CN106267351B - Graft for posterior elastic layer corneal endothelium transplantation and mechanical preparation method thereof - Google Patents
Graft for posterior elastic layer corneal endothelium transplantation and mechanical preparation method thereof Download PDFInfo
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- CN106267351B CN106267351B CN201610885272.3A CN201610885272A CN106267351B CN 106267351 B CN106267351 B CN 106267351B CN 201610885272 A CN201610885272 A CN 201610885272A CN 106267351 B CN106267351 B CN 106267351B
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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
- 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 graft for corneal endothelium transplantation of a posterior elastic layer and a mechanical preparation method thereof. The preparation method of the graft provided by the invention comprises the following steps: the porcine cornea is used as a raw material, and mechanical separation is carried out to obtain the graft consisting of a rear elastic layer and an endothelial cell layer. The invention takes the porcine eyeball as a raw material, prepares the graft consisting of the posterior elastic layer and the endothelial cell layer, is expected to effectively relieve the current situation that the corneal endothelial donor of the human is extremely deficient at present, brings the remainders hope for a large number of patients with corneal blindness, has great social value, very great industrialization and commercialization values and brings considerable economic value after being put into the clinical market.
Description
Technical Field
The invention relates to a graft for posterior elastic layer corneal endothelium transplantation and a mechanical preparation method thereof, in particular to a graft for posterior elastic layer corneal endothelium transplantation prepared from pig cornea and a mechanical preparation method thereof.
Background
According to statistics, 700000 patients who are blinded due to serious corneal diseases currently exist in China, and corneal blindness has become the second treatable blindness. Corneal transplantation is the only means of patient recovery. Approximately 50% of patients can be identified by corneal Endothelial transplantation (EK). As a recent corneal graft, the posterior elastic layer corneal graft (DMEK) has many advantages that other EKs do not have, and thus has a wide clinical application prospect. However, the lack of human corneal donor sources severely restricts the development of DMEK, and thus the search for alternative bioactive materials is inevitable.
Unlike traditional Penetrating Keratoplasty (PKP) which transplants a full layer of the cornea to the donor, EK only transplants the endothelial layer of the donor cornea to the patient, which largely remedies some of the inevitable drawbacks of PKP. EK mode is continuously innovated, so that clinical application prospect is wider, and the latest DMEK has the unique advantages of good postoperative visual quality recovery, very light postoperative inflammatory reaction and lower immunological rejection rate. The DMEK requires a graft comprising only a posterior elastic layer and a corneal endothelial layer, which has the characteristics of thin thickness (about 15 μm), fragility, easy breaking, easy curling, etc., and thus, it is difficult to completely separate the graft from the corneal stroma layer. On the other hand, due to the influence of traditional concepts, the restriction of laws and regulations, the shortage of publicity and education work donated by the public, etc., the present situation that the corneal donor source is extremely deficient in China also severely restricts the development of the DMEK, and because the porcine cornea has wide sources and the physical and optical properties and the shape and the function of endothelial cells are similar to those of the human cornea, the porcine cornea is the best choice for replacing the human corneal endothelium at present.
The cornea is divided into five layers which are sequentially from front to back: epithelial cell layer (Epithelium), pro-elastic layer (BowmanMembrane), stromal layer (Stroma), post-elastic layer (DM), and endothelial cell layer (Endothelium). The epithelial cell layer is about 50 μm thick and 10% of the total corneal thickness, and is composed of 5-6 layers of epithelial cells, the epithelial cell layer in the peripheral part of the cornea is thickened, and the cells are increased to 8-10 layers. The front elastic layer is a homogeneous transparent film without cell components, and is mainly composed of collagen fibers as shown by electron microscope observation. The stromal layer consists of about 200 sheets of collagen fiber bundles parallel to the corneal surface, about 500 μm thick, accounting for 90% of the total corneal thickness. The rear elastic layer is a tough transparent homogeneous film, is rich in elasticity and has strong resistance to chemical substances and pathological damage. The endothelial cell layer is composed of a layer of hexagonal flat cells and has the function of cornea-aqueous humor barrier.
Disclosure of Invention
The invention aims to provide a graft for posterior elastic layer corneal endothelium transplantation and a mechanical preparation method thereof, and particularly relates to a graft for posterior elastic layer corneal endothelium transplantation prepared from pig cornea and a mechanical preparation method thereof.
The invention provides a preparation method of a graft taking a porcine cornea as a material, which comprises the following steps:
(1) taking an isolated porcine eyeball, and cutting a cornea with a scleral ring at the outer edge;
(2) after the step (1) is finished, removing the trabecular meshwork;
(3) after the step (2) is completed, removing part or all of the trabeculae and exposing the edge of the rear elastic layer;
(4) marking the boundary of the circular cutting area after the step (3) is finished;
(5) after the step (4) is completed, gradually separating a membrane consisting of the rear elastic layer and the endothelial cell layer from the cornea from the middle of the rear elastic layer and the stroma layer to the center direction from the edge of the cornea until exceeding the boundary of the circular cutting area;
(6) after the step (5) is completed, resetting the membrane formed by the rear elastic layer and the endothelial cell layer;
(7) after the step (6) is finished, cutting along the boundary of the circular cutting area to obtain a circular material;
(8) and (3) taking the circular material, gradually separating the membrane consisting of the rear elastic layer and the endothelial cell layer along the edge at the rising edge between the rear elastic layer and the matrix layer which are separated in the step (5), and obtaining the pig DMEK graft consisting of the rear elastic layer and the endothelial cell layer.
In the step (1), the sclera in the cornea with the scleral ring at the outer edge is a part of the sclera.
In the step (1), the cornea with the scleral ring at the outer edge can be a cornea with a scleral ring at the outer edge of 1-2 mm.
In step (2), trypan blue staining may be performed first, and then the trabecular meshwork is removed (specifically, scraped).
In the step (2), the trypan blue staining can be performed on the whole cornea with a 1-2 mm scleral ring, or can be performed on the scleral groove of the cornea with a 1-2 mm scleral ring.
The step (2) also comprises the following steps: after trypan blue staining, the cornea was rinsed with PBS buffer before removal of the trabecular meshwork. The PBS buffer may specifically be PBS pH7.4basic (1X).
In the step (3), trypan blue staining may be performed first, and then a part or all of the trabeculae may be removed (specifically, torn off).
In the step (3), the exposed edge of the rear elastic layer accounts for more than one fifth, preferably one third to two thirds, and more preferably one half of the entire edge of the rear elastic layer.
The step (5) is as follows: after the completion of the step (4), the membrane composed of the posterior elastic layer and the endothelial cell layer is gradually separated from the cornea from the edge of the cornea toward the center from the middle of the posterior elastic layer and the stromal layer until reaching a position of one third to one half of the diameter of the circular cutting area.
The invention also provides a preparation method of the graft taking the porcine cornea as the material, which comprises the following steps:
(1) taking an isolated porcine eyeball, and cutting a cornea with a scleral ring at the outer edge;
(2) after the step (1) is finished, removing the trabecular meshwork;
(3) after the step (2) is completed, removing part or all of the trabeculae and exposing the edge of the rear elastic layer;
(4) after the step (3) is completed, marking the boundary of the cutting area;
(5) after the step (4) is finished, gradually separating a membrane consisting of the rear elastic layer and the endothelial cell layer from the cornea from the middle of the rear elastic layer and the stroma layer to the center direction from the edge of the cornea until the membrane exceeds the boundary of the cutting area;
(6) after the step (5) is completed, resetting the membrane formed by the rear elastic layer and the endothelial cell layer;
(7) after the step (6) is finished, cutting along the boundary of the cutting area, and taking the material of the cutting area part;
(8) and (5) taking the material obtained in the step (7), and gradually separating a membrane consisting of the rear elastic layer and the endothelial cell layer along the edge from the middle rising edge of the rear elastic layer and the matrix layer separated in the step (5) to obtain the pig DMEK graft consisting of the rear elastic layer and the endothelial cell layer.
In the step (1), the sclera in the cornea with the scleral ring at the outer edge is a part of the sclera.
In the step (1), the cornea with the scleral ring at the outer edge can be a cornea with a scleral ring at the outer edge of 1-2 mm.
In the step (2), trypan blue staining can be firstly carried out, and then the trabecular meshwork is removed (specifically, scraping can be carried out);
in the step (2), the trypan blue staining can be performed on the whole cornea with a 1-2 mm scleral ring, or can be performed on the scleral groove of the cornea with a 1-2 mm scleral ring.
The step (2) also comprises the following steps: after trypan blue staining, the cornea was rinsed with PBS buffer before removal of the trabecular meshwork. The PBS buffer may specifically be PBS pH7.4basic (1X).
In the step (3), trypan blue staining may be performed first, and then a part or all of the trabeculae may be removed (specifically, torn off).
In the step (3), the exposed edge of the rear elastic layer accounts for more than one fifth, preferably one third to two thirds, and more preferably one half of the entire edge of the rear elastic layer.
The step (5) is as follows: after the step (4) is completed, the membrane composed of the posterior elastic layer and the endothelial cell layer is gradually separated from the cornea from the edge of the cornea toward the center from the middle of the posterior elastic layer and the stroma until the separation region covers one fifth to three fifths of the area of the cutting region.
The invention also provides a preparation method of the graft taking the porcine cornea as the material, which comprises the following steps:
(1) taking an isolated porcine eyeball, and cutting a cornea with a scleral ring at the outer edge;
(2) after the step (1) is finished, removing the trabecular meshwork;
(3) after the step (2) is completed, removing part or all of the trabeculae and exposing the edge of the rear elastic layer;
(4) and (4) after the step (3) is completed, separating the membrane consisting of the rear elastic layer and the endothelial cell layer to obtain the pig DMEK graft consisting of the rear elastic layer and the endothelial cell layer.
In the step (1), the sclera in the cornea with the scleral ring at the outer edge is a part of the sclera.
In the step (1), the cornea with the scleral ring at the outer edge can be a cornea with a scleral ring at the outer edge of 1-2 mm.
In the step (2), trypan blue staining can be firstly carried out, and then the trabecular meshwork is removed (specifically, scraping can be carried out);
in the step (2), the trypan blue staining can be performed on the whole cornea with a 1-2 mm scleral ring, or can be performed on the scleral groove of the cornea with a 1-2 mm scleral ring.
The step (2) also comprises the following steps: after trypan blue staining, the cornea was rinsed with PBS buffer before removal of the trabecular meshwork. The PBS buffer may specifically be PBS pH7.4basic (1X).
In the step (3), trypan blue staining may be performed first, and then a part or all of the trabeculae may be removed (specifically, torn off).
In the step (3), the exposed edge of the rear elastic layer accounts for more than one fifth, preferably one third to two thirds, and more preferably one half of the entire edge of the rear elastic layer.
The invention also provides a preparation method of the graft taking the porcine cornea as the material, which comprises the following steps: the porcine cornea is used as a raw material, and mechanical separation is carried out to obtain the graft consisting of a rear elastic layer and an endothelial cell layer.
Any of the above-described cutting methods may be performed using a trephine.
The isolated porcine eyeball of any of the above is preferably fresh.
The graft prepared by any method described above also belongs to the protection scope of the invention.
The invention also protects the application of the graft in the preparation of products for the corneal endothelium transplantation of the posterior elastic layer.
The invention also protects a product for posterior elastic layer corneal endothelium transplantation, comprising the graft.
The invention also protects the application of the graft as a graft for posterior elastic layer corneal endothelium transplantation.
The invention combines the technical advantages of the DMEK operation and the huge potential of the pig cornea as a potential donor, and invents the mechanical separation preparation technology of the DMEK endothelial tissue sheet suitable for the structural characteristics of the pig cornea tissue for the first time so as to be used for the human posterior elastic layer corneal transplantation.
The graft provided by the invention has the following advantages for the corneal endothelium transplantation of the posterior elastic layer: 1) the porcine corneal endothelial tissue contains far fewer xenoantigens and antigen presenting cells than the whole layer and the lamellar layer, thereby avoiding the occurrence of hyperacute and delayed rejection to a great extent; 2) porcine corneal endothelial tissue, which is completely in aqueous humor, will be protected by a series of cytokines and mechanisms in the anterior chamber that inhibit the development of immune rejection (the state of immune excursion in the anterior chamber); 3) the endothelial graft does not require suture fixation, which avoids the possibility of neovascular rejection due to suture stimulation.
Pigs, the most common livestock, are widely available and inexpensive for clinical use. The invention takes the porcine eyeball as a raw material, prepares the graft consisting of the posterior elastic layer and the endothelial cell layer, is expected to effectively relieve the current situation that the corneal endothelial donor of the human is extremely deficient at present, brings the remainders hope for a large number of patients with corneal blindness, has great social value, very great industrialization and commercialization values and brings considerable economic value after being put into the clinical market.
Drawings
FIG. 1 is a photograph of step 1 of example 1.
FIG. 2 is a photograph of step 2 of example 1.
FIG. 3 is a photograph of step 4 of example 1.
FIG. 4 is a photograph of step 5 of example 1.
FIG. 5 is a photograph of step 6 of example 1.
FIG. 6 is a photograph of step 7 of example 1.
FIG. 7 is a photograph of the peeling process in step 8 of example 1.
FIG. 8 is a photograph of example 1 after complete peeling in step 8.
Detailed Description
The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged.
PBS buffer: PBS pH7.4basic (1X); gibco by Life technologiesTM。
Examples 1,
1. Taking out the separated pig eyeball, and cutting out the cornea with the outer edge provided with a scleral ring of 1-2 mm. The photograph is shown in FIG. 1 (endothelial cell layer placed up).
2. After completion of step 1, trypan blue staining was performed on the scleral groove (in practice, trypan blue staining was also performed on the whole cornea obtained in step 1), followed by washing with PBS buffer, and then scraping off the trabecular meshwork (with pigments attached thereto). The photograph after trypan blue staining is shown in FIG. 2 (endothelial cell layer placed up).
3. After step 2 is completed, trypan blue staining is performed, part or all of the trabeculae are torn off, and the edge of the rear elastic layer is exposed (the exposed edge of the rear elastic layer accounts for one half of the edge of the whole rear elastic layer; in actual operation, the exposed edge of the rear elastic layer accounts for more than one fifth of the edge of the whole rear elastic layer, and the most preferable half of the edge of the rear elastic layer). Because the trabecula is tightly connected with the posterior elastic layer of the cornea and the trabecula is tightly connected with the stroma layer of the cornea, when the trabecula is torn off, part of the posterior elastic layer and part of the stroma layer connected with the trabecula are torn off.
4. And (4) after the step 3 is finished, lightly pressing by using a trephine to display an annular indentation, namely the boundary needing to be cut in subsequent operation. The photograph is shown in FIG. 3 (endothelial cell layer placed up) with the arrows marking the circular indentations.
5. After step 4 was completed, the membrane consisting of the posterior elastic layer and the endothelial cell layer was carefully edged with a scleral tunnel knife (separation between the posterior elastic layer and the stroma layer), held with forceps, and slowly, gently and forcefully torn off evenly from the edge toward the center (tear-off area is 1/3, which is the diameter of the circular indentation; in practice, the tear-off area is larger on the basis that the posterior elastic layer does not break, but the posterior elastic layer is easily broken, and therefore the tear-off area is generally no more than 1/2, which is the diameter of the circular indentation). The photograph is shown in FIG. 4 (endothelial cell layer placed up).
6. After step 5 is completed, the membrane consisting of the torn-off post-elastic layer and the endothelial cell layer is repositioned. The photograph is shown in FIG. 5 (endothelial cell layer placed up).
7. After step 6 is completed, cutting at the annular indentation with a trephine. The photograph is shown in FIG. 6 (endothelial cell layer placed up).
8. After step 7, the membrane consisting of the rear elastic layer and the endothelial cell layer (starting from the original tearing region) was held by forceps and torn off along the edge ring shape slowly, gently and forcibly, to obtain the porcine DMEK graft consisting of the rear elastic layer and the endothelial cell layer. The photograph during the tearing off is shown in fig. 7. The photograph after complete tearing off is shown in FIG. 8.
Three repeated experiments are carried out, the operation is carried out on 100 isolated porcine eyeball in each repeated experiment, and the yield of the porcine DMEK graft is 85-90%.
Comparative examples 1,
1. Taking out the separated pig eyeball, and cutting out the cornea with the outer edge provided with a scleral ring of 1-2 mm.
2. And (3) after the step 1 is finished, lightly pressing by using a trephine to display an annular indentation, namely the boundary needing to be cut in subsequent operation.
3. After completion of step 2, the above procedure was completely impossible to achieve with a scleral tunnel knife carefully edged (separation between the posterior elastic and stromal layers).
Examples 2,
The porcine DMEK grafts prepared in example 1 were taken and examined for cell density and proportion of viable cells.
The specific method comprises the following steps:
1. taking a pig DMEK transplant, and staining the pig DMEK transplant for 2min by adopting 0.2% trypan blue staining solution.
2. After completion of step 1, wash 2 times with PBS buffer.
3. After completion of step 2, staining was performed with 1% alizarin red S staining solution for 3 min.
4. After completion of step 3, wash 2 times with PBS buffer.
5. After completion of step 4, the cell density (3 high power fields were counted at random and the sum was averaged) and the proportion of viable cells were counted under a microscope.
The porcine DMEK grafts prepared in example 1 were taken and all met the following criteria: cell density>2500/mm2Ratio of viable cells>90％。
The graft prepared in example 1 was used for grafting the endothelium of the posterior elastic layer on rhesus monkeys, and the rhesus monkeys were observed continuously for half a year to have a good living condition, the edematous cornea was transparent, the position of the DMEK graft was normal, and no significant rejection was observed.
Claims (7)
1. A preparation method of a graft comprises the following steps:
(1) taking an isolated porcine eyeball, and cutting a cornea with a scleral ring at the outer edge;
(2) after the step (1) is finished, removing the trabecular meshwork;
(3) after the step (2) is completed, removing part or all of the trabeculae and exposing the edge of the rear elastic layer;
(4) marking the boundary of the circular cutting area after the step (3) is finished;
(5) after the step (4) is completed, gradually separating a membrane consisting of the rear elastic layer and the endothelial cell layer from the cornea from the middle of the rear elastic layer and the stroma layer to the center direction from the edge of the cornea until exceeding the boundary of the circular cutting area;
(6) after the step (5) is completed, resetting the membrane formed by the rear elastic layer and the endothelial cell layer;
(7) after the step (6) is finished, cutting along the boundary of the circular cutting area to obtain a circular material;
(8) and (3) taking the circular material, gradually separating the membrane consisting of the rear elastic layer and the endothelial cell layer along the edge at the rising edge between the rear elastic layer and the matrix layer which are separated in the step (5), and obtaining the pig DMEK graft consisting of the rear elastic layer and the endothelial cell layer.
2. The method of claim 1, wherein: the step (5) is as follows: after the completion of the step (4), the membrane composed of the posterior elastic layer and the endothelial cell layer is gradually separated from the cornea from the edge of the cornea toward the center from the middle of the posterior elastic layer and the stromal layer until reaching a position of one third to one half of the diameter of the circular cutting area.
3. The method of claim 1 or 2, wherein: in the step (3), the exposed edge of the rear elastic layer accounts for more than one fifth of the edge of the whole rear elastic layer.
4. A preparation method of a graft comprises the following steps:
(1) taking an isolated porcine eyeball, and cutting a cornea with a scleral ring at the outer edge;
(2) after the step (1) is finished, removing the trabecular meshwork;
(3) after the step (2) is completed, removing part or all of the trabeculae and exposing the edge of the rear elastic layer;
(4) after the step (3) is completed, marking the boundary of the cutting area;
(5) after the step (4) is finished, gradually separating a membrane consisting of the rear elastic layer and the endothelial cell layer from the cornea from the middle of the rear elastic layer and the stroma layer to the center direction from the edge of the cornea until the membrane exceeds the boundary of the cutting area;
(6) after the step (5) is completed, resetting the membrane formed by the rear elastic layer and the endothelial cell layer;
(7) after the step (6) is finished, cutting along the boundary of the cutting area, and taking the material of the cutting area part;
(8) and (5) taking the material obtained in the step (7), and gradually separating a membrane consisting of the rear elastic layer and the endothelial cell layer along the edge from the middle rising edge of the rear elastic layer and the matrix layer separated in the step (5) to obtain the pig DMEK graft consisting of the rear elastic layer and the endothelial cell layer.
5. A graft prepared by the method of any one of claims 1 to 4.
6. Use of the graft of claim 5 in the preparation of a product for posterior elastic layer corneal endothelial grafting.
7. A product for posterior elastic layer corneal endothelial graft comprising the graft of claim 5.
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| "不同方法获取Desceme膜-角膜内皮细胞复合体的效果比较";叶向彧;《眼科研究》;20080930;第26卷(第9期);第645-648页 * |
| "后弹力层角膜内皮移植术植片制备研究进展";刘洋等;《国际眼科纵览》;20161031;第10卷(第5期);第321-327页 * |
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