CN114681688A

CN114681688A - Tissue regeneration membrane for promoting vascularization by using micro-channel and preparation method thereof

Info

Publication number: CN114681688A
Application number: CN202210394700.8A
Authority: CN
Inventors: 白晶; 沈佩琦; 程兆俊; 王先丽; 张越; 薛烽; 周健
Original assignee: Southeast University
Current assignee: Southeast University
Priority date: 2022-04-15
Filing date: 2022-04-15
Publication date: 2022-07-01
Anticipated expiration: 2042-04-15
Also published as: CN114681688B

Abstract

The invention discloses a tissue regeneration membrane for promoting vascularization by utilizing a microchannel and a preparation method thereof, wherein the regeneration membrane comprises a microchannel layer and a spinning layer; the microchannel layer is a regenerated membrane with a three-dimensional network channel, and the three-dimensional network channel in the regenerated membrane is prepared by corroding and eliminating a bent and fixed metal wire; the spinning layer is arranged on the compact side of the regenerated membrane. The preparation method comprises the following steps: dissolving the degradable polymer and injecting into a mould; winding a metal wire into a fixed three-dimensional net-shaped structure, flattening, putting the fixed three-dimensional net-shaped structure into a degradable polymer solution, and evaporating and forming to obtain a composite material; corroding the composite material by using a corrosive liquid, and washing away the metal wire to obtain a regenerated membrane with a microchannel; spinning the regenerated membrane with the dense side facing upwards on the surface to obtain the regenerated membrane. The tissue regeneration membrane effectively solves the problems of the drift diameter size and the structural controllability of the vascularization promoting microchannel, has good mechanical property and osteogenesis promoting property, meets the clinical requirement, and has wide application prospect.

Description

Tissue regeneration membrane for promoting vascularization by using micro-channel and preparation method thereof

Technical Field

The invention relates to a tissue regeneration membrane and a preparation method thereof, in particular to a tissue regeneration membrane promoting vascularization by utilizing a micro-channel and a preparation method thereof.

Background

At present, periodontal tissue damage and insufficient alveolar bone mass are a wide problem in clinical oral implantation. In order to obtain long-term good results after the implant surgery, periodontal tissue repair is required at the implant site before the tooth is implanted. The Guided Tissue Regeneration (GTR) technique is an important means for repairing periodontal defects of human body. Through guiding the implantation of the periodontal tissue regeneration membrane, the regeneration of related cells is guided, and the expression of normal functions of the cells is maintained. The periodontal tissue regeneration membrane needs to play a certain role in mechanical support, serving as a template, promoting vascularization, blocking fibroblast from growing in, providing places for adhesion, differentiation, proliferation and the like of osteocytes. Wherein the regeneration and formation of blood vessels play an important role in the repair process of periodontal tissues or organs, and based on the abundant vascular network system, oxygen, nutrients and the like are promoted to be transported to the damaged area, metabolic byproducts are removed, and the purpose of periodontal tissue regeneration is achieved. The properties of the membrane material, as well as the structural properties, are therefore critical to the repair of damaged periodontal tissues or organs. An ideal GTR film needs to have the following conditions: good mechanical supporting effect; good biocompatibility; degradability; controlled pore structure and high porosity.

The artificial polymer materials such as polycaprolactone, polylactic acid and the like, and some hydrogel materials all show good biocompatibility, so that the hydrogel material is widely applied to the fields of biomedicine, tissue regeneration and the like. At present, the vascularization promotion mainly has two research directions, firstly, for the construction of the structure, a plurality of technologies are applied to the preparation of porous GTR membranes with different structures, such as 3D printing, electrostatic spinning, phase separation, freeze-drying and other methods, although the technologies have different advantages, the methods with controllable pores and pore diameters cannot be constructed by other methods except the 3D printing, and the 3D printing can control the pore size but is limited by the current printing precision, and the pores are large and are not beneficial to the growth of blood vessels; the other direction is to load the angiogenesis promoting growth factors, such as VEGF, HGF, bFGF and the like, and some enzymes, ions and the like to promote the expression of the related genes and proteins of the blood vessels.

In a GTR membrane which is not vascularized, nutrients such as oxygen can only diffuse to a depth of less than 200 mu m in an implant membrane, and if the periodontal tissue regeneration membrane spontaneously induces vascularization only by the material itself, the induced blood vessels often grow into too shallow, and in both cases, cells in a repair area of a defect area die due to oxygen deficiency or lack of nutrients, thereby causing failure of the implant surgery. According to research reports, the pore size plays a crucial role in the angiogenesis rate and the size and maturity of the formed blood vessels, and the controllable structure micro-channel below 100 μm is difficult to form in the prior art.

Disclosure of Invention

The purpose of the invention is as follows: the invention aims to provide a tissue regeneration membrane which has good mechanical property and osteogenesis promoting property and promotes vascularization by utilizing a micro-channel;

the second purpose of the invention is to provide a preparation method of the tissue regeneration membrane which has adjustable drift diameter and controllable structure and can promote vascularization by using the micro-channel.

The technical scheme is as follows: the tissue regeneration membrane for promoting vascularization by utilizing the microchannel comprises a microchannel layer and a spinning layer; the microchannel layer is a regenerated membrane with a three-dimensional network channel, and the three-dimensional network channel in the regenerated membrane is prepared by corroding and eliminating bent and fixed metal wires; the spinning layer comprises high polymer fiber yarns, and is arranged on the compact side of the regenerated membrane.

Wherein, the metal wire is preferably a magnesium metal wire or a magnesium alloy wire. Wherein the regenerative film is composed of a degradable polymer; the degradable polymer material is at least one of polycaprolactone, polylactic acid, poly (lactic acid-glycolic acid), sodium alginate or chitosan.

And a supporting layer is also arranged between the micro-channel layer and the spinning layer.

Wherein, the inner surface of the microchannel layer is loaded with vascular endothelial growth factors.

The three-dimensional structure and the drift diameter of the microchannel layer are controllable, and vascular endothelial growth factors are loaded on the inner surface of the channel, so that the vascularization in a tissue regeneration membrane is promoted, and oxygen and nutrient substances are provided for seed cells; the supporting layer is a composite of a polymer compact layer and a metal sheet, so that the mechanical property of the tissue regeneration membrane is improved, and the growth of fibroblasts is prevented; the spinning layer is composed of disordered high-molecular fiber filaments, has a conveying porous structure and is beneficial to the adhesion of bone cells.

Wherein, the support layer is a composite layer of a metal sheet and a polymer. The thickness of the metal sheet is 80-200 μm; the metal in the metal sheet is one of magnesium, magnesium alloy, zinc or zinc alloy.

Wherein the thickness of the spinning layer is 1/3-2/5 of the tissue regeneration membrane.

Wherein the wire diameter range of the metal wire is 30-80 μm.

Wherein the porosity of the microchannel layer is 20% -50%.

The preparation method of the tissue regeneration membrane for promoting vascularization by utilizing the micro-channel comprises the following steps:

(1) dissolving a degradable polymer in an organic solvent to form a degradable polymer solution, and injecting the degradable polymer solution into a mold;

(2) winding micron-sized metal wires into a fixed three-dimensional net-shaped structure, pressing the structure to be flat, putting the structure into a degradable polymer solution, and obtaining a composite material through evaporation molding;

(3) corroding the composite material by corrosive liquid to remove metal wires in the composite material, and then cleaning and drying to obtain a regenerated membrane with a microchannel;

(4) and (3) enabling the dense side of the regenerated membrane to face upwards, coating a biological adhesive or spinning solution on the surface of the dense side, and then spinning to obtain the tissue regenerated membrane with an asymmetric structure.

(2) winding micron-sized metal wires into a fixed three-dimensional net-shaped structure, pressing the fixed three-dimensional net-shaped structure to be flat through a flattening device, putting the fixed three-dimensional net-shaped structure into a degradable polymer solution, and obtaining a composite material through evaporation molding;

(3) corroding the composite material by corrosive liquid, washing off metal wires in the composite material, and cleaning and drying to obtain a regenerated membrane with a microchannel;

(4) and coating spinning solution on the surface of the metal sheet, pressurizing and adhering the metal sheet to the compact side of the regenerated membrane, solidifying the metal sheet, coating biological adhesive or spinning solution on the solidified surface, and spinning to obtain the tissue regenerated membrane with the asymmetric structure.

In the step (2), the fixed three-dimensional net-shaped structure is pressed to be flat by a flattening device. The fixed three-dimensional net-shaped structure formed by winding is pressed to be smooth, so that the flatness is guaranteed, the thickness of the three-dimensional net-shaped structure can be adjusted on the other hand, and then the thickness of the microchannel layer is adjusted.

In the step (2), VEGF is dissolved in PBS solution and then added into degradable polymer solution to be stirred to obtain VEGF-polymer solution, then metal wires are wound into a fixed three-dimensional mesh structure, pressed to be flat and immersed into the VEGF-polymer solution, the metal wires are extracted to enable the surfaces of the metal wires to be attached with the VEGF-polymer solution, and finally the metal wires are placed into the degradable polymer solution in the step (1); wherein the concentration of the VEGF-polymer solution is 500-1000 ng/ml.

In the step (1), the concentration of the degradable polymer solution is 0.05-0.2 g/ml.

In the step (3), the metal wire material in the composite material is washed by hydrochloric acid with the concentration of 10% -20%.

The organic solvent used for dissolving the degradable polymer is at least one of dichloromethane, trichloromethane, hexafluoroisopropanol, N-dimethylformamide, tetrahydrofuran or ethanol.

Has the advantages that: compared with the prior art, the invention has the following remarkable effects: 1. the regeneration membrane has different structures and functional layers and has functional specificity, and one side of the regeneration membrane is of a micro-channel structure, so that the growth of blood vessels is promoted; the other side is a spinning porous structure which is beneficial to the growth and adhesion of bone cells. 2. The compact polymer and metal sheet composite layer between the microchannel layer and the spinning layer separates the growth of fibroblast, provides space for bone cell proliferation, and simultaneously improves the mechanical support performance for the whole tissue regeneration membrane. 3. The regenerated membrane is made of degradable materials, has good biocompatibility and functionality, can be gradually degraded in vivo, avoids secondary operation, and can reduce the injury and economic burden to patients. 4. By controlling the wire diameter of the micron-sized metal wire, constructing and fixing a formed structure by the wire, and accurately controlling the drift diameter of the blood vessel network template and the connectivity of the three-dimensional network structure, the problem that a micro-channel with the diameter less than 100 mu m is difficult to form is solved, and the constructed microstructure can promote tissue vascularization. 5. The vascular endothelial cell growth factor is loaded on the metal wire and introduced into the tissue regeneration membrane, and the metal wire is removed by acid cleaning, so that the vascular endothelial cell growth factor is loaded on the inner surface of the micro-channel, and the vascular endothelial cell growth factor can play a role in pertinence after being implanted into a body to guide the growth of blood vessels in the micro-channel.

Drawings

FIG. 1 is a cross-sectional view of a tissue regeneration membrane utilizing microchannel vascularization in accordance with the present invention;

FIG. 2 is a schematic view of a structure of a micrometer-sized magnesium or magnesium alloy wire wound in example 1 of the present invention;

FIG. 3 is a schematic view of a flattened structure of a micrometer-sized magnesium or magnesium alloy wire wound in example 1 of the present invention;

FIG. 4 shows the expression of the VEGF genes and proteins in the co-culture of the cells and membranes of examples 1-4 for 14 days;

fig. 5 is a mechanical test curve of examples 3 and 4 of the present invention.

Detailed Description

The invention is described in further detail below with reference to the drawings.

Example 1

As shown in figure 1, the invention provides a tissue regeneration membrane promoting vascularization by using a microchannel, which comprises a microchannel layer 1, a support layer 3 and a spinning layer 2; the microchannel layer is of a three-dimensional net structure, 4 in figure 1 is of a microchannel structure after magnesium wire elution, and the drift diameter of the microchannel is 40 μm. The preparation method comprises the following steps:

(1) selecting a polymer: selecting polycaprolactone with the molecular weight of 8 ten thousand, dissolving 2.5g of PCL in 50ml of hexafluoroisopropanol, namely the concentration of the PCL solution is 0.05g/ml, obtaining a PCL viscous solution, and injecting the PCL viscous solution into a mold;

(2) preparing a magnesium alloy wire loaded with vascular endothelial growth factors: dissolving VEGF in PBS solution at 5 μ g/ml, adding into PCL dichloromethane solution with concentration of 10%, stirring thoroughly to obtain VEGF-polymer solution with concentration of 500ng/ml, winding magnesium alloy wire drawn to wire diameter of 40 μm into three-dimensional grid structure, as shown in FIG. 2; flattening the magnesium wire into thin magnesium wire sheets by a flattening device, as shown in figure 3; then, immersing the magnesium wire sheet into the solution, and extracting at a constant speed to enable the surface of the magnesium wire sheet to be attached with the solution;

(3) forming a micro-channel structure: putting the magnesium wire sheet into the upper layer of the PCL viscous solution, evaporating at room temperature to form a film, demoulding, soaking the film in 10% hydrochloric acid solution for 2h, washing in distilled water, and drying to obtain a porosity of about 50%;

(4) obtaining a tissue regeneration membrane: putting 1.2g of polycaprolactone into 10ml of hexafluoroisopropanol, magnetically stirring for 3 hours to obtain an electrostatic spinning solution, building an electrostatic spinning instrument, adjusting the voltage to 8kv, the propelling speed to 0.5ml/h, the distance between a needle head and a collector to 5cm, pressurizing and adhering a pure magnesium sheet with the thickness of 80 mu m to a polymer compact layer by using the spinning solution, enabling the solidified polymer compact layer to face upwards, spraying the spinning solution with the same concentration on the surface of a film layer, placing the film layer on a spinning platform, and drying after 5 hours of spinning time to obtain the tissue regeneration film.

Through tests, the thickness of the regenerated membrane is 325 +/-23 mu m, wherein the thickness of a spinning layer is about 1/3 of the regenerated membrane, after the composite membrane and the cells are co-cultured for 14 days respectively by RT-qPCR and ELISA methods, the gene expression level of VEGF is shown as (a) in figure 4, the protein expression level in the supernatant of the mesenchymal stem cells of the bone marrow is shown as (b) in figure 4, and the regenerated membrane is found to enable the expression levels of the VEGF gene and the protein to be at relatively high levels.

Example 2

On the basis of example 1, the difference from example 1 is: the diameter of the micro-channel is 80 μm. The preparation method comprises the following steps:

(1) selecting a polymer: selecting 6 ten thousand molecular weight PLGA, dissolving 4g PLGA in 20ml mixed solution of chloroform and DMF to obtain 0.2g/ml PLGA viscous liquid, and injecting the viscous liquid into a mold; chloroform: the volume ratio of the DMF solution is 7: 3;

(2) preparing magnesium wires loaded with vascular endothelial growth factors: dissolving VEGF in a PBS solution by 10 mu g/ml, then adding the dissolved VEGF into a PLGA chloroform solution with the concentration of 10 percent, fully stirring to obtain a VEGF-polymer solution with the concentration of 800ng/ml, winding a magnesium alloy wire drawn to the diameter of 80 mu m on a plate with fine needles, forming a three-dimensional structure with a staggered network pattern, immersing the magnesium alloy wire into the solution, and extracting the magnesium alloy wire at a constant speed to enable the surface of the wire to be attached with the solution;

(3) formation of the microchannel structure: placing the fixed magnesium wire three-dimensional structure into the upper layer of a molten polymer, cooling the die at room temperature, solidifying, soaking the film in 10% hydrochloric acid solution for 2h, washing in distilled water, drying, and measuring the porosity to be about 20%;

(4) obtaining a tissue regeneration membrane: adding 1.5g of PLGA into 10ml of mixed solution of chloroform and DMF, magnetically stirring for 10h to obtain an electrostatic spinning solution, pressurizing and adhering a pure zinc sheet with the thickness of 100 mu m to a polymer compact layer by using a spinning solution, enabling the solidified polymer compact layer to face upwards, coating a layer of prepared electrostatic spinning solution with the surface of about 0.2ml, sucking the rest electrostatic spinning solution into a needle cylinder, building an electrostatic spinning instrument, regulating the voltage to be 20kv, the propelling speed to be 1.5ml/h, the distance from a needle head to a collection plate to be 12cm, spinning for 1h, and drying to obtain a regenerated membrane.

Through tests, the thickness of the regenerated membrane is 452 +/-13 mu m, wherein the thickness of a spinning layer is about 1/3 of the regenerated membrane, after the composite membrane and the cells are co-cultured for 14 days respectively by RT-qPCR and ELISA methods, the gene expression level of VEGF is shown as (a) in figure 4, the expression level of protein in the supernatant of the bone marrow mesenchymal stem cells is shown as (b) in figure 4, and the regenerated membrane is found to enable the expression levels of the VEGF gene and the protein to be at relatively high levels.

Example 3

A tissue regeneration membrane for promoting vascularization by using micro-channels, wherein the micro-channels are of a net structure, and the diameter of the micro-channels is 30 mu m. The preparation method comprises the following steps:

(1) selecting a polymer: selecting PLA with the molecular weight of 12 ten thousand, and mixing the materials in a mass-volume ratio of 1: 5 in dichloromethane to obtain 0.2g/ml of PLA viscous solution, magnetically stirring for 3 hours, and pouring the solution into a mold to be flatly laid;

(2) preparing a magnesium alloy wire material loaded with vascular endothelial growth factors: dissolving VEGF in a PBS solution by 20 mu g/ml, then adding the dissolved VEGF into a PLA dichloromethane solution with the concentration of 8%, fully stirring to obtain a VEGF-polymer solution with the concentration of 1000ng/ml, winding a magnesium alloy wire drawn to the wire diameter of 30 mu m on a plate with fine needles, forming a three-dimensional structure of an interlaminar interlaced network pattern, then immersing the magnesium alloy wire into the solution, and extracting the magnesium alloy wire at a constant speed to enable the surface of the wire to be attached with the solution;

(3) formation of the microchannel structure: placing the fixed three-dimensional structure of the magnesium wire into the upper layer of a molten polymer, cooling the die at room temperature, solidifying, soaking the film in 10% hydrochloric acid solution for 2h, washing in distilled water, and drying;

(4) obtaining a tissue regeneration membrane: putting 1g of polylactic acid into 10ml of hexafluoroisopropanol, magnetically stirring for 4h to obtain an electrostatic spinning solution, building an electrostatic spinning instrument, adjusting the voltage to 15kv, the propelling speed to 3ml/h, the distance between a needle head and a collector to 12cm, pressurizing and adhering a pure magnesium sheet with the thickness of 200 mu m to a polymer compact layer by using a spinning solution, enabling the solidified polymer compact layer to be upward, smearing biological adhesive on the surface of a film layer, placing the film layer on a spinning platform, and drying after 3h of spinning time to obtain the tissue regeneration film.

The test shows that the thickness of the regenerated membrane is 576 +/-26 microns, wherein the thickness of the spinning layer is about 2/5 of the regenerated membrane, the maximum stress of the composite membrane is about 35MPa through the tensile test, and the expression level of VEGF gene is shown as (a) in figure 4 after the composite membrane is co-cultured with cells for 14 days, and the expression level of protein in the supernatant of the mesenchymal stem cells is shown as (b) in figure 4, so that the two are in higher level.

Example 4

(3) formation of the microchannel structure: placing the fixed magnesium wire three-dimensional structure into the upper layer of a molten polymer, cooling the die at room temperature, solidifying, soaking the film in 10% hydrochloric acid solution for 2h, washing in distilled water, drying, and measuring the porosity to be 30%;

(4) obtaining a tissue regeneration membrane: putting 1g of polylactic acid into 10ml of hexafluoroisopropanol, magnetically stirring for 4h to obtain an electrostatic spinning solution, building an electrostatic spinning instrument, adjusting the voltage to 15kv, the propelling speed to 3ml/h, the distance between a needle and a collector to be 12cm, placing the compact side of the tissue regeneration membrane upwards on an electrostatic spinning platform, coating biological adhesive on the surface of the tissue regeneration membrane, placing the tissue regeneration membrane on the spinning platform, and drying after the spinning time is 3h to obtain the tissue regeneration membrane.

The test shows that the thickness of the regenerated membrane is 550 +/-32 mu m, the thickness of the spinning layer is about 2/5 of the regenerated membrane, and the maximum stress of the composite membrane is about 8MPa through the tensile test.

Claims

1. A tissue regeneration membrane for promoting vascularization by utilizing a microchannel is characterized by comprising a microchannel layer and a spinning layer; the microchannel layer is a regenerated film with a three-dimensional network channel, and the three-dimensional network channel in the regenerated film is prepared by corroding and eliminating a bent and fixed metal wire; the spinning layer comprises high polymer fiber yarns, and is arranged on the compact side of the regenerated membrane.

2. The membrane for promoting vascularization by means of microchannels according to claim 1, wherein a support layer is further provided between the microchannel layer and the spinning layer.

3. The membrane according to claim 2, wherein the support layer is a composite layer of a metal sheet and a polymer.

4. The membrane for tissue regeneration by means of microchannel vasopromotion according to claim 1, wherein the thickness of the spinning layer is 1/3-2/5 of the thickness of the tissue regeneration membrane.

5. The membrane for tissue regeneration using microchannel vasopromotion according to claim 1, wherein the wire diameter of the wire ranges from 30 μm to 80 μm.

6. The membrane for tissue regeneration by means of microchannel vasopromotion according to claim 1, wherein the porosity of the microchannel layer is 20% to 50%.

7. The membrane according to claim 1, wherein the inner surface of the microchannel layer is loaded with vascular endothelial growth factor.

8. A method for preparing a tissue regeneration membrane for promoting vascularization using microchannels according to claim 1, comprising the steps of:

(3) corroding the composite material by using corrosive liquid to remove metal wires in the composite material, and then cleaning and drying to obtain a regenerated membrane with a microchannel;

9. A method for preparing a tissue regeneration membrane for promoting vascularization using microchannels according to claim 2, comprising the steps of:

10. The method for preparing a tissue regeneration membrane using microchannel vasopromotion according to claim 8 or 9, wherein in the step (2), VEGF is dissolved in PBS solution and then added into degradable polymer solution to be stirred to obtain VEGF-polymer solution, then metal wires are wound into a fixed three-dimensional network structure and immersed into VEGF-polymer solution, and then the metal wires are provided with VEGF-polymer solution attached to surfaces thereof, and finally placed into the degradable polymer solution in the step (1).