CN109069696B - Acellular pig lamellar dry cornea and use method and application thereof - Google Patents
Acellular pig lamellar dry cornea and use method and application thereof Download PDFInfo
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Abstract
A decellularized porcine lamellar dry cornea is composed of a front elastic layer and a matrix layer of the porcine cornea; the matrix layer keeps a regular arrangement structure of collagen fibers; (ii) a moisture content of the dried cornea of no greater than 20% in the visible range; the light transmittance of the dried cornea was not less than 70%. The invention provides the optimal combination scheme of essential characteristics of the cornea product on the basis of taking the pig cornea as a raw material, and is favorable for realizing the standardization of the quality of the acellular pig dried cornea product.
Description
Field of the invention
The invention relates to a xenogenic artificial cornea used for cornea transplantation to replace human cornea and a use method and application thereof. In particular to an acellular lamellar dry cornea which adopts a pig cornea as a material source.
Background
The artificial cornea prepared by taking the pig cornea as a raw material is researched and developed by Chinese scientists for more than ten years, clinical tests are firstly carried out in the world, products of part of enterprises are examined and approved by related national departments, and the problem of human cornea shortage commonly existing in various domestic hospitals is solved to a certain extent. According to statistics, the number of patients with cornea injury caused by various reasons in China is up to 500 and more than ten thousand, and if the artificial cornea with the pig cornea as the raw material can be put into production in batches, the possibility of curing more patients can be provided.
The current patent and related literature reports that the artificial cornea taking the pig cornea as the raw material and the artificial cornea used in the clinical test are mostly non-dry corneas. Therefore, according to the results of the relevant published clinical trials, the final products obtained from such artificial corneas differ according to the different preparation methods, and thus the clinical effects obtained are different. The inventor of the invention has proved through a large number of tests and clinical tests that the artificial cornea as a product has the following main problems in large-scale popularization and use: the quality is different, and the final performance of the product has certain difference, so that the clinical use effect has larger difference. (for example, in Wuhan's cooperative hospital 40 clinical trial reports, the difference between the patient's post-transplant recovery effect and the recovery time is quite large.) among them, because the national biological characteristics of cornea for transplant do not set up specific standards, the products of artificial cornea entering clinical trial (partial use) in the market today include: there are great differences in biological properties, biomechanical properties, product morphology and methods of use, which are caused by differences in the methods of preparation. There is also a large difference in the products defined by the preparation methods in numerous patents and related documents.
The inventor of the present invention proposed in 2010, the prior chinese patent No. CN101947144, and the experimental data of the lamellar artificial cornea using the porcine cornea as the raw material was also the porcine lamellar cornea in the non-dried state. The recent research and clinical results of the inventor show that the product quality of the cornea for transplantation, particularly lamellar cornea, in clinical use is an important factor for determining the effect after transplantation. The stability of the quality of the product may determine the stability of the transplant effect, in other words, the transplant effect of a corneal product having the same characteristics is substantially the same. Therefore, from the perspective of mass production, there is a need for a lamellar corneal product that ensures safe and effective implantation. The quality of the product should remain consistent rather than varying with the method of preparation of the cornea. Therefore, in recent years, the inventor carries out a great amount of tests and clinics aiming at lamellar corneas with different performance indexes, so as to obtain the product characteristics which can be taken as lamellar cornea products and have need, overcome the defect that the lamellar cornea products taking pig corneas raw materials have different quality at present, and replace human cornea, thereby realizing the aim of large-scale clinical application of lamellar corneas.
The cornea is located in the front of the eyeball and is a highly regular, relatively acellular, hyalin collagen tissue. Most of the existing acellular lamellar corneal stroma consists of only a pre-elastic layer and a stromal layer. The heterologous cell components in the stroma layer are removed through necessary decellularization treatment, and the immunological rejection reaction of the porcine cornea transplantation is reduced. And then the sterilization treatment is carried out to control the sterile state of the corneal transplantation, so that the lamellar cornea reaches the biological index required by the corneal transplantation operation.
The corneal stroma layer is composed of nearly 150 sheets of regularly arranged collagen fiber bundles with corneal stromal cells in between. The stromal cells of the cornea are in a quiescent state, accounting for about 3-10% of the total volume, and are therefore called relatively acellular tissue by some scientists. The structure of the corneal stroma is characterized in that the three-strand helical I-type collagen is orderly and mutually parallel arranged, and the arrangement mode forms the structural basis of the physical properties of the corneal elastic mechanical strength, the transparency and the like. This important feature of the cornea induces the ordered and uniform ingrowth of stromal cells from the recipient cornea after implantation, keeping the cornea transparent.
Based on the analysis of the characteristics of the lamellar corneal stroma, the high rule of the collagen fiber arrangement structure of the corneal stroma layer is an important factor for realizing biophysical properties such as lamellar corneal transparency. However, current methods of decellularization inevitably suffer to varying degrees from the orderly arrangement of the matrix layers.
The conventional decellularization methods are mainly classified into physical methods, chemical methods, enzymatic methods, and electrophoretic methods. Physical methods are as follows: freeze thawing and drying, the cell removing efficiency is low, the residue of corneal cell components is more, and immunological rejection is easy to cause after implantation. The chemical method mainly comprises the following reagents: ionic detergents, nonionic detergents, alkaline reagents, acidic reagents; these agents, although highly effective in removing cells, destroy the structure of the cornea at the same time as removing cells, thereby causing a significant decrease in the transparency of the corneal stroma.
The enzyme method mainly comprises the following reagents: lipid enzymes, nucleases and proteases; different enzymes aim at specific cell components, lipid enzymes degrade lipid components of cell membranes, and proteases can degrade the cell membranes, but have obvious destructive effect on corneal extracellular matrix, so that the transparency of the cornea is reduced. The removal of cellular components within the corneal stroma using electrophoresis also has a greater effect on corneal transparency.
In addition to physical decellularization methods, other methods of decellularizing the cornea involve the use of decellularizing reagents. The residual acellular agent has an effect on the transparency of the acellular cornea after implantation, epithelial repair and corneal stromal reconstruction functions. The existing acellular technology can not well maintain the transparency of the acellular corneal stroma while completely removing cells, or the corneal reconstruction process is long due to excessive residues, so that the corneal neovascularization is easily caused, the transparency of the acellular corneal stroma is obviously reduced, or the transplantation fails.
It is known that the process of rehydration of the cornea is to soften the cornea so that it can be conveniently transplanted. However, the hydration process of the cornea greatly increases the water content of the cornea and greatly reduces its transparency. In clinic, the water content in the transplanted cornea gradually becomes transparent through a semi-dehydration mechanism of the human cornea background. Clinical data from existing products therefore show that the period of corneal transparency recovery after transplantation is longer than 3 months, and the time for corneal transparency in each case is also uncontrollable.
In addition, as a cornea product with mass production characteristics, the cornea product should have product attributes of convenient storage, transportation and use under the requirement of product quality identity. At present, the technology of replacing human cornea with acellular pig cornea enters a rapid development stage, and the quality identity and product attribute of cornea products are key points for solving the shortage of cornea transplantation donors in China. Therefore, there is a need to find an optimal combination of the necessary characteristics of the transplanted cornea product, such as the comprehensive biology, physics and product form thereof, without depending on the cornea preparation method, so as to realize the marketing of the cornea product.
Disclosure of Invention
The invention aims to provide an acellular porcine lamellar dry cornea, which is based on the angle of 'transparency' of the most basic physiological function of the cornea, provides the best scheme of the product characteristics of the acellular cornea by comprehensively considering the necessary characteristics of biology, physics, product form and the like of the cornea for transplantation on the basis of adopting the porcine cornea as a raw material, and realizes the quality identity of the acellular porcine lamellar dry cornea product. Therefore, the defect that cornea products in the prior art are different in quality due to different preparation methods is overcome, the technical requirement of mass production is met, the acellular lamellar cornea can be used as a substitute of a human cornea, the development of the ophthalmic medical level in China is promoted, and the problem that a plurality of patients cannot be effectively treated due to the lack of cornea donors is solved.
The invention also aims to provide the acellular porcine lamellar dry cornea, so that the acellular porcine lamellar dry cornea has a product form which is convenient to store, transport and use, improves the product attribute of the artificial cornea suitable for the market, and meets the requirement of large-scale popularization and use of artificial cornea products.
It is still another object of the present invention to provide a decellularized porcine lamellar dried cornea which is easy to standardize and simplifies the operation of corneal processing in clinical transplantation, making it convenient for clinical application.
The invention aims to realize the purpose, and the acellular porcine lamellar dry cornea provided by the invention consists of a front elastic layer and a matrix layer of the porcine cornea; the matrix layer keeps a regular arrangement structure of collagen fibers; (ii) the light transmittance of the dried cornea is not less than 70% in the visible range; the moisture content of the dried cornea is not more than 20%; the dried cornea is smooth in surface at least in the anterior elastic layer, and has no visible ridge-like protrusions or fine folds, so that the dried cornea is more favorable for epithelial cell growth.
The acellular pig sheet layer dries the cornea, and the residual DNA in the acellular stromal layer is not more than 100 ng/mg.
In an alternative embodiment of the present invention, the light transmittance is a measured value after sterilization, and is the product state of the dried cornea.
In an optional embodiment of the invention, the dry cornea is externally provided with a sealed package which can play a supporting role, so that the product state of the cornea after terminal sterilization can be effectively maintained, and pollution and damage to the cornea product in transportation and storage are avoided.
In an alternative embodiment of the invention, the sealed package is placed before or after terminal sterilization of the dry cornea.
According to the acellular pig lamina layer dry cornea, a matrix layer keeps a collagen fiber regular arrangement structure; means that the average interval of the type I collagen in the extracellular matrix of the dry cornea is 25nm +/-10 nm under the observation of a transmission electron microscope.
In an alternative embodiment of the invention, the corneal stroma layer maintains the original collagen fiber regular arrangement structure, namely that the I-type collagen in the corneal extracellular matrix has clear boundaries and consistent cross section size under the observation of a transmission electron microscope. The collagen fiber structure basically keeps the original regular arrangement structure of the porcine cornea stroma, thereby realizing that the porcine cornea can still keep the most basic physiological function of the cornea after the complex preparation processes such as decellularization and the like, and further achieving better transplantation effect.
The thickness of the prepared cornea lamina is 300-700 um.
In the present invention, the light transmittance of the lamellar dry cornea is selectively measured for absorbance values A at two or more different wavelengths according to the formula "T ═ (1/10)AX 100% "calculated.
In the preferred embodiment of the present invention, the light transmittance is selected to be within the measurement wavelength range of 380-780nm, and the measurement wavelength is not greater than 10 nm.
The application method of the acellular pig lamina dry cornea comprises the steps of opening a closed package before operation; taking out the dried cornea, and soaking in normal saline for 15-30 minutes to carry out transplantation operation.
In the application method of the dry cornea, the optimal time for corneal rehydration is 15-30 minutes.
The acellular porcine lamellar dry cornea is applied to corneal transplantation.
The acellular porcine lamellar dry cornea is applied to a refractive correction surgery.
When the acellular pig lamina dry cornea is applied to the refractive correction, the dry cornea with the adaptive diopter can be selected according to the refractive requirement before the refractive correction operation, or the dry cornea is precisely processed according to the diopter requirement before the operation, so that the required diopter requirement is met.
The technical effects of the invention are remarkable: firstly, the inventor provides an acellular porcine lamellar dry cornea based on a large number of tests of cornea prepared by different methods from the perspective of basic requirements of clinical cornea products by synthesizing necessary characteristics of cornea for transplantation and the influence of the characteristics on transplantation effect according to the statistical analysis results of a large number of test data obtained in the research of acellular lamellar corneas taking porcine corneas as materials for more than ten years. The dry cornea of the invention can achieve an optimal combination scheme on the necessary characteristics required by corneal transplantation such as comprehensive biology, physics, product morphology and the like, and realize uniform quality of the acellular pig dry cornea product, thereby overcoming the defects of inconsistent quality or inconsistent product performance of the cornea product due to different preparation methods in the prior art. The defect causes difficult regulation of clinical operation, and a predictable transplanting effect cannot be obtained, so that the large-scale clinical application of the porcine acellular cornea is hindered.
Throughout the research course of more than ten years, people originally thought that the porcine cornea replaces the human cornea to be used as a medical material for transplantation, and the first problem to be solved is to solve the immunogenicity of the heterogeneous material to the human body, and a plurality of cell removal methods and different combinations thereof are proposed by referring to the cell removal methods of other heterogeneous substitute materials (such as skin, bones, lung and other organs). As described above, the existing domestic decellularization technology can effectively remove cells of a corneal stroma layer, namely the DNA residual quantity is not more than the standard of 100 ng/mg. However, the cornea has distinct characteristics from all other tissues: transparency, which is also the most important basis for the cornea to perform its physiological functions. While corneal transparency results from the highly regular arrangement of collagen in the stromal layer. However, a great deal of facts prove that the existing acellular methods destroy the highly regularly arranged ultrastructure of the stroma of the cornea to different degrees while pursuing a high level of acellular effect, thereby neglecting the optical performance of the cornea which is the most important relative to the cornea, namely the "transparency". Accordingly, the applicant first proposed an acellular porcine lamellar dried cornea, starting from the most essential and most important property of the corneal product, namely "transparency". The dry cornea of the invention can achieve an optimal combination scheme on the necessary characteristics required by corneal transplantation such as comprehensive biology, physics, product form and the like, and achieve the quality consistency and traceability of the cornea product, so that the artificial cornea product can achieve the 'transparency' requirement required by transplantation clinic on the basis of ensuring the requirement of the heterogeneous material cornea on the immunogenicity of the human body, and the batch production can be realized to meet the large clinical requirements. The applicant proves that the cornea product provided by the invention can be used as a substitute of human cornea in current clinical cases.
Secondly, the applicant believes that dry cornea, as a cornea product that can be applied clinically in bulk, is undoubtedly the best product state of the cornea product, and is convenient for storage and transportation. Compared with a glycerol storage method, the quality and stable performance of the dried cornea product can be kept in the market links of storage, transportation and the like in the dry state of the cornea product. Meanwhile, the dried cornea has the water content of 5-20%, and has certain toughness while the inherent shape of the cornea is kept, so that the defect that the brittleness of a cornea product is increased due to overhigh dryness is reduced. The dried corneas of the present invention greatly extend the shelf life of the product relative to glycerin preserved corneas.
The other important technical effect of the dry cornea of the invention is that the rehydration operation before operation is simple and the rehydration time is short. In the rehydration process, the water content of the cornea can meet the surgical requirements, and the water content of the cornea after rehydration can be properly controlled, which is very important for controlling or shortening the corneal renaturation time. The invention can completely standardize the rehydration operation before the operation, and effectively control the water content of the cornea after rehydration. However, all the current clinical cases of the applicant can prove that in the transplantation operation after rehydration of the dried cornea, the time for the cornea to recover from transparency after transplantation can be controlled within 3 days, wherein 50% of the cases are that the cornea gradually recovers to be transparent during the operation process, and the transparent effect of at least 3 months after the operation is achieved after the operation (see clinical reports of Wuhan cooperation and medical colleges). In addition, the standard and simple rehydration operation can accurately control the moisture content of the rehydrated cornea before the operation, and therefore the difficulty of the operation is greatly reduced.
Third, tests and clinics prove that the dried cornea has the product morphological characteristic of high surface flatness, and is beneficial to the attachment and proliferation of epithelial layer cells. In particular, the flatness of the elastic layer before the cornea is favorable for improving the growth speed and the growth quality of epithelial cells after the cornea is implanted.
Fourth, no DNA residual standard is specified for the bio-artificial cornea which is made of pig cornea and enters the clinic in the market at present. The applicant found that cornea is different from other organs, and the stroma cells of the cornea are few and account for about 3-10% of the total volume, so that the cornea is called relatively acellular tissue by some scientists. This criterion is generally ignored in allogeneic transplants, i.e., human corneal transplants. Therefore, the DNA residue standard of the cornea is excessively pursued, and the original collagen regular arrangement in the corneal stroma is inevitably destroyed by increasing the force of corneal decellularization treatment in the process of preparing the cornea. The applicant believes that the proper selection of this biological indicator value of the cornea is highly necessary due to the less immunogenic biological nature of the cornea due to its absence of vascular structures. Accordingly, the applicant has conducted a large number of animal experiments, and based on the research results, comprehensively considering the special optical characteristics required for the corneal products, and setting the residual amount of DNA to be not more than 100ng/mg for the dried corneal products, so as to achieve the purpose of decellularization treatment in the preparation of the cornea, and to maximally maintain the ultrastructure of the regular arrangement of the collagen of the corneal stroma layer under the condition of satisfying the basic requirement of the corneal immunogenicity, thereby enabling the artificial corneal products to have "transparency" capable of ensuring the transplantation effect, as shown in fig. 2.
In summary, the present invention provides an acellular lamellar cornea made of porcine cornea, based on the optimal combination scheme aiming at the essential characteristics of the transplanted cornea product, such as the comprehensive biology, physics and product form. The acellular porcine cornea can be used as a substitute of a human cornea, the problems of different performances and non-uniform quality of cornea products due to the preparation method are solved, and the guarantee is provided for batch production and clinical application of the cornea products.
Drawings
The present invention, its embodiments and effects will be described briefly with reference to the accompanying drawings, which are only illustrative of some specific test examples selected for the present invention and are not all the present invention.
FIG. 1A is a cross-sectional arrangement of collagen alignment of a human cornea;
FIG. 1B shows a cross-sectional arrangement of acellular porcine corneal collagen;
FIG. 1C shows the arrangement of collagen cross-sections of the porcine cornea after decellularization according to the invention;
FIG. 2 is a photograph of a porcine acellular dry lamellar cornea product of the invention;
FIG. 3 is a photograph of HE staining of porcine acellular dry lamellar cornea according to the invention;
FIG. 4 is a photograph of a porcine acellular dry lamellar cornea of the present invention after transplantation into a New Zealand rabbit lamellar graft;
FIG. 5A is a pre-clinical transplant photograph of the present invention;
FIG. 5B is a photograph of FIG. 5A taken 3 days after clinical transplantation;
FIG. 5C is a photograph of FIG. 5A taken 2 months after clinical transplantation;
FIG. 5D is a photograph of FIG. 5A taken 6 months after clinical transplantation;
fig. 5E is a photograph of fig. 5A taken 1 year after clinical transplantation.
Detailed Description
The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments of the present invention, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
In this example, the decellularized porcine lamellar dry cornea was prepared from a fresh porcine cornea, and consisted of only the anterior elastic layer and the basal layer of the porcine cornea, and did not include the epithelial layer, the posterior elastic layer, and the endothelial layer. The matrix layer keeps a regular arrangement structure of collagen fibers; (ii) the light transmittance of the dried cornea is not less than 70% in the visible range; the moisture content of the dried cornea is no greater than 20%.
Specifically, in this example 1, the light transmittance measured for lamellar dried cornea with a moisture content of 15% + -2% was 86 + -5% prior to terminal sterilization. In the transmission electron microscope observation of the lamellar dry cornea given in this example, as shown in fig. 1C, the arrangement and gap of type I collagen in the corneal extracellular matrix are uniform, the boundary of type I collagen in the corneal extracellular matrix is clear, the cross-sectional sizes are uniform, and the average distance is about 25 ± 10 nm. The collagen arrangement is very similar to the human cornea in FIG. 1A and the porcine cornea in FIG. 1B without decellularization. A large number of animal experiments prove that the lamellar cornea structure provided by the invention can induce the recipient corneal stromal cells to grow in order and uniformly after being implanted, so that the cornea can quickly recover a transparent state after being implanted.
It is well known that the regular arrangement of collagen fibers of the corneal stroma and the "semi-dehydrated state" together maintain the transparency of the cornea: the regular arrangement of the corneal stroma layers acts as a diffraction grating, reducing scattering by disrupting interference; the light transmission of the cornea is related to the relative water content of the cornea, and the increase of the water content in the cornea leads to the decrease of the transparency of the cornea. Therefore, proper selection of "transparency" and "moisture content" parameter ranges for a corneal product is an important link in achieving the normalization of corneal product quality.
The inventor researches for more than ten years and through the results of statistical analysis of a large amount of experimental data, from the perspective of basic requirements of clinical cornea products, the transparency of the cornea and the moisture content of the cornea are important and indispensable basic characteristics for lamellar dry cornea products which can be applied to the clinic, otherwise, the cornea products have no clinical application value at all. The invention is based on the angle of the essential physiological function of the cornea, provides the most basic physical characteristics of the cornea, achieves an optimal combination scheme, and realizes the quality consistency of the acellular porcine dried cornea product. Undeniably, any of the methods currently used for preparing the cornea will have different effects on certain characteristics of the cornea, which is a main reason for the inconsistent quality of the current cornea products. However, the applicant believes that as a corneal product that can be used in large quantities in clinical applications, it is necessary to ensure consistent product quality or identity of product properties. Therefore, in selecting a corneal preparation method, the most basic requirements of the corneal product proposed by the present invention should be considered first.
In one possible embodiment of example 1, in lamellar corneal preparation, the epithelial, posterior and endothelial layers are removed, the anterior elastic layer is retained, and the thickness of the stromal layer from which fresh cornea is prepared is: 500 +/-50 um, and forms lamellar cornea.
In a preferred embodiment of this embodiment 1, the dried pre-corneal elastic layer is smooth and free of visible ridges or fine folds. A large number of animal tests and clinical tests prove that the smooth surface of the corneal elastic layer is more beneficial to the growth of epithelial layer cells. As shown in fig. 5A to 5E, the dried cornea described in this example 1 had an overall surface smoothness. The effect after transplantation shows that the dry cornea with integral surface smoothness has excellent transparency and is beneficial to the recovery of postoperative vision.
In another preferred embodiment of this embodiment 1, the amount of residual DNA in the matrix layer after decellularization is no greater than 100 ng/mg. Specifically, in example 1, the amount of DNA remaining in the matrix layer after decellularization was 47 ng/mg. The pig cornea is used as a medical appliance product for transplantation instead of a human cornea, and the problem to be solved is the immunogenicity problem of a heterogeneous material. In the research on xenogenic cornea replacement at home and abroad for more than ten years, the acellular treatment of xenogenic cornea is a central link of all preparation methods. Based on the findings of the studies on the transplantation of heterogeneous material organs in other fields, it is generally considered that the indicator of DNA residue in the stromal layer is one of the important factors for determining the effect of the xenotransplantation of cornea. Therefore, the treatment force for corneal acellular is increased by various methods, and the aim is to achieve a lower DNA residual level. However, the present inventors considered that, while seeking a lower DNA residual level, it is necessary to reduce the destruction of the original regular collagen arrangement structure in the corneal stromal layer as much as possible by paying attention to the decellularization process, ensure the transparency of the corneal product, and ensure a better recovery effect after the transplantation of the cornea.
In a large number of animal experiments, the lamellar cornea provided in this example 1 can meet the national requirement for DNA residues of animal-derived biomaterials, and can also meet the requirement for immune rejection rate in transplantation, and the original regular collagen fiber arrangement structure in the corneal stroma layer is maintained, so that a good "transparent" effect is obtained after product transplantation.
An alternative embodiment of this example 1 is where the dried cornea is placed in a closed package that provides support. Compared with the cornea preserved by glycerol, the dried cornea provided by the invention can greatly prolong the storage time of the product, and has the advantages of convenient storage and transportation. Since any sterilization method will have some effect on the clarity of the membrane product. Thus, in particular in this embodiment, the corneal light transmittance measured after sterilization using a 25kgy radiation terminal is 78% ± 5%.
The light transmittance of the lamellar dry cornea of the present invention is measured by selecting the absorbance value a of two or more different wavelengths, and calculated according to the formula "T: 1/10a × 100%". Wherein the selective spectrum scanning range is 380-780nm, and the step size is 10 nm. The "light transmittance" of the dried cornea of the present invention is not limited to the above-mentioned value, and may be measured by other optical methods, but the obtained light transmittance value is equivalent to the light transmittance proposed by the present invention.
The application method of the acellular pig lamina dry cornea has the characteristic of easy operation, and the sealed package is opened before operation; taking out the dried cornea, and soaking in normal saline for 15-30 minutes to carry out transplantation operation. Specifically, in this embodiment, the corneal rehydration time is 15-20 minutes.
The specific clinical case of this example proves that the applicant has proved that the product index of the lamellar dry cornea provided by the invention can achieve better transplantation effect in all the clinical cases at present. Fig. 5A to 5E show a clinical example of the present invention, wherein fig. 5A to 5E are a set of photographs of a human cornea transplantation clinical post-operation of a porcine acellular lamellar dry cornea according to example 1 of the present invention, which are photographs of a pre-operation case of fig. 5A, and fig. 5B is a post-operation 3 days; fig. 5C is 2 months after surgery; FIG. 5D is 6 months after surgery; fig. 5E is 1 year post-surgery. Compared with the clinical effects recorded in the existing documents, the lamellar dry cornea proposed in the example 1 is already in a transparent state 3 days after the operation, the corneal epithelium is basically repaired, no obvious rejection reaction is seen, the cornea is completely recovered to be transparent after 1 month after the operation, no new blood vessel grows in, and no rejection reaction is seen. After 1 year, the cornea is completely transparent, the effect is basically the same as that of cornea transplantation, and the corrected vision reaches 1.0.
Tests have shown that for corneal products for transplantation, the most important is the "transparency" property. The invention provides the technical index of the cornea product, ensures the mass production of the product on the basis of better transparency, and meets the clinical requirement.
Example 2
In this example, the decellularized porcine lamellar dry cornea was prepared from a fresh porcine cornea, and consisted of only the anterior elastic layer and the basal layer of the porcine cornea, and did not include the epithelial layer, the posterior elastic layer, and the endothelial layer. Through all the cell-removing preparation processes, the regular arrangement structure of the collagen fibers is still kept on the matrix layer; specifically, in example 2, the light transmittance measured for lamellar dried cornea with a moisture content of about 5% was 83 ± 5% before terminal sterilization. In the observation of the transmission electron microscope of the lamellar dry cornea given in this example 2, the arrangement gaps of the type I collagen in the corneal extracellular matrix are uniform, the boundary of the type I collagen in the corneal extracellular matrix is clear, the cross-sectional sizes are substantially the same, and the average distance is about 25 ± 10nm, and the collagen arrangement structure is substantially similar to that of the human cornea in fig. 1A and the porcine cornea which is not subjected to decellularization treatment in fig. 1B. The test results showed that the dried cornea of example 2 had a slightly increased brittleness in a state where the water content was close to 5%. The corneal light transmittance measured after terminal sterilization with 25kgy irradiation was 73% ± 2%.
In this example 2, in the preparation of lamellar cornea, the epithelial layer, the posterior elastic layer and the endothelial layer were removed, and the anterior elastic layer was retained and the thickness of the stroma layer from which fresh cornea was prepared was 600. + -.50. mu.m, which together constituted the lamellar corneal structure.
In this example 2, the dried pre-corneal elastic layer was smooth in surface, with no visible ridges or fine folds.
In example 2, 86ng/mg of DNA remained in the matrix layer after decellularization.
An alternative embodiment of this example 2 is that the outer surface of the dry cornea is provided with a sealed package that can act as a support, so that the lamellar dry cornea of the invention remains in a solid product form for ease of storage and transport.
The method for detecting the transmittance in this embodiment 2 is basically the same as that in embodiment 1, and therefore, the description thereof is omitted. The application method of the acellular pig lamina dry cornea comprises the steps of opening a sealed package before operation; the dried cornea is taken out and put into physiological saline to be soaked for 25 minutes, and then the transplantation operation can be carried out.
Example 3
In this example, the decellularized porcine lamellar dry cornea was prepared from a fresh porcine cornea, and consisted of only the anterior elastic layer and the basal layer of the porcine cornea, and did not include the epithelial layer, the posterior elastic layer, and the endothelial layer. The matrix layer still maintains the regular arrangement structure of collagen fibers; in this example 3 in particular, the light transmittance measured for lamellar dried cornea with a moisture content of about 20% was 85% ± 5% prior to terminal sterilization. In the observation of the transmission electron microscope of the lamellar dry cornea given in this example 3, the arrangement gaps of the type I collagen in the corneal extracellular matrix are uniform, the boundary of the type I collagen in the corneal extracellular matrix is clear, the cross-sectional sizes are substantially the same, and the arrangement structure of the collagen at an average pitch of about 25 ± 10nm is substantially similar to the arrangement structure of the collagen fibers of the human cornea in fig. 1A and the porcine cornea in fig. 1B which is not subjected to decellularization treatment. The test results show that the lamellar cornea of example 3 has better toughness in the state of approximately 20% moisture content of the dried cornea. The corneal light transmittance measured after terminal sterilization with 25kgy irradiation was 78% ± 2%.
In this example 3, in the preparation of lamellar cornea, the epithelial layer, the posterior elastic layer and the endothelial layer were removed, and the anterior elastic layer was retained and the thickness of the stroma layer from which fresh cornea was prepared was 600um ± 50um to constitute lamellar corneal structure.
In example 3, the dried pre-corneal elastic layer was smooth in surface, with no visible ridges or fine folds.
In example 3, 56ng/mg of DNA remained in the matrix layer after decellularization.
An alternative embodiment of this example 3 is that the dried cornea is placed in a sealed package that provides support so that the lamellar dried cornea of the invention retains a solid product form for ease of storage and shipping.
The method for detecting the transmittance in this embodiment 3 is basically the same as that in embodiment 1, and therefore, the description thereof is omitted.
In the using method of the acellular pig lamina dry cornea in the embodiment, a closed package is opened before operation; taking out the dried cornea, and soaking in normal saline for 20-30 minutes to carry out transplantation operation.
The lamellar dry corneas described in the above embodiments of the invention were obtained by different methods of preparation. From the test results and the effect of clinical transplantation, the dry cornea can achieve an optimal combination scheme on the necessary characteristics required by clinical cornea transplantation such as biology, physics, product morphology and the like, and realize the quality standardization of the acellular pig dry cornea product. It is essential to select or combine different preparation methods appropriately under the condition of ensuring the same quality and product performance of the cornea product.
The acellular porcine lamellar dry cornea is suitable for being applied to corneal transplantation.
The acellular porcine lamellar dry cornea is applied to a refractive correction surgery. The lamellar dry cornea has better processability, and can be precisely processed according to the diopter requirement before a refraction correction operation so as to meet the required diopter requirement.
The present invention is not limited to the above embodiments, and in particular, various features described in different embodiments can be arbitrarily combined with each other to form other embodiments, and the features are understood to be applicable to any embodiment except the explicitly opposite descriptions, and are not limited to the described embodiments.
Claims (13)
1. A decellularized porcine lamellar dry cornea is composed of a front elastic layer and a matrix layer of the porcine cornea; the matrix layer keeps a regular arrangement structure of collagen fibers; (ii) the light transmittance of the dried cornea is not less than 70% in the visible range; the water content of the dried cornea is 5-20%; at least the pre-elastic layer of the dried cornea is smooth in surface, free of visible ridges or fine folds; the dried cornea is used as a material for corneal transplantation after rehydration.
2. The decellularized porcine lamellar dried cornea of claim 1, wherein said decellularized porcine lamellar dried cornea has a DNA residual of no greater than 100 ng/mg.
3. The acellular porcine lamellar dry cornea of claim 1, wherein the light transmittance is measured after terminal sterilization.
4. The acellular porcine lamellar dry cornea of claim 1, characterized in that a supporting hermetic packaging is provided outside the dry cornea.
5. The decellularized porcine lamellar dried cornea of claim 4, wherein said sealed packaging is placed before or after terminal sterilization of the dried cornea.
6. The acellular porcine lamellar dry cornea of claim 1, wherein the stromal layer retains a regular arrangement of collagen fibers, meaning that the average collagen type I spacing in the extracellular matrix of the dry cornea is 25nm ± 10nm, as observed by transmission electron microscopy.
7. The acellular porcine lamellar dry cornea of claim 1 or 6, wherein the stromal layer maintains the original collagen fiber regular arrangement structure, which means that the type I collagen in the extracellular matrix of the cornea has clear boundaries and consistent cross-sectional dimensions under the observation of a transmission electron microscope.
8. The decellularized porcine lamellar dried cornea of claim 1, wherein said prepared dried anterior corneal lamellar thickness is 300um to 700 μm.
9. The decellularized porcine lamellar dried cornea of claim 1, wherein the light transmittance of said lamellar dried cornea is determined by selectively measuring the absorbance values a at two or more different wavelengths according to the formula "T ═ 1/10AX 100% "calculated.
10. The decellularized porcine lamellar dried cornea of claim 9, wherein said light transmittance is selected in the range of 380 to 780nm, measured at a wavelength not greater than 10 nm.
11. A method of using the decellularized porcine lamellae dried cornea of any of claims 1 to 10, wherein the closed package is opened prior to surgery; taking out the dried cornea, and putting the cornea into physiological saline for 15-30 minutes for rehydration.
12. The method of using a dried cornea of claim 11, wherein the corneal rehydration time is from 15 to 20 minutes.
13. Use of the decellularized porcine lamellar dried cornea of any of claims 1 to 10 for the preparation of a material for corneal transplantation.
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