CN107929811A - A kind of tissue engineering comea - Google Patents
A kind of tissue engineering comea Download PDFInfo
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- CN107929811A CN107929811A CN201711347993.XA CN201711347993A CN107929811A CN 107929811 A CN107929811 A CN 107929811A CN 201711347993 A CN201711347993 A CN 201711347993A CN 107929811 A CN107929811 A CN 107929811A
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- amnion
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/3604—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix characterised by the human or animal origin of the biological material, e.g. hair, fascia, fish scales, silk, shellac, pericardium, pleura, renal tissue, amniotic membrane, parenchymal tissue, fetal tissue, muscle tissue, fat tissue, enamel
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/3683—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment
- A61L27/3687—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment characterised by the use of chemical agents in the treatment, e.g. specific enzymes, detergents, capping agents, crosslinkers, anticalcification agents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
- A61L27/3804—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
- A61L27/3834—Cells able to produce different cell types, e.g. hematopoietic stem cells, mesenchymal stem cells, marrow stromal cells, embryonic stem cells
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2430/00—Materials or treatment for tissue regeneration
- A61L2430/16—Materials or treatment for tissue regeneration for reconstruction of eye parts, e.g. intraocular lens, cornea
Abstract
The invention discloses a kind of tissue engineering comea, including at least three amnion tissue layers, adjacent two amnion tissue interlayers growth has keratocyte, and contain the extracellular matrix for including keratocan, lumican, I-type collagen and collagen type v albumen secreted by above-mentioned keratocyte, and above-mentioned extracellular matrix queueing discipline, form the institutional framework similar to corneal stroma.Corneal epithelial cell is inoculated with respectively in this structure upper and lower surface and endothelial cell forms total corneal.The surface collagen fiber alignment rule of above-mentioned amnion tissue layer, it is parallel to each other between fiber, and just to intersect close-packed arrays into sheets of fibres, to guide the extracellular matrix of keratocyte and its secretion regularly arranged, there is above-mentioned at least three amnion tissue layers the characteristic for suppressing fibrosis to be broken up with keeping the characteristic of keratocyte and suppressing keratocyte to fibroblast.
Description
Technical field
The invention belongs to tissue engineering technique field, and in particular to a kind of tissue engineering comea.
Background technology
People's corneal stroma is made of compact arranged collagenous fibres thin slice, high with transparency, the good characteristic of mechanical tenacity,
It is difficult to reach in vitro, hence in so that vitro tissue engineering cornea faces huge challenge.The whole world there are about 2.85 hundred million people's visual impairments,
Wherein 39,000,000 people are blind, and disease of cornea is then the second largest reason for causing blindness inferior to cataract.At the same time it is reported that children blind
It is that disease of cornea causes probably to have 20% in reason.At present when other treatment method unsuccessfully after, corneal transplantation be disease of cornea most
Effectively and most common treatment method, but due to the shortage of high quality corneal donor, the postoperative appearance of corneal transplantation rejection,
Mortality of increased post-transplantation etc. year by year, all so that this optimal treatment method is restricted.
Keratocyte mainly synthesizes tropocollagen molecule and proteoglycans, maintains corneal stroma connective tissue slow generation
Thank.Due to the arrangement architecture of corneal stroma its collagen tissue high-sequential and the unstability of external keratocyte so that
Vitro tissue engineering cornea differs greatly with expected results.Have confirmed at present, the keratocyte of in vitro culture is easy to
Fibroblast is just divided into, and is further divided into fibroblast, causes keratocyte specific proteins such as angle egg
White glycan, CD34 and CHST6 expression reduce.Fibroblast can then secrete irregular arrangement, have the characteristics that the extracellular of scar
Matrix organization.Therefore, how to keep stroma cell phenotype to greatest extent in vitro, be the key that tissue engineering comea need to be inquired into
One of problem.
The content of the invention
It is an object of the invention to overcome prior art defect, there is provided a kind of tissue engineering comea.
Another object of the present invention is to provide the preparation method of above-mentioned tissue engineering comea.
Technical scheme is as follows：
A kind of tissue engineering comea, including at least three amnion tissue layers, two adjacent amnion tissue interlayers growth have cornea
Stroma cell, and include keratocan, lumican, I-type collagen containing what is secreted by above-mentioned keratocyte
With the extracellular matrix of collagen type v albumen, and above-mentioned extracellular matrix queueing discipline, forms the knot of tissue similar to corneal stroma
Structure, the arrangement of collagen fibers rule on the surface of above-mentioned amnion tissue layer, is parallel to each other between fiber, has a groove structure, and with
Just intersecting close-packed arrays are above-mentioned to guide the extracellular matrix of keratocyte and its secretion regularly arranged into sheets of fibres
At least three amnion tissue layers, which have, suppresses the characteristic of fibrosis to keep the characteristic of keratocyte and to suppress corneal stroma thin
Born of the same parents break up to fibroblast.
In a preferred embodiment of the invention, the keratocyte is by being inoculated at least three amnion groups
Stem cell differentiation between tissue layer forms, which includes corneal stroma stem cell, mescenchymal stem cell, neural crest origin
Stem cell and embryonic stem cell.
In a preferred embodiment of the invention, the amnion tissue layer for holostrome amnion or through protease digestion at
Reason removes the compacted zone retained after spongy layer and lamina reticularis.
In a preferred embodiment of the invention, the amnion tissue layer of at least the superiors of three amnion tissue layers
Upper surface be inoculated with and grow and have corneal epithelial cell.
It is further preferred that the corneal epithelial cell is from the corneal epithelial cell of original cuiture, secondary culture
The corneal epithelium that corneal epithelial cell, Characteristic Analysis of Corneal Epithelial Cell Line or stem cell differentiate substitutes cell.
In a preferred embodiment of the invention, the undermost amnion tissue layer of at least three amnion tissue layers
Lower surface be inoculated with and grow and have endothelial cell.
It is further preferred that the endothelial cell is from the endothelial cell of original cuiture, secondary culture
The corneal endothelium that endothelial cell, corneal endothelium cell system or stem cell differentiate substitutes cell
In a preferred embodiment of the invention, the oxygen content of its culture environment is 5~15%.
In a preferred embodiment of the invention, the culture medium used in it is to include nonessential amino acid, into fiber
Porcine HGF 2 and transforming growth factor β 3 have blood serum medium or serum free medium.
The beneficial effects of the invention are as follows：
1st, the present invention cultivates keratocyte (the arrangement of collagen fibers rule on amnion surface, fiber in certain circumstances
Between be parallel to each other, have a groove structure, guide keratocyte and the collagen of cell secretion regularly arranged；The collagen row of amnion
Row are closely similar with people's corneal stroma, i.e., just to intersect close-packed arrays into sheets of fibres, corneal stroma sample tissue between collagenous fibres
The arrangement of secretion collagen and remodeling can be guided in forming process as template；Amnion suppresses the characteristic of fibrosis, in vitro can be with
Keratocyte characteristic is kept well, is suppressed it and is broken up to fibroblast), above-mentioned keratocyte secretion produces thin
Extracellular matrix, these extracellular matrixs arrangement height rule, collagenous fibres are arranged in multi-layer fiber thin slice in just intersecting, form class
It is similar to the institutional framework of corneal stroma.
2nd, the present invention is laminated by least three amnion tissues plus forms laminated structure, simulates the multi-layer fiber of corneal stroma
Thin-slab structure, while substantially increase the thickness and mechanical tenacity of tissue engineering comea.
3rd, tissue engineering comea of the invention can be applied to cornea tissue development research, cornea drug research and keratonosus
The replacement therapy of change.
Brief description of the drawings
Fig. 1 is the preparation principle figure of the tissue engineering comea of the present invention.
Fig. 2 is that form and arrangement of the keratocyte of the present invention in amnion surface and Tissue Culture Dish are schemed.
Fig. 3 is mRNA and expressing quantity knot of the keratocyte of the present invention in amnion surface and Tissue Culture Dish
Fruit is schemed.
Fig. 4 is the optical transparency figure of the different number of plies amnions in the present invention.
Fig. 5 is the optical coherence tomography figure of different number of plies amnions in the present invention.
Fig. 6 is the stereoscan photograph of the tissue engineering comea in the present invention.
Fig. 7 is the HE colored graphs of the tissue engineering comea containing different number of plies amnions in the present invention.
Fig. 8 is the transmission electron microscope photo of the extracellular matrix of the tissue engineering comea in the present invention.
UAM in description above attached drawing refers both to remove what is retained after spongy layer and lamina reticularis using collagenase digesting processing
The ultra-thin amnion of compacted zone.
Embodiment
Technical scheme is further detailed and described below by way of embodiment combination attached drawing.
Ultra-thin amnion in following embodiments is above-mentioned to be protected after protease digestion processing removes spongy layer and lamina reticularis
The compacted zone stayed.
Material and method：
1st, Multilayer ultrathin amnion production process (as shown in Figure 1)
(1) placenta comes from Cesarean esction patient, through puerpera and its family members agree to and sign letter of consent, and passed through before Cesarean esction
The pathogenic examinations such as AIDS, syphilis, hepatitis B, hepatitis are crossed as feminine gender；In superclean bench, with chorial amnion from tire
Cut on disk；Remove chorion；Rinsed repeatedly with sterile PBS solution, until amnion without bloodstain, glossy clear, obtains amnion tissue；
It is placed in sterile centrifugation tube, -80 DEG C of preservations or stand-by.
(2) in an aseptic environment, the above-mentioned amnion tissue fresh above-mentioned amnion tissue or melt at room temperature frozen
Epithelial surface is in 0.02%EDTA, 37 DEG C, digestion 1h；Under disecting microscope, amniotic epithelial cells are carefully struck off with epithelium scraper；
After PBS rinses removal epithelial cell repeatedly, epithelium amnion must be removed, can be with -20 DEG C of preservations.
(3) by the above-mentioned matrix face for removing epithelium amnion after 0.05%Collagenase IV, 37 DEG C, digestion 75min,
Matrix and residual enzyme are removed in PBS rinsings, obtain ultra-thin amnion.
(4) corneal stroma stem cell is inoculated in above-mentioned ultra-thin amnion to cultivate in stem cell medium to merging, then
At least three layers are folded into, cell is then broken up nutrient solution with keratocyte between adjacent two layers of ultra-thin amnion
Culture 8 weeks (specific cell culture sees below " 2, cell culture "), makes corneal stroma stem cell therein be divided into cornea base
Cell plastid simultaneously secretes the enough corneal stromas of generation, forms multilayer amnion tissue.
(5) corneal epithelial cell and endothelial cell are seeded in above-mentioned multilayer amnion tissue upper surface and following table respectively
Face, and cultivate to fusion.
(6) part in the tissue obtained by step (5) is bored with 10mm diameters trepan it is stand-by, up to the tissue work
Journey cornea.
2nd, cell culture
From Xiamen, Eye Center obtains people's cornea tissue, and clostridiopetidase A IV digestion corneal limbus, isolated corneal stroma are done carefully
Born of the same parents (Human corneal stromal stem cells, hCSSCs).Stem cell medium culture cell reaches fusion (probably
Three days), stem cell medium formula：MCDB-201 (Sigma), 2%fetal bovine serum, 10ng/mL
Epidermal growth factor (Invitrogen), 10ng/mL platelet-derived growth factor
(PDGF-BB) (Peprotech, USA), 5mg/mL insulin (Sigma), 5mg/mL transferrin (Sigma), 5ng/
ML selenous acid (Sigma), lipid-rich bovine serum albumin (Thermo, USA), 0.1mM L-
Ascorbic acid-a-phosphate (Sigma), 10-8M dexamethasone (Sangon Biotech, Shanghai,
China), 100 μ g/ml Streptomycin (Thermo, USA) of 100IU/mL Penicillin and, 100ng/ml
cholera toxin(Sigma).When the cells reached confluence, keratocyte differentiation nutrient solution is changed, its formula is：
Advanced DMEM (Life Technologies), 0.1mM L-ascorbic acid-a-phosphate (Sigma
Aldrich), 10ng/ml basic fibroblast growth factor (FGF2, Invitrogen), 0.1ng/ml
Transforming growth factor-beta3 (TGF-β 3), 2.0mM L-Glutamine (Sigma Aldrich),
1.0mM Eagle′s minimum essential medium(MEM)non-essential amino acid(NEAA)
(Sigma Aldrich), 100 μ g/ml Streptomycin (Thermo) of 100IU/mL Penicillin and.Nutrient solution
Change once, cultivate 8 weeks every three days.
Corneal epithelial cell is seeded in multilayer amnion tissue upper surface described above, the culture of SHEM nutrient solutions is treated carefully for 7 days
After born of the same parents' fusion, solution-air contact culture 7 days.SHEM cultivates formula of liquid：DMEM/F12,5%FBS, 1.0mg/ml Insulin,
0.55mg/ml Transterring, 0.5 μ g/ml Selenium, 0.5ug/ml Hydrocortise, 0.5%DMSO,
10ng/ml EGF.
Endothelial cell sheet for cornea is seeded in the lower surface of multilayer amnion tissue, SHEM cultivate treat within 7 days endothelial cell migration,
Extension reaches fusion.
3rd, immunofluorescence dyeing
The sample for cultivating 1 week and 8 week respectively in ultra-thin amnion and Tissue Culture Dish is taken, abandons nutrient solution, 1 × PBS
Rinse cell and remove culture medium residual, blot and discard after jiggling；Add the green element of 500 μ L calcium in amnion ring and 12 orifice plates,
Room temperature, lucifuge are incubated 30 minutes；PBS washes away uncombined dyestuff；Fluorescence microscope result.
Frozen section be placed in cold acetone, -20 DEG C fix 10 minutes；PBS is rinsed 3 times；Mountant mounting with DAPI；It is glimmering
Light microscope observes result.
4、RT-PCR(qPCR)
Using real-time polymerase chain reaction (RT-PCR) analysis from people's cornea after ultra-thin amnion and culture dish culture 14 days
The changes in gene expression of stroma cell.Use ArcturusTMPicoPureTMRNA separating kits (Thermo
Scientific total serum IgE) is extracted；With RevertAid H Minus Reverse Transcriptase (Thermo
Scientific) by RNA reverse transcriptions into cDNA；According to specification, using SYBR Premix Ex Taq Kit (TAK-ARA,
Japan) kit, with StepOne plus Real-Time PCR detection systems (Applied Biosystems,
Darmstadt, Germany) carry out real-time amplification；Amplification program is included in the denaturation carried out at 95 DEG C 10 seconds, continues 95
DEG C denaturation 10 seconds, 60 DEG C anneal 30 seconds, carry out 40 circulation；By calculating 2-ΔΔCTCarry out relative quantitative assay.
Primer sequence：β-Actin, sense primer 5 '-CTCACCATGGATGATGATATCGC-3 ' (SEQ ID NO 01),
Anti-sense primer 5 '-AGGAATCCTTCTGACCCATGC-3 ' (SEQ ID NO 02)；Keratoc an, sense primer 5 '-
AAACTGCCTTCAGCTGCCTATT-3 ' (SEQ ID NO 03), anti-sense primer 5 '-ATCTTAGTGCCTACACAGCCC-3 '
(SEQ ID NO 04)；Lumican, sense primer 5 '-GCCCTGAAAGCTACCCAAGT-3 ' (SEQ ID NO 05), downstream
Primer 5 '-TGATTCCAGGAGGCACCATT-3 ' (SEQ ID NO 06)；Collagen-I, sense primer 5 '-
CCTGGGGCAAGACAGTGATT-3 ' (SEQ ID NO 07), anti-sense primer 5 '-AACGTCGAAGCCGAATTCCT-3 ' (SEQ
ID NO 08)；Collagen-V, sense primer 5 '-TGCGTCTTCCCTGACAAGAA-3 ' (SEQ ID NO 09), downstream is drawn
Thing 5 '-GAGCAGTTTCCCACGCTTGA-3 ' (SEQ ID NO 10)；α-SMA, sense primer 5 '-
CTTGTCCAGGAGTTCCGCTC-3 ' (SEQ ID NO 11), anti-sense primer 5 '-TTTCTTGGGCCTTGATGCGA-3 ' (SEQ
ID NO 12)。
5、western Blot
In ultra-thin amnion surface and culture dish culture people keratocyte 14 days, with containing protease and phosphatase suppresses
RIPA lysates (Sigma, USA) the extraction albumen of agent；Protein compression is measured by BCA protein determination kits (Thermo)
Degree；Polyacrylamide gel electrophoresis protein isolate is carried out per 30 microgram of hole loading；Primary antibody uses anti-Keratocan (Sigma), resists
Lumican (Abcam, Cambridge Science Park, UK), anti-CollagenI (Proteintech, Chicago,
Illinois), anti-Collagen V (Santa Cruz), anti alpha-SMA (Abcam, Cambridge Science Park, UK) and
Anti- beta-actin (Sigma, USA) antibody.Enhanced chemical luminescence reagent (Lulong Inc., Xiamen, China) is shown
Protein expression, and be imaged by gel imaging system (ChemiDoc XRS, Bio-Rad, Hercules, CA, USA).
Quantity One softwares carry out quantitative analysis to the optical density of protein band.
6th, the optical transparency of the ultra-thin amnion system of different layers is assessed
Prepare the ultra-thin amnion model of fresh different layers, blot nutrient solution, PBS rinsings, be placed on A4 paper, visually observe and compare
The writing form of alphabetical " A ", takes pictures at the same time under amnion.
7th, optical coherence tomography inspection
Fresh specimens are taken out from incubator, abandon nutrient solution, PBS rinsings；Dress preset lens, choose 3D cornea and
Pachymetry options；By amnion ring be placed on preset lens it is saturating before, adjust the angle and focal length, observation result, take pictures.
8th, scanning electron microscopy
2.5% glutaraldehyde room temperature fixes 2h, and amnion and glass slide are cut into general 3mm*3mm sizes；Various concentrations wine
Smart serial dehydration, be progressively transitioned into the pure tert-butyl alcohol；4 DEG C overnight to sample crystallization；Freeze-drying；Metal spraying；Scanning electron microscopic observation, bat
According to.
9th, Hematoxylin-eosin dyes
4% paraformaldehyde, 4 DEG C of fixed 12h；OCT is embedded；Liquid nitrogen frost, section；Hematoxylin-eosin staining kit contaminates
Color；Optical microphotograph sem observation is taken pictures.
10th, transmission electronic microscope checking method
2.5% glutaraldehyde room temperature fixes 2h, and 1% osmic acid fixes 2h；Alcohol serial dehydration；Embedding machine gradient penetration；Sample
Cure；Section；Dyeing；Transmission electron microscope observing, take pictures.
11st, result statistical analysis
Related experiment result mainly carries out statistical analysis using 5 softwares of Graphpad Prism, and comparison among groups uses Two-
Way ANOVA are analyzed, and are then thought with statistical significance when P values are less than 0.05.
As a result：
1st, form and arrangement of the cell in amnion surface and Tissue Culture Dish
Scanning electron microscope is the results show that the arrangement of collagen fibers rule on ultra-thin amnion surface, linear between fiber
Groove structure (Fig. 2A), can guide cell growth and extracellular matrix to arrange [7].Assessment corneal stroma is dyed with calcein
Cell viability and arrangement mode of the cell on ultra-thin amnion surface and culture dish, keratocyte differentiation nutrient solution culture 1
Zhou Hou, people's corneal stroma stem cell breed on ultra-thin amnion surface (Fig. 2 B) and culture dish (Fig. 2 D) and reach fusion, cell
In elongated fusiform form.On ultra-thin amnion surface, cell shows stronger multiplication capacity, its line along amnion surface
Property groove direction growth, queueing discipline it is consistent (Fig. 2 C).On Tissue Culture Dish, cell growth direction is random, no regular, and 8
Zhou Hou, is interweaved between cell, is disorganized.
2nd, gene expression.
As shown in Figure 3A, stem cell medium culture people corneal stroma stem cell changes corneal stroma differentiation nutrient solution after 3 days
Culture 7 days, qPCR detections change respectively from the cellular gene expression of ultra-thin amnion and culture dish.The results show that corneal stroma
Cell-specific genes, keratocan (Keratocan), lumican (Lumican), I-type collagen
(CollagenI) no significant change is expressed, collagen type v albumen (CollagenV) is reduced in ultra-thin amnion surface expression, together
When, fibroblast-like cell specific label, α types smooth muscle actin (α-SMA) are substantially reduced in ultra-thin amnion surface expression.
3rd, protein expression
It is seeded in the corneal epithelial cell propagation cladding of the upper surface of multilayer amnion tissue and shows polarity, is immunized glimmering
Light dyeing shows epithelial cell expressing K 3, K14, p63.
People's corneal stroma stem cell respectively after ultra-thin amnion surface and culture dish, serum-free differentiation nutrient solution culture 7 days,
Detected by Western blot (Western Blot) detects keratocyte specific proteins glycan and collagen expression change (figure
3B).The proteoglycans of corneal stroma is mainly Keratocan and Lumican, and CollagenI and CollagenV are to form collagen
The main component of fiber.Similar to gene expression results, the corneal stroma from ultra-thin amnion surface and Tissue Culture Dish is done carefully
Its keratocan of born of the same parents' cell, lumican and collagenI express no significant change, and collagenV is in the super of queueing discipline
Thin amnion surface expression reduces, and α-SMA are substantially reduced in ultra-thin amnion surface expression.
4th, the optical transparency of the ultra-thin amnion system of different layers
As shown in figure 4, observing by the naked eye the clarity of alphabetical A, the optical transparency of the ultra-thin amnion system of different layers is assessed.
Although with the increase of the amnion number of plies, Multilayer ultrathin amnion model optical transparency reduces after 8 week of culture, it still has preferable
Transparency.
5th, tissue engineering comea thickness
Observed flesh tissue engineering cornea cross section with optical coherence tomography inspection and measured its thickness.As a result show
Show that the ultra-thin amnion of individual layer for not planting cell is about 35 ± 6 μ m-thicks (Fig. 5 B), " sandwich " structure with cell among two layers
About 80 ± 10 μ m thicks (Fig. 5 D), three layers of ultra-thin amnion system thickness are 198 ± 18 μm (Fig. 5 F), four layers of ultra-thin amnion system
Thickness of uniting is about 250 ± 18 μm (Fig. 5 H).Therefore, the extracellular matrix thickness of average one layer of stroma cell secretion is about 30 μM.
Result displays that the homogenieity of the ultra-thin amnion system of holostrome at the same time, prompt can able person's corneal stroma stem cell secretion extracellular base
Matter is merged with ultra-thin amnion stroma well, or cell secretion extracellular matrix and ultra-thin amnion stroma with similar
Density.
6th, the micro-structure of extracellular matrix
6.1 scanning electron microscope
Scanning electron microscope, which is shown between ultra-thin amnion surface fiber queueing discipline, fiber, the consistent linear channel in direction
Structure.Figure Fig. 6 A show have the fine and close, collagenous fiber bundle of queueing discipline to be deposited on ultra-thin amnion surface (figure between cell
6B).At other positions of identical sample, Fig. 6 C, D show the collagenous fibres that cell surface is generated along cell major axis, fiber
Between it is parallel to each other.
Keratocyte only generates some sparse extracellular matrixs on the cover slip, and fiber direction is mixed and disorderly
(Fig. 6 E, F).In some compact areas, fiber alignment is without rule, the design feature (Fig. 6 G, H) with cicatricial tissue.
Multilayer amnion tissue lower surface is inoculated with endothelial cell, shows that cell is completely embedded, cell is in hexagonal under scanning electron microscope
Shape or polygon, surface have microvillus.
6.2 Hematoxylin-eosins dye
After different layers ultra-thin 8 week of amnion systematic cultivation, Hematoxylin-eosin dyeing and nuclear targeting are shown, single
The ultra-thin amnion of layer does not have cell in itself, and the cell after inoculation is evenly distributed on after breeding, migrating in Multilayer ultrathin amnion system
(Fig. 7 A, C, E, G).
6.3 transmission electron microscope
The extracellular matrix internal structure of cell secretion is further looked at by transmission electron microscope.(Fig. 8 A, B, C) and
(Fig. 8 D, E, F) shows people's corneal stroma and the arrangement of amnion stroma collagen respectively, it is mainly characterized by collagenous fibres with just intersecting
Mode is arranged in the band (also referred to as sheets of fibres) of tight spacing.People's cornea and collagen membrane fibre diameter be respectively 29 ±
5.7nm and 36.5 ± 8.4nm, fiber spacing are respectively 55.1 ± 7.4nm, 43.8 ± 6.5nm.External people's keratocyte exists
It is distributed in Multilayer ultrathin amnion system in the cell process of tabular morphology, its length in extracellular matrix (Fig. 8 G).Under high power lens,
Collagenous fibres in a manner of just intersecting close-packed arrays into sheets of fibres (Fig. 8 H, I), collagenous fibres diameter (36.3 ± 6nm) and fiber
Spacing (45.0 ± 5.8nm) uniformity, it is more slightly larger than people's corneal stroma fibre diameter, fiber gap ratio corneal stroma is small, but
It is similar to spacing to collagen membrane fibre diameter.
The foregoing is only a preferred embodiment of the present invention, therefore cannot limit the scope that the present invention is implemented according to this, i.e.,
The equivalent changes and modifications made according to the scope of the claims of the present invention and description, all should still belong in the range of the present invention covers.
Claims (9)
- A kind of 1. tissue engineering comea, it is characterised in that：Including at least three amnion tissue layers, two adjacent amnion tissue interlayers life Keratocan, lumican, I types are included with keratocyte, and containing what is secreted by above-mentioned keratocyte The extracellular matrix of collagen and collagen type v albumen, and above-mentioned extracellular matrix queueing discipline, formation are similar to corneal stroma Institutional framework.Corneal epithelial cell is inoculated with respectively in this structure upper and lower surface and endothelial cell forms full-shape Film.The arrangement of collagen fibers rule on the surface of above-mentioned amnion tissue layer, is parallel to each other between fiber, has a groove structure, and with Just intersecting close-packed arrays are above-mentioned to guide the extracellular matrix of keratocyte and its secretion regularly arranged into sheets of fibres At least three amnion tissue layers, which have, suppresses the characteristic of fibrosis to keep the characteristic of keratocyte and to suppress corneal stroma thin Born of the same parents break up to fibroblast.
- A kind of 2. tissue engineering comea as claimed in claim 1, it is characterised in that：The keratocyte is by being inoculated in State the differentiation of the stem cell between at least three amnion tissue layers to form, which includes corneal stroma stem cell, mesenchyma is done carefully Born of the same parents, the stem cell of neural crest origin and embryonic stem cell.
- A kind of 3. tissue engineering comea as claimed in claim 1, it is characterised in that：The amnion tissue layer for holostrome amnion or The compacted zone retained after protease digestion processing removes spongy layer and lamina reticularis.
- A kind of 4. tissue engineering comea as claimed in claim 1, it is characterised in that：At least three amnion tissue layers it is most upper The upper surface of the amnion tissue layer of layer, which is inoculated with and grows, corneal epithelial cell.
- A kind of 5. tissue engineering comea as claimed in claim 4, it is characterised in that：The corneal epithelial cell is from primary The angle that the corneal epithelial cell of culture, the corneal epithelial cell of secondary culture, Characteristic Analysis of Corneal Epithelial Cell Line or stem cell differentiate Film epithelium substitutes cell.
- A kind of 6. tissue engineering comea as claimed in claim 1, it is characterised in that：At least three amnion tissue layers it is most lower The lower surface of the amnion tissue layer of layer, which is inoculated with and grows, endothelial cell.
- A kind of 7. tissue engineering comea as claimed in claim 6, it is characterised in that：The endothelial cell is from primary The angle that the endothelial cell of culture, the endothelial cell of secondary culture, corneal endothelium cell system or stem cell differentiate Film endothelium substitutes cell.
- A kind of 8. tissue engineering comea as claimed in claim 1, it is characterised in that：The oxygen content of its culture environment for 5%~ 15%.
- A kind of 9. tissue engineering comea as claimed in claim 1, it is characterised in that：Culture medium used in it be include it is nonessential Amino acid, fibroblast growth factor 2 and transforming growth factor β 3 have blood serum medium or serum free medium.
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| CN201711347993.XA CN107929811A (en) | 2017-12-15 | 2017-12-15 | A kind of tissue engineering comea |
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| CN111235109A (en) * | 2019-12-26 | 2020-06-05 | 爱尔眼科医院集团股份有限公司 | Culture medium, corneal stroma tablet prepared from culture medium and preparation method |
| CN111235109B (en) * | 2019-12-26 | 2022-01-11 | 爱尔眼科医院集团股份有限公司 | Culture medium, corneal stroma tablet prepared from culture medium and preparation method |
| CN114259601A (en) * | 2021-12-27 | 2022-04-01 | 暨南大学 | Viable cell bionic cornea anterior lamina layer and preparation method thereof |
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