CN112295016A

CN112295016A - Collagen board layer matrix material and preparation method and application thereof

Info

Publication number: CN112295016A
Application number: CN202011094786.XA
Authority: CN
Inventors: 蒋波; 冯欢欢; 尹越; 王宁馨; 郭纪彤
Original assignee: Sichuan University
Current assignee: Sichuan University
Priority date: 2020-09-03
Filing date: 2020-10-14
Publication date: 2021-02-02

Abstract

The invention discloses a collagen plate substrate material and a preparation method and application thereof. The prepared collagen lamellar matrix material has a component structure similar to that of a natural cornea, has excellent optical characteristics and mechanical properties, and good cell compatibility, has great significance for reconstructing the cornea in vitro, and is expected to become one of ideal choices of cornea substitutes.

Description

Collagen board layer matrix material and preparation method and application thereof

Technical Field

The invention belongs to the technical field of biological materials, tissue engineering, artificial organs and medical instruments, and particularly relates to a collagen plate substrate material and a preparation method and application thereof.

Background

The cornea is located at the front end of the eyeball and is a protective window of the eye, which protects delicate intraocular tissues by withstanding intraocular pressure and preventing damage from external factors. The cornea is a highly structured transparent tissue whose edges are connected to an opaque sclera composed of densely interlaced fibers. The cornea is the main refractive organ in the eyeball, has the functions of light transmission and refraction, and plays an important role in forming vision. The diameter of a human cornea is about 12mm, and the radius of a central anterior plane is about 7.8 mm; the thickness of the cornea varies in each part, about 520 μm in the center and about 650 μm in the periphery. The cornea is divided into 5 layers, the outermost layer is an epithelial cell layer which is composed of 5-6 layers of epithelial cells and mainly plays a role in protection; bowman's layer is a layer of transparent tissue, evolved from the surface layer of the corneal stroma, inseparable from the stromal layer; the stroma layer occupies 90% of the thickness of the cornea, about 400-500 μm, and is a lamellar structure formed by stacking and arranging sheets with collagen fibers arranged in parallel in different directions, the stroma of the human cornea is about composed of 200 sheets, and the thickness of each sheet is about 0.2-2.5 μm; the rear part of the matrix is provided with a Descemet layer, cells are not contained in the Descemet layer, the Descemet layer is formed by the secretion of endothelial cells and can be easily separated from the matrix; the endothelial layer is composed of a monolayer of cells, has an active liquid pumping function, and is an important barrier for maintaining the transparency of the cornea.

Corneal lesions and corneal damage can cause corneal opacities to severely affect a patient's vision and even cause blindness. The corneal disease has high prevalence rate and strong blindness causing property, and is the second blindness causing eye disease in the world. Statistically, about 5500 tens of thousands of patients suffer eye trauma each year, of which 160 thousands are blind, and up to 92.8% of those suffering eye trauma require the rescue of residual vision through corneal transplant surgery. The endothelial cells of the cornea cannot be automatically repaired or replaced, so that patients with corneal diseases traditionally replace the damaged cornea by transplanting a donated healthy cornea, which results in great demand for cornea banks. In many areas, corneal donors are extremely in shortage, the operation cost is high, a large number of patients cannot be treated in time, or the operation fails due to complications such as immunological rejection reaction and corneal endothelium decompensation after the operation. Therefore, finding an ideal corneal substitute to help corneal patients restore vision has been the focus of research in the ophthalmology community.

With the continuous development of biomaterials, cell biology and molecular biology, tissue engineering technology is rapidly developed, and the application of tissue engineering technology to reconstruct tissue engineering artificial cornea in vitro as a cornea substitute becomes a research hotspot in recent years. The carrier scaffold materials for constructing the tissue engineering artificial cornea at present mainly comprise heterogenous acellular corneas, natural biological materials (including collagen, gelatin, chitosan, fibrin, silk fibroin, amnion and the like), and artificially synthesized materials (including polyvinyl alcohol, polylactic acid, polyglycolic acid and the like), but have certain defects and shortcomings, and a material which can meet the requirements of clinical corneal implantation treatment is not developed at present. For example, the detergent for decellularization of the xenogenic (animal sources such as pigs) acellular corneal stroma in the preparation process can destroy the original structure and components of the stroma, influence the transparency and mechanical strength of the stroma, and can cause inflammatory reaction after transplantation; the scaffold material made of natural biological materials such as collagen, chitosan, fibrin and the like has too high degradation rate and poor mechanical strength, and cannot meet the requirements of surgical suture; the artificially synthesized material has poor biocompatibility, and the degradation product may produce certain toxic effect on organisms. The defects can be overcome by adopting the cell sheet engineering technology without using a bracket material and utilizing the cell sheet engineering technology to reconstruct the cornea, but the technology has the defects that the seed cells are difficult to obtain when the tissue is reconstructed, the internal cells are necrotized due to the superposition of multiple layers of cell sheets, and a plurality of important problems exist in the technology. Therefore, the preparation of ideal scaffold materials with stable physicochemical properties, high transparency, good air permeability, sufficient mechanical strength to resist surgery and good biocompatibility with seed cells is still a difficult point to overcome in the field of tissue engineering corneas.

Disclosure of Invention

The invention aims to: aiming at the defects of the prior art, the collagen plate matrix material, the preparation method and the application thereof, the method for tissue engineering cornea and the device thereof are provided, and the prepared collagen plate matrix material has a component structure similar to that of a natural cornea, excellent optical property and mechanical property and good cell compatibility.

The technical scheme adopted by the invention is as follows:

a preparation method of a collagen plate substrate material comprises the following steps:

and etching the surface of the collagen sheet obtained after degreasing, decalcification and decellularization, rinsing, then placing the collagen sheet in a cross-linking mold with curvature for cross-linking, cleaning and sterilizing to obtain the collagen sheet.

Further, the method specifically comprises the following steps:

s1, placing a collagen sheet subjected to degreasing, decalcification and decellularization with the front side facing upwards, uniformly spraying etching liquid on the surface of the collagen sheet, and simultaneously keeping the collagen sheet in vibration;

s2, after the etching of S1 is finished, rinsing with pure water for 2-3 times, each time for 10-15min, and obtaining an etched collagen matrix;

s3, placing the etched collagen matrix obtained in the step S2 in a cross-linking mold with curvature, adding a cross-linking agent, cross-linking for 12-24h at room temperature, and rinsing for 2-3 times with a buffer solution for 10-15min each time to obtain a collagen matrix material;

s4, soaking the collagen matrix material obtained in the step S3 in pure water, and ultrasonically cleaning for 1-2 h; then sterilizing by ultraviolet, soaking in 75% ethanol for 10-20h, and cleaning.

Further, the collagen sheet is fish scale.

Further, a partition plate is placed above the collagen sheet in the S1 etching process, the middle part of the collagen sheet is etched for 16-20h, and the edge part of the collagen sheet is etched for 7-10 h.

Further, the etching solution in S1 is at least one of a hydrochloric acid solution, an acetic acid solution, a citric acid solution, papain, a neutral protease solution, a pepsin solution, a collagenase solution, a trypsin solution, and a papain solution; the concentration is 0.5-2.5 mg/mL.

Further, the crosslinking mold having a curvature in S3 includes an upper plate having a curvature radius of 6.8mm and a lower plate having a curvature radius of 7.8 mm.

Further, the crosslinking agent in S3 is 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide solution/N-hydroxysuccinimide solution, glutaraldehyde, genipin or a modified epoxy compound.

Further, the ultraviolet sterilization of S4 specifically comprises: placing the collagen matrix material in a cell culture plate, performing double-sided ultraviolet sterilization for 30-60min respectively, and performing ultraviolet irradiation with energy of 100-²。

The collagen lamina matrix material prepared by the method.

The collagen lamellar matrix material is applied to the preparation of tissue engineering cornea.

In summary, due to the adoption of the technical scheme, the invention has the beneficial effects that:

the decalcification and spray etching treatment can remove the main component structure of the fish scale surface layer which influences the transparency of the matrix material, and the obtained matrix material is very close to a natural cornea in component and structure, and has excellent optical characteristics and excellent mechanical properties. Through detection, the light transmittance of the scale matrix material prepared by the invention in the visible wavelength range is 90-95%, and the Young modulus is 414.9MPa, so that the requirement of the artificial cornea on the support material is met.

The preparation process of the invention avoids the phenomena that the fibers in the matrix layer are swelled and the fiber arrangement orderliness is deteriorated, which further influences the light transmittance and the mechanical strength of the material, caused by long-time soaking and other treatments, and keeps the natural regular compact structure of the scaly matrix biomaterial. The corneal endothelial cells cultured in vitro have excellent biocompatibility, no cytotoxicity and no antigenicity, and the seed cells can normally adhere to and grow on the surface of the material to form a compact cell monolayer. The prepared scale matrix material is an excellent cornea substitute, can be used for various corneal lamellar transplantation indications such as keratitis, corneal pannus, corneal dystrophy, corneal degeneration and the like, and can be directly applied to clinical transplantation and treatment of various keratoses. And the raw material source is wide, the production cost is low, the storage and the use are convenient, and the requirements of a large number of carrier supports of the tissue engineering cornea can be met.

The device of the invention adopts a special cross-linking mould, and can obtain a matrix membrane with the same curvature as the human cornea, thereby leading the material to have certain diopter, realizing the purpose of improving or recovering the vision and being more fit with tissues.

The preparation method is simple and easy to operate, the preparation efficiency can be greatly improved by combining the device, meanwhile, the self-made partition plate can be designed with the aperture size according to the needs, and the concentration of the etching solution and the etching time can be adjusted according to the needs. The fish scale matrix material prepared by the method meets the requirements of artificial cornea on scaffold materials in terms of optical and mechanical properties, has no toxicity and antigenicity to cells, has the advantage that other materials cannot be substituted, has great significance for reconstructing cornea in vitro, and is expected to become one of ideal choices of cornea substitutes.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.

FIG. 1 is a cross-sectional view of a specialized cross-linking mold;

FIG. 2 is a scanning electron micrograph of a cross-section of the product of the example;

FIG. 3 is a comparison of the light transmittance of the product of the example in the visible wavelength range with that of a contact lens;

FIG. 4 is an inverted microscope photograph of human corneal endothelial cells cultured on the product of example;

FIG. 5 is a fluorescent chart of FDA/PI staining of human corneal endothelial cells cultured on the product of the examples;

the labels in the figure are: 1-fish scale sample, 2-upper plate, 3-lower plate.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the detailed description and specific examples, while indicating the preferred embodiment of the invention, are intended for purposes of illustration only and are not intended to limit the scope of the invention. The components of embodiments of the present invention generally described and illustrated in the figures herein may be arranged and designed in a wide variety of different configurations.

Thus, the following detailed description of the embodiments of the present invention, presented in the figures, is not intended to limit the scope of the invention, as claimed, but is merely representative of selected embodiments of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments of the present invention without making any creative effort, shall fall within the protection scope of the present invention.

It is noted that relational terms such as "first" and "second," and the like, may be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Also, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. Without further limitation, an element defined by the phrase "comprising an … …" does not exclude the presence of other identical elements in a process, method, article, or apparatus that comprises the element.

The features and properties of the present invention are described in further detail below with reference to examples.

Example 1

The preferred embodiment of the invention provides a preparation method of a collagen plate layer matrix material, which comprises the following specific steps:

laying the decalcified fish scales on a bottom plate with holes, enabling each bottom plate hole to correspond to a sample, starting a vacuum device to enable the samples to be adsorbed on the bottom plate, and selecting a partition plate with a proper pore diameter as required to be laid above the fish scale samples. And uniformly spraying the etching solution on the surface of the decalcified fish scale, and standing. Spraying cleaning liquid at intervals, flushing the etching liquid on the surfaces of the scales, and spraying new etching liquid again. During the etching process, different partition plates are replaced to ensure that the etching time of the middle part of the sample reaches 17 hours and the etching time of the edge part reaches 8 hours. And after etching is finished, rinsing with ultrapure water for 3 times, and rinsing for 10min each time to obtain etched fish scales.

And then placing the etched fish scales in a cross-linking mold with curvature, adding cross-linking liquid, cross-linking for 20 hours, and then rinsing for 3 times with buffer solution for 10min each time to obtain the fish scale matrix material.

And finally, soaking the scale matrix material in ultrapure water, putting the scale matrix material into an ultrasonic cleaning machine for 1h, removing residual chemical reagents on the surface of the sample, and replacing liquid in the middle. Placing fish scale matrix material into 12-hole cell culture plate, performing double-sided ultraviolet sterilization for 30min respectively, and performing ultraviolet irradiation with energy of 300mj/cm². Then soaked with 75% ethanol overnight. Washing the sample with sterile buffer solution for 3 times in a clean bench, soaking in sterile buffer solution, changing the solution for several times to completely remove residual ethanol, and storing the obtained sterile fish scale matrix material in sterile buffer solution at 4 deg.C.

The etching solution is hydrochloric acid solution with the concentration of 1.0 mg/mL. Ultrapure water is used as the diluent. The crosslinking agent was 3mg/mL 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide solution (EDC), 0.8mg/mL N-hydroxysuccinimide solution (NHS). The cleaning liquid is ultrapure water. The sterile buffer was 10mM phosphate buffer, pH 7.4.

The cross-linking mold with curvature was designed according to the curvature of the anterior-posterior plane of a human cornea, as shown in fig. 1, in which the upper plate was designed with a radius of curvature of 6.8mm and the lower plate was designed with a radius of curvature of 7.8 mm. And placing the etched collagen matrix in each groove of the lower plate of the cross-linking mold, adding cross-linking liquid, immersing the sample, mounting the upper plate of the mold, and carrying out cross-linking.

The sectional view of the scanning electron microscope of the product obtained in the embodiment is shown in FIG. 2; the light transmittance of the contact lens is compared with that of a common corneal contact lens in a visible wavelength range, the result is shown in figure 3, the light transmittance in the visible wavelength range is 90-95%, the Young modulus is 414.9MPa, and the light transmittance of the product is high; human corneal endothelial cells were cultured thereon, as shown in FIGS. 4 and 5, and the cells grew well.

Example 2

laying the decalcified fish scales on a bottom plate with holes, enabling each bottom plate hole to correspond to a sample, starting a vacuum device to enable the samples to be adsorbed on the bottom plate, and selecting a partition plate with a proper pore diameter as required to be laid above the fish scale samples. And uniformly spraying the etching solution on the surface of the decalcified fish scale, and standing. Spraying cleaning liquid at intervals, flushing the etching liquid on the surfaces of the scales, and spraying new etching liquid again. During the etching process, different partition plates are replaced to ensure that the etching time of the middle part of the sample reaches 18 hours and the etching time of the edge part reaches 9 hours. And after etching is finished, rinsing with ultrapure water for 3 times, and rinsing for 15min each time to obtain etched fish scales.

And then placing the etched fish scales in a cross-linking mold with curvature, adding cross-linking liquid, cross-linking for 20 hours, and then rinsing for 3 times with buffer solution for 15min each time to obtain the fish scale matrix material.

And finally, soaking the scale matrix material in ultrapure water, putting the scale matrix material into an ultrasonic cleaning machine for 2 hours, removing residual chemical reagents on the surface of the sample, and replacing liquid in the middle. Placing fish scale matrix material into 12-hole cell culture plate, performing double-sided ultraviolet sterilization for 60min respectively, and performing ultraviolet irradiation with energy of 100mj/cm². Then soaked with 75% ethanol overnight. Washing the sample with sterile buffer solution for 3 times in a clean bench, soaking in sterile buffer solution, changing the solution for several times to completely remove residual ethanol, and storing the obtained sterile fish scale matrix material in sterile buffer solution at 4 deg.C.

The etching solution is acetic acid solution with the concentration of 1.5 mg/mL. The diluent is physiological saline. The cross-linking agent is glutaraldehyde. The cleaning solution is normal saline. The sterile buffer was 10mM phosphate buffer, pH 7.4.

Example 3

laying the decalcified fish scales on a bottom plate with holes, enabling each bottom plate hole to correspond to a sample, starting a vacuum device to enable the samples to be adsorbed on the bottom plate, and selecting a partition plate with a proper pore diameter as required to be laid above the fish scale samples. And uniformly spraying the etching solution on the surface of the decalcified fish scale, and standing. Spraying cleaning liquid at intervals, flushing the etching liquid on the surfaces of the scales, and spraying new etching liquid again. During the etching process, different partition plates are replaced to ensure that the etching time of the middle part of the sample reaches 19 hours and the etching time of the edge part reaches 8 hours. And after etching is finished, rinsing with ultrapure water for 3 times, and each time for 12min to obtain etched fish scales.

And then placing the etched fish scales in a cross-linking mold with curvature, adding cross-linking liquid, cross-linking for 18h, and then rinsing for 3 times with buffer solution for 12min each time to obtain the fish scale matrix material.

And finally, soaking the scale matrix material in ultrapure water, putting the scale matrix material into an ultrasonic cleaning machine for 1.5h, removing residual chemical reagents on the surface of the sample, and replacing liquid in the middle. Placing fish scale matrix material into 12-hole cell culture plate, performing double-sided ultraviolet sterilization for 40min respectively, and performing ultraviolet irradiation with energy of 200mj/cm². Then soaked with 75% ethanol overnight. Washing the sample with sterile buffer solution for 3 times in a clean bench, soaking in sterile buffer solution, changing the solution for several times to completely remove residual ethanol, and storing the obtained sterile fish scale matrix material in sterile buffer solution at 4 deg.C.

The etching solution is citric acid solution with the concentration of 1.2 mg/mL. Ultrapure water is used as the diluent. The cross-linking agent is genipin. The cleaning liquid is ultrapure water. The sterile buffer was 10mM phosphate buffer, pH 7.4.

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 within the scope of the present invention.

Claims

1. The preparation method of the collagen board layer matrix material is characterized by comprising the following steps:

2. The method for preparing a collagen lamellar matrix material according to claim 1, characterized in that the specific steps comprise:

3. The method for preparing a collagen lamellar matrix material according to claim 1 or 2, characterized in that said collagen sheet is fish scale.

4. The method for preparing a collagen lamellar matrix material according to claim 2, wherein a spacer is placed above the collagen sheet during the etching of S1, and the middle part of the collagen sheet is etched for 16-20h and the edge part is etched for 7-10 h.

5. The method for preparing a collagen plate layer matrix material according to claim 2, wherein the etching solution in S1 is at least one of a hydrochloric acid solution, an acetic acid solution, a citric acid solution, papain, a neutral protease solution, a pepsin solution, a collagenase solution, a trypsin solution and a papain solution; the concentration is 0.5-2.5 mg/mL.

6. The method for preparing a collagen lamellar matrix material according to claim 2, characterized in that said cross-linking mold having curvature in S3 comprises an upper plate having a curvature radius of 6.8mm and a lower plate having a curvature radius of 7.8 mm.

7. The method for preparing a collagen lamellar matrix material according to claim 2, characterized in that the cross-linking agent in S3 is 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide solution/N-hydroxysuccinimide solution, glutaraldehyde, genipin or a modified epoxy compound.

8. The method for preparing a collagen lamellar matrix material according to claim 2, characterized in that said S4 UV sterilization is in particular: placing the collagen matrix material in a cell culture plate, performing double-sided ultraviolet sterilization for 30-60min respectively, and performing ultraviolet irradiation with energy of 100-²。

9. A collagen lamellar matrix material produced by the method of any one of claims 1 to 8.

10. Use of the collagen lamellar matrix material according to claim 9 for the preparation of a tissue engineered cornea.