CN110029500B

CN110029500B - Functionalized oriented fiber for tissue engineering intravascular stent construction and preparation method thereof

Info

Publication number: CN110029500B
Application number: CN201910329619.XA
Authority: CN
Inventors: 张彦中; 易兵成; 沈炎冰; 唐寒; 王先流
Original assignee: Donghua University
Current assignee: Donghua University
Priority date: 2019-04-23
Filing date: 2019-04-23
Publication date: 2021-07-02
Anticipated expiration: 2039-04-23
Also published as: CN110029500A

Abstract

The invention relates to a functionalized oriented fiber for constructing a tissue engineering vascular scaffold and a preparation method thereof, wherein a DA/Lys solution is obtained by adding dopamine salt DA and lysine Lys into a buffer solution; immersing the oriented fiber in DA/Lys solution for reaction to obtain the fiber. The invention not only effectively enhances the coating effect of PDA by introducing the active ingredient Lys, but also obviously improves the function of the oriented fiber induced vascular endothelial regeneration after the coating is modified. The PDA/Lys coating method provided by the invention has the advantages of simple process and high efficiency, can effectively overcome the defects of poor biocompatibility, insufficient endothelial regeneration induction function and the like of the polymer-based tissue engineering intravascular stent, and promotes the research and development and clinical application transformation of the functional tissue engineering intravascular stent.

Description

Functionalized oriented fiber for tissue engineering intravascular stent construction and preparation method thereof

Technical Field

The invention belongs to the field of functionalized oriented fibers and preparation thereof, and particularly relates to a functionalized oriented fiber for tissue engineering intravascular stent construction and a preparation method thereof.

Background

The oriented fiber prepared by the electrospinning method has the structural characteristics of the extracellular matrix of the bionic natural vascular tissue, so that the oriented fiber is considered to be an important bionic scaffold construction material with the functions of regulating vascular cell behaviors and promoting the repair and functional regeneration of damaged blood vessels. The synthetic polymer has the characteristics of easy processing, controllable performance, wide material source and the like, is widely used for preparing the oriented fiber scaffold in the field of vascular tissue engineering, but has poor biocompatibility and no capacity of regulating and controlling adhesion, proliferation and differentiation of vascular cells, and needs to be modified properly when applied to the vascular tissue engineering, such as enhancing the endothelialization function of the vascular tissue engineering. The endothelial regeneration is one of the key factors for determining the success or failure of the clinical application of the tissue-engineered blood vessel, and has the important function of improving the long-term and short-term patency of the blood vessel. Therefore, how to conveniently and rapidly improve the biocompatibility of the synthetic polymer-based oriented fiber scaffold, enhance the interaction between scaffold and cells to induce the functional expression of vascular cells and promote the regeneration of vascular endothelial tissues is an urgent problem to be solved for biomimetic construction of tissue engineering blood vessels.

In recent years, formation of PDA coating on the surface of material by utilizing chemical diversity and affinity diversity of dopamine catechol group is considered to be a modification method for efficiently and rapidly improving biocompatibility of stent surface, thereby enhancing cell adhesion and tissue affinity of stent (Ding et al, Acta biomaterials, 2015.15: 150-. However, since DA forms non-covalent bonds during the self-polymerization to form PDA, the nano-sized PDA aggregates tend to deposit on the surface of the stent to form a non-uniform particulate state, which has a detrimental effect on the anticoagulant properties of the vascular stent (Liu et al, compositions Science and Technology,2017.151: 164-173). Research shows that the introduction of the molecules containing multi-amino functional groups (such as hexamethylenediamine, hexamethylenetetramine, polyethyleneimine and the like) can further successfully introduce active groups for subsequent grafting of bioactive molecules on the basis of effectively improving the coating effect of PDA (Fu et al, Journal of Chromatography A,2015.1416: 94-102). However, the subsequent process of grafting active ingredients complicates the preparation process of the scaffold, and the introduced molecules containing multi-amino functional groups often lack biological activity. Therefore, if bioactive molecules containing multiple amino functional groups can be selected to improve the coating effect of PDA, it will be of great significance to enhance the biological functionality of the oriented fiber.

Disclosure of Invention

The invention aims to solve the technical problem of providing a method for improving the coating effect of PDA by selecting bioactive Lys (lysine), modifying and modifying oriented fiber prepared from synthetic polymer and preparing the oriented fiber modified by PDA/Lys, wherein the defects that an active group needs to be further introduced on the basis of a PDA coating for grafting, the preparation process is complex, and the introduced polyamino functional group is lack of activity in the prior art.

The invention relates to a functionalized oriented fiber, which is characterized in that the fiber is oriented fiber modified by polydopamine PDA and lysine Lys.

The invention discloses a preparation method of functionalized oriented fibers, which comprises the following steps:

(1) dissolving a polymer in a solvent to obtain a spinning solution, and then carrying out electrospinning to prepare an oriented fiber;

(2) dissolving dopamine hydrochloride DA and lysine Lys in a buffer solution, and stirring for dissolving to obtain a DA/Lys solution;

(3) and dipping the oriented fiber into DA/Lys solution, and reacting to obtain the functionalized oriented fiber.

The preferred mode of the above preparation method is as follows:

the polymer in the step (1) is one or more of a copolymer PLCL of lactic acid and caprolactone, polyurethane PU, polycaprolactone PCL, polysebacic acid glyceride PGS, poly octyl glycol citrate POC, polylactic acid PLA, polymethyl methacrylate PMMA, a copolymer PHBV of 3-hydroxybutyrate and 3-hydroxyvalerate, and a copolymer PLGA of lactic acid and glycolic acid. The solvent in the step (1) is one or more of hexafluoroisopropanol, formic acid, acetic acid, dichloromethane, trichloromethane, acetone, dimethyl sulfoxide, trifluoroacetic acid, trifluoroethanol, methanol, ethanol and water.

The mass-to-volume ratio of the polymer in the spinning solution in the step (1) is 1-100 g/mL.

The electrospinning process parameters in the step (1) are as follows: spinning speed 0.01-100mL/h, applying voltage 1-100kV, receiving jet monofilament with roller at receiving distance 0.01-1m, winding speed of roller receiving device 1-10000rpm, ambient temperature 0-1000 deg.C, and ambient relative humidity 0-100%.

The mass ratio of the dopamine hydrochloride DA to the lysine Lys in the step (2) is 1: 0-100; the concentration of the dopamine salt DA in the DA/Lys solution is 0.1-10 mg/mL.

The buffer in the step (2) is alkaline Tris buffer, wherein the pH value is 8.5, and the concentration is 10 mM.

The reaction in the step (3) is as follows: the reaction time is 0-100 h; the reaction temperature is 0-100 ℃.

The invention relates to a functionalized oriented fiber prepared by the method.

The invention provides an application of the functionalized oriented fiber in the construction of a tissue engineering blood vessel scaffold.

Advantageous effects

(1) The PDA/Lys coating method provided by the invention has the advantages of simple process and high efficiency, can effectively overcome the defects of poor biocompatibility, insufficient regeneration function of the induced endothelium and the like of the polymer-based tissue engineering intravascular stent, and promotes the research and development and clinical application conversion of the functional tissue engineering blood vessel;

(2) the invention selects the method of regulating the PDA coating by Lys, is convenient and fast, has high economic effect, can effectively improve the coating effect of PDA, and can ensure that the coating has the special functional activity of Lys along with the addition of Lys;

(3) the PDA/Lys coating method of the invention can enhance the application efficacy of biological materials in the field of vascular tissue engineering, such as enhancing the bioactivity of a synthetic polymer with biological inertia in vascular injury repair;

(4) the preparation method of the functionalized oriented fiber for the construction of the tissue engineering vascular scaffold can effectively improve the potential application prospect of the oriented fiber with the bionic natural vascular extracellular matrix structure in the field of vascular tissue engineering.

Drawings

FIG. 1 is a scanning electron microscope image of a PLCL oriented fiber prepared according to the invention;

FIG. 2 is a scanning electron microscope image of pure PDA modified PLCL oriented fibers;

FIG. 3 is a scanning electron microscope image of oriented fibers of PDA/Lys (mass ratio of 1:0.1) modified PLCL;

FIG. 4 is a scanning electron microscope image of PDA/Lys (1: 0.5 mass ratio) modified PLCL oriented fibers;

FIG. 5 is a scanning electron microscope image of oriented fibers of PDA/Lys (1: 1 by mass) modified PLCL;

FIG. 6 is a Fourier transform infrared spectrum FTIR of PLCL, PDA modified PLCL, PDA/Lys modified PLCL oriented fibers; wherein PLCL-PDA/Lys (1-1), PLCL-PDA/Lys (1-0.5), PLCL-PDA/Lys (1-0.1) correspond to example 4, example 3 and example 2, respectively;

FIG. 7 is a fluorescent staining pattern of cell adhesion after 3 hours of culture of endothelial cell ECs on PLCL fibers;

FIG. 8 is a graph of cell adhesion fluorescence staining after 3 hours of culture of ECs on PDA modified PLCL fibers;

FIG. 9 is a fluorescent staining pattern of cell adhesion after 3 hours of culture of ECs on PDA/Lys (1: 0.5 mass ratio) modified PLCL fibers;

FIG. 10 is a photograph of immunofluorescence staining of the cell-specific protein VE-Cadherin after 3 days of culture of ECs on PLCL fibers;

FIG. 11 is a photograph of immunofluorescence staining of the cell-specific protein VE-Cadherin after 3 days of culture of ECs on PDA modified PLCL fibers;

FIG. 12 is an immunofluorescence staining pattern of the cell-specific protein VE-Cadherin after 3 days of culture of ECs on PDA/Lys (mass ratio of 1:0.5) modified PLCL fibers.

FIG. 13 is SEM images of PCL/CNF composite fiber scaffold before and after coating in comparative example: before coating (left panel a); after coating (right panel b).

Detailed Description

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Further, it should be understood that various changes or modifications of the present invention may be made by those skilled in the art after reading the teaching of the present invention, and such equivalents may fall within the scope of the present invention as defined in the appended claims.

Example 1

At room temperature, 0.57g of PLCL (the copolymerization ratio of lactic acid and caprolactone is 50:50, the viscosity is 2.9dl/g) is dissolved in 6mL of hexafluoroisopropanol, and the spinning solution with the mass fraction of 10% is obtained after stirring for 12 hours; electrospinning was carried out at room temperature under a humidity of 40-60%, with an applied voltage of 5kV, a take-up distance of 20cm, a drum take-up speed of 1000rpm, and an injection rate of 0.5 mL/h.

The scanning electron micrograph of the prepared pure PLCL-oriented fiber, as shown in fig. 1, shows that it has a highly oriented structure with a fiber diameter of 1.67 ± 0.17 μm.

The cell adhesion of the scaffolds after being planted with ECs for 3 hours is shown in FIG. 7, and the cell functional protein VE-Cadherin expression of the scaffolds after being cultured for 3 days is shown in FIG. 10, which indicates that although the PLCL oriented fibers can maintain the functional expression of ECs, the cell adhesion is not ideal.

20mg of DA and 0mg of Lys were dissolved in 10mL of Tris buffer solution having a concentration of 10mM and a pH of 8.5, and the solution was dissolved with sufficient stirring to prepare a DA/Lys solution. Immersing an oriented fiber-based support with the size of 1cm multiplied by 1cm in DA/Lys solution, and oscillating for 24 hours at normal temperature at the vibration rate of 100 times/minute to obtain the PDA modified oriented fiber. The scanning electron microscope image of the PDA modified oriented fiber shows that granular aggregates exist on the surface of the fiber, and the increase of the fiber diameter (1.89 +/-0.13 mu m) shows that polydopamine is successfully coated on the surface of the fiber, as shown in figure 2. FTIR spectra of the holder, as shown in fig. 6, the increase in-NH absorption peak further confirms successful coating of PDA. The cell adhesion condition of the scaffold after the ECs are planted for 3 hours is shown in FIG. 8, and the cell functional protein VE-Cadherin expression condition of the scaffold after the ECs are planted for 3 days is shown in FIG. 11, which shows that the PDA coating is favorable for promoting the adhesion and functional expression of the ECs.

Example 2

At room temperature, 0.57g of PLCL (the copolymerization ratio of lactic acid and caprolactone is 50:50, the viscosity is 2.9dl/g) is dissolved in 6mL of hexafluoroisopropanol, and the spinning solution with the mass fraction of 10% is obtained after stirring for 12 hours; electrospinning was carried out at room temperature under a humidity of 40-60%, with an applied voltage of 5kV, a take-up distance of 20cm, a drum take-up speed of 1000rpm, and an injection rate of 0.5 mL/h. 20mg of DA and 2mg of Lys were dissolved in 10mL of Tris buffer solution having a concentration of 10mM and a pH of 8.5, and the solution was dissolved with sufficient stirring to prepare a DA/Lys solution. Immersing an oriented fiber-based bracket with the size of 1cm multiplied by 1cm in DA/Lys solution, and oscillating for 24 hours at normal temperature at the vibration rate of 100 times/minute to obtain the PDA/Lys modified oriented fiber. Scanning electron microscopy of PDA/Lys modified oriented fibers, as shown in FIG. 3, shows that particulate aggregates begin to increase on the fiber surface, and a further increase in fiber diameter (1.98. + -. 0.11 μm) shows an increase in the coating effect of PDA. The FTIR spectrum of the holder, as shown in fig. 6, a further increase of the-NH absorption peak confirms the enhancement of the PDA coating effect.

Example 3

At room temperature, 0.57g of PLCL (the copolymerization ratio of lactic acid and caprolactone is 50:50, the viscosity is 2.9dl/g) is dissolved in 6mL of hexafluoroisopropanol, and the spinning solution with the mass fraction of 10% is obtained after stirring for 12 hours; electrospinning was carried out at room temperature under a humidity of 40-60%, with an applied voltage of 5kV, a take-up distance of 20cm, a drum take-up speed of 1000rpm, and an injection rate of 0.5 mL/h. 20mg of DA and 10mg of Lys were dissolved in 10mL of 10mM Tris buffer solution having a pH of 8.5, and the solution was dissolved with stirring sufficiently to prepare a DA/Lys solution. Immersing an oriented fiber-based bracket with the size of 1cm multiplied by 1cm in DA/Lys solution, and oscillating for 24 hours at normal temperature at the vibration rate of 100 times/minute to obtain the PDA/Lys modified oriented fiber. Scanning electron microscopy of PDA/Lys modified oriented fibers, as shown in FIG. 4, shows that the particle aggregates on the fiber surface begin to decrease, but the increase in fiber diameter (2.02. + -. 0.16 μm) confirms better coating effect of PDA. The FTIR spectrum of the mount is shown in FIG. 6, and the continued increase in the NH absorption peak indicates a further enhancement of the PDA coating effect. The cell adhesion of the scaffold after 3 hours of ECs implantation is shown in FIG. 9, and the cell function protein VE-Cadherin expression of the scaffold after 3 days of ECs implantation is shown in FIG. 12, which shows that the adhesion and function expression of ECs are further promoted after PDA/Lys coating.

Example 4

At room temperature, 0.57g of PLCL (the copolymerization ratio of lactic acid and caprolactone is 50:50, the viscosity is 2.9dl/g) is dissolved in 6mL of hexafluoroisopropanol, and the spinning solution with the mass fraction of 10% is obtained after stirring for 12 hours; electrospinning was carried out at room temperature under a humidity of 40-60%, with an applied voltage of 5kV, a take-up distance of 20cm, a drum take-up speed of 1000rpm, and an injection rate of 0.5 mL/h. 20mg of DA and 20mg of Lys were dissolved in 10mL of Tris buffer solution having a concentration of 10mM and a pH of 8.5, and the solution was dissolved with stirring sufficiently to prepare a DA/Lys solution. Immersing an oriented fiber-based bracket with the size of 1cm multiplied by 1cm in DA/Lys solution, and oscillating for 24 hours at normal temperature at the vibration rate of 100 times/minute to obtain the PDA/Lys modified oriented fiber. In a scanning electron microscope image of the PDA/Lys modified oriented fiber, as shown in FIG. 5, the fiber diameter was measured to be in a decreasing trend of 1.93. + -. 0.15. mu.m, indicating that the coating effect of PDA began to deteriorate. The FTIR spectrum of the holder is shown in FIG. 6, and the decrease of the NH absorption peak indicates a downward trend in the PDA coating effect.

Comparative example 1

The comparative example relates to a preparation method (application number: 201810182493.3) of a composite fiber scaffold based on a uniform polydopamine coating modified Polycaprolactone (PCL)/natural nanofiber (CNF), and the preparation steps are as follows: weigh 4g of PCL into 20mL of chloroform: dissolving in a dimethylformamide (4:1) mixed solution at normal temperature by shaking to obtain a PCL spinning solution; electrospinning at room temperature and humidity of 40% to obtain PCL disordered nano-fiber membrane, wherein the applied voltage is 15kV, the receiving distance is 15cm, and the injection rate is 1 mL/h. Weighing 0.08g of CNF, placing in 50mL of deionized water, and stirring for 30min to obtain a CNF suspension; placing the PCL fiber membrane in the CNF suspension for ultrasonic treatment for 10min to obtain a PCL/CNF composite fiber membrane; 0.5g of DA was dissolved in 250mL of Tris buffer having a concentration of 10mM and a pH of 8.5, and the solution was dissolved with sufficient stirring to prepare a DA solution. And immersing the PCL/CNF composite fiber membrane in the DA solution, and magnetically stirring for 20h at room temperature to obtain the composite fiber scaffold with the PDA uniform coating. Figure 13 shows SEM images of PCL/CNF composite fiber scaffolds before and after PDA coating. It can be seen that the surface roughness of the fiber scaffold after compounding is increased and the fiber diameter is increased. The results show that a uniform PDA coating is successfully formed on the surface of the PCL/CNF composite fiber.

The preparation method mainly solves the problems of poor uniformity, long coating time, poor coating stability and the like of the PDA coating by introducing natural nano cellulose on the surface of the fibers in disordered arrangement. However, the CNF ultrasonic treatment process involved in this method complicates PDA coating, and has defects such as residual of unnecessary material components such as CNF. The preparation method of the functionalized oriented fiber, which is related by the invention, introduces Lys component capable of promoting angiogenesis under the background of the tissue engineering blood vessel field, and not only successfully solves the PDA coating problem on the basis of simplifying the operation steps, but also further endows the fiber scaffold with the function of promoting angiogenesis, and has application prospect in the tissue engineering field.

Claims

1. A method of making a functionalized oriented fiber comprising:

2. The preparation method according to claim 1, wherein the polymer in step (1) is one or more of a copolymer of lactic acid and caprolactone PLCL, polyurethane PU, polycaprolactone PCL, PGS, POC, PLA, PMMA, PHBV, a copolymer of 3-hydroxybutyrate and 3-hydroxyvalerate, and PLGA, a copolymer of lactic acid and glycolic acid.

3. The preparation method according to claim 1, wherein the solvent in step (1) is one or more of hexafluoroisopropanol, formic acid, acetic acid, dichloromethane, trichloromethane, acetone, dimethyl sulfoxide, trifluoroacetic acid, trifluoroethanol, methanol, ethanol, and water.

4. The preparation method according to claim 1, wherein the electrospinning process parameters in the step (1) are as follows: spinning speed 0.01-100mL/h, applying voltage 1-100kV, receiving jet monofilament with roller at receiving distance 0.01-1m, winding speed of roller receiving device 1-10000rpm, ambient temperature 0-1000 deg.C, and ambient relative humidity 0-100%.

5. The production method according to claim 1, wherein the mass ratio of dopamine hydrochloride DA to lysine Lys in the step (2) is 1:0 to 100; the concentration of dopamine hydrochloride DA in the DA/Lys solution is 0.1-10 mg/mL.

6. The method of claim 1, wherein the buffer in step (2) is an alkaline Tris buffer, wherein the pH is 8.5.

7. The method according to claim 1, wherein the reaction in the step (3) is: the reaction time is 0-100 h; the reaction temperature is 0-100 ℃.

8. A functionalized oriented fiber prepared according to the method of claim 1.

9. Use of the functionalized oriented fiber prepared by the method of claim 1 in the construction of tissue engineered vascular scaffolds.