CN107715178B

CN107715178B - Multilayer high-strength artificial dura mater containing cell factors and preparation method thereof

Info

Publication number: CN107715178B
Application number: CN201711094328.4A
Authority: CN
Inventors: 李瑞锋; 王斐; 李典; 李翰林
Original assignee: Individual
Current assignee: Individual
Priority date: 2017-11-09
Filing date: 2017-11-09
Publication date: 2020-10-09
Anticipated expiration: 2037-11-09
Also published as: CN107715178A

Abstract

The invention provides a multilayer nanofiber artificial dura mater containing a cell growth factor, which comprises three layers, a hydrophobic inner layer facing the brain, a hydrophilic outer layer facing away from the brain and a woven middle layer between the two layers. The inner layer is a non-oriented fiber film, the outer layer is an oriented nano fiber film, cell growth factors are uniformly attached to fiber gaps of the outer layer, and the middle layer is formed by weaving thin strips made of hydrophilic materials and hydrophobic materials. The invention also provides a preparation method of the multilayer nanofiber artificial dura mater containing the cell growth factors, which comprises the following steps: preparing a hydrophobic inner layer by electrospinning; placing the middle layer woven in advance on the upper surface of the inner layer; preparing an outer layer by electrospinning; spraying cell growth factor on the outer layer; finally, sterilizing to obtain the artificial dura mater. The artificial dura mater effectively solves the problems of long period of dura mater damage repair and low utilization rate of cell growth factors, and has wide application prospect.

Description

Multilayer high-strength artificial dura mater containing cell factors and preparation method thereof

Technical Field

The invention relates to a multilayer high-strength artificial dura mater containing cytokines and a preparation method thereof, belonging to the technical field of biological micro-nano manufacturing.

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 the cells can be effectively prevented from entering the electrostatic spinning fiber film, and the 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.

Fibroblast Growth Factor (FGF) has several isoforms, and is mainly bFGF (basic fibroblast growth factor) which plays a role in arteriosclerosis foci and can be secreted by endothelial cells, smooth muscle cells and macrophages. It has effects in promoting migration of endothelial cells and proliferation of smooth muscle cells, promoting neovascularization, and repairing damaged endothelial cells.

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.

The invention patent CN103480042A discloses a composite artificial dura mater and a preparation method thereof, which comprises a gel layer and a woven layer, wherein the woven layer is arranged between two hydrogel layers, and the hydrogel layer contains cell growth factors. The invention has the beneficial effects that: the cell growth factor is beneficial to wound repair and accelerates the healing of dura mater injury. But has the following defects that 1) the outer layer of the meninges is of a colloid structure instead of a nano-fiber scaffold, which is not beneficial to cell attachment and has long meninges repairing period, and 2) the cell growth factors are dissolved in the colloid, only the cell growth factors on the surface layer contact with cells, and the promotion effect on cell production is limited.

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 directional 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 can promote the healing of brain injury, is reliable in combination of the inner layer and the outer layer, is convenient to use and has a wide industrialization prospect 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 high-strength artificial dura mater containing cytokines, and the artificial dura mater is different from the traditional artificial dura mater in that:

(1) 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 are combined with the outer layer through the gaps of the silk ribbons made of the hydrophobic material, the thin strips made of the hydrophobic material are combined with the inner layer through the gaps of the silk ribbons made of the hydrophilic material, 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.

(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 strength of the artificial dura mater is structurally improved.

(3) The artificial dura mater outer layer is prepared by adopting an electrospinning process to obtain the oriented nanofiber film, and fibroblasts are convenient to attach to fiber gaps.

(4) The cell growth factor is scattered in the middle of the electrospinning fiber, so that the contact probability of the cell growth factor and meninges cells is effectively improved, and the function of the cell growth factor is fully exerted.

Another object of the invention is to provide a method for preparing a multilayer high-strength artificial dura mater containing cytokines.

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

a multilayer high-strength artificial dura mater containing cytokine comprises three layers, a hydrophobic inner layer facing the brain, a hydrophilic outer layer facing away from the brain, and a woven middle layer 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 a hydrophilic material with good biocompatibility, and 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 hydrophobic materials and hydrophilic materials.

A multilayer high-strength artificial dura mater containing cell factors 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 electrostatic spinning is carried out for 4 hours;

(2) and preparing the intermediate layer.

Carboxymethyl chitosan (Nantong Lvshen bioengineering Co., Ltd.) was electrospun to a film thickness of 40 μm. The film was made into thin strips of 3mm in width and 1000mm in length.

Polycaprolactone (Shenzhen Bo Li biomaterial Co., Ltd.) was prepared into a film with a thickness of 40 μm by electrostatic spinning. The film was made into thin strips of 3mm in width and 1000mm in length.

Weaving the two thin strips by adopting a diamond weaving method to obtain the woven middle layer.

(3) The woven middle layer is placed on the upper surface of the inner layer before the inner layer is cured. And (3) downwards extruding the woven 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 (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 oriented electrostatic spinning process is adopted for preparation for 2h, an oriented nanofiber layer is obtained, and the longitudinal direction of the fiber is parallel to any side.

(5) Fibroblast growth factor (rh-bFGF dry powder, trade name: recombinant human basic fibroblast growth factor for external use, south China sea Lantai peptide pharmaceuticals, Inc.) was dissolved with water for injection (south China sea Industrial chemical, Inc.), and uniformly sprayed onto the epidural layer. The optimal dosage is 150IU/cm²。

(6) Freeze-drying the prepared artificial meninges, vacuum packaging, sterilizing by 25kGy drill-60, and storing at low temperature of-20 ℃.

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:

Drawings

FIG. 1 is a schematic diagram of the structure of an artificial dura mater, wherein 1 is an inner layer, 2 is an intermediate layer, 3 is an outer layer, and 4 is a cell growth factor.

Fig. 2 is a schematic view of the structure of the intermediate layer. 2-1 is a fine strip made of carboxymethyl chitosan, and 2-2 is a fine strip made of polycaprolactone.

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: reagents, materials and the like used in the following examples are commercially available unless otherwise specified.

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 electrostatic spinning 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 oriented electrostatic spinning process is used for preparing for 3h, an oriented nanofiber layer is obtained, and the longitudinal direction of the fiber 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.

(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 longitudinal direction of the fiber is parallel to any side.

Claims

1. A multilayered nanofiber artificial dura mater containing a cell growth factor, 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 content of the first and second substances,

the inner layer is 80 μm +/-10 μm thick and is prepared from fibers formed by non-oriented electrostatic spinning;

the thickness of the outer layer is 30 mu m +/-10 mu m, and the film is prepared by an oriented electrostatic spinning process;

the outer layer is sprayed with fibroblast growth factor solution;

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 formed by weaving strips which are made of hydrophilic materials and have the width of 3mm and the thickness of 40 mu m and thin strips which are made of hydrophobic materials and have the width of 3mm and the thickness of 40 mu m by a diamond weaving method.

2. The multilayer nanofiber-artificial dura mater containing cell growth factors as claimed in claim 1, wherein the first layer of fine strands is cut after being prepared into a film with a thickness of 40 μm by electrospinning; the second layer of thin strips is prepared by cutting after being prepared into a film with the thickness of 40 mu m by an electrospinning process.

3. The multilayered nanofiber artificial dura mater according to claim 1, wherein the middle layer is placed on the upper surface of the inner layer before the inner layer is cured, and the whole middle layer is pressed down with a pressure of 50N for 1 min.

4. The multilayered nanofiber artificial dura mater containing a cell growth factor according to claim 1, wherein the fibroblast growth factor is dissolved in water for injection, uniformly sprayed on the epidural layer, and uniformly adhered to the fiber space of the epidural layer, and the optimal dosage is 150IU/cm²。

5. A method for preparing the multi-layered nanofiber artificial dura mater containing the cell growth factor as recited in any one of claims 1 to 4, comprising the steps of:

(1) preparation of the inner layer: the inner layer material component is polycaprolactone, the 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 electrostatic spinning, wherein the thickness of the film is 40 mu m;

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

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

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

making the two strips face downwards at a thickness of 3mmx1000mm, transversely arranging the hydrophilic strips, longitudinally arranging the hydrophobic strips, and weaving by a diamond weaving method to obtain a woven intermediate layer;

(3) before the inner layer is solidified, the woven middle layer is placed on the upper surface of the inner layer, the woven middle layer placed on the upper side is extruded downwards by the pressure of 50N, and the time is kept 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;

(5) the fibroblast growth factor is dissolved by water for injection, and uniformly sprayed on the outer layer of dura mater, and the optimal dosage is 150IU/cm²；

(6) Freeze-drying the prepared artificial dura mater, vacuum-packaging, sterilizing by 25kGy cobalt-60, and storing at-20 deg.C.