阅读说明：本技术 一种基于共轭同轴静电纺丝技术的生物型心室辅助泵及其制备方法 (Biological type ventricular assist pump based on conjugate coaxial electrostatic spinning technology and preparation method thereof ) 是由 何晓敏 张晓阳 郑景浩 祝忠群 石博中 罗凯 于 2020-12-25 设计创作，主要内容包括：本发明涉及医学组织工程领域,具体地说,是一种基于共轭同轴静电纺丝技术的生物型心室辅助泵及其制备方法。包括如下步骤：1)制备含有复壁碳纳米管的取向静电纺丝心肌支架；2)通过同轴静电纺丝技术构建负载VEGF细胞因子的心肌支架；3)将分离的动物心肌细胞作为种子细胞种于膜片上；4)生物型心室辅助泵的构建；将膜片上种植心肌细胞后,将该细胞材料复合物按照心肌三层的排列方向叠加为三层,然后,制作成圆锥形袖套状结构。本发明袖套状生物型心室辅助泵可弥补传统机械辅助泵的不足,有望为广大终末期心衰患儿从根本上赋予心室持续收缩的动力性,从而彻底改善患儿预后及提高生存质量,降低患者住院费用及远期成本。(The invention relates to the field of medical tissue engineering, in particular to a biological type ventricular assist pump based on a conjugate coaxial electrostatic spinning technology and a preparation method thereof. The method comprises the following steps: 1) preparing an oriented electrostatic spinning myocardial scaffold containing the double-walled carbon nanotube; 2) constructing a cardiac muscle scaffold loaded with VEGF cell factors by a coaxial electrostatic spinning technology; 3) using the separated animal myocardial cells as seed cells to be planted on the membrane; 4) constructing a biotype ventricular assist pump; after the myocardial cells are planted on the membrane, the cell material compound is overlapped into three layers according to the arrangement direction of the three layers of the myocardial cells, and then the conical oversleeve-shaped structure is manufactured. The oversleeve-shaped biological heart chamber auxiliary pump can make up the defects of the traditional mechanical auxiliary pump, and is expected to fundamentally endow the heart chambers with continuous contraction dynamic property for vast end-stage heart failure patients, thereby thoroughly improving the prognosis of the patients, improving the life quality, and reducing the hospitalization cost and the long-term cost of patients.)

1. A preparation method of a biological type ventricular assist pump based on a conjugate coaxial electrospinning technology is characterized by comprising the following steps:

1) preparation of oriented electrostatic spinning myocardial scaffold containing double-walled carbon nanotubes

Dissolving the double-walled carbon nanotube with conductivity in hexafluoroethanol to prepare a MWCNT-containing spinning solution; preparing an oriented electrostatic spinning myocardial scaffold by adopting a conjugate spinning technology;

2) VEGF (vascular endothelial growth factor) -loaded myocardial scaffold constructed by coaxial electrostatic spinning technology

Adopting a coaxial electrostatic spinning technology, wherein a VEGF solution is adopted in a core layer, and a PLCL/collagen and WNCNT dissolved spinning solution is adopted in a shell layer; constructing an electrostatic spinning myocardial scaffold with a shell layer of col and PLCL and a core layer of VEGF cytokine solution;

3) the separated animal cardiac muscle cell is used as seed cell to be planted on the membrane

Taking myocardial cells for isolated culture, and redistributing the cell suspension on a sterilized membrane;

4) construction of biological type ventricular assist pump

After the myocardial cells are planted on the membrane, the cell material compound is overlapped into three layers according to the arrangement direction of the three layers of the myocardial cells, and then the conical oversleeve-shaped structure is manufactured.

2. A method for preparing a bio-type ventricular assist pump according to claim 1, wherein the MWCNT spinning solution concentration of step 1) is 4% and 8%.

3. A method for preparing a biologic ventricular assist pump according to claim 1, wherein the parameters of the conjugate spinning in step 1) are as follows: the voltage is 25kv, the anode is connected with a spinning needle, the cathode is connected with a receiving roller, the distance from the needle to the receiving roller is 20cm, and the rotating speed of the receiving roller is 2000 r/min.

4. A method for preparing a biologic ventricular assist pump according to claim 1, wherein the PLCL/collagen concentration in step 2) is 10%.

5. A method for preparing a biological ventricular assist pump according to claim 1, wherein the concentration of the VEGF solution in the step 2) is 1%.

6. A method for preparing a biologic ventricular assist pump according to claim 1, wherein the parameters of the coaxial electrospinning in the step 2) are as follows: the voltage during spinning is 25kv, and the distance is 20 cm.

7. A method for manufacturing a biologic pump for assisting heart pumping according to any one of claims 1 to 6, wherein said step 3) comprises cutting myocardial tissue into a homogenate, digesting with 0.05% trypsin for 20 minutes, stopping digestion with serum, repeatedly filtering the suspension through a tissue filter to retain the underlying cell suspension, and centrifuging to obtain a cell pellet.

8. The biological ventricular assist pump prepared by the preparation method of any one of claims 1 to 6, which is a myocardial scaffold with myocardial cells, and is divided into an inner longitudinal structure, a middle ring structure and an outer oblique three-layer structure according to myocardial fibers, wherein the three-layer structure is arranged in a layered manner, and the whole structure is in a conical oversleeve-shaped structure.

9. A biological type ventricular assist pump based on a conjugate coaxial electrostatic spinning technology is characterized in that the assist pump is integrally in a conical oversleeve-shaped structure, the upper end of the pump is open, the lower end of the pump is closed, and the pump is formed by superposing three myocardial fiber membrane supports, namely an inner longitudinal layer, a middle annular layer and an outer inclined layer; the fibers of the inner longitudinal layer myocardial fiber membrane support are longitudinally oriented, the fibers of the middle ring layer myocardial fiber membrane support are transversely oriented, and the fibers of the outer oblique layer myocardial fiber membrane support are obliquely oriented; myocardial cells are respectively planted on the inner longitudinal layer, the middle annular layer and the outer oblique layer myocardial fiber membrane support.

10. The biological ventricular assist pump based on the conjugate coaxial electrospinning technology as claimed in claim 9, wherein the spinning is composed of a core layer and a shell layer, the core layer is VEGF solution, and the shell layer is MWCNT/col/PLCL polymer material.

Technical Field

The invention relates to the field of medical tissue engineering, in particular to a biological ventricular assist pump based on a conjugate coaxial electrospinning technology and a preparation method thereof.

Background

Congenital heart disease (called congenital heart disease for short) is the first birth defect in China, and seriously harms physical and mental health of children. The incidence rate of the congenital heart disease is about 0.8 percent, and about 15 million children with the congenital heart disease are newly added in China every year, wherein the complicated congenital heart disease accounts for 30 to 40 percent. In recent years, the number of various palliative operations and radical operations for treating complicated congenital heart disease is also obviously increased, and the incidence rate of postoperative heart failure (heart failure for short) is also obviously increased. Among them, it is reported in literature that 1-5% of children suffering from heart failure are in the terminal stage, and seriously threaten life because of difficult control of the medicine. Although heart transplantation is an effective means for treating end-stage heart failure, because of the serious shortage of donors, only a small number of children patients have the opportunity to receive transplantation, and most children patients need to use a ventricular assist device to maintain life to wait for heart transplantation. At present, the ventricular assist device is an important means for saving the life of children suffering from end-stage heart failure.

The ventricular assist device mainly has the function of reducing the ventricular burden by draining blood in the left or right ventricle of a patient into the ventricular assist device and infusing the blood into an aorta or a pulmonary artery through a mechanical pump so as to replace the ejection function of part of the ventricles. At present, the ventricular assist device is widely applied to clinical treatment of the end-stage heart disease of children, and obtains better clinical curative effect. However, these ventricular assist devices have limited applications, and are limited to short-term circulation support, and their bulky external devices and monitoring devices are not suitable for long-term or lifetime circulation support of children, and the children often have limited activities and cannot leave the intensive care unit, and more complications occur. In view of the disadvantages of these ventricular assist devices, if a biological ventricular pump assist device can be provided, the device can provide the ventricular contraction power for a long time by means of the rhythmic contraction of the myocardial tissue, so as to fundamentally provide the ventricular circulation power, and thus, the device is expected to thoroughly correct the end-stage heart failure, and even avoid the final heart transplantation. With the development of tissue engineering technology in recent years, the construction of tissue-engineered myocardium with bioactivity by using tissue engineering methods has become a research hotspot.

Disclosure of Invention

The invention aims to provide a preparation method of a biological ventricular assist pump based on a conjugate coaxial electrospinning technology, aiming at the defects in the prior art.

The second object of the present invention is to provide a biotype ventricular assist pump prepared by the above preparation method.

The third purpose of the invention is to provide a biological ventricular assist pump based on the conjugate coaxial electrospinning technology.

In order to achieve the first purpose, the invention adopts the technical scheme that:

a preparation method of a biological type ventricular assist pump based on a conjugate coaxial electrostatic spinning technology comprises the following steps:

1) preparation of oriented electrostatic spinning myocardial scaffold containing double-walled carbon nanotubes

Dissolving the double-walled carbon nanotube with conductivity in hexafluoroethanol to prepare a MWCNT-containing spinning solution; preparing an oriented electrostatic spinning myocardial scaffold by adopting a conjugate spinning technology;

2) VEGF (vascular endothelial growth factor) -loaded myocardial scaffold constructed by coaxial electrostatic spinning technology

Adopting a coaxial electrostatic spinning technology, wherein a VEGF solution is adopted in a core layer, and a PLCL/collagen and WNCNT dissolved spinning solution is adopted in a shell layer; constructing an electrostatic spinning myocardial scaffold with a shell layer of col and PLCL and a core layer of VEGF cytokine solution;

3) the separated animal cardiac muscle cell is used as seed cell to be planted on the membrane

Taking myocardial cells for isolated culture, and redistributing the cell suspension on a sterilized membrane;

4) construction of biological type ventricular assist pump

After the myocardial cells are planted on the membrane, the cell material compound is overlapped into three layers according to the arrangement direction of the three layers of the myocardial cells, and then the conical oversleeve-shaped structure is manufactured.

In the above method for preparing the bio-type ventricular assist pump, preferably, the MWCNT spinning dope concentration of the step 1) is 4% and 8%.

In the above method for preparing the biotype ventricular assist pump, preferably, the parameters of the conjugate spinning in the step 1) are as follows: the voltage is 25kv, the anode is connected with a spinning needle, the cathode is connected with a receiving roller, the distance from the needle to the receiving roller is 20cm, and the rotating speed of the receiving roller is 2000 r/min.

In the above method for preparing the biotype ventricular assist pump, preferably, the PLCL/collagen concentration in the step 2) is 10%.

In the above method for preparing the biotype ventricular assist pump, preferably, the concentration of the VEGF solution in the step 2) is 1%.

In the above method for manufacturing the bio-type ventricular assist pump, preferably, the parameters of the coaxial electrospinning in the step 2) are as follows: the voltage during spinning is 25kv, and the distance is 20 cm.

In the above method for manufacturing the biotype ventricular assist pump, preferably, in the step 3), the myocardial tissue is first cut into a homogenate and digested with 0.05% pancreatin for 20 minutes, then the digestion is terminated with serum, the suspension is repeatedly filtered through a tissue filter screen, the lower layer cell suspension is retained, and the cell precipitate is obtained after centrifugation.

In order to achieve the second object, the invention adopts the technical scheme that:

the biological ventricular assist pump prepared by the preparation method is a myocardial scaffold with myocardial cells, is divided into an inner longitudinal structure, a middle ring structure and an outer oblique three-layer structure according to myocardial fibers, is arranged in a layered mode, and is integrally in a conical oversleeve-shaped structure.

In order to achieve the third object, the invention adopts the technical scheme that:

a biological type ventricle auxiliary pump based on a conjugate coaxial electrostatic spinning technology is characterized in that the whole auxiliary pump is in a conical sleeve-shaped structure, the upper end of the auxiliary pump is opened, the lower end of the auxiliary pump is closed, and the auxiliary pump is formed by superposing three myocardial fiber membrane supports, namely an inner longitudinal layer, a middle annular layer and an outer inclined layer; the fibers of the inner longitudinal layer myocardial fiber membrane support are longitudinally oriented, the fibers of the middle ring layer myocardial fiber membrane support are transversely oriented, and the fibers of the outer oblique layer myocardial fiber membrane support are obliquely oriented; myocardial cells are respectively planted on the inner longitudinal layer, the middle annular layer and the outer oblique layer myocardial fiber membrane support.

In the above biological ventricular assist pump based on the conjugate coaxial electrospinning technology, preferably, the spinning is composed of a core layer and a shell layer, the core layer is a VEGF solution, and the shell layer is an MWCNT/col/PLCL polymer material.

The CNT/Collagen/PLCL conductive sensing myocardial scaffold based on the CNT modification and orderly arranged VEGF-loaded CNT/Collagen/PLCL can better simulate the myocardial extracellular matrix structure and guide the directional arrangement of cells. The biological type ventricular assist pump with the bioactivity and the pulsable cardiac muscle forming the oversleeve-shaped structure is constructed on the basis of the cardiac muscle support, and the biological type ventricular assist pump is wrapped on the outer layer of the heart and can be used for assisting the contraction of the heart, so that the dynamic property of continuous contraction of the ventricle can be fundamentally endowed to vast children suffering from end-stage heart failure, the prognosis of the children is thoroughly improved, the life quality is improved, and even the heart transplantation can be avoided.

The ventricular assist device is an important means for saving the life of children suffering from end-stage heart failure. The sleeve-shaped biological ventricular assist pump can make up the defects of the traditional mechanical assist pump. The whole experiment is implemented based on animal experiments, a certain achievement is obtained, and the dynamic property of continuous contraction of ventricles is expected to be fundamentally endowed to vast children with end-stage heart failure, so that the prognosis of the children is thoroughly improved, the life quality is improved, and the hospitalization cost and the long-term cost of patients are reduced.

Drawings

FIG. 1 shows an optical microscope and a scanning electron microscope image of MWCNT-containing oriented electrospun myocardial scaffold (A.4% MWCNT myocardial scaffold optical microscope image, FIG. 1 B.8% MWCNT myocardial scaffold optical microscope image, FIG. 1℃ scaffold scanning electron microscope image).

FIG. 2 shows the transmission electron microscope image of VEGF cell factor solution cardiac muscle scaffold in core layer.

Figure 3 properties of the myocardial membrane (a. elastic modulus of myocardial scaffolds containing different concentrations of MWCNT, b. conductivity of myocardial scaffolds containing different concentrations of MWCNT).

FIG. 4 shows a photoscope image and immunofluorescence assay (A. photoscope image, B. alpha. MHC fluorescence assay) of primary cardiomyocytes seeded on a patch.

FIG. 5 survival of cardiomyocytes on different myocardial scaffolds. A.4% survival of cardiomyocytes on MWCNT scaffolds (green viable cells; red dead cells) and B.

Fig. 6 is a schematic diagram of a biotype ventricular assist pump construction. The CNT/collagen/PLCL myocardial scaffolds with the cardiac muscle cells and the ordered VEGF load are arranged in a layered mode according to the three-layer structure of the inner longitudinal layer, the middle ring and the outer oblique layer of the myocardial fibers.

Fig. 7A is a schematic diagram of the inner longitudinal layer myocardial scaffold structure.

Fig. 7B is a schematic view of the inner longitudinal layer myocardial stent wrapped around the outer layer of the heart.

Fig. 8A is a schematic diagram of a medium-ring layer myocardial scaffold structure.

Fig. 8B is a schematic view of a mid-annulus myocardial scaffold wrapped around the outside of the heart.

Fig. 9A is a schematic structural diagram of an outer oblique layer myocardial scaffold.

FIG. 9B is a schematic view of an outer oblique layer myocardial stent wrapped around the outside of the heart.

FIG. 10 is a schematic plan view of the inner longitudinal layer, the middle annular layer and the outer oblique layer of the myocardial stent being staggered.

Detailed Description

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

Example 1 preparation of a biotype ventricular assist pump based on conjugated coaxial electrospinning technology

1. Preparation of oriented electrostatic spinning myocardial scaffold containing double-walled carbon nanotubes

The method comprises the following steps: modified and attenuated double-walled carbon nanotubes (MWCNTs) produced by cheaptube, singapore were dissolved in hexafluoroethanol at different ratios, and then mixed well with a magnetic stirrer to prepare 4% and 8% MWCNTs-containing spinning solutions. The method adopts a conjugate spinning technology to prepare an oriented electrostatic spinning myocardial scaffold, the voltage is 25kv, the anode is connected with a spinning needle, and the cathode is connected with a receiving roller. The distance from the needle head to the receiving roller is 20cm, and the rotating speed of the receiving roller is 2000 r/min. And fully drying the spun membrane in a vacuum drying oven for 1 month, and observing the arrangement of the spinning by using a light mirror and a scanning electron microscope respectively after the organic solvent is fully volatilized.

As a result: under a light mirror and a scanning electron microscope, the MWCNTs in the bracket are uniformly oriented and distributed among parallel filatures, so that the MWCNTs have good conductivity. As the concentration of WNCNT increased, the color of the membrane darkened. This provides the basis for the orientation of cardiomyocytes on the patch (FIG. 1).

2. VEGF (vascular endothelial growth factor) -loaded myocardial scaffold constructed by coaxial electrostatic spinning technology

The method comprises the following steps: the coaxial electrostatic spinning technology is adopted, the core layer adopts 1% VEGF solution, and the shell layer adopts spinning solution dissolved with 10% PLCL/collagen and WNCNT with different concentrations. Constructing a shell layer of col and PLCL, and a core layer of VEGF cell factor solution electrostatic spinning myocardial scaffold. The voltage during spinning is 25kv, and the distance is 20 cm. And observing the spinning structure of the diaphragm by a transmission electron microscope.

As a result: the VEGF solution is taken as the middle core layer of the spinning, and the degradable MWCNT/col/PLCL high polymer material is taken as the shell layer. The preparation method combined with the high molecular degradable material can realize the slow release of the VEGF cell factor with activity in the application process of the stent (figure 2).

3. Mechanical properties and conductivity of myocardial scaffolds containing different MWCNTs

The method comprises the following steps: the mechanical properties of the myocardial scaffolds were tested with an XT2i texture Analyzer (texture Technologies, Hamilton, MA, USA). The samples were measured from the slope of the linear region corresponding to 5% -15% strain at 0.1 mm/s. The obtained compression modulus was averaged. The conductivity of the myocardial scaffold was assessed by a three-point probe (Oakton Instruments, Vernon Hills, IL, USA). The myocardial scaffolds were immersed in PBS solution and four conductivity readings were taken for each sample at a frequency of 27kHz for each scaffold.

As a result: each set of experiments was repeated three times. The differences between groups were statistically significant (P <0.01) with increasing MWCNT content increasing the elastic modulus of the myocardial scaffold (fig. 3A). The differences between groups were statistically significant (P <0.01) with increasing MWCNT content with increasing conductivity of the myocardial scaffold (fig. 3B).

4. Isolated cultured mouse cardiac muscle cell as seed cell to be planted on the membrane

The method comprises the following steps: the left ventricle of the suckling mouse is taken to carry out isolated culture of the myocardial cells. The obtained left ventricular myocardial tissue was minced and homogenized, and digested with 0.05% trypsin for 20 minutes, and then the digestion was terminated with serum. Repeatedly filtering the suspension by a tissue filter screen, reserving the cell suspension at the lower layer, and centrifuging to obtain cell sediment. The membrane subjected to repeated ultraviolet irradiation and alcohol soaking sterilization is placed at the bottom of a 100mm culture dish, and the cell suspension is redistributed on the membrane.

As a result: after about 7 days of culture, the primary myocardial cells reach 100% confluence rate and are arranged orderly, and can jump in pieces, so that excitation contraction coupling of the whole myocardial tissue is realized, synchronous rhythmic contraction is achieved, and the myocardial cells under the microscope are polygonal (figure 4). The cultured primary cardiomyocytes were subjected to fluorescent labeling with α MHC (fig. 4).

5. Membrane biocompatibility identification

The method comprises the following steps: the MWCNT oriented electrostatic spinning membrane is soaked in alcohol under an ultraviolet lamp for 2 hours for complete disinfection, and then repeatedly washed by PBS for cell culture. Primary cells were plated at 1 × 10⁶Planting on the membrane at different concentrations, and changing the liquid every other day. After 4 days, the upper layer culture solution is discarded, and dead and live cell dye is added to identify the cell activity on the membrane. The survival of the myocardial cells on the myocardial scaffolds containing different concentrations was counted by using image J software.

As a result: with increasing MWCNT content, cell survival decreased. Cells adhered less on the 8% MWCNT sheet, while more adhered and more active on the 4% MWCNT sheet (fig. 5A).

6. Construction of biological type ventricular assist pump

The method comprises the following steps: after the myocardial cells are planted on the membrane, the cell material compound is overlapped into three layers according to the arrangement direction of the three layers of the myocardial cells, and then the conical oversleeve-shaped structure is manufactured.

As a result: the myocardial rack with myocardial cells is divided into three layers of inner longitudinal, middle ring and outer inclined layers according to myocardial fiber (figure 6).

EXAMPLE 2 biological ventricular Assist Pump

As shown in fig. 7-10, fig. 7A is a schematic diagram of an inner longitudinal-layer myocardial stent structure, fig. 7B is a schematic diagram of an inner longitudinal-layer myocardial stent wrapped on an outer layer of a heart, fig. 8A is a schematic diagram of an intermediate-layer myocardial stent structure, fig. 8B is a schematic diagram of an intermediate-layer myocardial stent wrapped on an outer layer of a heart, fig. 9A is a schematic diagram of an outer oblique-layer myocardial stent structure, fig. 9B is a schematic diagram of an outer oblique-layer myocardial stent wrapped on an outer layer of a heart, and fig. 10 is a schematic diagram of a plane where the inner longitudinal-layer, the intermediate-layer and the outer oblique.

The biological type ventricular assist pump based on the conjugate coaxial electrostatic spinning technology is characterized in that the assist pump is integrally in a conical oversleeve-shaped structure, the upper end of the assist pump is provided with an opening 1, the lower end of the assist pump is closed 2, the assist pump is formed by overlapping three myocardial fiber membrane supports, namely an inner longitudinal layer 3, a middle ring layer 4 and an outer inclined layer 5, when the pump is used, a heart is placed into the assist pump from the opening 1, the three layers of the inner longitudinal layer 3, the middle ring layer 4 and the outer inclined layer 5 are sequentially overlapped and wrapped on the outer layer of the heart, and the ventricular contraction is powered by the rhythmic contraction of myocardial tissues planted on the three myocardial fiber membrane supports, so that the heart contraction. In this example, the fibers of the inner longitudinal layer of the myocardial fiber membrane scaffold are oriented longitudinally (fig. 7A), the fibers of the middle ring layer of the myocardial fiber membrane scaffold are oriented transversely (fig. 8A), and the fibers of the outer oblique layer of the myocardial fiber membrane scaffold are oriented obliquely (fig. 9A); myocardial cells are respectively planted on the myocardial fiber membrane stents of the inner longitudinal layer, the middle ring layer and the outer oblique layer.

The myocardial fiber membrane scaffold is prepared by an electrostatic spinning technology, wherein spinning is composed of a core layer and a shell layer, the core layer is a VEGF solution, and the shell layer is an MWCNT/col/PLCL high polymer material. In the embodiment, the CNT/Collagen/PLCL conductive sensing myocardial scaffold based on CNT modification and capable of orderly arranging VEGF-loaded CNT/Collagen/PLCL can better simulate the myocardial extracellular matrix structure and guide the directional arrangement of cells.

The above description is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, several modifications and additions can be made without departing from the method of the present invention, and these modifications and additions should also be regarded as the protection scope of the present invention.