CN112057672A

CN112057672A - PCL-lignin nanofiber scaffold material and preparation method thereof

Info

Publication number: CN112057672A
Application number: CN202010677165.8A
Authority: CN
Inventors: 闵永刚; 庞贻宇; 廖松义; 刘荣涛; 肖天华; 李达
Original assignee: Guangdong University of Technology; Dongguan South China Design and Innovation Institute
Current assignee: Guangdong University of Technology; Dongguan South China Design and Innovation Institute
Priority date: 2020-07-14
Filing date: 2020-07-14
Publication date: 2020-12-11

Abstract

The embodiment of the invention provides a PCL-lignin nanofiber scaffold material and a preparation method thereof, wherein alkyl lignin, caprolactone and 2-stannous ethyl hexanoate are mixed to form a mixture; after purging with nitrogen, stirring the mixture at high temperature to form a lignin-PCL copolymer; cooling the lignin-PCL copolymer, dissolving the cooled lignin-PCL copolymer with chloroform to form a first solution, and removing unreacted lignin by centrifugation; pouring the supernatant of the first solution into hexane, collecting the precipitate and drying the precipitate in a vacuum oven; dissolving PCL in 1, 1, 1, 3, 3, 3-hexafluoro-2-propanol (HFP) together with the precipitate; and (3) carrying out electrostatic spinning to prepare a fiber mat, and drying the fiber mat in a vacuum oven to form the PCL-lignin nanofiber scaffold material. Compared with other nanofiber scaffold materials, the PCL-lignin nanofiber scaffold material provided by the invention has the characteristics of good oxidation resistance, good biocompatibility, good biodegradability, good mechanical properties and the like.

Description

PCL-lignin nanofiber scaffold material and preparation method thereof

Technical Field

The invention relates to the technical field of biological scaffold materials, in particular to a PCL-lignin nanofiber scaffold material and a preparation method thereof.

Background

The nervous system loses its function due to disease, trauma or aging. Nerve damage is always associated with inflammation and oxidative stress. The human nervous system is particularly vulnerable to damage associated with oxidative stress for a number of reasons, including high oxygen consumption, excessive Reactive Oxygen Species (ROS) production, high polyunsaturated fatty acid content, specific neuronal transmission and synaptic transmission activity (high Ca2 flux). Tissue Engineered (TE) nerve grafts show their potential as replacements for autologous nerve grafts for peripheral nerve repair. However, many synthetic polymeric biomaterials, such as Polycaprolactone (PCL) and poly (lactic acid), invariably induce an inflammatory response, initiating oxidative stress and generating ROS and other free radicals in local tissues. Excessive amounts of ROS can cause damage to cell membrane lipids, proteins and DNA, leading to graft cell death and implant failure. Therefore, nerve TE grafts with antioxidant properties may be a suitable solution to overcome these problems. Various antioxidants, including melatonin, acetyl-L-carnitine, beta-carotene, vitamin E, and B-complex vitamins, have been successfully applied to support nerve repair by counteracting the deleterious effects of ROS. However, all of these conventional antioxidants have limitations such as risk of enzymatic degradation, difficulty in reaching the desired destination, and short half-life.

Disclosure of Invention

The invention provides a PCL-lignin nanofiber scaffold material and a preparation method thereof, aiming at solving the technical problem that the existing biological scaffold is not beneficial to use.

The embodiment of the invention provides a preparation method of a PCL-lignin nanofiber scaffold material, which comprises the following steps:

s1, mixing alkyl lignin, caprolactone and 2-ethyl stannous caproate to form a mixture;

s2, after nitrogen purging, stirring the mixture at high temperature to form a lignin-PCL copolymer;

s3, cooling the lignin-PCL copolymer, dissolving the cooled lignin-PCL copolymer with chloroform to form a first solution, and removing unreacted lignin by centrifugation;

s4, pouring the supernatant of the first solution into hexane, collecting the precipitate and drying the precipitate in a vacuum oven;

s5, dissolving PCL and the precipitate in 1, 1, 1, 3, 3, 3-hexafluoro-2-propanol (HFP);

s6, carrying out electrostatic spinning to prepare a fiber mat, and drying the fiber mat in a vacuum oven to form the PCL-lignin nanofiber scaffold material.

Further, in step S2, the time for purging with nitrogen is 30 to 60 minutes and the mixture is stirred at 130 ℃ for 12 to 24 hours.

Further, in step S3, the lignin-PCL copolymer is dissolved in 100ml of chloroform, centrifuged at 5000rpm, and dried in a vacuum oven at 50-110 ℃ for 12-24 hours.

Further, in step S5, the mass ratio of PCL to precipitate is 95: 5 to 90: 10; the mass concentration range of the first solution is 5-10% w/v.

Further, in step S6, pumping out the solution at a rate of 1ml/h in the electrostatic spinning process, and applying a voltage in the range of 10-15 kV on the needle; collecting the formed fibers on an aluminum foil covered platform to form a fiber mat; the resulting fiber mat was dried in a vacuum oven.

On the other hand, the invention also provides a PCL-lignin nanofiber scaffold material, and the PCL-lignin nanofiber scaffold material is prepared by adopting the preparation method.

The invention has the beneficial effects that: examples of the invention lignin-PCL copolymers (with different lignin content) were synthesized onto alkali lignin by solvent-free ring-opening polymerization (ROP) of caprolactone. In addition, this antioxidant lignin-PCL copolymer was blended with PCL and then engineered into composite fibers by electrospinning. Electrospun nanofibers with a structure similar to the native extracellular matrix (ECM) are considered as potential biomaterial implants for nerve TEs. The PCL-lignin nanofiber scaffold material provided by the invention can increase cell viability, especially after oxidative stress; in addition, Schwann cells and Dorsal Root Ganglion (DRG) neurons can be cultured on the PCL-lignin nanofiber scaffold material to evaluate the nerve regeneration possibility of the PCL-lignin nanofiber scaffold material, and the results show that the PCL-lignin nanofiber scaffold material containing lignin promotes the cell proliferation of BMSCs and Schwann cells, enhances the myelin basic protein expression of Schwann cells and stimulates the neurite outgrowth of DRG neurons; compared with other nanofiber scaffold materials, the PCL-lignin nanofiber scaffold material has the characteristics of good oxidation resistance, good biocompatibility, good biodegradability, good mechanical property and the like.

Detailed Description

In the description of the present invention, it is to be understood that the terms "central," "longitudinal," "lateral," "length," "width," "thickness," "upper," "lower," "front," "rear," "left," "right," "vertical," "horizontal," "top," "bottom," "inner," "outer," "clockwise," "counterclockwise," and the like are used merely for convenience in describing and simplifying the description, and do not indicate or imply that the device or element so referred to must have a particular orientation, be constructed and operated in a particular orientation, and thus are not to be considered as limiting.

Furthermore, the terms "first", "second" and "first" are used for descriptive purposes only and are not to be construed as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. In the description of the present invention, "a plurality" means two or more unless specifically defined otherwise.

In the present invention, unless otherwise expressly specified or limited, the terms "mounted," "connected," "secured," and the like are to be construed broadly and can, for example, be fixedly connected, detachably connected, or integrally connected; can be mechanically or electrically connected; they may be connected directly or indirectly through intervening media, or they may be interconnected between two elements. The specific meanings of the above terms in the present invention can be understood by those skilled in the art according to specific situations.

In the present invention, unless otherwise expressly stated or limited, "above" or "below" a first feature means that the first and second features are in direct contact, or that the first and second features are not in direct contact but are in contact with each other via another feature therebetween. Also, the first feature being "on," "above" and "over" the second feature includes the first feature being directly on and obliquely above the second feature, or merely indicating that the first feature is at a higher level than the second feature. A first feature being "under," "below," and "beneath" a second feature includes the first feature being directly under and obliquely below the second feature, or simply meaning that the first feature is at a lesser elevation than the second feature.

The present invention will be described in further detail with reference to the following embodiments.

Examples of the invention lignin-PCL copolymers (with different lignin content) were synthesized onto alkali lignin by solvent-free ring-opening polymerization (ROP) of caprolactone. In addition, this antioxidant lignin-PCL copolymer was blended with PCL and then engineered into composite fibers by electrospinning. Electrospun nanofibers with a structure similar to the native extracellular matrix (ECM) are considered as potential biomaterial implants for nerve TEs. The PCL-lignin nanofiber scaffold material provided by the invention can increase cell viability, especially after oxidative stress; in addition, Schwann cells and Dorsal Root Ganglion (DRG) neurons can be cultured on the PCL-lignin nanofiber scaffold material to evaluate the nerve regeneration possibility of the PCL-lignin nanofiber scaffold material, and the results show that the PCL-lignin nanofiber scaffold material containing lignin promotes the cell proliferation of BMSCs and Schwann cells, enhances the myelin basic protein expression of Schwann cells and stimulates the neurite outgrowth of DRG neurons; compared with other nanofiber scaffold materials, the PCL-lignin nanofiber scaffold material has the characteristics of good oxidation resistance, good biocompatibility, good biodegradability, good mechanical property and the like.

Lignin is a well-known oxygen radical scavenger used to stabilize reactions initiated by oxygen radicals. Such complex aromatic macromolecules contain a large number of hydroxyl and methoxy functional groups that can provide hydrogen to terminate oxidative propagation reactions. Many research groups have reported that lignin may be used as an antioxidant for healthcare applications, but lignin remains a "rather unexplored" biomaterial. In our previous studies, we designed and developed functional lignin-based materials that exhibit excellent antioxidant properties and biocompatibility.

PCL is a biodegradable polyester approved by the U.S. Food and Drug Administration (FDA), and PCL-based scaffolds have been widely used as scaffolds for various tissue engineering applications due to their biocompatibility, slow degradation rate and ease of processing. Thus, PCL is used as a matrix material and polymerized with lignin to obtain a copolymer, which can overcome the disadvantages of using PCL and lignin alone, including mechanical properties, biocompatibility and resistance to oxidative damage. The morphology, mechanical properties and antioxidant activity of the blended nanofibers were evaluated. In addition, neurons and schwann cells were cultured on the fibers to evaluate their growth behavior. The lignin-PCL copolymer provided by the embodiment of the invention can provide mechanical enhancement and oxidation resistance for the obtained nano-fiber, which can increase the activity of nerve cells against oxidative stress and promote the neurite outgrowth of the nerve cells and the growth of Schwann cells for nerve regeneration.

In an alternative embodiment, the lignin-PCL copolymer comprises an alkyl lignin, caprolactone and tin (II) 2-ethylhexanoate, and 0.5 wt% by weight of tin (II) 2-ethylhexanoate monomer as catalyst for the reaction is added to a round bottom flask and stirred at high temperature.

In an alternative embodiment, in step S2, the nitrogen purge time is 30 to 60 minutes and the mixture is stirred at 130 ℃ for 12 to 24 hours.

In an alternative embodiment, in step S3, the lignin-PCL copolymer is dissolved with 100ml chloroform, centrifuged at 5000rpm, and dried in a vacuum oven at 50-110 ℃ for 12-24 hours.

In an alternative embodiment, in step S5, the mass ratio of PCL to precipitate is 95: 5 to 90: 10; the mass concentration range of the first solution is 5-10% w/v.

In an alternative embodiment, in step S6, the solution is pumped out by the electrostatic spinning process at 1ml/h, and a voltage in the range of 10-15 kV is applied to the needle; collecting the formed fibers on an aluminum foil covered platform to form a fiber mat; the resulting fiber mat was dried in a vacuum oven.

Compared with other nanofiber scaffolds, the invention has the characteristics of good oxidation resistance, good biocompatibility, good biodegradability, good mechanical property and the like.

The specific embodiment is as follows:

example 1

The lignin-PCL copolymer is synthesized by solvent-free polymerization.

1. Alkyl lignin (4 g), caprolactone (6 g) and tin (II) 2-ethylhexanoate (0.5 wt% of monomer as catalyst) were added to a round bottom flask. After purging with N2 for 30 minutes, the mixture was stirred at 500rpm for 24 hours at 130 ℃.

2. The mixture was cooled to room temperature and dissolved with 100ml of chloroform, the synthesized lignin-PCL copolymer was dissolved in chloroform, and unreacted lignin was removed by centrifugation (5000 rpm, 5 minutes). The supernatant was poured into excess hexane, and the precipitate (lignin-PCL copolymer) was collected and dried in a vacuum oven at 50 ℃ for 24 hours to form PCL-lignin nanofiber scaffold material.

3. PCL was dissolved in 1, 1, 1, 3, 3, 3-hexafluoro-2-propanol (HFP) together with lignin-PCL copolymer. The mass ratio of the PCL to the lignin-PCL copolymer is 95: 5. the total concentration of the solution was 10% (w/v). Stirred for 24 hours.

4. The homogeneous solution was loaded into a 5 mL syringe using a 22 gauge blunt needle. The mixture was pumped out of the solution at a rate of 1ml/h and a voltage of 15kV was applied to the needle. The formed fibers were collected on an aluminum foil covered platform. (10 cm from the tip). The resulting fiber mat was dried in a vacuum oven overnight.

Example 2

The lignin-PCL copolymer is synthesized by solvent-free polymerization.

1. The alkyl lignin (2 g), caprolactone (8 g) was added to a two-necked 150 mL round bottom flask for reaction. Tin (II) 2-ethylhexanoate (0.5 wt% of the monomer as catalyst) was added and the mixture was stirred at 110 ℃ and 350rpm for 24 h.

2. The mixture was cooled and chloroform (100 mL) was added to dissolve the synthesized lignin copolymer (unreacted lignin would not dissolve). Unreacted lignin was removed by centrifugation and the supernatant was precipitated twice in diethyl ether. The lignin-PCL copolymer was dried overnight in a vacuum oven at 50 ℃.

3. The prepared lignin-PCL copolymer and PCL were dissolved together in a mixed solvent of Dichloromethane (DCM) and Dimethylformamide (DMF) in a solvent ratio of 7: 3. the mixture was stirred well overnight to obtain a homogeneous mixture. The mass ratio of the PCL to the lignin-PCL copolymer is 90: 10, the total concentration of the solution was 8% (w/v). Stirred for 24 hours.

4. The homogeneous solution was loaded into a 5 mL syringe using a 22 gauge blunt needle. The mixture was withdrawn at a rate of 1mL/h and a voltage of 10kV was applied to the needle. The nanofibers were collected on an aluminum foil wrapped around the collector, the distance between the aluminum foil and the needle tip was 15 cm. And after the electrostatic spinning is finished, drying the obtained nano fibers in a vacuum oven overnight to form the PCL-lignin nano fiber scaffold material.

Example 3

The lignin-PCL copolymer is synthesized by solvent-free polymerization.

1. Alkyl lignin (3 g), caprolactone (7 g) and tin (II) 2-ethylhexanoate (0.5 wt% of the monomer as catalyst) were added to a round bottom flask. After purging with N2 for 30 minutes, the mixture was stirred at 450rpm for 24 hours at 120 ℃.

2. The mixture was cooled to room temperature and dissolved with 100ml of chloroform, the synthesized lignin-PCL copolymer was dissolved in chloroform, and unreacted lignin was removed by centrifugation (5000 rpm, 5 minutes). The supernatant was poured into excess hexane and the precipitate (lignin-PCL copolymer) was collected and dried in a vacuum oven at 50 ℃ for 24 hours.

3. PCL was dissolved in 1, 1, 1, 3, 3, 3-hexafluoro-2-propanol (HFP) together with lignin-PCL copolymer. The mass ratio of the PCL to the lignin-PCL copolymer is 90: 10. the total concentration of the solution was 12% (w/v). Stirred for 24 hours.

4. The homogeneous solution was loaded into a 5 mL syringe using a 22 gauge blunt needle. The mixture was pumped out of the solution at a rate of 1ml/h and a voltage of 12kV was applied to the needle. The formed fibers were collected on an aluminum foil covered platform. (12 cm from the tip). The resulting fiber mat was dried in a vacuum oven overnight to form PCL-lignin nanofiber scaffold material.

In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples" or the like, mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above do not necessarily refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.

The foregoing is a more detailed description of the present invention that is presented in conjunction with specific embodiments, and the practice of the invention is not to be considered limited to those descriptions. It will be apparent to those skilled in the art that a number of simple derivations or substitutions can be made without departing from the inventive concept.

Claims

1. A preparation method of a PCL-lignin nanofiber scaffold material is characterized by comprising the following steps:

2. The method for preparing PCL-lignin nanofiber scaffold material according to claim 1, wherein in step S2, the time of purging with nitrogen is 30-60 minutes and the mixture is stirred at 130 ℃ for 12-24 hours.

3. The method for preparing PCL-lignin nanofiber scaffold material according to claim 1, wherein in step S3, the lignin-PCL copolymer is dissolved in 100ml of chloroform, centrifuged at 5000rpm, and dried in a vacuum oven at 50-110 ℃ for 12-24 hours.

4. The method for preparing PCL-lignin nano fiber scaffold material according to claim 1, wherein in step S5, the mass ratio of PCL to the precipitate is 95: 5 to 90: 10; the mass concentration range of the first solution is 5-10% w/v.

5. The method for preparing PCL-lignin nanofiber scaffold material according to claim 1, wherein in step S6, the solution is pumped out by 1ml/h in the electrostatic spinning process, and a voltage in the range of 10-15 kV is applied on the needle; collecting the formed fibers on an aluminum foil covered platform to form a fiber mat; the resulting fiber mat was dried in a vacuum oven.

6. A PCL-lignin nanofiber scaffold material, which is characterized by being prepared by the preparation method of any one of claims 1-5.