CN114225111A

CN114225111A - Preparation method of extracellular matrix support material with controllable pore structure

Info

Publication number: CN114225111A
Application number: CN202111587767.5A
Authority: CN
Inventors: 朱美峰; 李雯; 孔德领
Original assignee: Nankai University
Current assignee: Nankai University
Priority date: 2021-12-23
Filing date: 2021-12-23
Publication date: 2022-03-25

Abstract

The invention discloses a preparation method of an extracellular matrix support material with a controllable pore channel structure, which particularly takes a tubular or film-shaped artificially synthesized high molecular polymer fiber support with a pseudo-natural structure as a sacrificial template material, fills a cell-free matrix solution among pores of the fiber template support, then carries out crosslinking, and washes away the fiber-free template material, thus obtaining the extracellular matrix support with the controllable pore structure. The invention can realize the preparation in vitro, has short period, and the obtained material has a controllable pore structure and can induce the oriented regeneration of tissues so as to recover the appearance and the function of the tissues.

Description

Preparation method of extracellular matrix support material with controllable pore structure

Technical Field

The invention relates to the field of preparation of scaffold materials for tissue engineering, in particular to a preparation method of an extracellular matrix scaffold material with a controllable pore structure.

Background

Trauma, disease and congenital abnormalities often result in tissue dysfunction or organ damage, requiring rapid recovery of lost function and morphology. The development of tissue engineering and biomaterials provides new strategies for repairing defects of human tissues and organs, and scientists have developed various tissue engineering scaffold materials for promoting tissue defect repair, including those of bones, cartilages, nerves, blood vessels, skins and artificial organs, such as liver, kidney, spleen, urethra, etc. In fact, many tissues in the human body are anisotropic, with specific spatial arrangements of their cells and the extracellular matrix (ECM) that they secrete. For example, in oriented tissues such as Achilles tendon, muscle, nerve, the orientation of cells and extracellular matrix can be clearly observed. Thus, an important factor in the functional regeneration of oriented tissues is the induction of oriented regeneration of cells and tissues. This can be improved by controlling the physical parameters of the scaffold such as the direction of pore arrangement, pore size, porosity, bioactivity, hardness, etc.

At present, the preparation of tissue regeneration scaffold materials using synthetic or natural materials has been used to improve tissue injury repair, but the effect still remains to be improved. Synthetic polymers have more controllable physicochemical properties and processability than natural materials having low mechanical strength such as collagen, chondroitin, hyaluronic acid and the like. However, the synthetic polymer material has poor biocompatibility, and the degradation product is acidic, which can cause chronic inflammation and immune rejection, thereby affecting tissue regeneration and integration. In recent years, the extracellular matrix materials are more and more emphasized, and a plurality of researches show that the extracellular matrix has a remarkable regulation effect on various aspects such as adhesion, proliferation and differentiation of cells, the topological structure of the matrix can also regulate the behaviors of the cells, and more importantly, the extracellular matrix can improve a local immune microenvironment after being implanted into a body so as to promote tissue repair. However, most of the acellular scaffold materials prepared by the existing methods generally have the problems of small pore size and poor controllability, so that cells are difficult to migrate into the scaffold, and the scaffold materials lack the guiding effect on the cells, thereby limiting tissue remodeling and function integration. In addition, our previous studies constructed extracellular matrix scaffolds with controlled pore structure using animal subcutaneous implantation combined with fibrotemplate leaching, however, this approach has several problems such as: the culture time is too long, and the extracellular matrix components lack tissue specificity; animal subcutaneous tissues are required to be used as reactors, so that animal resources are wasted, and the preparation cost is high; in addition, there is a disadvantage that the sample preparation is unstable due to the implantation position and the individual animal difference. Based on the method, a novel method for quickly preparing the extracellular matrix scaffold with the controllable pore structure in vitro is provided, the extracellular matrix material used in the method has wide sources, and the obtained scaffold has strong tissue specificity; in addition, the method obviously shortens the preparation period of the stent and reduces the preparation cost.

Disclosure of Invention

The invention aims to provide a preparation method of an extracellular matrix support material with a controllable pore channel structure, which aims to solve the problems in the prior art.

In order to achieve the purpose, the invention provides the following technical scheme:

the first technical scheme is as follows: an extracellular matrix scaffold material with a controllable pore structure is prepared by taking a tubular or film-shaped polymer fiber scaffold with a pseudo-natural structure as a sacrificial template material, pouring acellular matrix solution between pores of the fiber template scaffold, then performing crosslinking, and washing off the fiber template material to obtain the extracellular matrix scaffold with the controllable pore structure.

Further, the tubular or film-shaped polymer fiber scaffold with the pseudo-natural structure is prepared by using biodegradable high molecules as raw materials and adopting electrostatic spinning, wet spinning, melt spinning or 3D printing technology, and the obtained product is a nano-scale or micron-scale ordered or disordered arrangement structure.

When electrostatic spinning is adopted, the distance between the needle head and the receiver is set to be 1-30cm, the solution flow rate is 1-30mL/h, the direct current voltage is 1-30kV, the rotating speed is 10-3000rpm, and the spinning time is 10-400 min.

When wet spinning is adopted, the solution flow rate is set to be 1-30mL/h, the rotating speed of a receiving rod is 10-1000rpm, and the spinning time is 1-400 min.

When melt spinning is adopted, the advancing speed of the charging barrel is set to be 1-60mL/h, the moving speeds of the x axis and the y axis of the receiving plate are respectively 1-100mm/s, the moving distance is 1-100cm, and the spinning time is 1-300 min.

When the 3D printing technology is adopted, the set printing program (the length, the width and the height of the printing support are respectively 1-100cm, 1-100cm and 1-100cm, and the fiber intersection angle is 0-90 degrees).

Further, the raw materials are selected from one or more of synthetic high molecular polylactic acid (PLA), polylactic acid-polyglycolic acid copolymer (PLGA), Polycaprolactone (PCL), Polyhydroxyalkanoate (PHA), polyglycolic acid (PGA), Polyurethane (PU), poly L-lactide-caprolactone (PLCL) and poly-p-dioxanone (PDS).

Furthermore, the acellular matrix solution is prepared by carrying out acellular treatment on different tissues of animals, removing antigens and dissolving with acetic acid.

Further, the tissue source is a tissue or organ of an artificially fed animal, comprising: brain, heart, liver, spleen, lung, kidney, muscle, skin, meninges, diaphragm, amniotic membrane, pericardium, heart valve, small intestine submucosa, muscle, blood vessel, tendon, ligament, cartilage, esophagus, stomach, nerve, and bladder. The artificial feeding animals include cattle, pig, dog, sheep, rabbit, rat, and mouse.

The second technical scheme is as follows: a preparation method of an extracellular matrix scaffold material with a controllable pore channel structure comprises the following steps:

pouring the acellular matrix solution into pores of the tubular or membranous polymer fiber scaffold material with the pseudo-natural structure, freezing, freeze-drying, placing the freeze-dried compound in a cross-linking agent for cross-linking overnight, taking out, washing with sterile water, dehydrating with gradient ethanol, placing in dichloromethane, and oscillating to remove the tubular or membranous polymer fiber scaffold with the pseudo-natural structure to obtain the extracellular matrix scaffold material with the controllable pore structure.

Furthermore, the freezing temperature is-20 ℃ to-100 ℃, the time is 12-48h, and the freezing drying temperature is-10 ℃ to-100 ℃, and the time is 24-72 h.

Further, the cross-linking agent is obtained by mixing (1-ethyl- (3-dimethylaminopropyl) carbodiimide) and N-hydroxysuccinimide according to the mass ratio of 4: 1.

The third technical scheme is as follows: an application of an extracellular matrix scaffold material with a controllable pore structure as a tissue repair material.

Compared with the prior art, the invention has the beneficial effects that:

1. the polymer scaffold is used as a template, and the extracellular matrix scaffold with the porous channel simulated natural structure is prepared in vitro by simulating the natural structure of a target tissue, so that a guide structure can be provided for oriented tissue regeneration, and the problem of pore formation of an extracellular matrix scaffold material is solved;

2. the tissue engineering scaffolds with different pseudo-natural structures can be prepared by regulating the diameter, angle, pore, size, structure and the like of the polymer template fiber; the microporous structure among pore channels can be regulated and controlled by regulating the freezing temperature; the tissue engineering scaffold with specificity can be prepared by obtaining extracellular matrix materials of different tissue sources. Such as: blood vessel, nerve, muscle, tendon and other tissue engineering scaffolds.

3. As all the scaffold materials prepared by the invention are prepared in vitro, compared with the prior in vivo implantation, the extracellular matrix scaffold with high tissue specificity can be prepared by using the extracellular matrix materials from corresponding tissues for repairing specific damaged organs or tissues, thereby realizing targeted and efficient treatment. In addition, the preparation time of the bracket is short, the cost is low, the material uniformity is high and the process stability is high.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings without creative efforts.

FIG. 1 is a scanning electron microscope image of extracellular matrix scaffold material with controllable pore structure for different tissue repair; wherein, a picture A is a cross-sectional view of an artificial blood vessel which can be used in example 2, a picture B is a cross-sectional view of a stent material which can be used in tendon repair which can be used in example 1, a picture C is a structural view of a membrane stent material with a parallel micro-channel structure which can be used in example 3, and a picture D is a structural view of a membrane stent material with a cross-angle micro-channel structure which can be prepared in example 4;

FIG. 2 is a diagram of an extracellular matrix scaffold for repairing rat Achilles tendon for 3 months, in which A is the extracellular matrix scaffold prepared in example 1 of the present invention, and B is the extracellular matrix scaffold prepared by a subcutaneous implantation method;

FIG. 3 shows the results of H & E staining of an extracellular matrix scaffold for repairing rat Achilles tendon for 3 months, wherein A is the extracellular matrix scaffold prepared in example 1 of the present invention, and B is the extracellular matrix scaffold prepared by a subcutaneous implantation method;

FIG. 4 shows that the extracellular matrix scaffold repairs rat Achilles tendon for 3 months, and the diameter of collagen fiber of the regenerated Achilles tendon is detected by a transmission electron microscope, wherein A is the extracellular matrix scaffold prepared in the embodiment 1 of the invention, and B is the cell matrix scaffold prepared by a subcutaneous implantation method;

FIG. 5 is a photograph showing the regeneration of rat sciatic nerve by extracellular matrix scaffolds for 3 months, wherein A is the extracellular matrix scaffold prepared in example 2 of the present invention, and B is the cell matrix scaffold prepared by subcutaneous implantation;

FIG. 6 is a Transmission Electron Microscope (TEM) observation of myelin sheath after the rat sciatic nerve is repaired by the extracellular matrix scaffold for 3 months, wherein A is the extracellular matrix scaffold prepared in example 2 of the invention, and B is the cell matrix scaffold prepared by the subcutaneous implantation method;

FIG. 7 is a diagram of compound muscle action potentials of rat sciatic nerve repaired by extracellular matrix scaffolds after 3 months, wherein A is the extracellular matrix scaffold prepared in example 2 of the invention, and B is the cell matrix scaffold prepared by a subcutaneous implantation method.

Detailed Description

Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but as a more detailed description of certain aspects, features and embodiments of the invention.

It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. Further, for numerical ranges in this disclosure, it is understood that each intervening value, between the upper and lower limit of that range, is also specifically disclosed. Every smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in a stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference herein for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.

It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the present disclosure without departing from the scope or spirit of the disclosure. Other embodiments will be apparent to those skilled in the art from consideration of the specification. The specification and examples are exemplary only.

As used herein, the terms "comprising," "including," "having," "containing," and the like are open-ended terms that mean including, but not limited to.

The organizations used in the invention are all purchased in the market.

Example 1

Achilles tendon extracellular matrix support with parallel orientation micron pore canal prepared by using PCL micron fiber as template

Preparation of PCL micrometer fiber scaffold in parallel orientation: the template scaffold was prepared using melt spinning at room temperature. Adding 20g of PCL into a charging barrel, heating the charging barrel to 100 ℃, keeping the temperature for 5h, adjusting the distance between a needle head of the charging barrel and a receiving plate to be 1cm, setting the propelling speed of the charging barrel to be 6mL/h, the moving speeds of an x axis and a y axis of the receiving plate to be 1mm/s, the moving distance to be 15cm and the spinning time to be 60 min. And after the preparation is finished, drying the parallel orientation micron fiber support in vacuum for later use.

Preparation of bovine achilles tendon tissue acellular solution: taking fresh bovine Achilles tendon tissue, removing surrounding connective tissue, cutting into sheet structure with thickness of about 500 μm, immersing in liquid nitrogen for 2min, then placing into 40mL physiological saline, shaking at 100rpm for 15min in shaking table at 37 deg.C, and repeating freeze thawing for 5 times. To remove residual nuclei, after the final washing, the sample was placed in a reaction solution containing 50U/mL of DNase I and 1U/mL of RNase A, and reacted at 37 ℃ for 24 hours on a shaker at 150 rpm. Followed by washing with sterile PBS to remove the enzyme solution. Then freeze-drying the obtained acellular bovine achilles tendon tissue for 12h, shearing into pieces, weighing 100mg of bovine achilles tendon extracellular matrix, dissolving in 0.01mol/L hydrochloric acid solution containing 10mg of pepsin, and stirring overnight to obtain the acellular bovine achilles tendon solution.

Preparation of PCL polymer-bovine achilles tendon extracellular matrix complex: rolling the prepared PCL micron fiber into a cylinder with the length of 1cm and the diameter of 2mm, fastening two ends, soaking the cylinder into the decellularized bovine achilles tendon solution, freezing the solution in a refrigerator at the temperature of-20 ℃ for 12h, and then freeze-drying the solution in a freeze-drying machine for 24h to obtain the polymer-bovine achilles tendon extracellular matrix composite.

Cross-linking of PCL polymer-bovine achilles tendon extracellular matrix complex: placing the freeze-dried compound in a container containing the components in a mass ratio of 4:1 EDC/NHS in 80% ethanol at 4 ℃ overnight, and after removal with sterile water to remove residual crosslinker.

Preparation of bovine achilles tendon extracellular matrix scaffold with parallel micron orientation: and (3) dehydrating the compound obtained in the step (a) by gradient ethanol (volume fractions of 70%, 80%, 90% and 100%) for 30min, placing the compound in chloroform, oscillating for 48h, changing liquid every 12h to remove the polymer template, thus obtaining the bovine achilles tendon extracellular matrix scaffold material with the pseudo-natural achilles tendon structure, and then sterilizing by ethylene oxide for later use.

Example 2

Nerve extracellular matrix support with parallel orientation micron pore canal prepared by taking PC micron fiber as template

Preparation of PCL micrometer fiber scaffold in parallel orientation: the scaffold was prepared using melt spinning at room temperature. Adding 20g of PCL into a charging barrel, heating the charging barrel to 100 ℃, keeping the temperature for 5h, adjusting the distance between a needle head of the charging barrel and a receiving plate to be 2cm, setting the propelling speed of the charging barrel to be 5mL/h, the moving speeds of an x axis and a y axis of the receiving plate to be 1mm/s, the moving distance to be 20cm and the spinning time to be 20 min. And after the preparation is finished, drying the parallel orientation micron fiber support in vacuum for later use.

Preparation of a solution for removing pig nerve cells: cleaning peripheral nerve tissue of fresh pig with sterile distilled water, soaking in 0.1% peroxyacetic acid solution, sterilizing at room temperature for 2 hr, and cleaning with sterile water for 1 hr for 3 times. The neural tissue was then placed in a solution containing 1% (w/v) Sodium Dodecyl Sulfate (SDS), shaken in a shaker at room temperature for 24h, and washed with sterile distilled water for 72h, with changes every 6 h. Then, the nerve tissue was put into a PBS (pH 7.4) buffer containing 4.00U/mLDNA enzyme and 1U/mLRNA enzyme, and stirred with a shaker at 37 ℃ for 24 hours to remove nucleic acids. Freeze-drying the obtained acellular nerve tissue for 24h, grinding the acellular nerve tissue into powder at low temperature, dissolving the extracellular matrix into 0.1mol/L acetic acid solution in proportion, and stirring the solution overnight to obtain the acellular nerve extracellular matrix solution.

Preparation of PCL polymer-porcine neuronal extracellular matrix complex: rolling the prepared PCL micron fibers into a cylinder with the length of 2cm and the diameter of 1.8mm, fastening two ends, soaking the cylinder into a pig acellular matrix solution, freezing the solution at-20 ℃ for 12h in a refrigerator, and then freeze-drying for 24h to obtain the polymer-pig nerve extracellular matrix compound.

Cross-linking of PCL polymer-porcine neuronal extracellular matrix complex: placing the freeze-dried compound in a container containing the components in a mass ratio of 4:1 EDC/NHS in 80% ethanol at 4 ℃ overnight, and after removal with sterile water to remove residual crosslinker.

Preparation of porcine neural extracellular matrix scaffolds with parallel micron orientation: and (3) dehydrating the obtained compound by using gradient ethanol (volume fractions of 70%, 80%, 90% and 100%) for 30min, placing the dehydrated compound in dichloromethane, oscillating for 48h, changing liquid every 12h to remove the polymer template, thus obtaining the porcine neural extracellular matrix scaffold material, and then sterilizing the porcine neural extracellular matrix scaffold material by using ethylene oxide for later use.

Example 3

Preparation of vascular cell matrix scaffold with circumferentially oriented micron pore canal by using PLA as template

Preparation of circumferentially oriented PLA micrometer fiber scaffolds: the scaffold was prepared using wet spinning in a room temperature fume hood. 1g of PLA was dissolved in 10mL of hexafluoroisopropanol and stirred at room temperature until the solution was clear. A metal rod having a diameter of 2mm was connected to a rotary motor. The PLA spinning solution was drawn into a syringe, which needle was placed in the ethanol coagulation bath 1cm from the receiving cylinder. The solution flow rate was set at 2mL/h, the receiving rod rotation speed was 500rpm, and the spinning time was 40 min. After the preparation is finished, the circumferentially oriented micron fiber tubular scaffold is dried in vacuum for standby.

Preparing a pig aortic vascular tissue acellular solution: taking a fresh pig aorta blood vessel, peeling off the aorta blood vessel from surrounding tissues, cutting into blocks with the size of 0.8cm multiplied by 0.8cm, cleaning with sterile distilled water, soaking in 0.1% peracetic acid solution, sterilizing at room temperature for 2h, and cleaning with a sterile water shaking table for 3 times, 1h each time. The vessels were then placed in a solution containing 1% (w/v) Sodium Dodecyl Sulfate (SDS), shaken in a shaker at room temperature for 24h, and washed with sterile distilled water for 72h, changing the solution every 6 h. The vascular tissue was then placed in PBS (pH 7.4) buffer containing 4.00U/mLDNA enzyme and 1U/mLRNA enzyme and shaken at 37 ℃ for 24h to remove nucleic acids. Freeze-drying the obtained vascular acellular machine for 24h, grinding the vascular acellular machine into powder at low temperature, dissolving extracellular matrix into 0.1mol/L acetic acid solution in proportion, and stirring the solution overnight to obtain the vascular extracellular matrix solution.

Preparation of PLA Polymer-porcine vascular extracellular matrix Complex: fully soaking the prepared circumferential-oriented PLCL micron fibers in a decellularized pig blood vessel solution, freezing the solution at the temperature of-20 ℃ for 12 hours in a refrigerator, and then freeze-drying for 24 hours to obtain the polymer-pig blood vessel extracellular matrix compound.

Cross-linking of PLA polymer-porcine vascular extracellular matrix complex: placing the freeze-dried compound in a container containing the components in a mass ratio of 4:1 EDC/NHS in 80% ethanol at 4 ℃ overnight, and after removal with sterile water to remove residual crosslinker.

Preparation of porcine vascular extracellular matrix scaffold with circumferentially oriented microchannel structure: and (3) dehydrating the obtained compound by using gradient ethanol (volume fractions of 70%, 80%, 90% and 100%) for 30min respectively, placing the dehydrated compound in dichloromethane, oscillating for 48h, changing liquid every 12h to remove the polymer fiber template, thus obtaining the extracellular matrix scaffold material with the circumferentially oriented micron channel structure of the pseudo-natural vascular structure, and then sterilizing the extracellular matrix scaffold material by using ethylene oxide for later use.

Example 4

Muscle cell matrix scaffold with parallel-oriented nano-pore channels prepared by using PLGA as template

Preparation of a parallel-oriented PLGA micrometer fiber scaffold: the scaffold was prepared using electrospinning in a room temperature fume hood. 1.0g PLGA with molecular weight of 60000 is weighed, added to 10mL dichloromethane, and dissolved at room temperature with stirring until clear to prepare 100g/L PLGA solution. Electrospinning was carried out at room temperature with a room relative humidity of 60%. The aluminum foil paper was covered on the electrospinning roller receiving device and grounded, 100g/L of PLGA spinning solution was filled into a disposable syringe having a diameter of about 14.95mm, and a high voltage DC power source was connected to the syringe needle. The needle of the syringe is adjusted to be aligned with the center of the cylindrical receiver, the distance between the needle and the receiver is set to be 12cm, the solution flow rate is 1mL/h, the direct-current voltage is 16kV, the rotating speed is 3000rpm, and the spinning time is 40 min. The membrane scaffold material with the parallel orientation nanofiber structure can be prepared, and then the obtained membrane scaffold material is dried in vacuum for 48 hours at room temperature so as to completely volatilize the solvent. Preparation of porcine skeletal muscle tissue acellular solution: taking fresh pig skeletal muscle tissue, cutting into blocks of 1cm multiplied by 1cm, placing into a 1L reagent bottle, adding sterile water, placing in a shaking table, shaking and washing, soaking in 0.1% peroxyacetic acid solution after washing till no blood water exists, sterilizing at room temperature for 2h, and washing with sterile water for 3 times, each time for 1 h. The tissue was then placed in a 1% SDS solution, shaken on a shaker at room temperature for 12h, and washed with sterile distilled water for 72h, changing the solution every 6 h. The muscle tissue was then placed in PBS (pH 7.4) buffer containing 6.25U/ml dna enzyme and 1U/ml rna enzyme and shaken at 37 ℃ for 24h to remove nucleic acids. And freeze-drying the obtained muscle extracellular matrix for 24h, crushing the muscle extracellular matrix into powder at low temperature, dissolving the extracellular matrix (the mass ratio of the extracellular matrix to the pepsin is 10:1) in 0.01mol/L hydrochloric acid solution according to a proportion, and stirring the solution overnight to obtain the muscle solution without cells.

Preparation of PLGA polymer-porcine muscle extracellular matrix complex: fully soaking the prepared PLGA nano fibers in parallel orientation in a pig extracellular matrix scaffold solution, freezing the solution in a refrigerator at the temperature of 20 ℃ below zero for 12h, and then freeze-drying for 24h to obtain the polymer-pig muscle extracellular matrix compound.

Cross-linking of PLGA polymer-porcine muscle extracellular matrix complex: placing the freeze-dried compound in a container containing the components in a mass ratio of 4:1 EDC/NHS in 80% ethanol at 4 ℃ overnight, and after removal with sterile water to remove residual crosslinker.

Preparation of porcine muscle extracellular matrix scaffolds with parallel-oriented nanochannels: and (3) dehydrating the obtained compound by using gradient ethanol (volume fractions of 70%, 80%, 90% and 100%) for 30min respectively, placing the dehydrated compound in dichloromethane, oscillating for 48h, changing liquid every 12h to remove the polymer template, thus obtaining the muscle extracellular matrix scaffold material with the nanopore structure, and then sterilizing the muscle extracellular matrix scaffold material by using ethylene oxide for later use.

Example 5

Preparation of fibrous ring cell matrix scaffold with cross-angled micron pore canal by using PLA as template

Cross-angle PLA micron fiber layer scaffold preparation: the scaffold was prepared using 3D printing technology at room temperature. Firstly, a printing program set by 3Dsimplify (the length, the width and the height of a printing support are respectively 2cm, 1.5cm and 1cm, the fiber crossing angle is 60 degrees) is used, then a PLA printing material is installed in a 3D printer (end-3S), after the temperature is heated to 200 ℃, the printing time is 30 minutes, and the micron fiber support with 60-degree angle crossing can be prepared.

Preparation of rabbit fiber ring tissue acellular solution: after the experimental rabbit auricular edge vein air injection is killed, the material-taking part is disinfected, the spine is separated, the surrounding muscle and fascia tissues are removed, the intervertebral disc tissues are cut between two vertebrae, and then the fibrous ring is separated from the intervertebral disc tissues. The obtained fiber ring tissue was soaked in 0.1% peracetic acid solution, sterilized at room temperature for 2 hours, and then washed with sterile water for 3 times, each for 1 hour. Then immersing the fiber ring tissue in liquid nitrogen for 2min, then placing the fiber ring tissue in 40mL of physiological saline, shaking the fiber ring tissue in a shaking table at 100rpm and 37 ℃ for 15min, and repeatedly freezing and thawing for 5 times in the way. To remove residual nuclei, after the final washing, the sample was placed in a reaction solution containing 50U/mL of DNase I and 1U/mL of RNase A, and reacted at 37 ℃ for 24 hours on a shaker at 150 rpm. Followed by washing with sterile PBS to remove the enzyme solution. And freeze-drying the decellularized rabbit fiber ring tissue for 12h, grinding the decellularized rabbit fiber ring tissue at low temperature into powder, finally weighing 100mg of rabbit fiber ring extracellular matrix, dissolving the rabbit fiber ring extracellular matrix into 0.01mol/L hydrochloric acid solution containing 10mg of pepsin, and stirring the solution overnight to obtain the rabbit fiber ring extracellular matrix solution.

Preparation of PLA Polymer-Rabbit fiber Ring extracellular matrix Complex: the prepared PLA micron fibers with the crossing angle (60 degrees) are fully soaked in the decellularized bovine fiber ring solution, and then the solution is frozen in a refrigerator at the temperature of-20 ℃ for 12 hours and then is frozen and dried for 24 hours, so that the polymer-bovine fiber ring extracellular matrix composite can be obtained.

Cross-linking of PLA polymer-rabbit fiber loop extracellular matrix complex: placing the freeze-dried compound in a container containing the components in a mass ratio of 4:1 EDC/NHS in 80% ethanol at 4 ℃ overnight, and after removal with sterile water to remove residual crosslinker.

Preparation of rabbit fiber loop extracellular matrix scaffold with cross-angled microchannels: the composite obtained in the above step is dehydrated by gradient ethanol (volume fraction is 70%, 80%, 90%, 100%) for 30min, and then placed in dichloromethane for oscillation for 48h, and the solution is changed every 12h to remove the polymer template, so as to obtain the extracellular matrix scaffold material with the pseudo-natural fiber ring structure, and then ethylene oxide is sterilized for standby.

FIG. 1 is a scanning electron microscope image of extracellular matrix scaffold material with controllable pore structure for different tissue repair; wherein, a picture A is a cross-sectional view of an artificial blood vessel which can be used in example 2, a picture B is a cross-sectional view of a scaffold material which can be used in tendon repair which can be used in example 1, a picture C is a structural view of a scaffold material with a parallel micro-channel structure which can be used in example 3, and a picture D is a structural view of a scaffold material with a cross-angle micro-channel structure which can be prepared in example 4. The process can be used for preparing corresponding extracellular matrix scaffold with tissue specificity by simulating the structure of natural tissue for repairing corresponding damaged tissue.

The cell matrix scaffold material prepared by the subcutaneous embedding method is concretely as follows: (same operation mode for different parts)

Preparation of cell matrix scaffold material with micron pore size:

preparing a PCL micron fiber scaffold: add 20g pcl to the barrel. Heating the charging barrel to 90 ℃, keeping the temperature for 5h, adjusting the distance between the needle head of the charging barrel and the receiving plate, setting the propelling speed and the flow speed of the charging barrel to be 6ml/h, setting the moving speed of the x axis and the y axis of the receiving plate to be 1mm/s, setting the moving distance to be 15cm, and setting the spinning time to be 60 min. And after the preparation is finished, drying the crude fiber support in vacuum for later use. The method for implanting the stent subcutaneously comprises the following steps: cutting the PCL micrometer fiber support into 2cm × 1cm strips, sterilizing with 75% medical alcohol, and replacing with sterile physiological saline. Anesthetizing a rat by using 10% (m/v) chloral hydrate, depilating two sides of the back to form an area of 1.5cm multiplied by 1.0cm, opening a 1cm long incision by using scissors, separating a cortex from muscle tissues by using flat-head scissors to form a cavity of 2.5cm multiplied by 1.5cm, flatly placing a PCL micron fiber support material into the cavity, suturing the incision, and sterilizing and raising the rat.

Preparation of cell matrix scaffold material with micron pore size: when the subcutaneous implantation time reached 1 month, rats were anesthetized with isoflurane, an incision was made on the other side of the implanted scaffold material, and the material was removed after being peeled from the surrounding tissue. And (5) sewing and sterilizing the cut, and then feeding. And dehydrating the obtained stent by using 50 percent, 70 percent and absolute ethyl alcohol for 3 hours respectively, placing the stent in chloroform, oscillating for 72 hours, changing liquid once in 24 hours, and washing off the polymer PCL. Vacuum drying, and sterilizing with ethylene oxide.

FIG. 2A, B shows the regenerated Achilles tendon after the in situ implantation of the porous Achilles tendon extracellular matrix scaffold and the extracellular matrix scaffold prepared by the method of example 1 and the subcutaneous implantation, respectively, into the defect site of the Achilles tendon of rat for 3 months. It can be seen that the two scaffolds implanted in vivo the nascent achilles tendon was integrated with the host.

FIG. 3A, B shows the results of H & E staining of a porous Achilles tendon extracellular matrix scaffold and an extracellular matrix scaffold prepared by the method of example 1 and subcutaneous implantation, respectively, after being implanted in situ in a rat Achilles tendon defect for 3 months. It can be seen that the extracellular matrix of the nascent achilles tendon scaffold prepared in the embodiment 1 of the invention is denser, and the cell orientation ratio is high.

FIG. 4A, B shows the diameter of regenerated collagen fibers measured by transmission electron microscopy after the porous Achilles tendon extracellular matrix scaffold and the extracellular matrix scaffold prepared by the method of example 1 and subcutaneous implantation, respectively, were implanted in situ in rat Achilles tendon defect site for 3 months. It can be seen that the regenerated Achilles tendon scaffold prepared in example 1 of the present invention has a high collagen fiber density and a larger fiber diameter.

FIG. 5A, B shows the regenerated nerve after the porous channel nerve extracellular matrix scaffold and extracellular matrix scaffold prepared by the subcutaneous implantation method and example 2 of the present invention, respectively, were implanted in situ in the sciatic nerve defect of rat for 3 months. It can be seen that the new nerves after the in vivo implantation of the scaffolds prepared by the two processes are well integrated with the host.

FIG. 6A, B shows the new myelin sheaths of the rat sciatic nerve after the porous neural extracellular matrix scaffold and the extracellular matrix scaffold prepared by the subcutaneous implantation method and example 2 of the present invention, respectively, were implanted in situ for 3 months. It can be seen that the number and thickness of myelin sheaths of the new nerves of the neural extracellular matrix scaffold prepared in example 2 of the invention are superior to those of extracellular matrix scaffold materials prepared by previous subcutaneous implantation;

FIG. 7A, B is the composite muscle action potential map (CMAP) of the new nerve 3 months after the porous channel neural extracellular matrix scaffold and the extracellular matrix scaffold prepared by the subcutaneous implantation method and example 2 of the present invention, respectively, were implanted in situ into the sciatic nerve defect site of rat. It can be seen that the amplitude of CAMP stent prepared in example 2 of the invention is significantly higher than that of the stent prepared by subcutaneous implantation.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included therein.

Claims

1. An extracellular matrix scaffold material with a controllable pore structure is characterized in that a tubular or film-shaped polymer fiber scaffold with a pseudo-natural structure is used as a sacrificial template material, a cell-free matrix solution is poured among pores of the fiber template scaffold and then is crosslinked, and the fiber template material is washed and removed, so that the extracellular matrix scaffold with the controllable pore structure is obtained.

2. The extracellular matrix scaffold material with the controllable pore structure according to claim 1, wherein the tubular or film-shaped polymer fiber scaffold with the pseudo-natural structure is prepared by using biodegradable high molecules as raw materials and adopting electrostatic spinning, wet spinning, melt spinning or 3D printing technology.

3. An extracellular matrix scaffold material with a controllable pore channel structure according to claim 1, wherein the raw material is selected from one or more of synthetic polymer polylactic acid, polylactic acid-polyglycolic acid copolymer, polycaprolactone, polyhydroxyalkanoate, polyglycolic acid, polyurethane, poly-L-lactide-caprolactone and poly-p-dioxanone.

4. The extracellular matrix scaffold material with a controlled pore structure according to claim 1, wherein the acellular matrix solution is prepared by carrying out acellular treatment on different tissues of animals and then dissolving the acellular matrix solution with acetic acid.

5. An extracellular matrix scaffold material with a controlled pore structure according to claim 4, wherein the tissue source is brain, heart, liver, spleen, lung, kidney, muscle, skin, meninges, diaphragm, amnion, pericardium, heart valve, small intestine submucosa, muscle, blood vessel, tendon, ligament, cartilage, esophagus, stomach, nerve and bladder of an artificial farm animal.

6. A method for preparing an extracellular matrix scaffold material with a controlled pore structure according to any one of claims 1 to 5, comprising the following steps:

7. The method according to claim 6, wherein the crosslinking agent is a mixture of (1-ethyl- (3-dimethylaminopropyl) carbodiimide) and N-hydroxysuccinimide in a mass ratio of 4: 1.

8. The preparation method according to claim 6, wherein the freezing temperature is-20 ℃ to-100 ℃ for 12 to 48 hours, and the freeze-drying temperature is-10 ℃ to-100 ℃ for 24 to 72 hours.

9. Use of an extracellular matrix scaffold material with a controlled pore structure according to any one of claims 1 to 5 as a tissue repair material.