CN108273138B

CN108273138B - Intelligent-control absorbable peripheral nerve repair catheter and preparation method thereof

Info

Publication number: CN108273138B
Application number: CN201810109693.6A
Authority: CN
Inventors: 陶秀梅; 冷鸿飞; 徐小雨; 陈鹏; 刘培岩
Original assignee: Beijing Nuokangda Pharmaceutical Technology Co ltd
Current assignee: Beijing Nuokangda Pharmaceutical Technology Co ltd
Priority date: 2018-02-05
Filing date: 2018-02-05
Publication date: 2021-01-05
Anticipated expiration: 2038-02-05
Also published as: CN108273138A

Abstract

The invention provides an intelligently-controlled absorbable peripheral nerve repair catheter and a preparation method thereof, wherein the nerve repair catheter has a four-layer structure, wherein a first layer and a second layer of supporting materials are loaded with plasmin sensitive microspheres, a third layer and a fourth layer of supporting materials are supporting layers with mechanical properties at the later stage of catheter implantation, and the catheter is prepared by a 3D spinning printing technology. The high-sensitivity self-regulating slow-release microsphere 3D printing composite gradient catheter provided by the invention can automatically regulate and release nerve growth factors according to nerve growth characteristics, realizes synchronous nerve growth and material degradation, solves the problems of narrow tube caused by in vivo implantation swelling and the like of the existing nerve repair catheter, and avoids adverse effects on biocompatibility due to long-time slow degradation after nerve regeneration is completed.

Description

Intelligent-control absorbable peripheral nerve repair catheter and preparation method thereof

Technical Field

The invention belongs to the technical field of medicine, particularly relates to a peripheral nerve repair catheter and a preparation method thereof, and particularly relates to an intelligently-controlled absorbable peripheral nerve repair catheter and a preparation method thereof.

Background

Peripheral nerve injury is a common type of injury with a high incidence and disability rate, with defective injuries being the most common type of injury. While small defects can be sutured with tension-free ends, autologous nerve transplantation remains the current method of choice for clinical treatment for a wide range of defects, the results of this repair are less than ideal, especially when the nerve defect segment is long (>6 mm). In recent years, the repair of peripheral nerve defects by using a tissue engineering method becomes a research hotspot of people, namely, a nerve scaffold catheter is used for bridging the damaged broken end of the peripheral nerve to provide a regeneration path for the damaged nerve.

Although nerve bridging catheters have achieved some effectiveness in promoting nerve regeneration, the functional recovery of the regenerating nerve is often undesirable. Attempts have been made to improve the repair by loading neurotrophic factors or cells that secrete neurotrophic factors into the bridging material. The patent CN 102343112A prepares a porous chitosan catheter stent, and absorbs neurotrophic factors through a pore structure; patent CN 1241656a prepares a nerve conduit with a longer sustained release time by blending polylactic acid and nerve growth factor. The nerve conduit prepared by directly blending the neurotrophic factors or preparing the neurotrophic factor slow-release microspheres and then blending can promote the regeneration of nerves to a certain extent, however, the demand of the nerve regeneration for different trophic factors in local micro-environments is different at different times, and the local concentration of the exogenous neurotrophic factors (3mg/d) is too high, so that the regeneration speed of the nerves at early stage is reduced. Therefore, how to release neurotrophic factors according to the regeneration needs of nerves is a problem that needs to be researched and solved urgently.

Research shows that plasminogen activator secreted by the nerve regeneration growth cone head can promote the conversion of plasminogen into plasmin. The preparation of plasmin-sensitive delivery systems can control the continuous and local release of trophic factors along with nerve regeneration, and general hospitals of people's liberation force in China have reports on the preparation of plasmin-sensitive microspheres (preparation and evaluation of plasmin-sensitive hydrogel microspheres, liberation of military medical colleges, master academic papers), but the reported studies have the following disadvantages: 1) the preparation method of the microsphere is complex, and the microsphere is prepared by modifying polypeptide with PEG derivative and then polymerizing free radicals; 2) the sensitive slow release period is long, 93.85% is released after 120h, and the release period is far longer than the concentration change period in the nerve growth process (for example, the interval between the two-phase expression peak and the wave trough of mRNA of nerve growth factor NGF at the damaged part is less than or equal to 48h), so that the problem of regulation lag exists; 3) the amido bond and the ester bond in the sustained-release microsphere can be hydrolyzed in the absence of plasmin, thereby leading to the release of growth factors; 4) the entrapment rate of the nerve growth factor is only 73-75%;

patent CN105457103A discloses a 3D prints peripheral nerve pipe, adopts biodegradable PGA and PLA to prepare nerve pipe body and hexagonal tubular column to embedding the growth factor carrier ball that carries different amount growth factors in the hexagonal tubular column, realize the accurate control of nerve growth factor's quantity and gradient. However, this invention has the following disadvantages: (1) neglecting the degradation period of the high polymer material, the catheter material is difficult to avoid water absorption and expansion after the nerve catheter is implanted into the body, and the swelling catheter can cause the stenosis and even the closure in the tube, so that the nerve growth is hindered. (2) The gradient release of the nerve growth factor is completely realized according to the concentration of the growth factor carrier sphere, the concentration of the nerve growth factor carrier sphere can be only speculated according to theory, and the gradient release cannot be realized according to the actual requirement of the nerve growth cycle.

Disclosure of Invention

In order to solve the problems in the prior art, the invention provides the nerve bridging catheter with four layers of gradient distribution, which is prepared by the 3D printing technology, so that the synchronization of the nerve growth rate and the material degradation rate is realized, and the phenomenon that the nerve repairing catheter is implanted and swelled in a body to cause the stenosis in the tube and further block the nerve growth is avoided. The slow release microspheres loaded on the first layer and the second layer of the catheter have simple preparation method, and the design of the multi-arm structure improves the enzymolysis sensitivity of the microspheres to the micro-change of plasmin in different periods of nerve growth, and can sensitively regulate and release nerve growth factors.

The invention provides an intelligently-controlled absorbable peripheral nerve repair catheter which has a four-layer structure, wherein plasmin sensitive microspheres are loaded on a first layer of support material and a second layer of support material, and the third layer of support material and the fourth layer of support material are support layers for mechanical properties at the later stage of catheter implantation.

Further, the plasmin-sensitive microsphere comprises a polypeptide modifier.

Further, the polypeptide modifier is selected from one or more of double-arm or multi-arm PEG-CHO, and the average molecular weight is 1000-30000; the multi-arm is 4-16 arms.

Further, the plasmin-sensitive microsphere further comprises a sensitive polypeptide and a nerve growth factor.

Further, the sensitive polypeptide is selected from one or more of MHVRRR, TQRRLRK and IRYKGKH; the growth factor is selected from one or more of nerve growth factor, brain-derived neurotrophic factor, colloidal neurotrophic factor and neurotrophic factor-3.

Further, in the sensitive microsphere, the polypeptide modifier, the sensitive polypeptide and the nerve growth factor are as follows according to the parts by weight: 2-300: 0.5-1.0: 0.001-0.1

Further, the supporting materials of the first layer and the second layer of the nerve conduit and the loaded plasmin-sensitive microspheres thereof are 100-500: 0.5-9.5.

Furthermore, the supporting materials of the first layer and the second layer of the catheter and the plasmin-sensitive microspheres are mixed according to the weight ratio of 200-: 4-8.

Further, the support material of the first layer of the catheter has a viscosity of 0.05 to 0.5 dl-g^-1The supporting material of the second layer is 0.2-1.0 dl.g^-1The third layer of supporting material of Polycaprolactone (PCL) has viscosity of 0.5-3.0 dl.g^-1The fourth layer of support material is selected from polyglycolic acid (PGA) having a viscosity of 1.0 to 6.0dl · g^-1The polylactic acid (PLA), the Polycaprolactone (PCL), the polylactic-co-glycolic acid (PLGA) and the polylactic-co-polyglycolic acid (PLA-PCL).

Further, the nerve conduit is prepared by 3D spinning printing, the first layer is 0.05-0.2 dl-g^-1The viscosity gradient per cm is distributed from the near end to the far end of nerve growth in an increasing way, and plasmin sensitive microspheres distributed in a gradient decreasing way according to the weight part of 0.1-2 parts per cm are loaded; the second layer has a dl.g value of 0.1-0.5^-1The viscosity gradient of/cm is distributed from the proximal end to the distal end of the nerve growth in an increasing way, and the load is in terms of weightThe plasmin sensitive microspheres are distributed in a gradient descending manner with the amount of 0.1-2 parts per cm.

Furthermore, the four layers of supporting materials of the conduit are subjected to 3D spinning printing with the diameter of 5-50 μm, and the spinning pore diameter of the prepared porous conduit is 5-30 μm.

The invention further provides a preparation method of the plasmin sensitive microsphere, which comprises the following steps: dissolving glucan in a phosphate buffer solution to obtain a glucan solution; dissolving nerve growth factor, sensitive polypeptide and polypeptide modifier in deionized water, and adding NaHCO₃Adjusting pH, adding NaCNBH₃And adding the mixture into a glucan solution, washing with water, centrifuging, and freeze-drying to obtain the plasmin sensitive microspheres.

Furthermore, the preparation method of the plasmin sensitive microsphere comprises the following preparation steps: weighing 50-100 parts of dextran T-500, dissolving in 100-300mL PBS buffer solution, and fully swelling to obtain dextran solution; weighing 0.001-0.1 part of nerve growth factor and 0.5-10 parts of sensitive polypeptide, dissolving in 100-200mL deionized water, adding 2-300 parts of polypeptide modifier, fully dissolving, adding NaHCO₃Adjusting the pH value to 5-7, stirring for 1h, and adding NaCNBH₃10-30 parts of the plasmin sensitive microsphere, continuously stirring for 5 hours, adding the solution into a glucan solution, vortexing at 3000r/min for 2 minutes, standing for 2 hours, repeating the steps for three times, washing with water, centrifuging, collecting the microsphere, and freeze-drying to obtain the plasmin sensitive microsphere.

The invention further provides a preparation method of the intelligently controlled absorbable peripheral nerve repair catheter, which comprises the following steps:

(1) gradient printing of catheter first layer and enzyme sensitive microspheres

Adding PCL with viscosity of 0.05-0.5dl/g into the mixture according to the ratio of 0.05-0.2dl g^-1A viscosity gradient of/cm, placed in 2-9 different print heads respectively; the enzyme sensitive microspheres are respectively mixed into corresponding printing heads in the axial length direction from the nerve growth near end to the nerve growth far end according to the gradient decreasing of (0.1-2 parts)/cm; mixing uniformly, and axially extending from proximal end to distal end of nerve growth according to dl.g of 0.05-0.2 dl.g^-13D printing of a first layer of the catheter from low to high viscosity with a gradient of viscosity change/cm;

(2) catheter second layer and enzyme sensitive microsphere gradient printing

Adding PCL with viscosity of 0.2-1dl/g at a ratio of 0.1-0.5dl g^-1A viscosity gradient of/cm, placed in 2-9 different print heads respectively; the enzyme sensitive microspheres are respectively mixed into corresponding printing heads in the axial length direction from the nerve growth near end to the nerve growth far end according to the gradient decreasing of (0.1-2 parts)/cm; mixing uniformly, and axially extending from proximal end to distal end of nerve growth according to 0.1-0.5 dl.g^-13D printing of a first layer of the catheter from low to high viscosity with a gradient of viscosity change/cm;

(3) third layer printing of catheters

Carrying out spinning printing on PGA with the viscosity of 0.5-3.0dl/g on the wall of the second layer of guide pipe;

(4) conduit fourth layer printing

One or more of PLA, PCL, PLGA and PLA-PCL with the viscosity of 1.0 to 6.0dl/g is subjected to spinning printing on the wall of the third layer of catheter;

the invention has the beneficial effects that:

1) the design of the multi-arm structure in the plasmin sensitive slow-release microsphere increases the degradation sites of the plasmin sensitive microsphere, on one hand, the enzymolysis sensitivity of the plasmin sensitive microsphere to the trace changes of the plasmin in different periods of nerve growth is improved, and the release rate of the nerve growth factor of 10h is more than 86 percent; on the other hand, the crosslinking degree of the microspheres is improved, and the encapsulation rate of the enzyme-sensitive slow-release microspheres to NGF and the like is more than or equal to 95 percent; the sensitive polypeptide in the plasmin sensitive slow-release microsphere is rich in lysine, arginine, histidine and other basic amino acids, and along with the degradation of the catheter material, the basic amino acids in the sensitive polypeptide are released, so that acidic substances generated by the degraded material are neutralized, and the biocompatibility of the catheter is improved.

2) 3D gradient printing of the first layer of pipe wall and the second layer of pipe wall of the pipe realizes synchronization of nerve growth rate and material degradation rate, not only solves the problems of narrow pipe and the like caused by in vivo implantation swelling of the existing nerve repair pipe, but also avoids nerve growth obstruction; but also greatly shortens the distance between the nerve growth tip (the release of plasmin) and the sustained-release microspheres, is more beneficial to the enrichment of growth factors at the nerve growth end, and accelerates the regeneration of nerves.

3) 3D gradient printing of the enzyme sensitive microspheres meets the requirement of nerve regeneration, and can avoid high-concentration nerve growth factor inhibiting nerve regeneration; the invention realizes the double regulation and control of the nerve growth factor through the gradient distribution of the microspheres and the sensitive degradation of the plasmin, improves the release accuracy of the nerve growth factor and realizes the 'precise self-regulation and control release as required' of the nerve growth factor in nerve repair.

4) The third layer and the fourth layer of the catheter are support layers of mechanical properties at the later stage of catheter implantation, PGA, PLA, PCL, PLGA and PLA-PCL basically have no quality degradation loss at the early stage of nerve growth, and rapid degradation is realized at an excellent degradation rate after the nerve growth is finished, so that on one hand, effective mechanical support is provided for the nerve growth period, and on the other hand, adverse effects on biocompatibility caused by slow degradation for a long time after the nerve regeneration is finished are avoided.

Drawings

FIG. 1 is a graph showing staining of nerve fibers of a rat regenerated after the nerve catheter operation prepared in example 1

FIG. 2 is a graph showing staining of nerve fibers of regenerated nerves after a nerve conduit operation in rats prepared by using a comparative example

Detailed Description

Example 1 peripheral nerve repair catheter loaded with four-arm PEG-CHO modified polypeptide sustained-release microspheres

Structural distribution of ducts

The preparation method of the catheter comprises the following steps:

(1) preparation of four-arm PEG-CHO modified polypeptide sustained-release microspheres

Weighing 50 parts of glucan T-500, dissolving in 150ml of PBS buffer solution, and fully swelling; 0.001 part of nerve growth factor and 2 parts of sensitive polypeptide MHVRRR are weighed and dissolved in 100mlAdding 20 parts of four-arm PEG-CHO with average molecular weight of 10000 into ionized water, dissolving completely, and adding NaHCO₃Adjusting the pH value of the solution to 6, stirring for 1h, and adding NaCNBH₃And 15 parts of the plasmin sensitive microsphere, continuously stirring for 5 hours, adding the solution into a glucan solution, vortexing at 3000r/min for 2 minutes, standing for 2 hours, repeating the steps for three times, washing with water, centrifuging, collecting the microsphere, and freeze-drying to obtain the plasmin sensitive microsphere.

(2) Gradient printing of catheter first layer and enzyme sensitive microspheres

250 parts of PCL with the viscosity of 0.05-0.3dl/g are divided into 5 parts according to the viscosity interval of 0.05 dl/g: 50 parts of 0.05-0.1dl/g, 0.1-0.15dl/g, 0.15-0.2dl/g, 0.2-0.25dl/g and 0.25-0.3dl/g which are respectively placed in 5 different printing heads; respectively mixing 1.5 parts of the enzyme sensitive microspheres prepared in the step (1) into 5 printing heads, wherein the parts are 0.5 part, 0.4 part, 0.3 part, 0.2 part and 0.1 part; uniformly mixing, and making into support material with a density of 0.1 dl.g^-1Increasing the viscosity per cm, decreasing the number of the microspheres by 0.2 part per cm, and performing 3D printing on a first layer of the conduit, wherein the diameter of a spinning printing filament is 5 micrometers, the diameter of a spinning pore is 15 micrometers, and the length of the conduit is 25 mm;

(3) catheter second layer and enzyme sensitive microsphere gradient printing

250 parts of PCL with the viscosity of 0.2-0.7dl/g are divided into 5 parts according to the viscosity interval of 0.1 dl/g: 50 parts of 0.2-0.3dl/g, 0.3-0.4dl/g, 0.4-0.5dl/g, 0.5-0.6dl/g and 0.6-0.7dl/g which are respectively placed in 5 different printing heads; 3.5 parts of the enzyme sensitive microspheres prepared in the step (1) are divided into 0.8 part, 0.75 part, 0.7 part, 0.65 part and 0.6 part which are respectively mixed into 5 printing heads; mixing well, and axially extending from proximal end to distal end of nerve growth according to 0.2dl g^-1Increasing the viscosity per cm, decreasing the amount of the microspheres by 0.1 part per cm, and performing 3D printing on a second layer of the catheter, wherein the diameter of a spinning printing filament is 10 micrometers, and the diameter of a spinning pore is 20 micrometers;

(4) the third layer of the conduit is subjected to spinning printing on the wall of the second layer of conduit by PGA with the viscosity of 1.5dl/g, the diameter of a spinning yarn is 5 mu m, and the pore diameter of the spinning yarn is 5 mu m;

(5) the fourth layer of the guide pipe is spun and printed on the wall of the third layer of the guide pipe by PLA-PCL copolymer with the viscosity of 3.0dl/g, the diameter of the spinning yarn is 50 mu m, and the diameter of the spinning hole is 30 mu m;

example 2: peripheral nerve repair catheter loaded with eight-arm PEG-CHO modified polypeptide sustained-release microspheres

Structural distribution of ducts

The preparation method of the repair catheter comprises the following steps:

(1) preparation of eight-arm PEG-CHO modified polypeptide sustained-release microspheres

Weighing 100 parts of glucan T-500, dissolving in 200ml of PBS buffer solution, and fully swelling; weighing 0.01 part of nerve growth factor and 10 parts of sensitive polypeptide MHVRRR, dissolving in 150ml of deionized water, adding 100 parts of eight-arm PEG-CHO with average molecular weight of 8000, fully dissolving, adding NaHCO₃Adjusting the pH value of the solution to 6, stirring for 1h, and adding NaCNBH₃And 20 parts of the plasmin sensitive microsphere, continuously stirring for 5 hours, adding the solution into a glucan solution, vortexing at 3000r/min for 2 minutes, standing for 2 hours, repeating the steps for three times, washing with water, centrifuging, collecting the microsphere, and freeze-drying to obtain the plasmin sensitive microsphere.

(2) Gradient printing of catheter first layer and enzyme sensitive microspheres

270 parts of PCL with the viscosity of 0.05-0.5dl/g are divided into 9 parts according to the viscosity interval of 0.05 dl/g: 0.05-0.10dl/g, 0.10-0.15dl/g, 0.15-0.20dl/g, 0.20-0.25dl/g, 0.25-0.30dl/g, 0.30-0.35dl/g, 0.35-0.40dl/g, 0.40-0.45dl/g, 0.45-0.50dl/g, each 30 parts are respectively placed in 9 different printing heads; dividing 4.5 parts of the enzyme sensitive microspheres prepared in the step (1) into 0.9 part, 0.8 part, 0.7 part, 0.6 part, 0.5 part, 0.4 part, 0.3 part, 0.2 part and 0.1 part, and respectively mixing the parts into 9 printing heads; after being mixed evenly, the supporting material is 0.1dl g along the axial length direction from the nerve growth near end to the far end^-1The viscosity gradient of the per cm is increased progressively, the microspheres are decreased progressively by 0.2 part per cm for 3D printing of the first layer of the conduit, the diameter of a spinning printing filament is 10 mu m, the pore diameter of the spinning filament is 15 mu m, and the length of the conduit is 45 mm;

(3) catheter second layer and enzyme sensitive microsphere gradient printing

200 parts of PCL with the viscosity of 0.5-1.0dl/gThe viscosity interval 0.1dl/g is divided into 5 parts: 40 parts of 0.5-0.6dl/g, 0.6-0.7dl/g, 0.7-0.8dl/g, 0.8-0.9dl/g and 0.9-1.0dl/g which are respectively placed in 5 different printing heads; respectively mixing 5 parts of the enzyme sensitive microspheres prepared in the step (1) into 5 printing heads, wherein the parts are 1.8 parts, 1.4 parts, 1 part, 0.6 part and 0.20 part; mixing, and axially extending from proximal to distal ends along nerve growth direction to obtain a solution with dl-g of 0.1^-1The viscosity gradient of the per cm is increased progressively, the microspheres are decreased progressively by 0.4 part per cm for 3D printing of the first layer of the catheter, the diameter of a spinning printing filament is 30 mu m, and the pore diameter of the spinning filament is 10 mu m;

(4) the third layer of the conduit is subjected to spinning printing on the wall of the second layer of conduit by PGA with the viscosity of 3.0dl/g, the diameter of a spinning yarn is 10 mu m, and the pore diameter of the spinning yarn is 30 mu m;

(5) the fourth layer of the guide pipe is spun and printed on the wall of the third layer of the guide pipe by PLA with the viscosity of 6.0dl/g, the diameter of the spinning yarn is 35 mu m, and the pore diameter of the spinning is 25 mu m;

example 3 peripheral nerve repair catheter loaded with double-arm PEG-CHO modified polypeptide sustained release microspheres

Structural distribution of ducts

The preparation method of the repair catheter comprises the following steps:

(1) preparation of double-arm PEG-CHO modified polypeptide sustained-release microspheres

Weighing 60 parts of glucan T-500, dissolving in 100ml of PBS buffer solution, and fully swelling; weighing 0.05 part of brain-derived neurotrophic factor and 0.5 part of sensitive polypeptide TQRRLRK, dissolving in 120ml deionized water, adding 300 parts of double-arm PEG-CHO with average molecular weight of 1000, dissolving completely, adding NaHCO₃Adjusting the pH value of the solution to 5, stirring for 1h, and adding NaCNBH₃And 10 parts of the plasmin sensitive microsphere, continuously stirring for 5 hours, adding the solution into a glucan solution, vortexing at 3000r/min for 2 minutes, standing for 2 hours, repeating the steps for three times, washing with water, centrifuging, collecting the microsphere, and freeze-drying to obtain the plasmin sensitive microsphere.

(2) Gradient printing of catheter first layer and enzyme sensitive microspheres

Mixing 50 parts of viscosity0.1-0.2dl/g PCL is divided into 5 parts according to the interval of viscosity of 0.02 dl/g: 10 parts of 0.1-0.12dl/g, 0.12-0.14dl/g, 0.14-0.16dl/g, 0.16-0.18dl/g and 0.18-0.20dl/g which are respectively placed in 5 different printing heads; respectively mixing 3 parts of the enzyme sensitive microspheres prepared in the step (1) into 5 printing heads, wherein the parts are 0.68 part, 0.64 part, 0.60 part, 0.56 part and 0.52 part; mixing uniformly, and making into support material of 0.05 dl.g^-1And 3D printing of the first layer of the catheter is carried out by gradient increasing of/cm and decreasing of 0.1 part/cm of microspheres, the diameter of a spinning printing filament is 30 micrometers, the diameter of a spinning pore is 30 micrometers, and the length of the catheter is 20 mm.

(3) Catheter second layer and enzyme sensitive microsphere gradient printing

50 parts of PCL with the viscosity of 0.5-1.0dl/g are divided into 5 parts according to the viscosity interval of 0.1 dl/g: 10 parts of 0.5-0.6dl/g, 0.6-0.7dl/g, 0.7-0.8dl/g, 0.8-0.9dl/g and 0.9-1.0dl/g which are respectively placed in 5 different printing heads; dividing the 5 parts of the enzyme sensitive microspheres prepared in the step (1) into 1.4 parts, 1.2 parts, 1 part, 0.8 part and 0.6 part, and respectively mixing the parts into five printing heads; uniformly mixing, and making into support material of 0.25 dl.g^-1And 3D printing of the first layer of the catheter is carried out by increasing the gradient of/cm and decreasing the microspheres by 0.5 part/cm, the diameter of a spinning printing filament is 5 mu m, and the pore diameter of the spinning filament is 5 mu m.

(4) The third layer of the guide tube was spin-printed with PGA having a viscosity of 0.5dl/g on the wall of the guide tube of the second layer, the diameter of the spun yarn was 30 μm, and the diameter of the spun yarn was 10 μm.

(5) The fourth layer of the guide tube is spun and printed on the wall of the guide tube of the third layer by PCL with the viscosity of 1.0dl/g, the diameter of the spinning yarn is 5 mu m, and the diameter of the spinning pore is 10 mu m.

Example 4 peripheral nerve repair catheter loaded with sixteen-arm PEG-CHO modified polypeptide sustained-release microspheres

Structural distribution of ducts

The preparation method of the peripheral nerve repair catheter loaded with the sixteen-arm PEG-CHO modified polypeptide sustained-release microspheres comprises the following steps:

(1) preparation of sixteen-arm PEG-CHO modified polypeptide sustained-release microspheres

Weighing 80 parts of glucan T-500, dissolving in 300ml of PBS buffer solution, and fully swelling; weighing 0.1 part of colloidal neurotrophic factor and 5 parts of sensitive polypeptide IRYKGKH, dissolving in 200ml of deionized water, adding 2 parts of sixteen-arm PEG-CHO with average molecular weight of 30000, fully dissolving, adding NaHCO₃The pH value of the solution is adjusted to 7, the solution is stirred for 1h, and NaCNBH is added₃And 30 parts of the plasmin sensitive microsphere, continuously stirring for 5 hours, adding the solution into a glucan solution, vortexing at 3000r/min for 2 minutes, standing for 2 hours, repeating the steps for three times, washing with water, centrifuging, collecting the microsphere, and freeze-drying to obtain the plasmin sensitive microsphere.

(2) Gradient printing of catheter first layer and enzyme sensitive microspheres

50 parts of PCL with the viscosity of 0.30-0.50dl/g are divided into 2 parts according to the viscosity interval of 0.1 dl/g: 25 parts of 0.30-0.40dl/g and 0.40-0.50dl/g respectively, which are respectively placed in 2 different printing heads; dividing 2 parts of the enzyme sensitive microspheres prepared in the step (1) into 1.5 parts and 0.5 part, and respectively mixing the parts into 2 printing heads; uniformly mixing, and making into support material of 0.2 dl.g^-1The viscosity gradient of the/cm is increased gradually, 2 parts of microspheres/cm are decreased gradually to perform 3D printing on the first layer of the catheter, the diameter of a spinning printing filament is 50 mu m, the diameter of a spinning pore is 5 mu m, and the length of the catheter is 10 mm.

(3) Catheter second layer and enzyme sensitive microsphere gradient printing

150 parts of PCL with the viscosity of 0.30-0.55dl/g are divided into 5 parts according to the viscosity interval of 0.05 dl/g: 30 parts of 0.30-0.35dl/g, 0.35-0.40dl/g, 0.4-0.45dl/g, 0.45-0.50dl/g and 0.50-0.55dl/g which are respectively placed in 5 different printing heads; dividing 2.0 parts of the enzyme sensitive microspheres prepared in the step (1) into 0.2 part, 0.3 part, 0.4 part, 0.5 part and 0.6 part, and respectively mixing the parts into 5 printing heads; uniformly mixing, and making into support material of 0.25 dl.g^-1And (3) performing 3D printing on the second layer of the catheter by increasing the viscosity gradient of the per cm and decreasing the microspheres by 2 parts per cm, wherein the diameter of a spinning printing filament is 50 mu m, and the diameter of a spinning pore is 30 mu m.

(4) The third layer of the guide tube was spin-printed with PGA having a viscosity of 2.5dl/g on the wall of the second layer of the guide tube, the diameter of the spinning yarn was 25 μm, and the diameter of the spinning hole was 20 μm.

(5) The fourth layer of the catheter was printed on the wall of the third layer of catheter with PLGA having a viscosity of 4.0dl/g, the diameter of the spun yarn was 20 μm and the diameter of the spun yarn was 5 μm.

Example 5 peripheral nerve repair catheter loaded with ten-arm PEG-CHO modified polypeptide sustained-release microspheres

Structural distribution of ducts

The preparation method of the repair catheter comprises the following steps

(1) Preparation of ten-arm PEG-CHO modified polypeptide sustained-release microspheres

Weighing 100 parts of glucan T-500, dissolving in 300ml of PBS buffer solution, and fully swelling; weighing 0.05 part of neurotrophic factor-3 and 7 parts of sensitive polypeptide IRYKGKH, dissolving in 200ml of deionized water, adding 150 parts of ten-arm PEG-CHO with the average molecular weight of 20000, fully dissolving, adding NaHCO₃Adjusting the pH value of the solution to 6, stirring for 1h, and adding NaCNBH₃And 20 parts of the plasmin sensitive microsphere, continuously stirring for 5 hours, adding the solution into a glucan solution, vortexing at 3000r/min for 2 minutes, standing for 2 hours, repeating the steps for three times, washing with water, centrifuging, collecting the microsphere, and freeze-drying to obtain the plasmin sensitive microsphere.

(2) Gradient printing of catheter first layer and enzyme sensitive microspheres

300 parts of PCL with the viscosity of 0.15-0.30dl/g are divided into 3 parts according to the viscosity interval of 0.05 dl/g: 100 parts of each of 0.15-0.20dl/g, 0.20-0.25dl/g and 0.25-0.30dl/g are respectively placed in 3 different printing heads; dividing 0.3 part of the enzyme sensitive microspheres prepared in the step (1) into 0.15 part, 0.10 part and 0.05 part, and mixing the parts into 3 printing heads respectively; uniformly mixing, and making into support material of 0.15 dl.g^-1The viscosity gradient of the/cm is increased gradually, the microspheres are decreased by 0.15 part/cm to perform 3D printing on the first layer of the catheter, the diameter of a spinning printing filament is 25 mu m, the pore diameter of the spinning filament is 10 mu m, and the length of the catheter is 10 mm.

(3) Catheter second layer and enzyme sensitive microsphere gradient printing

50 parts of PCL with the viscosity of 0.25-0.75dl/g are divided into 2 parts according to the viscosity interval of 0.25 dl/g: 25 parts of 0.25-0.5dl/g and 0.5-0.75dl/g respectively, which are respectively placed in 2 different printing heads; dividing 0.2 part of the enzyme sensitive microspheres prepared in the step (1) into 0.15 part and 0.05 part, and mixing the parts into 2 printing heads respectively; uniformly mixing, and making into support material of 0.5 dl.g^-1And (3) performing 3D printing on the second layer of the catheter by increasing the viscosity gradient of the per cm and decreasing the microspheres by 0.2 part per cm, wherein the diameter of a spinning printing filament is 30 mu m, and the pore diameter of the spinning filament is 10 mu m.

(4) The third layer of the guide tube was spin-printed with PGA having a viscosity of 1.0dl/g on the wall of the guide tube of the second layer, the diameter of the spun yarn was 40 μm, and the diameter of the spun yarn was 25 μm.

(5) The fourth layer of the guide pipe is spun and printed on the wall of the guide pipe of the third layer by PLA-PCL with the viscosity of 5.0dl/g, the diameter of the spinning yarn is 10 mu m, and the diameter of the spinning hole is 30 mu m.

Comparative example

Structural distribution of ducts

The preparation method of the catheter comprises the following steps:

50 parts of dextran T-500 are weighed and dissolved in 150ml of sodium bicarbonate buffer (pH 8.5) and fully swelled; weighing 0.001 part of nerve growth factor and 2 parts of sensitive polypeptide MHVRRR, dissolving in 100ml of deionized water, adding 20 parts of four-arm PEG-CHO with average molecular weight of 10000, fully dissolving, adding NaHCO₃Adjusting the pH value of the solution to 6, stirring for 1h, and adding NaCNBH₃And 15 parts of the plasmin sensitive microsphere, continuously stirring for 5 hours, adding the solution into a glucan solution, vortexing at 3000r/min for 2 minutes, standing for 2 hours, repeating the steps for three times, washing with water, centrifuging, collecting the microsphere, and freeze-drying to obtain the plasmin sensitive microsphere. (2) Catheter first layer and enzyme sensitive microsphere printing

And (2) uniformly mixing 250 parts of PCL with the viscosity of 0.2dl/g and 1.5 parts of the sustained-release microspheres prepared in the step (1), and performing 3D printing on the first layer of the catheter, wherein the diameter of a spinning printing filament is 5 micrometers, the diameter of a spinning pore is 15 micrometers, and the length of the catheter is 25 mm.

(3) Catheter second layer and enzyme sensitive microsphere printing

And (2) uniformly mixing 250 parts of PCL with the viscosity of 0.45dl/g and 3.5 parts of the sustained-release microspheres prepared in the step (1), and performing 3D printing on the second layer of the catheter, wherein the spinning diameter is 10 microns, and the spinning aperture is 20 microns.

(4) The third layer of the guide tube was spin-printed with PGA having a viscosity of 1.5dl/g on the wall of the second layer of the guide tube, the diameter of the spun yarn was 5 μm, and the diameter of the spun yarn was 5 μm.

(5) The fourth layer of the conduit is spun and printed on the wall of the third layer of the conduit by PLA-PCL copolymer with the viscosity of 3.0dl/g, the diameter of the spinning yarn is 50 mu m, and the diameter of the spinning pore is 30 mu m.

EXAMPLE 6 measurement of nerve growth factor Release Rate and Encapsulated Rate in sustained Release microspheres

6.1 determination of nerve growth factor Release Rate in Slow Release microspheres

10mg of the sustained-release microspheres prepared in examples 1 to 5 and the sustained-release microspheres in the control group were precisely weighed, extracted in 1ml of PBS buffer solution with pH7.4 at 37 ℃, sampled lml at 10 hours and 96 hours respectively, and supplemented with a corresponding volume of newly formulated PBS buffer solution. The concentration of nerve growth factor is measured by enzyme-linked immunosorbent assay (ELISSA), and the cumulative release amount is calculated.

The preparation method of the slow release microspheres (few sensitive polypeptide plasmin sensitive sites) of the control group comprises the following steps: weighing 50 parts of glucan T-500, dissolving in 150ml of PBS buffer solution, and fully swelling; weighing 0.001 part of nerve growth factor and 2 parts of sensitive polypeptide GGVRNGGK, dissolving in 100ml of deionized water, and adding NaHCO₃Adjusting pH to 6, adding 20 parts of four-arm PEG-CHO with average molecular weight of 10000, dissolving, stirring for 1 hr, and adding NaCNBH₃And 15 parts of the plasmin sensitive microsphere, continuously stirring for 5 hours, adding the solution into a glucan solution, vortexing at 3000r/min for 2 minutes, standing for 2 hours, repeating the steps for three times, washing with water, centrifuging, collecting the microsphere, and freeze-drying to obtain the plasmin sensitive microsphere.

6.2 the method for measuring the encapsulation rate of the growth factors in the sustained-release microspheres comprises the following steps:

encapsulation ratio (%) [ (amount of total NGF charged-amount of NGF in liquid medium)/amount of total NGF charged ] × 100%, wherein the amount of NGF in liquid medium was measured by an enzyme-linked immunosorbent assay (elisas method).

The results of the release rate and the encapsulation rate of the nerve growth factor in the sustained-release microspheres are shown in table 1, and the experimental results show that the sustained-release microspheres prepared in examples 1-5 have the release rate of more than 86% in 10 hours and much higher than the release rate of 68% in a control group, and have significant difference; at 96h, the release rate of the sustained-release microspheres prepared in the embodiments 1 to 5 of the invention is close to 100%, while the release rate of the control group sustained-release microspheres is only 87%, and the release period is far longer than the concentration change period in the nerve growth process, so that the nerve repair is not facilitated. The encapsulation rate of the sustained-release microspheres prepared in examples 1-5 to the growth factor is more than or equal to 95%, while the encapsulation rate of the sustained-release microspheres in the control group to the nerve growth factor is only 73%, which have significant difference. The slow-release microspheres provided by the invention contain the polypeptide modifier, so that on one hand, the enzymolysis sensitivity of the slow-release microspheres to the micro-change of plasmin in different periods of nerve growth is improved, on the other hand, the crosslinking degree of the microspheres is improved, the release rate and the encapsulation rate of the microspheres are increased, and the slow-release microspheres are more suitable for promoting nerve regeneration.

TABLE 1 Release and encapsulation rates of the sustained-release microspheres for nerve growth factor

Example 7 study of the repair Effect of nerve conduits

8 wistar rats were randomly divided into 4 groups of 4 neuroductal groups (group A) prepared in example 1 and 4 neuroductal groups (group B) prepared in comparative example. 10% chloral hydrate is anesthetized by intraperitoneal injection of 0.4mL/100g, and fixed in prone position. Preparing skin of left hind leg, sterilizing with iodophor, cutting skin and muscle, exposing sciatic nerve, and cutting sciatic nerve 6-7mm away to allow it to naturally retract to 10mm defect. Under an XTS-4A surgical microscope, sciatic nerves are cut off, A, B groups of two cut ends are respectively inserted into the catheter for suturing 2mm adventitia and the catheter, the nerve defect gap is kept for 10mm, 11-0 noninvasive suture lines are used for suturing incisions, and the sciatic nerves are raised in cages. And observing the growth condition of nerves, histology and sciatic nerve function detection after 90 days.

(1) Nerve regeneration conditions: the A group of nerve anastomotic stoma has good growth condition, no adhesion and no neuroma formation; the nerves in group B did not grow completely, and adhesions occurred, with 1 neuroma formation.

(2) And (3) histological observation: cutting the far regenerated nerve at the sciatic nerve defect part, fixing with 10% formaldehyde solution for over 24 hr, HE staining, and observing with optical microscope. The results are shown in the attached figures 1-2, the periphery of the nerve cell membrane of the ductal group has a very small amount of lymphocytes, a large amount of Schwann cells are seen in nerve bundles, and neutrophil and lymphocyte infiltration is seen outside the nerve fiber membrane of the B group, which shows that the repairing effect of the A group is better than that of the B group, and the repairing effect is good.

(3) And (3) testing the function of sciatic nerves: the repairing condition of the damaged sciatic nerve function of the rat is detected through myodynamia measurement, electromyogram analysis, gait analysis and electrophysiology. The results are shown in table 2, compared with the group B, the latency of the group A is obviously shortened, the amplitude is obviously increased, and the motor conduction rate is obviously increased, which shows that the sciatic nerve function of the group A is well recovered, and the sciatic nerve recovery effect of the rats in the group B is poorer than that of the group A.

TABLE 2 electrophysiological measurements

	Incubation period (ms)	Wave amplitude (mv)	Speed of motion conduction (m/s)
				Group A	1.89±0.02	10.70±0.44	39.01±1.71
Group B	2.57±0.06*	7.57±0.98*	21.61±2.48*

P is less than 0.05 compared with group A

The results of nerve growth condition, histological observation and sciatic nerve function detection are integrated, and the nerve repair catheter provided by the invention is shown to be capable of promoting nerve growth and repair to be fast and high in anastomosis degree, so that the catheter swelling is prevented from causing in-tube stenosis, and neuroma is not easy to form.

The above detailed description is specific to one possible embodiment of the present invention, and the embodiment is not intended to limit the scope of the present invention, and all equivalent implementations or modifications without departing from the scope of the present invention should be included in the technical scope of the present invention.

Claims

1. An intelligently regulated and controlled absorbable peripheral nerve repair catheter is characterized in that the peripheral nerve repair catheter has a four-layer structure, a first layer and a second layer are loaded with plasmin sensitive microspheres containing nerve growth factors, and a third layer and a fourth layer are support layers of mechanical properties at the later stage of catheter implantation;

the supporting material of the first layer of the catheter is polycaprolactone, the supporting material of the second layer of the catheter is polycaprolactone, the supporting material of the third layer of the catheter is polyglycolic acid, and the supporting material of the fourth layer of the catheter is one or more of polylactic acid, polycaprolactone, polylactic acid-glycolic acid copolymer and polylactic acid-polyglycolic acid copolymer;

the first layer of supporting material is distributed from the nerve growth near end to the far end in an increasing way according to the viscosity gradient and loads plasmin sensitive microspheres distributed in a gradient decreasing way according to the parts by weight; the viscosity gradient of the second layer is distributed from the near end to the far end of the nerve growth in an increasing way, and plasmin sensitive microspheres distributed in a gradient way according to the parts by weight are loaded.

2. The intelligently regulated absorbable peripheral nerve repair catheter of claim 1, wherein the plasmin-sensitive microspheres comprise a polypeptide modifier.

3. The intelligently regulated absorbable peripheral nerve repair catheter of claim 2, wherein the polypeptide modifying agent is selected from one or more of double-arm or multi-arm PEG-CHO with an average molecular weight of 1000-30000; the multi-arm is 4-16 arms.

4. The intelligently regulated absorbable peripheral nerve repair catheter of claim 2, wherein the plasmin-sensitive microspheres further comprise a sensitive polypeptide and a nerve growth factor; the sensitive polypeptide is selected from one or more of MHVRRR, TQRRLRK and IRYKGKH; the growth factor is selected from one or more of nerve growth factor, brain-derived neurotrophic factor, colloidal neurotrophic factor and neurotrophic factor-3; the polypeptide modifier, the sensitive polypeptide and the nerve growth factor are as follows according to the parts by weight: 2-300: 0.5-10: 0.001-0.1.

5. The intelligently regulated absorbable peripheral nerve repair catheter of any one of claims 1-4, wherein the first and second layers of support material and its loaded plasmin-sensitive microspheres are in a weight ratio of 100-: 0.5-9.5.

6. The smart regulated absorbable peripheral nerve repair catheter of claim 5, wherein the support material of the first layer of the catheter has a viscosity of 0.05-0.5 dl-g^-1The supporting material of the second layer is 0.2-1.0 dl.g^-1The third layer of supporting material is polycaprolactone with viscosity of 0.5-3.0 dl.g^-1The fourth layer of support material is selected from the group consisting of polyglycolic acid having a viscosity of 1.0 to 6.0 dl-g^-1The polylactic acid, polycaprolactone, polylactic acid-glycolic acid copolymer and polylactic acid-polyethyleneOne or more alkyd copolymers.

7. The smart regulated absorbable peripheral nerve repair catheter of claim 6, wherein the nerve catheter is prepared by 3D spin printing with the first layer as 0.05-0.2 dl-g^-1The viscosity gradient per cm is distributed from the near end to the far end of nerve growth in an increasing way, and plasmin sensitive microspheres distributed in a gradient decreasing way according to the weight part of 0.1-2 parts per cm are loaded; the second layer has a dl.g value of 0.1-0.5^-1The viscosity gradient of/cm is distributed from the near end to the far end of nerve growth in an increasing way, and plasmin sensitive microspheres distributed in a gradient descending way according to the weight part of 0.1-2 parts/cm are loaded.

8. The intelligently regulated absorbable peripheral nerve repair catheter of claim 7, wherein the four layers of support materials of the catheter are all 3D spun printed with 5-50 μm filament diameter spinning, and the prepared porous catheter has 5-30 μm spinning pore diameter.

9. The intelligently regulated absorbable peripheral nerve repair catheter of claim 8, wherein the preparation method of said plasmin-sensitive microspheres comprises the steps of: dissolving glucan in a phosphate buffer solution to obtain a glucan solution; dissolving nerve growth factor, sensitive polypeptide and polypeptide modifier in deionized water, and adding NaHCO₃Adjusting pH, adding NaCNBH₃And adding the mixture into a glucan solution, washing with water, centrifuging, and freeze-drying to obtain the plasmin sensitive microspheres.

10. A method of making the smart regulated absorbable peripheral nerve repair catheter of claim 1 comprising the steps of:

(1) gradient printing of catheter first layer and enzyme sensitive microspheres

Adding polycaprolactone in the amount of 0.05-0.2 dl-g^-1A viscosity change gradient of/cm, respectively placed in different print heads; respectively mixing plasmin sensitive microspheres into corresponding printing heads according to the gradient decrease of 0.1-2 parts/cm; after being mixed evenly, are3D printing of a first layer of the catheter is carried out in the axial length direction from the nerve growth near end to the far end;

(2) catheter second layer and enzyme sensitive microsphere gradient printing

Adding polycaprolactone in the amount of 0.1-0.5dl g^-1A viscosity change gradient of/cm, respectively placed in different print heads; respectively mixing plasmin sensitive microspheres into corresponding printing heads according to the gradient decrease of 0.1-2 parts/cm; 3D printing a second layer of the catheter in the axial length direction from the nerve growth near end to the nerve growth far end after uniform mixing;

(3) third layer printing of catheters

Carrying out spinning printing on polyglycolic acid on the wall of the second layer of the conduit;

(4) conduit fourth layer printing

And (3) one or more of polylactic acid, polycaprolactone, polylactic acid-glycolic acid copolymer and polylactic acid-polyglycolic acid copolymer is subjected to spinning printing on the wall of the third layer of the conduit.