CN110613862B - Tissue engineering cornea and preparation method thereof - Google Patents
Tissue engineering cornea and preparation method thereof Download PDFInfo
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- CN110613862B CN110613862B CN201910888009.3A CN201910888009A CN110613862B CN 110613862 B CN110613862 B CN 110613862B CN 201910888009 A CN201910888009 A CN 201910888009A CN 110613862 B CN110613862 B CN 110613862B
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- 238000002360 preparation method Methods 0.000 title claims abstract description 8
- 210000001519 tissue Anatomy 0.000 claims abstract description 57
- 239000000835 fiber Substances 0.000 claims abstract description 38
- 210000002536 stromal cell Anatomy 0.000 claims abstract description 34
- 210000002569 neuron Anatomy 0.000 claims abstract description 27
- 102000008186 Collagen Human genes 0.000 claims abstract description 22
- 108010035532 Collagen Proteins 0.000 claims abstract description 22
- 229920001436 collagen Polymers 0.000 claims abstract description 22
- 239000011159 matrix material Substances 0.000 claims abstract description 19
- 210000002919 epithelial cell Anatomy 0.000 claims abstract description 14
- 238000010041 electrostatic spinning Methods 0.000 claims abstract description 13
- 210000002744 extracellular matrix Anatomy 0.000 claims abstract description 13
- 238000010146 3D printing Methods 0.000 claims abstract description 12
- 102000010834 Extracellular Matrix Proteins Human genes 0.000 claims abstract description 12
- 108010037362 Extracellular Matrix Proteins Proteins 0.000 claims abstract description 12
- 108010022355 Fibroins Proteins 0.000 claims abstract description 12
- 210000000399 corneal endothelial cell Anatomy 0.000 claims abstract description 11
- 238000000016 photochemical curing Methods 0.000 claims abstract description 11
- 239000002131 composite material Substances 0.000 claims abstract description 9
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- 238000003501 co-culture Methods 0.000 claims description 7
- 210000000130 stem cell Anatomy 0.000 claims description 6
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- 238000012258 culturing Methods 0.000 claims description 4
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- 210000002889 endothelial cell Anatomy 0.000 claims description 3
- 239000008273 gelatin Substances 0.000 claims description 3
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- A61F2/00—Filters implantable into blood vessels; Prostheses, i.e. artificial substitutes or replacements for parts of the body; Appliances for connecting them with the body; Devices providing patency to, or preventing collapsing of, tubular structures of the body, e.g. stents
- A61F2/02—Prostheses implantable into the body
- A61F2/14—Eye parts, e.g. lenses, corneal implants; Implanting instruments specially adapted therefor; Artificial eyes
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Abstract
A tissue engineering cornea and a preparation method thereof are disclosed, the tissue engineering cornea comprises a laminated structure of at least three layers, wherein each layer is a composite layer formed by solidifying and compounding a fibrous scaffold prepared by electrostatic spinning or electrostatic direct writing or photocuring 3D printing and one or more of collagen, acellular matrix and silk fibroin, corneal stromal cells and neurons are inoculated between two adjacent layers, the corneal stromal cells and the neurons can grow along the direction of fibers of the scaffold, the corneal stromal cells between two adjacent layers secrete extracellular matrix to form a tissue structure similar to human corneal stroma, and the upper surface and the lower surface of the laminated structure are respectively inoculated with corneal epithelial cells and corneal endothelial cells. The tissue engineering cornea can well simulate the structure of the cornea, is simple to prepare, has good biocompatibility and is easy to fuse with tissues.
Description
Technical Field
The invention relates to the technical field of tissue engineering, in particular to a tissue engineering cornea and a preparation method thereof.
Background
Corneal disease is a common frequently encountered disease in ophthalmology, the blindness rate of corneal disease is ranked second in blind epidemiological investigation, and corneal transplantation is the only effective method for treating corneal blindness. However, the cornea donation is highly insufficient, so that most patients cannot get cornea transplantation and cannot be clarified. In recent years, the rise and development of corneal tissue engineering create conditions for the in vitro reconstruction and clinical application of tissue engineering cornea, and bring new hopes for patients with keratopathy blindness through the transplantation of tissue engineering cornea, but how to obtain an ideal carrier bracket is still a hotspot and difficulty of the current tissue engineering cornea in vitro reconstruction research.
The cornea is structurally divided into 5 layers, and the corneal stroma layer is composed of human corneal stromal cells and 250 collagen fiber lamina layers of 200-. Corneal stromal cells mainly synthesize extracellular matrix. The collagen fibers of the stroma layer are regularly and uniformly arranged and are in a sheet shape, the stroma layers are closely overlapped layer by layer, the fiber layers are in a cross arrangement, and the light transmittance of the cornea is determined by the structure of the corneal stroma, so that the tissue engineering cornea is of great importance whether to have a three-dimensional structure similar to that of a human cornea.
Disclosure of Invention
The invention mainly aims to overcome the defects of the prior art and provide a tissue engineering cornea and a preparation method thereof, so that the cornea in the human environment can be better simulated and the performance of the tissue engineering cornea can be improved.
In order to achieve the purpose, the invention adopts the following technical scheme:
a tissue engineered cornea comprises a laminated structure of at least three layers, wherein each layer is a composite layer formed by solidifying and compounding a fibrous scaffold prepared by electrostatic spinning or electrostatic direct writing or photocuring 3D printing and one or more of collagen, acellular matrix and silk fibroin, corneal stromal cells and neurons are seeded between two adjacent layers, the corneal stromal cells and the neurons can grow along the direction of fibers of the scaffold, the corneal stromal cells between two adjacent layers secrete extracellular matrix to form a tissue structure similar to human corneal stroma, and the upper surface and the lower surface of the laminated structure are respectively seeded with corneal epithelial cells and corneal endothelial cells.
Further:
the material of the fiber support is a single material or a mixed material.
The single material or the mixed material is selected from one or more of PCL, PLGA, gelatin, methacrylate PDC (mPDC) prepolymer and GelMA.
The fibers of two adjacent layers of the scaffold are aligned or stacked at any angle.
The fibers of two adjacent layers of the bracket are arranged orthogonally.
The corneal stromal cells are differentiated from stem cells seeded between two adjacent layers, and preferably, the stem cells are corneal stromal cells.
The neuron is rat or mouse hippocampal neuron.
The corneal epithelial cells and corneal endothelial cells inoculated on the upper surface and the lower surface of the laminated structure are derived from primary or subcultured corneal epithelial cells and endothelial cells.
The fibrous scaffold is an ordered or unordered cornea-imitating substrate plate layer structure or a nerve-imitating structure scaffold.
The preparation method of the tissue engineering cornea comprises the following steps:
preparing a fiber support through electrostatic spinning, electrostatic direct writing or photocuring 3D printing;
placing the prepared scaffold in a mould, then pouring one or more of prepared collagen solution, acellular matrix hydrogel solution and silk fibroin solution, and solidifying and forming, wherein the collagen solution is compressed and dehydrated to finally obtain a layer;
inoculating corneal stromal cells and neurons on the obtained layer, and culturing to ensure that the corneal stromal cells secrete extracellular matrix so as to realize the co-culture of the corneal stromal cells and the neurons;
and stacking at least three layers, wherein the upper surface and the lower surface of the stacked structure are respectively inoculated with corneal epithelial cells and corneal endothelial cells to obtain the tissue engineering cornea.
The invention has the following beneficial effects:
the tissue engineering cornea provided by the invention adopts a fiber bracket prepared by electrostatic spinning, electrostatic direct writing or photocuring 3D printing and a composite layer formed by collagen/acellular matrix/silk fibroin to stack at least three layers, the three layers are superposed to form a plywood structure, a multilayer fiber sheet structure simulating the cornea matrix is formed, the fiber stacks of different layers can be arranged at any angle or regularly, such as aligned or orthogonally arranged, and the corneal stromal cells between the layers secrete highly-regular extracellular matrix to form a tissue structure similar to the cornea matrix, and neurons are introduced between the layers at the same time, so that the co-culture of the corneal cells and the neurons is realized, and the cornea in a human body environment can be better simulated. Meanwhile, the composite layer laminated structure formed based on the fibrous scaffold has good mechanical property, and the thickness and the mechanical toughness of the tissue engineering cornea are greatly improved.
The tissue engineering cornea can well simulate the structure of the cornea, is simple to prepare, has good biocompatibility, is easy to fuse with tissues, can be applied to corneal tissue development research and substitution treatment of corneal drug research, and can reduce the occurrence probability of rejection reaction after corneal transplantation and improve the transplantation success rate.
Drawings
FIG. 1 is a general schematic view of a tissue engineered cornea according to an embodiment of the present invention, wherein 1a, 1b, and 1c are a three-dimensional structure, a left-view structure, and a top-view structure of the tissue engineered cornea, respectively;
FIG. 2 is a schematic diagram of a tissue engineered cornea of an embodiment of the present invention before and after four fiber scaffolds are seeded with corneal stromal cells and neurons, wherein 2a and 2b are electrostatic direct-writing fiber scaffolds, 2c and 2d are ordered electrostatic spinning fiber scaffolds, 2e and 2f are ordered nerve-like structure fiber scaffolds, and 2g and 2h are disordered nerve-like structure fiber scaffolds;
fig. 3 is a schematic diagram of a method for preparing a tissue-engineered cornea according to an embodiment of the invention.
Detailed Description
The embodiments of the present invention will be described in detail below. It should be emphasized that the following description is merely exemplary in nature and is not intended to limit the scope of the invention or its application.
Referring to fig. 1-3, in one embodiment, a tissue engineered cornea 1a, 1b, 1c, comprises a laminated structure of at least three layers, wherein each layer 11, 12 is a composite layer formed by solidifying and compounding fiber scaffolds 2a, 2b, 2c, 2D, 2e, 2f, 2g, 2h prepared by electrostatic spinning or electrostatic direct writing or photocuring 3D printing and one or more of collagen, acellular matrix and silk fibroin, the corneal stroma cells 21, 23, 25, 27 and the neurons 22, 24, 26, 28 are seeded between the two adjacent layers, the corneal stromal cells 21, 23, 25, 27 and the neurons 22, 24, 26, 28 can grow along the direction of the fibers of the scaffold, the corneal stromal cells between two adjacent layers secrete extracellular matrix, forming a tissue structure similar to human corneal stroma, the upper and lower surfaces of the laminated structure are seeded with corneal epithelial cells and corneal endothelial cells, respectively. The fiber support can be an electrostatic direct-writing or electrostatic spinning fiber support, and also can be an ordered or disordered cornea-imitating substrate plate layer structure or a nerve-imitating structure support.
The tissue engineering cornea provided by the embodiment of the invention adopts a fiber support prepared by electrostatic spinning, electrostatic direct writing or photocuring 3D printing and a composite layer formed by collagen/acellular matrix/silk fibroin to stack at least three layers, and the three layers are superposed to form a plywood structure to form a multilayer fiber sheet structure simulating the corneal matrix, and the fiber stacks of different layers can be arranged at any angle or regularly, such as aligned or orthogonally arranged, and the corneal stromal cells between the layers secrete and arrange highly regular extracellular matrix to form an organizational structure similar to the corneal matrix, and simultaneously neurons are introduced between the layers to realize the co-culture of the corneal cells and the neurons. The obtained tissue engineering cornea can better simulate the cornea in human body environment. Meanwhile, the composite layer laminated structure formed based on the fibrous scaffold has good mechanical property, and the thickness and the mechanical toughness of the tissue engineering cornea are greatly improved. The tissue engineering cornea of the invention can be applied to corneal tissue development research and substitution treatment of corneal drug research, has good biocompatibility, is easy to fuse with tissues, can reduce the occurrence probability of rejection reaction after corneal transplantation and improve the success rate of transplantation.
The tissue engineering cornea of the invention not only can well simulate the structure of the cornea, but also has simple preparation.
Referring to fig. 1 to 3, in another embodiment, a method for preparing a tissue engineered cornea includes the following steps:
preparing a fiber support through electrostatic spinning, electrostatic direct writing or photocuring 3D printing;
placing the prepared scaffold in a mould, then pouring one or more of prepared collagen solution, acellular matrix hydrogel solution and silk fibroin solution, and solidifying and forming, wherein the collagen solution is compressed and dehydrated to finally obtain a layer;
inoculating corneal stromal cells and neurons on the obtained layer, and culturing to ensure that the corneal stromal cells secrete extracellular matrix so as to realize the co-culture of the corneal stromal cells and the neurons;
and stacking at least three layers, wherein the upper surface and the lower surface of the stacked structure are respectively inoculated with corneal epithelial cells and corneal endothelial cells to obtain the tissue engineering cornea.
As shown in fig. 1 to 2, in some embodiments, a tissue engineered cornea 1a, 1b, 1c includes at least three layers, wherein each layer 11, 12 is prepared by compounding an electrostatic direct-writing fiber scaffold 2a, 2b, an electrospun fiber scaffold 2c, 2d, an ordered neuromorphic fiber scaffold 2e, 2f, one of disordered neuromorphic fiber scaffolds 2g, 2h and collagen/acellular matrix/silk fibroin, and the scaffold fibers of two adjacent layers can be aligned or stacked at any angle. The adjacent two layers of the tissue engineering cornea are inoculated with corneal stroma cells 21, 23, 25, 27 and neurons 22, 24, 26, 28, the corneal stroma cells 21, 23, 25, 27 and the neurons 22, 24, 26, 28 can well grow along the direction of the scaffold fibers, and the corneal stroma cells between the two layers secrete regularly arranged extracellular matrixes to form a tissue structure similar to the corneal stroma. The collagen can be rat tail type collagen/humanized collagen.
In some embodiments, the electrostatically direct written fibrous scaffold 2a, 2b resulting from electrostatic direct writing, or said electrospun scaffold 2c, 2D resulting from electrospinning, or the neuromorphic fibrous scaffold 2e, 2f, 2g, 2h prepared from photocured 3D printing, may be selected from a single or mixed material. A certain degree of controllable degradation can be achieved by controlling the proportion and the degree of crosslinking of the mixed materials. Preferably, the material may be selected from one or more of PCL, PLGA, PCL/gelatin, methacrylate pdc (mpdc) prepolymer, GelMA.
The corneal stromal cells are differentiated from stem cells seeded between two layers of tissue engineered cornea, and in a preferred embodiment, the stem cells are corneal stromal stem cells.
The neuron is inoculated between two layers of tissue engineering cornea, preferably rat or mouse hippocampal neuron.
The corneal epithelial cells and corneal endothelial cells inoculated on the upper surface and the lower surface of the tissue engineering cornea are derived from primary or subcultured corneal epithelial cells and endothelial cells.
As shown in fig. 3, a method for preparing a tissue-engineered cornea according to an embodiment includes:
preparing a fiber support through electrostatic spinning, electrostatic direct writing or photocuring 3D printing;
placing the prepared scaffold in a designed mould 3a, then pouring the prepared collagen solution/acellular matrix hydrogel solution/silk fibroin solution 3c, solidifying and forming under a certain condition, and compressing and dehydrating the collagen solution to obtain a layer 3 e;
inoculating cells to culture for a period of time, secreting extracellular matrix from corneal stromal cells, and stacking at least three layers after the corneal stromal cells and neurons realize good co-culture to obtain the tissue engineering cornea 1 a.
The foregoing is a more detailed description of the invention in connection with specific/preferred embodiments and is not intended to limit the practice of the invention to those descriptions. It will be apparent to those skilled in the art that various substitutions and modifications can be made to the described embodiments without departing from the spirit of the invention, and these substitutions and modifications should be considered to fall within the scope of the invention.
Claims (10)
1. A tissue engineering cornea is characterized by comprising a laminated structure of at least three layers, wherein each layer is a composite layer formed by solidifying and compounding a fibrous scaffold prepared by electrostatic spinning or electrostatic direct writing or photocuring 3D printing and one or more of collagen, acellular matrix and silk fibroin, the composite layers are stacked to form a plywood structure, a multi-layer fiber sheet structure simulating the corneal matrix is formed, corneal stromal cells and neurons are inoculated between the two adjacent layers, the corneal stromal cells and the neurons can grow along the fiber direction of the scaffold, the corneal stromal cells between the two adjacent layers secrete extracellular matrix to form a tissue structure similar to the human corneal matrix, and the upper surface and the lower surface of the laminated structure are respectively inoculated with corneal epithelial cells and corneal endothelial cells; wherein, the tissue engineering cornea is obtained by the following preparation method: preparing a fiber support through electrostatic spinning, electrostatic direct writing or photocuring 3D printing; placing the prepared scaffold in a mould, then pouring one or more of prepared collagen solution, acellular matrix hydrogel solution and silk fibroin solution, and solidifying and forming, wherein the collagen solution is compressed and dehydrated to finally obtain a layer; inoculating corneal stromal cells and neurons on the obtained layer, and culturing to ensure that the corneal stromal cells secrete extracellular matrix so as to realize the co-culture of the corneal stromal cells and the neurons; stacking at least three layers, wherein the upper surface and the lower surface of the stacked structure are respectively inoculated with corneal epithelial cells and corneal endothelial cells.
2. The tissue engineered cornea of claim 1, wherein the material of said fibrous scaffold is a single material or a mixture of materials.
3. The tissue engineered cornea of claim 2, wherein the single material or the hybrid material is selected from one or more of PCL, PLGA, gelatin, methacrylate PDC prepolymer, GelMA.
4. A tissue engineered cornea as in any one of claims 1 to 2, wherein the fibers of two adjacent layers of the scaffold are aligned or stacked at any angle.
5. A tissue engineered cornea as in any one of claims 1 to 2, wherein the fibers of two adjacent layers of said scaffold are orthogonally arranged.
6. A tissue engineered cornea as in any one of claims 1 to 2, wherein said corneal stromal cells are differentiated from corneal stromal stem cells seeded between two adjacent layers.
7. A tissue engineered cornea as in any one of claims 1 to 2, wherein said neurons are rat or mouse hippocampal neurons.
8. A tissue engineered cornea as in any one of claims 1 to 2, wherein the upper and lower surfaces of the laminated structure are seeded with corneal epithelial cells and corneal endothelial cells derived from primary or subcultured corneal epithelial cells and endothelial cells.
9. The tissue engineered cornea of any one of claims 1 to 2, wherein the fibrous scaffold is an ordered or unordered keratomimetic matrix plate layer structure or a neuro-mimetic structure scaffold.
10. A method of preparing a tissue engineered cornea as in any one of claims 1 to 9, comprising the steps of:
preparing a fiber support through electrostatic spinning, electrostatic direct writing or photocuring 3D printing;
placing the prepared scaffold in a mould, then pouring one or more of prepared collagen solution, acellular matrix hydrogel solution and silk fibroin solution, and solidifying and forming, wherein the collagen solution is compressed and dehydrated to finally obtain a layer;
inoculating corneal stromal cells and neurons on the obtained layer, and culturing to ensure that the corneal stromal cells secrete extracellular matrix so as to realize the co-culture of the corneal stromal cells and the neurons;
and stacking at least three layers, wherein the upper surface and the lower surface of the stacked structure are respectively inoculated with corneal epithelial cells and corneal endothelial cells to obtain the tissue engineering cornea.
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| CN112222436B (en) * | 2020-04-13 | 2022-08-12 | 广东佳悦美视生物科技有限公司 | Preparation method of artificial corneal mirror column |
| CN111603609B (en) * | 2020-05-25 | 2022-05-03 | 医工瑞思(福建)工程研究中心有限公司 | Bionic tissue engineering scaffold and preparation method thereof |
| CN112316219B (en) * | 2020-09-29 | 2022-05-10 | 浙江大学 | Anti-adhesion hydrogel-silk scaffold composite membrane and preparation and application thereof |
| CN113456895B (en) * | 2021-07-20 | 2022-05-06 | 广州市朴道联信生物科技有限公司 | GelMA-collagen double-network antibacterial cornea repair material and preparation method and application thereof |
| CN114259601B (en) * | 2021-12-27 | 2023-05-26 | 暨南大学 | Living cell bionic cornea anterior lamina and preparation method thereof |
| CN116196475B (en) * | 2023-03-17 | 2024-09-17 | 华南理工大学 | Preparation method of 3D printing curvature biological cornea and application of preparation method in preventing cornea stroma fibrosis |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2011109712A3 (en) * | 2010-03-04 | 2012-03-01 | University Of Pittsburgh-Of The Commonwealth System Of Higher Education | Bioengineered human corneal stromal tissue |
| CN107929811A (en) * | 2017-12-15 | 2018-04-20 | 厦门大学 | A kind of tissue engineering comea |
| CN107979992A (en) * | 2015-06-08 | 2018-05-01 | 科尔尼特视觉有限公司 | Artificial cornea and application thereof |
| CN109010918A (en) * | 2018-08-22 | 2018-12-18 | 清华-伯克利深圳学院筹备办公室 | A kind of compound rest and preparation method thereof |
| CN109157305A (en) * | 2018-09-25 | 2019-01-08 | 清华大学深圳研究生院 | Combined artificial cornea and preparation method thereof |
| CN110029059A (en) * | 2019-04-22 | 2019-07-19 | 清华大学深圳研究生院 | A kind of cell co-culture system based on nano fibrous membrane |
-
2019
- 2019-09-19 CN CN201910888009.3A patent/CN110613862B/en not_active Expired - Fee Related
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2011109712A3 (en) * | 2010-03-04 | 2012-03-01 | University Of Pittsburgh-Of The Commonwealth System Of Higher Education | Bioengineered human corneal stromal tissue |
| CN107979992A (en) * | 2015-06-08 | 2018-05-01 | 科尔尼特视觉有限公司 | Artificial cornea and application thereof |
| CN107929811A (en) * | 2017-12-15 | 2018-04-20 | 厦门大学 | A kind of tissue engineering comea |
| CN109010918A (en) * | 2018-08-22 | 2018-12-18 | 清华-伯克利深圳学院筹备办公室 | A kind of compound rest and preparation method thereof |
| CN109157305A (en) * | 2018-09-25 | 2019-01-08 | 清华大学深圳研究生院 | Combined artificial cornea and preparation method thereof |
| CN110029059A (en) * | 2019-04-22 | 2019-07-19 | 清华大学深圳研究生院 | A kind of cell co-culture system based on nano fibrous membrane |
Non-Patent Citations (1)
| Title |
|---|
| 胶原支架在组织工程角膜中的研究进展;刘多静等;《生物医学工程与临床》;20140506;第18卷(第3期);论文第292-293页2.2.3 * |
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