CN115337222A

CN115337222A - Hollow collagen structure body and preparation method and application thereof

Info

Publication number: CN115337222A
Application number: CN202210900831.9A
Authority: CN
Inventors: 赵琪; 王志伟; 陈雄伟
Original assignee: Imeik Technology Development Co ltd
Current assignee: Imeik Technology Development Co ltd
Priority date: 2022-07-28
Filing date: 2022-07-28
Publication date: 2022-11-15
Anticipated expiration: 2042-07-28
Also published as: CN115337222B

Abstract

The invention discloses a hollow collagen structural body and a preparation method and application thereof. The hollow collagen structure body with a specific shape is prepared by an electrostatic spinning method so as to meet different application requirements, the operation is simple, the structure is easy to control, particularly, silk threads are redissolved into viscous gel after humidification post-treatment, the compactness of the external structure of the hollow collagen structure body is improved, and the crosslinking post-treatment is carried out after redissolution, so that the crosslinking effectiveness can be improved. The hollow collagen structure prepared by the invention has the advantages of smooth surface structure, good stability, high strength, toughness and good biocompatibility, and has important clinical significance and application value.

Description

Hollow collagen structure body and preparation method and application thereof

Technical Field

The invention relates to the field of biomedical materials and medical instruments, in particular to a hollow collagen structure body and a preparation method and application thereof.

Background

Collagen is a fibrous, macromolecular protein secreted by connective tissue cells and other cell types in mammals (e.g., liver, lung, spleen, and brain tissue cells), and is a major component of bone and skin. Collagen has not only biomechanical effects but also functions such as signal transduction, transport of growth factors and cytokines; in addition, the compound has low cost, low immunogenicity, biodegradability, biocompatibility and large-scale production, and is widely applied to the industries of food, cosmetics, medicines and the like. Among them, collagen is widely used in the pharmaceutical industry in the fields of medical and beauty treatment, burns, wounds, dentistry, ophthalmology, tissue repair, etc., and is most commonly in the form of a film and a sponge. But the treatment or alleviation of certain diseases often requires biomedical materials with hollow structures, for example, peripheral vascular reconstruction in microsurgery such as arterial anastomosis by using small-diameter tissue-engineered vascular grafts; glaucoma caused by refractory ocular hypertension is treated by implanting a diversion tube; and the artificial lacrimal canaliculus is inserted by the combination of laser probing to treat diseases such as lacrimal duct obstruction and the like. Therefore, the preparation method of the hollow collagen structure body with simple and efficient preparation method and excellent mechanical strength is particularly important when the hollow collagen structure body is applied to millimeter-scale or even micron-scale catheters used in the processes of artificial blood vessel reconstruction with small caliber and treatment of ophthalmic diseases.

In the past, the method of die method, electrochemical deposition and the like are adopted to prepare the collagen material with a hollow structure, but the methods are limited in practical application because of the problems of difficulty in controlling and processing the fine structure, low production efficiency and the like.

Chinese patent CN111318237B discloses a collagen-chitosan hydrogel and a preparation method thereof, wherein the diameter and the shape of the hollow part of a gel fiber are controlled by changing the diameter and the cross section shape of a stainless steel needle by utilizing the principle of electrochemical deposition, but the hollow collagen-chitosan hydrogel prepared by the method has the defects of insufficiently compact structure, insufficiently smooth surface and uneven thickness.

JP3221690B2 discloses a technique for producing a collagen structure by molding a tubular or planar shape while concentrating a collagen solution, which is contacted with a concentrating agent such as polyethylene glycol via a permeable member, concentrated to a collagen concentration of 50 to 100mg/ml, and molded into a ring shape, thereby forming a collagen structure having a ring shape.

Li et al propose to pour a Collagen solution into a cylindrical mold 8mM in Diameter and 12mM in height, place 0.5 or 1mM Diameter bovine serum albumin coated glass or stainless steel in the center of the mold, prepare a Collagen tube structure of Diameter less than or equal to 1mM, and cross-link with 20mM genipin (Li X, xu J, nicolescu CT, marinelli JT, tien J. Generation, endothelialization, and microscopic surgery analysis of Strong 1-mM-Diameter Collagen tubes tissue end Part A.2017Apr;23 (7-8): 335-344.), but with a Collagen concentration of 6mg/ml, it takes 100 times more time to dehydrate to obtain a Collagen tube, but with a larger concentration factor, the Collagen tube Diameter and surface tend to be non-uniform and longer.

Chinese patent CN103046225B discloses a technology for preparing a nano collagen membrane by an electrostatic spinning technology, but the spun collagen membrane is not subjected to a cross-linking treatment, and the collagen membrane is prepared only by the electrostatic spinning technology, and a collagen structure body with a hollow structure is not prepared.

Disclosure of Invention

In order to solve the above problems, the present invention provides a novel method for preparing a hollow collagen structure using electrospinning, and a hollow collagen structure prepared by the method and use thereof.

In a first aspect of the present invention, there is provided a hollow collagen material, also referred to herein as a hollow collagen structure, comprising a hollow structure and an outer structure. The structure is produced from collagen by spinning (e.g., phase separation spinning, flash spinning, electrospinning, liquid crystal spinning or reaction spinning, in particular electrospinning), post-treatment (e.g. wet treatment, cross-linking treatment, etc.) (in particular by the method of preparation according to the second aspect of the invention). The structure can control the flow by changing the appearance of the hollow structure, and can adjust the adaptation degree with the application scene by changing the appearance of the external structure. The hollow collagen structure has the advantages of stable structure, good biocompatibility, high strength, good toughness and good leakage resistance.

Specifically, the collagen may be a native collagen derived from any aquatic or other animal containing collagen (e.g., cattle, pigs, fish and chicken); the collagen may be recombinant active collagen or a collagen derivative.

Specifically, the collagen includes, but is not limited to, type I, type III collagen.

Specifically, the collagen derivative refers to a product obtained by modifying the molecular structure of the collagen, and includes, but is not limited to, acylated collagen such as succinylated collagen, phthaloylated collagen, and maleylated collagen, or esterified collagen.

Specifically, the hollow structure may be adjusted according to actual requirements, and may be wholly or partially preferably a cylindrical structure, a conical structure, a polygonal prism structure (e.g., a quadrangular prism, a pentagonal prism, a hexagonal prism structure), a polygonal pyramid structure, or the like.

In some embodiments of the invention, the hollow structure is a cylindrical structure, the diameter of the cylinder being 40-4000 μm (e.g. 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000 μm), preferably 50-2000 μm.

In further embodiments of the present invention, the hollow structure is a polygonal prism structure, such as a quadrangular prism or a hexagonal prism structure, having a cross-sectional side length of 40 to 5000 μm (e.g., 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000 μm), preferably 200 to 4000 μm.

Specifically, the outer structure may be adjusted according to actual needs, and may be wholly or partially preferably a cylindrical structure, an annular structure, a conical structure, a polygonal prism structure (e.g., a quadrangular prism, a pentagonal prism, a hexagonal prism structure), a polygonal cone structure, a spherical structure, or the like.

In some embodiments of the invention, the outer structure is a cylindrical structure having a diameter of 50-8000 μm (50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000 μm), preferably 100-4000 μm.

In further embodiments of the invention, the outer structure is a multi-prismatic structure, e.g. a quadrangular prism, a hexagonal prism structure, having a cross-sectional side length of 50-8000 μm (50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000 μm), preferably 500-5000 μm.

In particular, the outer structure has an average thickness of 100-2000 μm (e.g. 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1200, 1400, 1500, 1600, 1800, 2000 μm), preferably 200-1200 μm.

The hollow collagen structure may be formed into a ring shape or other shapes as required.

In a second aspect of the present invention, there is provided a method for preparing a hollow collagen structure, comprising the steps of preparing a collagen spinning solution, spinning, and post-treating.

Specifically, the collagen spinning solution is a collagen solution prepared by dissolving collagen in a solvent.

Specifically, the concentration of collagen in the collagen spinning solution is 10 to 150mg/ml (e.g., 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150 mg/ml), preferably 20 to 100mg/ml. The concentration of collagen in the collagen spinning solution should not be too high or too low, otherwise too high or too low viscosity may result in failure to spin.

Specifically, the solvent in the collagen spinning solution is selected from: one or more of hexafluoroisopropanol, trifluoroethanol, and an aqueous solution of an organic acid such as acetic acid or citric acid, and an ionic liquid (e.g., 1-butyl-3-methylimidazolium chloride or 1-ethyl-3-methylimidazolium acetate); in some embodiments of the invention, the solvent is an aqueous organic acid solution having a concentration of 20-60wt% (e.g., 20wt%, 25wt%, 30wt%, 35wt%, 40wt%, 45wt%, 50wt%, 60 wt%), particularly 20-40wt%; in one embodiment of the invention, the solvent is a 20 to 60wt% acetic acid solution.

Specifically, the spinning is phase separation spinning, flash spinning, electrostatic spinning, liquid crystal spinning or reaction spinning, especially electrostatic spinning.

Specifically, the electrospinning step comprises: spinning the collagen spinning solution through an electrostatic spinning device, wherein the electrostatic spinning device comprises a rotating receiving device. The tubular collagen body obtained by electrostatic spinning is also called as a nascent collagen fiber tube.

In some embodiments of the invention, the electrospinning step comprises: transferring the collagen spinning solution into a spinning injector, placing the injector filled with the spinning solution on an electrostatic spinning machine after centrifugal deaeration, adjusting parameters of the electrostatic spinning machine (including voltage, injection speed, rotating speed of a roller of a receiving device, moving distance of a sliding table, scanning speed of the sliding table and the like), starting operation, and taking down the tubular body on the collecting device after spinning is finished.

In particular, the receiving means comprise a thin wire, which may be of any material, preferably a metal wire, such as a steel wire, a tungsten wire, a gold wire, a platinum wire or a copper wire.

Specifically, the structure of the receiving device can be adjusted according to actual requirements, and a cylindrical structure, a conical structure, a polygonal prism structure (such as a quadrangular prism, a pentagonal prism, and a hexagonal prism structure), a polygonal cone structure, and the like can be preferred in whole or part.

In some embodiments of the invention, the receiving means is a cylindrical structure having a diameter of 40-4000 μm (e.g. 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000 μm), preferably 50-2000 μm.

In some embodiments of the invention, the receiving means is a multi-prismatic structure, such as a quadrangular prism, a hexagonal prism structure, with a cross-sectional side length of 40-5000 μm (e.g. 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000 μm), preferably 200-4000 μm.

Specifically, in the electrospinning step, the voltage is 2 to 65kV (e.g., 2, 4, 5, 6, 8, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65 kV), preferably 4 to 30kV.

Specifically, in the electrospinning step, the receiving distance is 0.5 to 15cm (e.g., 0.5, 1,2,3,4, 5, 7.5, 10, 12.5, 15 cm), preferably 2 to 10cm.

Specifically, in the electrospinning step, the bolus rate is 0.1 to 3ml/h (e.g., 0.1, 0.15, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.75, 0.8, 0.9, 1, 1.5, 2, 2.5, 3 ml/h), preferably 0.1 to 1.5ml/h.

Specifically, in the electrospinning step, the rotating speed of the drum of the receiving device is 100-2000r/min (e.g., 100, 500, 1000, 1500, 2000 r/min), preferably 500-1500r/min.

Specifically, in the electrospinning step, the moving distance of the slide table is 20 to 300mm (e.g., 50 to 230mm, 50 to 200 mm), preferably 50 to 230mm.

Specifically, in the electrospinning step, the scanning speed of the slide table is 10 to 50mm/s (e.g., 10, 15, 20, 25, 30, 35, 40, 50 mm/s), preferably 15 to 25mm/s.

Specifically, the duration of electrospinning is 0.5 to 8 hours (e.g., 0.5, 1,2,3,4, 5, 6, 7, 8 hours), preferably 1 to 5 hours.

In a preferred embodiment of the present invention, the post-treatment comprises a humidification treatment step; specifically, the humidification treatment includes, but is not limited to, steam humidification, electrothermal humidification, electrode humidification, or high-pressure spray humidification; preferably, the humidification treatment is steam humidification, in particular water steam humidification.

Specifically, the temperature of the humidification treatment is 25 to 125 ℃ (e.g., 25, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 125 ℃), particularly 60 to 90 ℃.

Specifically, the humidification treatment time is 5 to 50min (e.g., 5, 10, 15, 20, 25, 30, 35, 40, 50, 60 min), particularly 10 to 30min.

In a preferred embodiment of the present invention, the post-treatment comprises a crosslinking treatment step; more specifically, the crosslinking treatment is treatment by a crosslinking agent, for example, soaking a material to be crosslinked (for example, a nascent collagen fiber tube subjected to humidification treatment) in a crosslinking agent solution.

Specifically, the crosslinking agent may be one or more of genipin, an aldehyde crosslinking agent (e.g., formaldehyde, glutaraldehyde), a carbodiimide-hydroxysuccinimide (e.g., EDC-NHS), dialdehyde starch, chitosan, transglutaminase, or an epoxide.

Specifically, the epoxide may be ethylene oxide, propylene oxide, 1,2-butylene oxide or 1,4-butylene oxide, or may be a diepoxide (e.g., 1,4-butanediol diglycidyl ether, 1,2,3,4, diepoxybutane) or a polyepoxide (up to 3 or more, such as glycerol tris (1,2-epoxy) propyl ether).

In some embodiments of the invention, the crosslinker is a combination of 1-ethyl- (3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS).

In other embodiments of the invention, the crosslinking agent is 1,4-butanediol diglycidyl ether.

In other embodiments of the present invention, the crosslinking agent is genipin.

In other embodiments of the present invention, the cross-linking agent is dialdehyde starch.

In other embodiments of the present invention, the crosslinking agent is glutaraldehyde.

In one embodiment of the present invention, the solution of the diepoxide as the crosslinking agent further contains a catalyst; specifically, the catalyst is selected from alkali metal hydroxide or alkali metal carbonate. Wherein the alkali metal is selected from the group consisting of lithium, sodium, potassium, rubidium, cesium, and francium. The alkali metal hydroxide is selected from the group consisting of lithium hydroxide, sodium hydroxide, potassium hydroxide, rubidium hydroxide, cesium hydroxide, and francium hydroxide. The carbonate may be a normal salt, an acid salt or a basic salt, such as sodium carbonate, potassium carbonate, zinc carbonate, calcium carbonate, magnesium carbonate, iron carbonate, copper carbonate, and the like.

In some embodiments of the invention, the catalyst is selected from sodium hydroxide, potassium hydroxide, or sodium carbonate.

Specifically, the concentration of the crosslinker solution is 1-500mM (e.g., 1, 10, 20, 40, 50, 100, 200, 300, 400, 500 mM), preferably 10-200mM.

Specifically, the soaking time of the material to be crosslinked in the crosslinking agent solution is 2-48h (e.g., 2, 4, 6, 8, 10, 12, 18, 24, 30, 36, 42, 48 h), preferably 4-24h.

Specifically, the amount of the crosslinking agent may be 0.01 to 2.5 times (e.g., 0.01, 0.02, 0.04, 0.05, 0.06, 0.08, 0.1, 0.2, 0.4, 0.5, 0.6, 0.8, 1, 1.2, 1.4, 1.5, 1.6, 1.8, 2, 2.2, 2.4, 2.5 times), particularly 0.2 to 2.25 times, the mass of the collagen material (i.e., the mass of collagen in the collagen spinning solution used).

Preferably, after post-treatment, the obtained hollow collagen structure has a smooth, compact and stable surface structure, high strength, good toughness and good leakage resistance.

In a more preferred embodiment of the invention, the post-treatment comprises the following steps carried out in sequence: humidifying and crosslinking; specifically, the steps are as described above.

In particular, the post-treatment may also comprise a washing step; more specifically, the washing solvent may be a combination of one or more of alcohols (such as ethanol), physiological saline, water, and the like; more specifically, the number of washing may be one or more; in some embodiments of the present invention, the washing step is a plurality of washes with an ethanol solution, physiological saline.

In particular, the post-treatment may also comprise a demoulding step.

In particular, the post-treatment may also comprise a drying step; more specifically, the drying includes, but is not limited to, lyophilization, vacuum drying; in some embodiments of the invention, the drying is vacuum drying, the drying temperature is 4-37 ℃ (e.g., 4, 6, 8, 10, 15, 20, 25, 26, 30, 35, 37 ℃), preferably 20-26 ℃, and the drying time is 4-48h (e.g., 4, 6, 8, 10, 12, 18, 24, 30, 36, 42, 48 h), preferably 8-24h.

In particular, the post-treatment may also comprise a sterilization step, such as radiation sterilization.

In some embodiments of the invention, the post-processing comprises the following steps performed in sequence: humidifying, cross-linking, washing, demolding, drying and sterilizing; specifically, the steps are as described above.

In a third aspect of the invention, there is provided an implant comprising the hollow collagen structure of the first aspect or the hollow collagen structure prepared by the method of the second aspect.

Specifically, the implant includes an external structure in addition to the hollow structure of the hollow collagen structure, and the external structure may be shaped according to the implantation site in order to reduce exfoliation and the like.

In particular, the implant may be a cosmetic implant, but also an implant for the treatment of diseases.

In particular, the cosmetic implants include, but are not limited to, nasal implants, ocular implants, contact lenses, subcutaneous implants (e.g., facial or neck injections may reduce smoothing wrinkles).

In particular, the disease treatment implants include, but are not limited to, ocular implants (e.g., drainage tubes, artificial tear ducts), cardiac implants (e.g., heart valves), oral shields, denture padding, tissue substitutes, ureteral prostheses, tendon and ligament substitutes, bandages, sutures, vascular implants (e.g., vascular prostheses), orthopedic plates or staples, prosthetic joints, or staplers. Such as skin staplers, circular staplers for the alimentary tract (esophagus, stomach and intestine, etc.), rectal staplers, circular hemorrhoid staplers, circumcision staplers, vascular staplers, hernia staplers, lung cutting staplers, etc.

In a fourth aspect of the present invention, there is provided a cell culture material comprising the hollow collagen structure according to the first aspect or the hollow collagen structure produced by the method according to the second aspect.

Specifically, the cell culture material has a three-dimensional structure, so that cells can grow, proliferate and migrate in a three-dimensional space, the growth environment of the cells in vivo can be better simulated, and the cell culture material can be used for disease pathology, pharmacological research, three-dimensional culture of in vitro organoids, in vivo transplantation and the like.

Specifically, the cells may be, for example, tumor cells (e.g., for studying invasion and migration of tumor cells, formation and culture of tumor spheroids (sphenoids), pharmacogenomics, pharmacodynamic studies, etc.), stem cells (e.g., induced pluripotent stem cells, embryonic stem cells, adult stem cells, immortalized cell lines, primary cell lines, etc.), neural cells (e.g., for nerve cell culture and transplantation), and the like.

In a fifth aspect of the invention, there is provided the use of a hollow collagen structure according to the first aspect or a hollow collagen structure produced by the method according to the second aspect in the manufacture of a product for the treatment of disease and a cell culture material.

In particular, the disease treatment products include, but are not limited to, ocular implants (e.g., drainage tubes, artificial tear ducts), cardiac implants (e.g., heart valves), oral shields, denture padding, tissue substitutes, ureteral prostheses, tendon and ligament substitutes, bandages, sutures, vascular implants (e.g., vascular prostheses), orthopedic plates or staples, prosthetic joints, or staplers. Such as skin staplers, circular staplers for the alimentary tract (esophagus, stomach and intestine, etc.), rectal staplers, circular hemorrhoid staplers, circumcision staplers, vascular staplers, hernia staplers, lung cutting staplers, etc.

In a sixth aspect of the present invention, there is provided a use of the hollow collagen structure according to the first aspect or the hollow collagen structure obtained by the method according to the second aspect in the preparation of a cosmetic or health product.

In particular, the cosmetic products include, but are not limited to, nasal implants, ocular implants, contact lenses, subcutaneous implants (e.g., facial or neck injections may reduce smoothing wrinkles).

The invention has the outstanding characteristics that the hollow collagen structure body with a specific shape is prepared by an electrostatic spinning method so as to meet the requirements of different applications, the operation is simple, the structure is easy to control, particularly, the silk threads are redissolved into viscous gel after humidification post-treatment, the compactness of the external structure of the hollow collagen structure body is improved, the effectiveness of crosslinking can be improved by crosslinking post-treatment after redissolution, compared with direct crosslinking after spinning, the structure of the invention is more compact, the crosslinking degree is higher, the strength is higher, and the complete appearance of the hollow collagen structure body can be kept. The hollow collagen structure prepared by the invention has the advantages of smooth surface structure, good stability, high strength, toughness and good biocompatibility, and has important clinical significance and application value.

Drawings

Fig. 1 is a microphotograph showing a hollow collagen catheter obtained in example 1.

FIG. 2 is a diagram showing the external appearance of the catheter prepared in example 1 observed by a Field Emission Scanning Electron Microscope (FESEM).

FIG. 3 is a graph showing the external appearance of the catheter prepared in comparative example 2, as observed by a Field Emission Scanning Electron Microscope (FESEM).

Fig. 4 shows the results of the examination of the morphology of the conduit after rehydration for the samples obtained in example 1 and comparative example 2.

FIG. 5 is a graph showing in vitro degradation curves of hollow collagen tubes prepared according to examples of the present invention and comparative examples.

Detailed Description

Unless defined otherwise, all scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

An "implant" according to the present invention is an implantable article that is placed in a body cavity created by a surgical procedure or otherwise physiologically present and remains for a period of time. It can be adjusted in shape, length, thickness, etc. according to the type of disease or the shape of body cavity caused by surgical operation.

The cross-linking of the invention is the process of forming the net-shaped or body-shaped macromolecule by covalent bond connection among the linear or branch-shaped macromolecule chains, and comprises chemical cross-linking and physical cross-linking.

The disclosures of the various publications, patents, and published patent specifications cited herein are hereby incorporated by reference in their entirety.

The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments of the present invention, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without inventive step based on the embodiments of the present invention, are within the scope of protection of the present invention.

The experimental methods in the examples, in which specific conditions are not specified, are generally performed under the conditions described in the manual and the conventional conditions, or under the conditions recommended by the manufacturer; the materials, reagents and the like used are commercially available unless otherwise specified.

Example 1

Weighing 0.5g of I type collagen sponge extracted from pigskin, adding 5ml glacial acetic acid and 5ml purified water in sequence, stirring until completely dissolving, transferring the solution into an injector, placing the injector filled with spinning solution on an electrostatic spinning machine after centrifugal deaeration, and adjusting parameters of the electrostatic spinning machine: spinning positive voltage: 4.0KV; spinning negative voltage: 4.0KV; the bolus injection speed: 0.15ml/h; distance of nozzle from collector: 5cm; moving distance of the sliding table: 50-230 mm; filament receiver drum speed 1000r/min, cylindrical filament diameter: 60 mu m, the scanning speed of a sliding table is 20mM/s, the instrument is closed after 1h of operation, the silk thread on a rotor is taken down, the silk thread is humidified by steam at 80 ℃ for 10min, the silk thread is transferred into 200mM EDC solution (prepared by adding 0.620g EDC and 0.124g NHS into 20ml of purified water), the silk thread is soaked for 12h, then the silk thread is washed by ethanol solution and normal saline for a plurality of times, the obtained conduit is taken down from the silk thread, the conduit is placed in a vacuum drying box, the temperature is set to be 26 ℃, the drying is carried out for 24h, the hollow collagen tube is obtained after 25kGy radiation sterilization after packaging, the average inner diameter of the conduit is 58-60 mu m, and the average outer diameter is 360-370 mu m. The specific topography of the catheter is shown in fig. 1.

Example 2

Weighing 0.4g of I type collagen sponge extracted from bovine achilles tendon tissue, adding 4ml of hexafluoroisopropanol, stirring until the hexafluoroisopropanol is completely dissolved, transferring the solution into an injector, placing the injector filled with spinning solution on an electrostatic spinning machine after centrifugal deaeration, and adjusting parameters of the electrostatic spinning machine: spinning positive voltage: 10.0KV; spinning negative voltage: 10.0KV; the bolus injection speed: 0.75ml/h; distance of nozzle from collector: 10cm; moving distance of the sliding table: 50-230 mm; filament receiver drum speed 1500r/min, cylindrical filament diameter: 2000 mu m, the scanning speed of a sliding table is 25mM/s, the instrument is closed after the operation is carried out for 3h, the silk thread on the rotor is taken down, the silk thread is humidified by steam at 60 ℃ for 30min, the silk thread is transferred into 1mM 1,4-butanediol diglycidyl ether solution (BDDE) (prepared by adding 10.1mg of BDDE into 50ml of 15wt.% sodium hydroxide solution) to be soaked for 2h, the silk thread is washed by ethanol solution and normal saline for a plurality of times, the obtained conduit is taken down from the silk thread and placed in a vacuum drying box, the temperature is set to be 20 ℃, the drying is carried out for 24h, and the hollow collagen tube is obtained after the packaging by 25kGy radiation sterilization, wherein the average inner diameter of the conduit is 1990-2000 mu m, and the average outer diameter is 3000-3100 mu m.

Example 3

Weighing 0.5g of recombinant human-derived type III collagen sponge, adding 40wt% of citric acid solution to prepare 20mg/ml collagen spinning solution, stirring until the solution is completely dissolved, transferring the solution into an injector, placing the injector filled with the spinning solution on an electrostatic spinning machine after centrifugal deaeration, and adjusting parameters of the electrostatic spinning machine: spinning positive voltage: 30.0KV; spinning negative voltage: 30.0KV; the bolus injection speed: 1.5ml/h; distance of nozzle from collector: 15cm; moving distance of the sliding table: 50-200 mm; the rotating speed of a roller of the filament receiver is 500r/min, and the side length of the cross section of the quadrangular filament is as follows: 500 mu m, the scanning speed of a sliding table is 20mm/s, the instrument is closed after 5 hours of operation, the silk thread on a rotor is taken down, the silk thread is humidified by steam at 90 ℃ for 5 minutes, the silk thread is transferred to 100ml of 50mM genipin solution (obtained by weighing 1.125g of genipin and dissolving the genipin in 100ml of water at 37 ℃) and soaked for 24 hours, the silk thread is washed by ethanol solution and normal saline for a plurality of times, the obtained conduit is taken down from the silk thread and placed in a vacuum drying box, the temperature is set to be 30 ℃, the drying is carried out for 24 hours, the hollow collagen tube is obtained after 25kGy side length radiation sterilization after packaging, the hollow structure of the conduit is a hollow quadrangular prism, the cross section is 490 to 500 mu m, the external structure is a quadrangular prism, and the average side length is 1500 to 1600 mu m.

Example 4

Weighing 1.0g of succinylated collagen, adding 40wt% of acetic acid solution to prepare 150mg/ml collagen spinning solution, stirring until the solution is completely dissolved, transferring the solution into an injector, placing the injector filled with the spinning solution on an electrostatic spinning machine after centrifugal deaeration, and adjusting parameters of the electrostatic spinning machine: spinning positive voltage: 15.0KV; spinning negative voltage: 15.0KV; the bolus injection speed: 0.1ml/h; distance of nozzle from collector: 2cm; moving distance of the sliding table: 50-230 mm; filament receiver drum speed 2000r/min, cylindrical filament diameter: 100 mu m, the scanning speed of a sliding table is 25mm/s, the instrument is closed after 8 hours of operation, the silk thread on a rotor is taken down, the silk thread is humidified by steam at 60 ℃ for 30 minutes, the silk thread is transferred into 50ml of 10mM dialdehyde starch solution (obtained by weighing 0.17g of dialdehyde starch and adding the dialdehyde starch solution into 50ml of water for dissolving), after 48 hours of soaking, the silk thread is washed by ethanol solution and normal saline for multiple times, the obtained conduit is taken down from the silk thread, the conduit is placed in a vacuum drying box, the temperature is set at 25 ℃, the drying is carried out for 24 hours, after the packaging, 25kGy radiation sterilization is carried out, and the hollow collagen tube is obtained, the average inner diameter of the conduit is 90-100 mu m, and the average outer diameter is 400-500 mu m.

Example 5

Weighing 0.5g of esterified collagen, adding trifluoroethanol to prepare 1mg/ml collagen spinning solution, stirring until the esterified collagen spinning solution is completely dissolved, transferring the solution into an injector, placing the injector filled with the spinning solution on an electrostatic spinning machine after centrifugal deaeration, and adjusting parameters of the electrostatic spinning machine: spinning positive voltage: 5.0KV; spinning negative voltage: 5.0KV; the bolus injection speed: 0.30ml/h; distance of nozzle from collector: 7.5cm; moving distance of the sliding table: 50-200 mm; the rotating speed of a roller of the filament receiver is 100r/min, and the side length of the customized hexagonal stainless steel filament is as follows: 4000 mu m, the scanning speed of a sliding table is 25mm/s, the instrument is closed after the operation is carried out for 0.5h, the silk thread on the rotor is taken down, the humidification is carried out for 15min by 70 ℃ steam, the silk thread is transferred into 50ml of 20mM glutaraldehyde solution (0.10 g of glutaraldehyde is weighed and is added into 50ml of water to obtain the silk thread, the silk thread is soaked for 4h, then the silk thread is washed by ethanol solution and normal saline for many times, the obtained conduit is taken down from the silk thread, the conduit is placed in a vacuum drying box, the temperature is set to be 25 ℃, the drying is carried out for 24h, the hollow collagen tube is obtained after 25kGy radiation sterilization after packaging, the side length of the hexagonal prism hollow structure of the conduit is 3990-4000 mu m, and the average side length of the outside of the conduit is 4400-4500 mu m.

Comparative example 1

Weighing 0.5g of I type collagen sponge extracted from pigskin, adding 6ml of glacial acetic acid and 54ml of purified water in sequence, stirring until the mixture is completely dissolved, transferring the solution into an injector, placing the injector filled with spinning solution on an electrostatic spinning machine after centrifugal deaeration, and adjusting parameters of the electrostatic spinning machine: spinning positive voltage: 4.0KV; spinning negative voltage: 4.0KV; the bolus injection speed: 0.15ml/h; distance of nozzle from collector: 5cm; moving distance of the sliding table: 50-230 mm; filament receiver drum speed 1000r/min, cylindrical filament diameter: 60 mu m, the scanning speed of a sliding table is 20mm/s, and the spinning solution is found to have low viscosity and drop in the form of liquid drops in the running process, so that the spinning can not be carried out.

Comparative example 2

The process steps are the same as in example 1, and only after spinning is finished, the process step of humidifying with steam at 80 ℃ for 10min is not carried out. The average inner diameter of the conduit is 58 to 60 μm, and the average outer diameter is 400 to 420 μm.

Comparative example 3

The process steps are the same as example 1, after spinning, the silk thread is transferred to 200mM EDC solution (prepared by adding 0.620g EDC and 0.124g NHS into 20ml purified water) and soaked for 12h, then washed by ethanol solution and normal saline for many times without carrying out the process steps of humidifying for 10min by steam at 80 ℃, the catheter is directly taken down from the silk thread, the process steps of drying and sterilizing adopt the operation method of example 1, and the average inner diameter of the prepared catheter is 58-60 mu m, and the average inner diameter of the outer diameter is 420-430 mu m.

Density detection

The surface morphology of the hollow collagen structures prepared in example 1 and comparative example 2 was observed by FESEM, and is shown in fig. 2 to 3, respectively. As can be seen from fig. 3, the surface structure of the hollow collagen structure body without steam humidification is a multi-fiber overlapping porous structure, and the gaps among the silk threads are large, because the spun silk threads are difficult to dissolve in water, and are only generated in the nano silk threads during soaking and crosslinking, and the silk threads are difficult to crosslink with each other; and fig. 2 shows that the surface structure of the hollow collagen structure body after steam humidification is changed from a multi-fiber overlapped porous structure into a non-gap and non-fiber filamentous integral uniform form, the surface is smooth, the fact that the silk threads can be redissolved in a solvent system through steam humidification post-treatment is shown, the surface of a high-density layer is formed, the void ratio is far lower than that (or the void ratio) of the implant silk threads without steam humidification post-treatment, and the tensile strength of the structure body and the stability of the structure are favorably improved.

Detection of tensile Strength Properties

The samples obtained in examples 1 to 5 and comparative examples 2 to 3 were immersed in physiological saline at a set temperature of 37 ℃ for 5min, taken out, and the surface free water was removed, and the tensile strength was measured by a UTM6202 electronic universal tensile testing machine from shenzhen, mitsubishi, shrinktech, ltd. The results of the sample measurements are shown in table 1:

table 1 tensile Strength test results of samples

Sample (I)	Tensile Strength (MPa)
		Example 1	15
Example 2	13
		Example 3	17
Example 4	16
		Example 5	13
Comparative example 1	Sample could not be spun
		Comparative example 2	9
Comparative example 3	6

As can be seen from the results of the tensile strength measurements in table 1, the tensile strength of the crosslinked catheters (i.e., catheters obtained in examples 1 to 5) is greater than that of the catheters not subjected to steam humidification treatment (i.e., catheters obtained in comparative example 2), because steam humidification allows the non-crosslinked nanofibers to be redissolved under the action of water vapor, which is beneficial for the subsequent crosslinking reaction and increases the crosslinking degree of the whole structure, compared with the method in which the spun nanofibers are directly soaked for crosslinking, the structure of the present invention is denser and the crosslinking degree is higher. The above results demonstrate that post-humidification crosslinking in the present invention can improve the mechanical strength of the catheter, particularly after rehydrating the tube for wetting, the crosslinked polymer gel after humidification has a higher tensile strength than without steam humidification.

Catheter morphology detection after rehydration

The samples obtained in example 1 and comparative example 2 were completely immersed in purified water in an appropriate length, immersed for 60min at room temperature, and the change in morphology was observed and recorded at 5min and 60min, and the results are shown in fig. 4.

As can be seen from fig. 4, the sample obtained in example 1 can still maintain the original shape after being soaked for 60min, and is a cylinder with a smooth surface (as shown in fig. 2), but the sample obtained in comparative example 2 has obvious swelling under the same soaking time, the size of the catheter is increased, the shape is seriously bent and deformed, the stability of the structure cannot be maintained, and the surface is looser. Therefore, the hollow collagen structure prepared by the method has a smooth surface structure and is more stable after rehydration.

Stability test

0.1g of each of the samples of examples 1 to 5 and comparative examples 2 to 3 was weighed, precisely weighed, and the mass (m) of each sample was recorded ₁ ) Completely immersing the weighed sample in 10% acetic acid solution (v/v), soaking for 30min, taking out and drying to constant weight, and recording the dried mass (m) ₂ ) The loss rate of the sample = (m) was calculated ₁ -m ₂ )/m ₁ X 100%, the results are shown in table 2 below:

TABLE 2 results of the measurement of the loss rate of the sample

Sample name	Loss ratio (%)
		Example 1	1.3
Example 2	0.7
		Example 3	0.9
Example 4	1.1
		Example 5	0.8
Comparative example 2	33.2
		Comparative example 3	99.3

As can be seen from Table 2, the samples of examples 1 to 5 all have lower dissolution amounts in the acetic acid solution than the samples of comparative examples 2 to 3, wherein the loss rate of the sample of comparative example 2 is lower than that of comparative example 3 because the sample of comparative example 2 is subjected to the soaking crosslinking reaction and the sample subjected to chemical crosslinking is not dissolved in the acetic acid solution, so that the loss rate is lower; however, the loss rate of the sample of comparative example 2 is much higher than that of example 1, because the steam humidification treatment is performed before soaking and crosslinking in example 1, the tube wall structure is more compact, the probability of intermolecular crosslinking reaction is increased, and the possibility of dissolution in the soaking process is reduced. Therefore, the hollow collagen structure prepared by the present invention has a high degree of crosslinking, and can improve the stability of the catheter.

In vitro cytotoxic assay (MTT method)

Experimental groups: the hollow collagen tube prepared in examples 1 to 5 was cut into 12 mm-long pieces, 2g was weighed, 10ml of a culture medium (45 ml of DMEM (manufacturer: gibco, lot number: 2360346) culture solution, 5ml of newborn bovine serum (manufacturer: gibco, lot number: 2396297P) was mixed) was added and extracted at 37 ℃ for 24 hours, and the extract was collected and used.

At about 1X 10 ⁴ Cells plated in 96-well plates at 37 ℃ and 5% CO ₂ (humidity > 90%) for 24h to form a semi-confluent monolayer, aspirating the supernatant, adding 100ul of the experimental sample, adding 100ul of medium to the blank control, adding 100ul of incomplete medium to the negative control (mixing 50ml of DMEM medium with 10ml of 0.9% sodium chloride injection), and continuing the incubation for 24h. The 96-well plate was removed, the supernatant was aspirated, 50. Mu.l of MTT (0.50 g of thiazole blue (manufacturer: aladdin, lot: B1505012) was weighed out and added to 100ml of sterilized PBS to dissolve and filter-sterilize the solution for further use) was added to each well, and the mixture was left to stand in the dark at 37 ℃ and 5% CO ₂ After incubation in an incubator for 2H, the cells were removed, the supernatant was aspirated, 150. Mu.l DMSO (manufactured by Aladdin, lot: H2120207) was added to each well, the 96-well plate was placed in a decolorization shaker in the dark at 60r/min, and 5min later, the absorbance at 570nm and 630nm was measured in a microplate reader. According to cytotoxicity = (Experimental group OD) ₅₇₀ -OD ₆₃₀ ) /(blank group OD ₅₇₀ -OD ₆₃₀ ) X 100%, the test results are shown in Table 3.

TABLE 3 cytotoxicity assay results for samples

	Example 1	Example 2	Example 3	Example 4	Example 5
						Cytotoxicity	98％	89％	100％	95％	87％

As can be seen from the table 2, according to the judgment of the cytotoxicity grade, the interval of the relative proliferation rate of 80% -99% is grade 1 cytotoxicity, and the relative proliferation rate is more than or equal to 100, is grade 0 cytotoxicity, which indicates that the hollow collagen tube prepared by the invention is not more than grade 1 and has good biocompatibility.

In vitro degradation assay

Bacterial collagenases from Clostridium histolyticum were used for the in vitro degradation of hollow collagen structures. The samples obtained in examples 1 to 5 and comparative examples 2 to 3 were weighed, respectively, recorded for initial mass, immersed in 0.05M Tris buffer pH7.4, having a collagenase concentration of 40U/ml, and placed in a 37 ℃ incubator. After a defined time interval, the samples were removed, rinsed with distilled water, dried and weighed. The degree of in vitro degradation was calculated as the percentage of the weight of the dried gel before and after collagenase treatment. The results of the sample testing are shown in FIG. 5.

As can be seen from FIG. 5, the degradation curve of the catheter in example 1 in the in vitro degradation experiment is relatively flat, the residual mass percentage of the catheter after 10h in vitro degradation is about 81%, and the residual mass percentage of the catheter after 24h in vitro degradation can still be maintained at 78%, and has no obvious downward trend. The catheter of the comparative example 2 has low crosslinking degree and larger gaps on the surface due to no steam humidification process, so that the contact area of collagen and enzyme liquid is increased, the residual quantity of the catheter is obviously reduced, the degradation is fast, and the residual mass percent of the catheter is lower than 10 percent after the catheter is degraded in vitro for 6 hours. The catheter of comparative example 3 was not subjected to steam humidification and cross-linking treatment, and after only 2h of in vitro degradation, the residual amount of the catheter was very low, with the worst stability.

It can be seen from the degradation curves of examples 1 to 5 that samples prepared by selecting different cross-linking agents and cross-linking reaction conditions have different degradation times, for example, the sample of example 3 is soaked in 500mM genipin for cross-linking for 24h, and in an in vitro degradation test, the residual mass percentage still reaches 85% after 24h degradation, and the cross-linking degree is high. Therefore, the hollow collagen structure prepared by the method of the present invention has better stability, and the degradation degree can be realized by selecting different cross-linking agents and cross-linking processes according to clinical use requirements.

In conclusion, the hollow collagen structure body with a specific shape is prepared by the electrostatic spinning method, the operation is simple, the structure is easy to control, and the prepared hollow collagen structure body is high in strength, high in toughness, good in stability and good in biocompatibility.

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 the like that are within the spirit and principle of the present invention are included in the present invention.

The foregoing embodiments and methods described in this disclosure may vary based on the abilities, experience, and preferences of those skilled in the art.

The mere order in which the steps of a method are listed in the present invention does not constitute any limitation on the order of the steps of the method.

Claims

1. A hollow collagen structural body comprises a hollow structure and an external structure, and is characterized in that the hollow collagen structural body is prepared from collagen through spinning and post-treatment; the post-treatment comprises a humidification treatment.

2. The hollow collagen structure according to claim 1, wherein the humidification treatment is steam humidification, electrothermal humidification, electrode humidification, or high-pressure spray humidification, preferably steam humidification, more preferably water vapor humidification.

3. A hollow collagen structure according to claim 2, wherein the temperature of the humidification treatment is 25-125 ℃, preferably 60-90 ℃; and/or the presence of a gas in the gas,

the humidifying treatment time is 5-50min, preferably 10-30min.

4. The hollow collagen structure according to claim 2, wherein the post-treatment further comprises a cross-linking treatment after the humidifying treatment;

preferably, the crosslinking treatment is treatment by a crosslinking agent;

more preferably, the cross-linking agent is selected from: one or more of genipin, an aldehyde crosslinker, carbodiimide-hydroxysuccinimide, dialdehyde starch, chitosan, transglutaminase, or epoxide;

further preferably, the aldehyde crosslinker is formaldehyde or glutaraldehyde, the carbodiimide-hydroxysuccinimide is EDC-NHS, and the epoxide is selected from: ethylene oxide, propylene oxide, 1,2-butylene oxide, 1,4-butylene oxide, 1,4-butanediol diglycidyl ether, 1,2,3,4, butylene oxide, glycerol tris (1,2-epoxy) propyl ether.

5. The hollow collagen structure according to claim 1, wherein the hollow structure is a cylindrical structure, a conical structure, a polygonal pyramid structure or a polygonal pyramid structure in whole or in part; and/or the presence of a gas in the gas,

the external structure is wholly or partially a cylindrical structure, a ring texture structure, a conical structure, a polygonal prism structure, a polygonal cone structure or a spherical structure;

preferably, the hollow structure is a cylindrical structure, and the diameter of the cylinder is 40-4000 μm;

preferably, the outer structure is a cylindrical structure, the diameter of the cylinder being 50-8000 μm.

6. A hollow collagen structure according to any one of claims 1 to 5, wherein said collagen is type I or type III collagen;

the collagen is natural collagen, recombinant active collagen or a collagen derivative;

preferably, the collagen derivative is an acylated collagen or an esterified collagen;

more preferably, the acylated collagen is succinylated collagen, phthalated collagen or maleylated collagen.

7. A preparation method of a hollow collagen structure body comprises the steps of preparation of a collagen spinning solution, spinning and post-treatment, wherein the post-treatment comprises humidification treatment;

preferably, the humidification treatment is steam humidification, electrothermal humidification, electrode humidification or high-pressure spray humidification, preferably steam humidification, more preferably water vapor humidification.

8. The method of claim 7, wherein the temperature of the humidification treatment is 25 to 125 ℃, preferably 60 to 90 ℃; and/or the presence of a gas in the atmosphere,

the humidifying treatment time is 5-50min, preferably 10-30min.

9. The method according to claim 7, wherein the post-treatment further comprises a crosslinking treatment after the humidifying treatment;

preferably, the crosslinking treatment is to soak the material to be crosslinked in a crosslinking agent solution;

further preferably, the aldehyde crosslinker is formaldehyde or glutaraldehyde, the carbodiimide-hydroxysuccinimide is EDC-NHS, and the epoxide is selected from: ethylene oxide, propylene oxide, 1,2-butylene oxide, 1,4-butylene oxide, 1,4-butanediol diglycidyl ether, 1,2,3,4, butylene dioxide, glycerol tris (1,2-epoxy) propyl ether.

10. The method according to claim 9, wherein the amount of the crosslinking agent is 0.01 to 2.5 times, preferably 0.2 to 2.25 times, the mass of the collagen raw material; and/or the soaking time is 2-48h.

11. The preparation method according to any one of claims 7 to 10, wherein the concentration of collagen in the collagen spinning solution is 10 to 150mg/ml, preferably 20 to 100mg/ml.

12. The method of any one of claims 7 to 10, wherein the solvent in the collagen spinning solution is selected from the group consisting of: one or more of hexafluoroisopropanol, trifluoroethanol, an organic acid aqueous solution and an ionic liquid;

preferably, the organic acid is acetic acid or citric acid;

preferably, the solvent is a 20-60wt% aqueous solution of an organic acid;

more preferably, the solvent is a 20-60wt% acetic acid solution.

13. The method of any one of claims 7 to 10, wherein the spinning is phase separation spinning, flash spinning, electrospinning, liquid crystal spinning, or reaction spinning, preferably electrospinning.

14. The method of claim 7, wherein the post-treating further comprises: one or more steps of washing, demolding, drying and sterilizing.

15. An implant or a cell culture material comprising the hollow collagen structure according to any one of claims 1 to 6 or the hollow collagen structure produced by the production method according to any one of claims 7 to 14.

16. Use of the hollow collagen structure according to any one of claims 1 to 6 or the hollow collagen structure obtained by the production method according to any one of claims 7 to 14 for the production of a product for treating diseases, a cell culture material, a cosmetic product or a health product;

preferably, the cosmetic product is selected from nasal implants, ocular implants, contact lenses, subcutaneous implants;

preferably, the disease treatment product is selected from an ocular implant, an oral shield, a denture padding, a tissue replacement, a ureteral prosthesis, a tendon and ligament replacement, a bandage, a suture, a cardiac implant or a vascular implant.