CN109363801B - Method for preparing porcine corneal endothelial implant under assistance of femtosecond laser technology - Google Patents
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- 230000003511 endothelial effect Effects 0.000 title claims abstract description 38
- 239000007943 implant Substances 0.000 title claims abstract description 15
- 238000000034 method Methods 0.000 title claims abstract description 10
- 238000005516 engineering process Methods 0.000 title claims abstract description 9
- 210000004087 cornea Anatomy 0.000 claims abstract description 21
- 230000008961 swelling Effects 0.000 claims abstract description 8
- 238000005422 blasting Methods 0.000 claims abstract description 7
- 238000010257 thawing Methods 0.000 claims abstract description 7
- 238000005520 cutting process Methods 0.000 claims abstract description 6
- 210000000871 endothelium corneal Anatomy 0.000 claims abstract description 6
- 238000007605 air drying Methods 0.000 claims abstract description 5
- 241000446313 Lamella Species 0.000 claims abstract description 4
- 230000002452 interceptive effect Effects 0.000 claims abstract description 4
- 238000002309 gasification Methods 0.000 claims abstract description 3
- 238000004519 manufacturing process Methods 0.000 claims abstract description 3
- 238000002360 preparation method Methods 0.000 claims description 11
- 210000001519 tissue Anatomy 0.000 claims description 11
- 239000001963 growth medium Substances 0.000 claims description 10
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 10
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims description 6
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 claims description 6
- 239000012894 fetal calf serum Substances 0.000 claims description 6
- 238000007710 freezing Methods 0.000 claims description 6
- 230000008014 freezing Effects 0.000 claims description 6
- 239000000243 solution Substances 0.000 claims description 6
- 239000008223 sterile water Substances 0.000 claims description 6
- 239000008055 phosphate buffer solution Substances 0.000 claims description 5
- 238000002791 soaking Methods 0.000 claims description 4
- 210000003560 epithelium corneal Anatomy 0.000 claims description 3
- 238000004108 freeze drying Methods 0.000 claims description 3
- 230000001954 sterilising effect Effects 0.000 claims description 3
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- 238000005406 washing Methods 0.000 claims description 3
- 238000004659 sterilization and disinfection Methods 0.000 claims description 2
- 239000003826 tablet Substances 0.000 claims description 2
- 238000012014 optical coherence tomography Methods 0.000 claims 2
- 238000002054 transplantation Methods 0.000 abstract description 13
- 210000003683 corneal stroma Anatomy 0.000 abstract description 4
- 230000005847 immunogenicity Effects 0.000 abstract description 3
- 238000000053 physical method Methods 0.000 abstract description 2
- 238000002591 computed tomography Methods 0.000 description 4
- 210000000399 corneal endothelial cell Anatomy 0.000 description 4
- 238000000338 in vitro Methods 0.000 description 4
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- 230000002980 postoperative effect Effects 0.000 description 4
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- 102000008186 Collagen Human genes 0.000 description 3
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- 210000004027 cell Anatomy 0.000 description 3
- 229920001436 collagen Polymers 0.000 description 3
- 201000009310 astigmatism Diseases 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 230000004410 intraocular pressure Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 102000010834 Extracellular Matrix Proteins Human genes 0.000 description 1
- 108010037362 Extracellular Matrix Proteins Proteins 0.000 description 1
- 206010030113 Oedema Diseases 0.000 description 1
- 206010053159 Organ failure Diseases 0.000 description 1
- 229930182555 Penicillin Natural products 0.000 description 1
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 1
- QTENRWWVYAAPBI-YZTFXSNBSA-N Streptomycin sulfate Chemical compound OS(O)(=O)=O.OS(O)(=O)=O.OS(O)(=O)=O.CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@H]1[C@H](N=C(N)N)[C@@H](O)[C@H](N=C(N)N)[C@@H](O)[C@@H]1O.CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@H]1[C@H](N=C(N)N)[C@@H](O)[C@H](N=C(N)N)[C@@H](O)[C@@H]1O QTENRWWVYAAPBI-YZTFXSNBSA-N 0.000 description 1
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- 201000004180 corneal endothelial dystrophy Diseases 0.000 description 1
- 238000011161 development Methods 0.000 description 1
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- 210000003038 endothelium Anatomy 0.000 description 1
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- 238000011081 inoculation Methods 0.000 description 1
- 238000002844 melting Methods 0.000 description 1
- 230000008018 melting Effects 0.000 description 1
- 208000030159 metabolic disease Diseases 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 230000000149 penetrating effect Effects 0.000 description 1
- 229940049954 penicillin Drugs 0.000 description 1
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- 230000000007 visual effect Effects 0.000 description 1
Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61F—FILTERS IMPLANTABLE INTO BLOOD VESSELS; PROSTHESES; DEVICES PROVIDING PATENCY TO, OR PREVENTING COLLAPSING OF, TUBULAR STRUCTURES OF THE BODY, e.g. STENTS; ORTHOPAEDIC, NURSING OR CONTRACEPTIVE DEVICES; FOMENTATION; TREATMENT OR PROTECTION OF EYES OR EARS; BANDAGES, DRESSINGS OR ABSORBENT PADS; FIRST-AID KITS
- 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
- A61F2/142—Cornea, e.g. artificial corneae, keratoprostheses or corneal implants for repair of defective corneal tissue
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61F—FILTERS IMPLANTABLE INTO BLOOD VESSELS; PROSTHESES; DEVICES PROVIDING PATENCY TO, OR PREVENTING COLLAPSING OF, TUBULAR STRUCTURES OF THE BODY, e.g. STENTS; ORTHOPAEDIC, NURSING OR CONTRACEPTIVE DEVICES; FOMENTATION; TREATMENT OR PROTECTION OF EYES OR EARS; BANDAGES, DRESSINGS OR ABSORBENT PADS; FIRST-AID KITS
- A61F2240/00—Manufacturing or designing of prostheses classified in groups A61F2/00 - A61F2/26 or A61F2/82 or A61F9/00 or A61F11/00 or subgroups thereof
- A61F2240/001—Designing or manufacturing processes
- A61F2240/002—Designing or making customized prostheses
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- Oral & Maxillofacial Surgery (AREA)
- Heart & Thoracic Surgery (AREA)
- Vascular Medicine (AREA)
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Abstract
The invention discloses a method for preparing a porcine corneal endothelial graft under the assistance of a femtosecond laser technology, which comprises the following steps: s1, pretreating the porcine cornea; s2, setting operation parameters of a femtosecond laser according to a pig cornea thickness value measured before the operation after entering a femtosecond laser machine interactive interface, accurately focusing the laser on a set corneal surface, flattening, performing laser blasting and gasification cutting to finish the manufacture of a corneal posterior lamella implant, wherein the corneal endothelial plate with a posterior elastic layer has the thickness of 100 mu m and the diameter of 8.5 mm; s3, carrying out cell removal operation by physical methods such as swelling, repeated freeze thawing, shaking, air drying and the like; s4, preparing a porcine corneal endothelial implant. The invention utilizes the instantaneous laser blasting technology to finish the accurate cutting of the cornea, reduces the immunogenicity of the porcine corneal stroma slice, and simultaneously can prepare the ultrathin porcine corneal endothelium graft with the rear elastic layer, thereby greatly reducing the transplantation difficulty caused by uneven thickness.
Description
Technical Field
The invention relates to the field of preparation of corneal endothelium, in particular to a method for preparing a porcine corneal endothelium implant under the assistance of a femtosecond laser technology.
Background
In recent years, great progress has been made in Tissue Engineering (TE) corneas, which have attracted attention because of their ability to alleviate organ failure and transplant donor organ shortage to a great extent, and because human corneal endothelial cells cannot be repaired after being damaged, it is important how to establish a corneal endothelial cell tissue in vitro and perform a biological function similar to that of corneal endothelial cells to treat patients with endothelial blindness. Corneal endothelial transplantation, which is a replacement of diseased endothelium with healthy donor tissue, is widely regarded as the only effective option for treating corneal endothelial dystrophy, and at present, improvement of surgical modes such as penetrating corneal transplantation, DSEK, DMEK and the like needs to provide a corneal donor, so that the problem of donor shortage cannot be fundamentally solved, endothelial cell transplantation becomes a research hotspot in recent years, and in-vitro culture of autologous cells or allogeneic cells is greatly advanced.
With the rapid development and popularization of refractive surgery, the quality of possible cornea material donors in the future can not be guaranteed, the failure rate of cornea transplantation finally reaches more than 18 percent due to the unstable quality of donor grafts, immune rejection reaction or endothelial metabolic disorder after transplantation and the like, the immunogenicity of acellular porcine cornea matrixes is very low, the original limbal extracellular matrix microenvironment can be well reserved, APCM has been proved to have biomechanical properties suitable for being used as one of the choices of tissue engineering scaffolds, most functions of corneal tissues can be provided, and the strength, toughness and tissue compatibility brought by densely arranged collagen make the porcine cornea matrixes become the focus of attention of most researchers in tissue engineering, so the porcine cornea matrixes are selected as the experimental cell transplantation carriers.
Meanwhile, on the other hand, the femtosecond laser-assisted suture anterior lamellar corneal transplantation is proved to be safe and effective for treating the superficial corneal diseases, and a patient can obtain a better visual effect after the operation, not only in the field of refraction, but also the femtosecond laser-assisted corneal transplantation is greatly developed, because the falling of corneal endothelial cells is very easily caused in the operation processes of cell culture, inoculation and the like, compared with the method of preparing APCM by artificial cutting, the needed cutting thickness and layer can be determined by accurately measuring the corneal thickness before the APCM prepared by femtosecond assistance, so that the surface is smoother, the characteristics of the cornea can be approached to the next step of transplantation as far as possible, and the influences of transplantation difficulty, postoperative intraocular pressure measurement distortion, postoperative astigmatism and the like caused by uneven implant thickness are reduced.
Disclosure of Invention
In order to solve the problems, the invention provides a method for preparing a porcine corneal endothelial implant under the assistance of a femtosecond laser technology.
In order to achieve the purpose, the invention adopts the technical scheme that:
a method for preparing a porcine corneal endothelial implant assisted by a femtosecond laser technology comprises the following steps:
s1, pretreatment of porcine cornea: repeatedly washing and soaking a transparent pig cornea by PBS (phosphate buffer solution) containing double antibodies, selecting a pig eye with complete corneal epithelium and no scratch, observing the anterior segment of the eye by using a slit lamp conventionally, measuring the central corneal thickness by using a corrvis corneal biomechanics analyzer and anterior segment 0CT (computed tomography), and checking the corneal curvature by using a corneal topography;
s2 femtosecond laser-assisted preparation of corneal endothelial sheet
Setting operation parameters of a femtosecond laser according to a preoperatively measured pig cornea thickness value after entering a femtosecond laser machine interactive interface, accurately focusing the laser on a set corneal surface, flattening, performing laser blasting and gasification cutting, and finishing the manufacture of a corneal posterior lamina implant, wherein the corneal endothelial plate is 100 microns thick and 8.5mm in diameter and is provided with a rear elastic layer;
s3 preparation of acellular porcine corneal endothelium tablet
Under the aseptic condition, quickly transferring the prepared corneal endothelial sheet to a laboratory, placing the corneal endothelial sheet in sterile water containing double antibodies for swelling for 12h at 4 ℃, freezing for 1h at-80 ℃, thawing for 30min in a water bath kettle at 37 ℃, repeatedly freezing and thawing for 3 times to fully swell tissues, shaking a 0.5% SDS centrifuge tube for 24h at 4 ℃, rinsing with sterile water for at least 3 times, placing the corneal endothelial surface upwards on an ultraclean workbench for air drying, sterilizing for 12h by ultraviolet disinfection, and freeze-drying to obtain a decellularized porcine corneal endothelial sheet for later use;
s4, preparation of the porcine corneal endothelial implant:
and flatly paving the acellular porcine corneal endothelial slices in a 24-hole culture plate, adding a DMEM culture medium containing 5% fetal calf serum for rehydration, immersing, standing overnight in an incubator, observing the corneal endothelial slice swelling the next day, completely sucking the culture medium, and adding a freshly prepared DMEM culture medium containing 10% fetal calf serum, namely completing the preparation of the porcine corneal endothelial graft.
Adding a double-antibody solution into all the prepared solutions in the step S3 for avoiding pollution: 200u/ml penicillin and 200 u/streptomycin solution.
The invention has the following beneficial effects:
the cornea can be accurately cut by utilizing the instantaneous laser blasting technology, the obtained corneal endothelial sheet is thinner, and the complications such as transplantation difficulty caused by uneven thickness, postoperative intraocular pressure distortion, postoperative astigmatism and the like are greatly reduced. Meanwhile, the ultra-thin porcine corneal endothelial implant prepared by the femtosecond laser assistance can show good biocompatibility and is suitable for wide popularization and application.
The invention is different from other acellular porcine corneal stroma, a porcine corneal posterior elastic layer is reserved when materials are taken, documents prove that the posterior elastic layer is a support for good cell adhesion and growth, the acellular porcine corneal stroma on the market at home at present can only be used for lamellar transplantation, the blindness problem cannot be solved for patients with endothelial lesion, and the acellular porcine corneal stroma sheet with the posterior elastic layer can play an important role in constructing a tissue engineering cornea in vitro to treat patients with endothelial blindness.
The invention carries out the decellularization operation by physical methods of swelling, repeated freeze thawing, shaking, air drying and the like, can greatly reduce the immunogenicity of the cornea stroma collagen when completely preserving the cornea stroma collagen, and simultaneously, the porcine corneal endothelial implant prepared under the assistance of femtosecond laser is thinner, and the edema degree of the material obtained by soaking in a culture medium during in vitro culture is lighter than that of the material obtained by a traditional keratome, thereby being more beneficial to planting culture and observation under a microscope.
Detailed Description
The present invention will be described in detail with reference to specific examples. The following examples will assist those skilled in the art in further understanding the invention, but are not intended to limit the invention in any way. It should be noted that variations and modifications can be made by persons skilled in the art without departing from the spirit of the invention. All falling within the scope of the present invention.
Example 1
S1, repeatedly washing and soaking the transparent pig cornea with PBS containing double antibodies, selecting the pig eye with complete corneal epithelium and no scratch, placing the pig eye in an ice box for storage, observing the anterior segment of the eye by using a slit lamp conventionally, measuring the central corneal thickness by using a corrvis corneal biomechanics analyzer and a 0CT (computed tomography) anterior segment of the eye, and checking the corneal curvature by using a corneal topography.
S2, entering a femtosecond laser machine interactive interface to set processing parameters, flattening and performing laser blasting after laser is accurately gathered on a set corneal surface, taking an operation incision of about 3mm at the corneal scleral edge after the laser blasting, taking out the corneal endothelium layer with the rear elastic layer with the thickness of 100 and 200um, and rapidly transferring to a laboratory under an aseptic condition;
s3, placing the prepared corneal lamella in sterile water containing double antibodies for swelling for 12h at 4 ℃, freezing for 1h at-80 ℃, melting for 30min in a water bath kettle at 37 ℃, and repeatedly freezing and thawing for 3 times to fully swell the tissue; shaking a 0.5% SDS centrifuge tube for 24h at 4 ℃, rinsing with sterile water for at least 3 times, placing the APCM with the rear elastic layer on a clean bench with the inner surface facing upwards, air-drying, sterilizing by ultraviolet rays for 12h, and freeze-drying for later use, wherein a double-antibody solution is added into all preparation solutions to avoid pollution.
S4, flatly paving the acellular porcine corneal endothelial sheet in a 24-hole culture plate, adding a DMEM culture medium containing 5% fetal calf serum for rehydration, at least immersing the sheet, standing overnight in an incubator, showing the swelling of the corneal endothelial sheet the next day, after completely absorbing the culture medium, adding a freshly prepared DMEM culture medium containing 10% fetal calf serum again, and completing the preparation of the porcine corneal endothelial sheet.
The foregoing description of specific embodiments of the present invention has been presented. It is to be understood that the present invention is not limited to the specific embodiments described above, and that various changes or modifications may be made by one skilled in the art within the scope of the appended claims without departing from the spirit of the invention. The embodiments and features of the embodiments of the present application may be combined with each other arbitrarily without conflict.
Claims (1)
1. A method for preparing a porcine corneal endothelial graft assisted by a femtosecond laser technology is characterized by comprising the following steps: the method comprises the following steps:
s1, pretreatment of porcine cornea: repeatedly washing and soaking a transparent pig cornea by PBS (phosphate buffer solution) containing double antibodies, selecting a pig eye with complete corneal epithelium and no scratch, observing the anterior segment of the eye by using a slit lamp, measuring the central corneal thickness by using a corrvis corneal biomechanics analyzer and anterior segment OCT (optical coherence tomography), and checking the corneal curvature by using a corneal topography;
s2 femtosecond laser-assisted preparation of corneal endothelial sheet
Setting operation parameters of a femtosecond laser according to a preoperatively measured pig cornea thickness value after entering a femtosecond laser machine interactive interface, accurately focusing the laser on a set corneal surface, flattening, performing laser blasting and gasification cutting, and finishing the manufacture of a corneal posterior lamella implant, wherein the corneal posterior lamella implant is a corneal endothelial sheet with a thickness of 100 mu m and a diameter of 8.5mm and a posterior elastic layer;
s3 preparation of acellular porcine corneal endothelium tablet
Under the aseptic condition, quickly transferring the prepared corneal endothelial sheet to a laboratory, placing the corneal endothelial sheet in sterile water containing double antibodies for swelling for 12h at 4 ℃, freezing for 1h at-80 ℃, thawing for 30min in a water bath kettle at 37 ℃, repeatedly freezing and thawing for 3 times to fully swell tissues, shaking a 0.5% SDS centrifuge tube for 24h at 4 ℃, rinsing with sterile water for at least 3 times, placing the corneal endothelial surface upwards on an ultraclean workbench for air drying, sterilizing for 12h by ultraviolet disinfection, and freeze-drying to obtain a decellularized porcine corneal endothelial sheet for later use; adding a double-antibody solution into all the prepared solutions in the step S3 to avoid pollution;
s4, preparation of the porcine corneal endothelial implant:
flatly paving the acellular porcine corneal endothelial slices in a 24-hole culture plate, adding a DMEM culture medium containing 5% fetal calf serum for rehydration, immersing, standing overnight in an incubator, observing the acellular porcine corneal endothelial slice swelling the next day, completely sucking the culture medium, adding a freshly prepared DMEM culture medium containing 10% fetal calf serum, and thus completing the preparation of the porcine corneal endothelial plant.
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Granted publication date: 20210126 |