the ductal group had significant regenerating axons and myelin sheaths, with effects close to that of autologous suture, and the results are shown in fig. 3.

Example 6: experimental result of sciatic nerve repair by using multi-dimensional bifurcated silk fibroin nerve graft conduit obtained in example 2

1. Remyelination

Myelin sheaths of distal regenerated nerves in the silk fibroin catheters were observed for 3 months by a transmission electron microscope, and the observation was mainly performed after the nerve materials were fixed with glutaraldehyde and ultrathin sections were stained. The result of the transmission electron microscope shows that the collagen fibers and the myelin sheaths coexist in the defect group, while no collagen fibers exist in the autologous and ductal groups, which indicates that the regenerative nerve effect of the ductal group and the autologous group is better than that of the defect group. Furthermore, the thickness of the myelin sheath of the defect is relatively thin, the thickness of the myelin sheath of the autologous group is the thickest, and the thickness of the myelin sheath of the catheter group is slightly thinner than that of the autologous group, but significantly thicker than that of the defect group. Myelin is a structure essential for the development of the nervous system and is mainly composed of lipids and proteins arranged in layers with each other, so that thicker layers of myelin indicate better myelination. Thus, the transmission results also indicate that the regenerated sciatic nerve myelin sheath in the silk fibroin catheter works well.

2. Wet weight ratio of target muscle

The gastrocnemius and tibialis anterior muscles on the healthy and operative sides were perfused and harvested 3 months after the catheter bridging operation, and the wet-to-weight ratio was calculated. As can be seen from the results, at 12w, the wet-weight ratio of tibialis anterior muscle and gastrocnemius muscle of the defect group is significantly smaller than that of the autologous group and the silk fibroin catheter group, which indicates that the muscle atrophy recovery effect of the catheter group and the autologous group is significantly better than that of the defect group, and the wet-weight ratio of target muscle of the catheter group and the autologous group is not significantly different, which is also consistent with the myelin sheath result, and which collectively shows the nerve regeneration promotion application value of the silk fibroin catheter, as shown in fig. 6.

Claims (9)

1. A method for preparing multi-dimensional multi-branch microporous polymer nerve graft catheter is characterized in that wax is adopted to prepare an inner core of the multi-dimensional multi-branch catheter, the inner core is uniformly coated with natural biological macromolecule frozen glass state, and the multi-dimensional multi-branch microporous polymer nerve graft catheter is formed by demolding, drying and assembling through a hot melting wax method after molding.

2. The method for preparing the multi-dimensional multi-branch microporous polymer nerve graft conduit according to claim 1, wherein the natural macromolecule frozen glassy state is an amorphous solid state of a high-concentration biological macromolecule material solution under a low-temperature condition; the biological macromolecule is chitosan, vegetarian protein or collagen.

3. The method for preparing a multi-dimensional multi-branched microporous polymer nerve graft conduit according to claim 2, wherein the biomacromolecule solution is a chitosan acetic acid aqueous solution formed by dissolving chitosan in an acetic acid aqueous solution, or a silk fibroin or collagen aqueous solution formed by dissolving silk fibroin or collagen in water.

4. The method of claim 1, wherein the biopolymer solution is added with frozen wax particles and then removed by heating in a subsequent process to obtain micropores, the pore size of which is controlled to 10-40 μm by adjusting the size of the frozen wax particles and the processing technique.

5. The method for preparing the multi-dimensional multi-branch microporous polymer nerve graft conduit according to claim 2, wherein the concentration of the chitosan acetic acid aqueous solution is 4-6%, the concentration of the silk fibroin aqueous solution is 10-20%, and the low temperature is 0 +/-4 ℃.

6. The method of claim 1, wherein the assembly is performed by locally heating the single wax core to bond and assemble the multi-dimensional multi-branched polymer nerve graft conduit.

7. The method for preparing the multi-dimensional multi-branch microporous polymer nerve graft conduit according to claim 1, comprising the following steps:

step 1), manufacturing a multi-dimensional multi-bifurcation catheter inner core with different bifurcation structures;

melting wax blocks, pouring the melted wax blocks into metal molds of different specifications, standing the melted wax blocks at high temperature to eliminate bubbles, cooling the melted wax blocks until the melted wax blocks are completely solidified, demolding the melted wax blocks to obtain wax cylinders of different diameters, and then bonding and assembling the wax cylinders into the multidimensional multi-forked inner core by a local heating method;

step 2), sufficiently dissolving biological macromolecules, standing at a low temperature, and then cooling the solution to a frozen glassy state solution; adding the frozen wax particles, and uniformly mixing;

step 3) utilizing the characteristic that the natural macromolecular frozen glass state has solid and liquid states, and uniformly coating the biological macromolecular solution on the prepared multi-dimensional multi-branched wax inner core under the low-temperature operation condition, wherein the thickness is controllable;

step 4), rapidly shaping;

step 5), heating to remove the wax inner core, and fully washing with water until the wax is completely removed;

and 6) drying to obtain the multi-dimensional multi-branch microporous natural polymer nerve graft conduit.

8. The method for preparing the multi-dimensional multi-branch microporous polymer nerve graft conduit according to claim 6, wherein the inner diameter of the metal mold is 2 mm-4 mm.

9. The method for preparing the multi-dimensional multi-branch microporous polymer nerve graft conduit according to claim 6, wherein the drying comprises vacuum drying or freeze drying technology, the vacuum drying is performed at room temperature for 48 hours, and the freeze drying technology is performed for 24 hours by using a freeze dryer.

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