CN107648671B

CN107648671B - Multilayer anti-infection high-strength artificial dura mater and preparation method thereof

Info

Publication number: CN107648671B
Application number: CN201711094466.2A
Authority: CN
Inventors: 李瑞锋; 王斐; 李典
Original assignee: Individual
Current assignee: Individual
Priority date: 2017-11-09
Filing date: 2017-11-09
Publication date: 2021-02-26
Anticipated expiration: 2037-11-09
Also published as: CN107648671A

Abstract

The invention provides a multilayer anti-infection high-strength artificial dura mater, which comprises three layers, a hydrophobic inner layer facing the brain, a water-soluble chitosan outer layer facing away from the brain and a woven middle layer. The invention also provides a preparation method of the multilayer anti-infection high-strength artificial dura mater, which comprises the following steps: preparing a hydrophobic inner layer by electrospinning; preparing water-soluble chitosan thin strips; preparing hydrophobic strips; preparing a woven middle layer; preparing an outer layer on the middle layer by using a directional electrospinning process, and finally obtaining the artificial dura mater. The artificial dura mater of the invention effectively solves the infection problem in the dura mater repair process, simultaneously improves the strength of the traditional artificial dura mater, can be cut randomly according to requirements, and has safety, reliability and wide application prospect.

Description

Multilayer anti-infection high-strength artificial dura mater and preparation method thereof

Technical Field

The invention relates to a multilayer anti-infection high-strength artificial dura mater and a preparation method thereof, belonging to the technical field of electrostatic spinning.

Background

Dural defects are common in neurosurgical clinical work, and open craniocerebral injuries (industry, traffic, war and the like), tumor erosion, congenital meningeal defects and other craniocerebral disease reasons can cause dural defects. The dural defect needs to be repaired in time to prevent cerebrospinal fluid from overflowing, prevent the swelling of brain and the pressure of atmospheric pressure, otherwise, the life of a human body is endangered. The dural defect can also cause intracranial infection, brain adhesion, subcutaneous effusion and other complications, and often causes diseases such as headache, brain dysfunction and the like.

Currently, artificial dura mater made of various materials is in clinical use and can be mainly divided into two main categories, i.e., biologically derived materials and artificially synthesized polymer materials. The biological derived materials mainly comprise allogenic human dura mater, xenogenic porcine/bovine pericardium, dermal matrix, biological membrane prepared by bovine myokey type I collagen, and the like. The artificially synthesized polymer material mainly comprises polyester degradable polymers, such as polylactic acid, polyglycolic acid, polycaprolactone, polyurethane and the like. In addition, the material also comprises non-degradable high molecular materials such as polytetrafluoroethylene and the like.

At present, the meningeal injury repair effect is ideal for multilayer artificial dura mater. The multilayer artificial dura mater achieves the aim of simulating the physiological function of the dura mater through layering. Such artificial dura mater is generally composed of an inner layer and an outer layer. Wherein the inner layer is made of hydrophobic material and the outer layer is made of hydrophilic material. Due to the fact that the hydrophobic and hydrophilic properties of the two layers of materials are different, the reliability of connection between the outer layer and the inner layer is low, the peeling phenomenon occurs, and the life health of a patient is seriously threatened.

The meninges are mainly distributed with fibroblasts and collagen fibers secreted by the fibroblasts. Typically, the fibroblasts are between 20 and 30 μm in diameter. The literature reports that the average pore size can reach 2 μm when the electrospun fiber has a diameter between 50 and 1000 nm. The average pore diameter is less than 3 mu m, so that cells can be effectively prevented from entering the electrostatic spinning fiber film, and brain adhesion can be effectively generated. When the diameter of the fiber is between 5 and 200 mu m, the average pore diameter reaches 20 to 100 mu m. This facilitates the migration, adhesion, proliferation and growth differentiation of fibroblasts.

Chitosan is the only alkaline polysaccharide with cations found in nature, has good biocompatibility, degradability and tissue adhesion prevention characteristics, and more importantly, a large number of researches prove that the chitosan has excellent broad-spectrum antibacterial characteristics and is widely applied clinically, so the chitosan is an ideal material for preparing the anti-infection artificial dura mater.

A double layer electrospun membrane was described in Kyle Kurpinski et al, Nanomedicine, using a PLCL/PPG solution. The article obtained electrospun membranes arranged in parallel using a rotating shaft collection device, tested the mechanical strength of electrospun membranes in the directions parallel and perpendicular to the arrangement of fibers, with a sample size of 6 × 1cm, and measured maximum loads in both directions of 12.08 ± 1.15N and 3.01 ± 0.54N, respectively. It can be seen that the strength of the film in the direction coincident with the direction of the fibers is poor.

Chinese patent CN 103480042A, the name of which is "an artificial dura mater and a preparation method and a use method thereof", discloses an artificial dura mater with a double-layer structure, which is composed of an oriented polylactic acid/glycolic acid copolymer fibrous membrane inner layer and a non-oriented polylactic acid/glycolic acid copolymer-chitosan fibrous membrane outer layer. The artificial dura mater spinalis can realize the purposes of dura mater regeneration and preventing paralysis and adhesion, but has the defects that (1) outer layer fibers are compounded by polylactic acid/glycollic acid copolymer and chitosan, and when the content of the chitosan is lower, the anti-infection activity is inevitably limited, but the anti-infection performance of the artificial dura mater spinalis is not mentioned in the patent, and (2) the outer layer fibers and the inner layer fibers are required to be separately placed at the defect position of the dura mater spinalis in the clinical use process, and a binding agent is required to be coated between the two layers, so that the inconvenience in the clinical use process is greatly increased.

The invention patent CN 103656749A of China, entitled "a compound degradable antibacterial artificial dura mater and its preparation method", discloses an antibacterial artificial dura mater containing vancomycin or ofloxacin hydrochloride injection, chitosan and beta-sodium glycerophosphate, but the product mainly utilizes antibiotic drugs in the material composition to realize the antibacterial purpose, and the chitosan is only used as a drug carrier. Meanwhile, the artificial dura mater is difficult to register and report as a drug and instrument combination product, and industrial development and clinical application are not easy to realize.

The invention relates to Chinese patent CN106943634A entitled absorbable artificial dura mater with anti-infection function and a preparation method and application thereof, and discloses a nano-fiber membrane consisting of a degradable poly-acetate nano-fiber membrane and a chitosan nano-fiber membrane double-layer structure. The absorbable artificial dura mater has good anti-infection performance, can effectively prevent postoperative intracranial infection, can bear certain suturing force, and is not torn or stripped. However, two layers of the product are both prepared by adopting a roller orientation spinning process, two meninges layers are both oriented nano fibers, and the strength of the fiber film is far lower than that of a non-oriented fiber film. Meanwhile, the artificial dura mater has only two layers, and the bonding strength between the two layers is not enough.

Therefore, the artificial dura mater which has high strength, high brain pressure resistance, reliable combination of the inner layer and the outer layer, strong infection resistance and convenient use is urgently needed clinically at present.

Disclosure of Invention

In order to overcome the defects of the prior art, the invention aims to provide a multilayer anti-infection high-strength artificial dura mater, which is different from the traditional anti-infection artificial dura mater in that:

(1) the outer layer of the artificial dura mater is prepared from chitosan through a directional electrostatic spinning process, has reliable anti-infection performance, can be degraded, is non-toxic, and is safe and reliable.

(2) The artificial dura mater inner layer is prepared by adopting an electrospinning process to obtain the non-oriented nanofiber film. The tear strength of the film is far greater than that of an oriented film, and the integral tear strength of the artificial dura mater is improved.

(3) The woven middle layer is additionally arranged between the inner layer and the outer layer of the artificial dura mater, the middle layer is woven by adopting a hydrophobic material and a hydrophilic material, according to the principle of 'similar compatibility', the thin strips made of the hydrophobic material in the woven layer are reliably combined with the inner layer (made of the hydrophobic material by electrospinning), the thin strips made of the hydrophilic material are reliably combined with the outer layer (made of the hydrophilic material by electrospinning), the two thin strips are overlapped and interwoven together, the thin strips made of the hydrophilic material penetrate through the gaps of the thin strips made of the hydrophobic material and are combined with the outer layer, the thin strips made of the hydrophobic material penetrate through the gaps of the thin strips made of the hydrophilic material and are combined with the inner layer, and the inner layer and the outer layer are reliably combined together. In a word, the artificial dura mater with the structure improves the binding force between the inner layer and the outer layer, and is not easy to peel off from each other under the action of external force.

The invention also aims to provide a preparation method of the multilayer anti-infection high-strength artificial dura mater.

The technical scheme adopted by the invention for solving the problems is as follows:

a multilayer anti-infection high-strength artificial dura mater comprises three layers, wherein one layer facing to the brain is a hydrophobic inner layer, the other layer facing away from the brain is a water-soluble chitosan outer layer, and a woven middle layer is arranged between the two layers.

The hydrophilic outer layer is made by electrostatic spinning, namely, in order to effectively induce stem cells and fibroblasts to migrate, the hydrophilic outer layer adopts water-soluble chitosan with good biocompatibility, and furthermore, the average pore diameter reaches 20-100 μm and the diameter of the fibroblasts is generally between 20-30 μm by adjusting electrostatic spinning parameters. This facilitates the migration, adhesion, proliferation and growth differentiation of fibroblasts.

The inner layer is made of hydrophobic materials, so that cells are prevented from migrating, and the purpose of preventing adhesion is achieved; the electrostatic spinning parameters for preparing the layer material are adjusted, so that the pore diameter of pores is below nanometer and is one to two orders of magnitude smaller than cells (the diameter of general fibroblasts is between 20 and 30 mu m), thereby preventing the cells from entering and preventing the brain adhesion.

The middle layer is woven by a hydrophobic material and a water-soluble chitosan material.

A multilayer anti-infection high-strength artificial dura mater comprises an inner layer, a middle layer and an outer layer, and the preparation method comprises the following steps:

(1) preparation of the inner layer: the inner layer material component is polycaprolactone (Bolii biomaterial, Inc., Shenzhen), dissolved in hexafluoroisopropanol (Bolii biomaterial, Inc., Suzhou Hao Sai), the concentration of the spinning solution is 7% (wt), the stirring is carried out for 4 hours, the electrospinning parameters are that the flow rate is 0.9 ml/h, the voltage is 13kV, the receiving distance is 20cm, and the electrospinning is carried out for 4 hours.

(2) And preparing the intermediate layer.

The film prepared by electrostatic spinning of carboxymethyl chitosan (Nantong Lvsheng bioengineering Co., Ltd.) has a thickness of 40 μm. The film was made into thin strips of 3mm in width and 1000mm in length.

The film is prepared by electrostatic spinning of polycaprolactone (Bolii biomaterial, Inc. of Shenzhen), and the thickness of the film is 40 μm. The film was made into thin strips of 3mm in width and 1000mm in length.

The two kinds of strips are 3mmx1000mm face downwards, the hydrophilic strips are transversely arranged, the hydrophobic strips are longitudinally arranged, and the weaving type middle layer is obtained by weaving through a Hercules weaving process.

(3) The woven middle layer is layered on the upper surface of the inner layer before the inner layer is cured.

Finally, the intermediate layer was pressed down with a pressure of 50N for 1 min.

(4) Preparing an outer layer:

the outer layer material is carboxymethyl chitosan (Nantong Green bioengineering Co., Ltd.), dissolved in purified water (Jinan sea Industrial chemical Co., Ltd.), the concentration of the spinning solution is 6% (wt), stirred for 1 hour, and the electrospinning parameters are as follows: the flow rate is 0.4 ml/h, the voltage is 0.9kV, the receiving distance is 1mm, the orientation electrostatic spinning process is used for preparing for 2h, an orientation nanofiber layer is obtained, and the fiber arrangement direction is parallel to any side.

(5) Taking out the artificial meninges from the culture dish, rinsing with distilled water for 3 times, freeze-drying, vacuum-packaging, sterilizing with 25kGy of cobalt-60, and storing at-20 deg.C.

The thickness of each layer of the prepared multilayer electrostatic spinning artificial dura mater is as follows: the thickness of the inner layer is 80 μm +/-10 μm, the thickness of the outer layer is 30 μm +/-10 μm, and the thickness of the middle layer is 80 μm +/-20 μm.

When the inner layer is prepared, the diameter of the electrostatic spinning fiber is controlled between 50 nm and 1000nm, and the average pore diameter is 2 mu m;

when the outer layer is prepared, the diameter of the electrostatic spinning fiber is controlled between 5 and 200 mu m, and the average pore diameter is 20 to 100 mu m.

The invention has the beneficial effects that:

(1) the artificial dura mater adopts a three-layer structure, and the bonding strength of the inner layer and the outer layer is enhanced through the middle layer.

(2) The mechanical properties of the artificial dura mater can meet the requirements of the adaptation on tensile strength and flexibility, and the artificial dura mater has reliable strength and good flexibility and elasticity; can support a suture needle, is waterproof, can bear certain force and generate deformation without damage when and after surgical repair, prevents cerebrospinal fluid from leaking, plays a role in protecting brain tissue, and can be cut at will according to requirements.

(3) The inner side surface (namely the surface facing the brain) of the artificial dura mater is smooth, can prevent adhesion and can play a role in well isolating scalp soft tissues and brain tissues, and the outer side surface (namely the surface back to the brain) and the side edges are of porous structures and allow cells to grow in; can promote autologous fibroblasts and some undifferentiated cells to infiltrate into the collagen for growth as soon as possible, and promote early repair of meninges. Is beneficial to the skull repairing operation in the future.

(4) The dura mater of the invention is made of a material which is proved to be completely absorbed after the generation of the new tissue, and the carcinogenesis of the diaphragm is avoided.

(5) The material used in the invention is a safe biological material which is proved to be nontoxic and harmless to human bodies at present, has good biological tissue compatibility, no foreign matter rejection reaction, no toxic reaction and no carcinogenic and teratogenic effects. The method does not bring a plurality of risks of immunological rejection, virus transmission and disease infection, and does not bring other toxic effects.

(6) The preparation method of the artificial meninges has the advantages of simplified process steps, short production time, capability of effectively preventing products from being polluted in the processing process, easiness in controlling the product quality, easiness in realizing the product standard and capability of realizing low-cost and high-efficiency industrial production of the products.

(7) The artificial meninges prepared according to the invention is simple in clinical application, toxic substances such as glutaraldehyde and the like are not introduced in the treatment, and strict soaking and cleaning are not required in the clinical application, so that the condition that products are scrapped due to insufficient preselected size caused by early soaking is avoided.

Drawings

Fig. 1 is a schematic structural diagram of the prepared artificial dura mater, wherein 1 is an inner layer, 2 is an intermediate layer, and 3 is an outer layer.

Fig. 2 is a schematic view of the structure of the intermediate layer. 4 is a thin strip cut from a carboxymethyl chitosan film, and 5 is a thin strip cut from a polycaprolactone film.

FIG. 3 is a graph showing the effect of oriented nanofibers.

FIG. 4 is a graph showing the effect of the non-directional nanofibers.

Detailed Description

The present invention will be described below with reference to specific examples, but the present invention is not limited thereto.

The experimental procedures used in the following examples are, unless otherwise specified, conventional ones, and reagents, materials and the like used in the following examples are commercially available.

Example 1

(1) Preparation of the inner layer: the inner layer material component is polycaprolactone (Bolii biomaterial, Inc., Shenzhen), dissolved in hexafluoroisopropanol (Bolii biomaterial, Inc., Suzhou Hao Sai), the concentration of the spinning solution is 5% (wt), the stirring is carried out for 1 hour, the electrospinning parameters are that the flow rate is 1.0 ml/h, the voltage is 12kV, the receiving distance is 15cm, and the electrospinning is carried out for 2 hours.

(2) And preparing the intermediate layer.

(4) Preparing an outer layer:

the outer layer material is carboxymethyl chitosan (Nantong Green bioengineering Co., Ltd.), dissolved in purified water (Jinan sea Industrial chemical Co., Ltd.), the concentration of the spinning solution is 6% (wt), stirred for 1 hour, and the electrospinning parameters are as follows: the flow rate is 0.2 ml/h, the voltage is 0.8kV, the receiving distance is 1mm, the orientation electrostatic spinning process is used for preparing for 3h, an orientation nanofiber layer is obtained, and the fiber arrangement direction is parallel to any side.

Example 2

(1) Preparation of the inner layer: the inner layer material component is polycaprolactone (Bolii biomaterial, Inc., Shenzhen), dissolved in hexafluoroisopropanol (Bolii biomaterial, Inc., Suzhou Hao Sai), the concentration of the spinning solution is 7 wt, the stirring is carried out for 4 hours, the electrospinning parameters are that the flow rate is 0.9 ml/h, the voltage is 13kV, the receiving distance is 20cm, and the electrostatic spinning is carried out for 4 hours.

(2) And preparing the intermediate layer.

(4) Preparing an outer layer:

Example 3

(1) Preparation of the inner layer: the inner layer material component is polycaprolactone (Bolii biomaterial, Inc., Shenzhen), dissolved in hexafluoroisopropanol (Bolii biomaterial, Inc., Suzhou Hao Sai), the concentration of the spinning solution is 5% wt, the stirring is carried out for 4 hours, the electrospinning parameters are that the flow rate is 1.0 ml/h, the voltage is 15kV, the receiving distance is 25cm, and the electrospinning is carried out for 5 hours.

(2) And preparing the intermediate layer.

(3) The woven middle layer is placed on the upper surface of the inner layer before the inner layer is cured.

(4) Preparing an outer layer:

the outer layer material is carboxymethyl chitosan (Nantong Green bioengineering Co., Ltd.), dissolved in purified water (Jinan sea Industrial chemical Co., Ltd.), the concentration of the spinning solution is 6% (wt), stirred for 1 hour, and the electrospinning parameters are as follows: the flow rate is 0.6 ml/h, the voltage is 1kV, the receiving distance is 2mm, the oriented electrostatic spinning process is used for preparing for 3h, an oriented nanofiber layer is obtained, and the fiber arrangement direction is parallel to any one side.

Claims

1. A multilayer anti-infective high strength artificial dura mater comprising three layers: an inner layer facing the brain, an outer layer facing away from the brain and an intermediate layer between the two layers;

wherein, the thickness of the inner layer is 80 μm +/-10 μm, and the inner layer is prepared from hydrophobic materials by a non-oriented electrostatic spinning process;

the outer layer has a thickness of 30 mu m +/-10 mu m and is prepared from water-soluble chitosan by using an oriented electrostatic spinning process;

the thickness of the middle layer is 80 μm +/-20 μm, and the middle layer is formed by weaving a first layer of thin strips and a second layer of thin strips made of two different materials;

the middle layer is woven by a Hercules weaving process, wherein the thin strips are made of hydrophilic materials and 40 mu m thick, the thin strips are made of hydrophobic materials, the thin strips are 3mm wide and 40 mu m thick, and the Hercules weaving process is adopted.

2. The multilayer anti-infective high strength artificial dura mater according to claim 1, wherein the first layer of fine strands is prepared by electrospinning a hydrophobic material to form a film with a thickness of 40 μm, and cutting the film; the second layer of strips is prepared by preparing a film with the thickness of 40 mu m from a water-soluble chitosan material through an electrospinning process and then cutting.

3. The multilayer anti-infective high strength artificial dura mater according to claim 1, wherein the strands are woven with two strands facing 3mmx1000mm down, hydrophilic strands arranged laterally and hydrophobic strands arranged longitudinally.

4. The multilayer anti-infective high strength artificial dura mater according to claim 1, wherein the intermediate layer is placed on the upper surface of the inner layer before the inner layer is cured, and the placed intermediate layer is pressed down with a pressure of 50N for 1 min.

5. A method of preparing the multilayer anti-infective high strength artificial dura mater of any one of claims 1-4, comprising the steps of:

(1) preparation of the inner layer: the inner layer material is polycaprolactone, is dissolved in hexafluoroisopropanol, the concentration of a spinning solution is 7 percent (wt), the stirring is carried out for 4 hours, the electrospinning parameters are that the flow rate is 0.9 ml/h, the voltage is 13kV, the receiving distance is 20cm, and the electrostatic spinning is carried out for 4 hours;

(2) preparing an intermediate layer:

preparing the carboxymethyl chitosan into a film by an electrostatic spinning process, wherein the thickness of the film is 40 mu m;

making the film into strips with the width of 3mm and the length of 1000 mm;

preparing polycaprolactone into a film by an electrostatic spinning process, wherein the thickness of the film is 40 mu m;

making the film into strips with the width of 3mm and the length of 1000 mm;

the two thin strips are 3mmx1000mm face downwards, the hydrophilic thin strips are transversely arranged, the hydrophobic thin strips are longitudinally arranged, and a weaving type middle layer is obtained by weaving through a Hercules weaving process;

(3) before the inner layer is solidified, the woven middle layer is placed on the upper surface of the inner layer,

finally, vertically and downwardly extruding the middle layer arranged on the upper side by using the pressure of 50N, and keeping for 1 min;

(4) preparing an outer layer:

the outer layer material is carboxymethyl chitosan, which is dissolved in purified water, the concentration of the spinning solution is 6 percent (wt), the stirring is carried out for 1 hour, and the electrospinning parameters are as follows: the flow rate is 0.4 ml/h, the voltage is 0.9kV, the receiving distance is 1mm, the orientation electrostatic spinning process is used for preparing for 2h, an orientation nanofiber layer is obtained, and the fiber arrangement direction is parallel to any one side;