WO2018107485A1

WO2018107485A1 - Decellularized dried swine lamellar cornea, used method for same, and uses thereof

Info

Publication number: WO2018107485A1
Application number: PCT/CN2016/110462
Authority: WO
Inventors: 董晓鸥; 刘靖; 李志寒; 詹晓亮; 李洁
Original assignee: 厦门大开生物科技有限公司
Priority date: 2016-12-16
Filing date: 2016-12-16
Publication date: 2018-06-21
Also published as: CN109069696B; CN109069696A

Abstract

A decellularized dried swine lamellar cornea, constituted by the anterior elastic lamina and the stroma of swine cornea. The stroma retains a regularly arranged structure of collagen fibers. In the visible light range, the water content of the dried cornea is no greater than 20%, and the transmittance of the dried cornea is no less than 70%. Based on the employment of swine cornea as a raw material, provided is a solution for the optimal combination of essential characteristics of a cornea product, and facilitated is the implementation of normativeness in the quality of a decellularized dried swine cornea product.

Description

Decellularized pig layer dry cornea and use method and use thereof

Field

The present invention relates to a heterologous artificial cornea for corneal transplantation, replacing human cornea, and a method and use thereof. Specifically, it refers to a decellularized lamellar dried cornea using porcine cornea as a material source.

Background technique

After more than ten years of efforts by Chinese scientists to develop artificial corneas made from porcine corneas, they have taken the lead in clinical trials all over the world. Some of the products of the enterprises have been approved by the relevant state departments, and to some extent, they have solved the current domestic The problem of corneal scarcity in people with large hospitals. According to statistics, there are more than 5 million patients with corneal damage caused by various reasons in China. If the artificial cornea with porcine cornea as raw material can be put into production in batches, it can provide more possibilities for curing more patients.

Current patents and related literature reports that artificial corneas using porcine cornea as raw materials and artificial corneas used in clinical trials are mostly non-dried corneas. Therefore, according to the results of the relevant published clinical trials, due to the different preparation methods of such artificial corneas, the products obtained in the end are different, and the clinical effects obtained are also different. The inventors have proved through a large number of experiments and clinical trials that the main problem of artificial cornea as a product for large-scale promotion and use is that the quality is different, the final performance of the product has certain differences, and thus the clinical use effect also exists. There are big differences. (For example, according to more than 40 clinical trial reports of Wuhan Union Hospital, the difference between the effect of post-transplant recovery and the time of recovery delay is quite large.) Among them, there is no specific standard for the biological characteristics of cornea for transplantation. The products of artificial corneas that have entered clinical trials (partially used) on the market today have significant differences in biological characteristics, biomechanical properties, product morphology and methods of use, and these differences are due to different preparation methods. Caused. Among the numerous patents and related offerings, there are also significant differences in the products defined by the preparation methods.

The test data of the lamellar artificial cornea using porcine cornea as raw material, which was proposed by the inventor in the prior Japanese Patent No. CN101947144, also uses the pig lamellar cornea in an undried state. The research and clinical results of the present inventors in recent years have shown that the quality of the cornea for transplantation, especially the lamellar cornea, in clinical use is an important factor in determining the effect after transplantation. The stability of the product quality can determine the stability of the transplant effect, in other words, the corneal products with the same characteristics, the transplantation effect is basically the same. Therefore, from the perspective of mass products, it is necessary to provide a way to ensure the safety of transplantation and transplantation. Lamellar corneal products. The quality of the product should be consistent, rather than the method of preparation of the cornea. Accordingly, in recent years, the inventors conducted a large number of tests and clinical trials on lamellar corneas with different performance indexes, in order to obtain the product characteristics that can be possessed as lamellar corneal products, and overcome the current use of porcine cornea as a raw material. The defect of the quality of lamellar corneal products, the product can replace the human cornea, thus achieving the goal of large-scale clinical application of lamellar cornea.

Located at the front of the eyeball, the cornea is a highly structured, relatively cell-free, transparent collagenous tissue. Most of the existing decellularized lamellar corneal stroma contains only the front elastic layer and the matrix layer. After the necessary decellularization treatment, the heterologous cellular components in the stromal layer are removed, and the immune rejection of porcine corneal transplantation is reduced. The sterility of the corneal graft is then controlled by sterilization to achieve the biomarker required for corneal transplantation.

The corneal stroma layer consists of nearly 150 layers of regular collagen fiber bundle sheets with corneal stromal cells in between. The stromal cells of the cornea are at rest, about 3% to 10% of the total volume, and are therefore referred to by some scientists as relatively acellular tissue. The structural feature of the corneal stroma is that the three-helix type I collagen is ordered and parallel to each other. This arrangement constitutes the structural basis for the physical properties of the corneal elastic mechanical strength and its transparency. This important feature of the cornea can induce the orderly and uniform growth of the recipient corneal stromal cells after implantation, keeping the cornea transparent.

Based on the analysis of the characteristics of the above-mentioned lamellar corneal stroma, the high degree of regularity of the collagen matrix arrangement structure of the corneal stroma is an important factor for realizing the biophysical properties such as lamellar corneal transparency. However, current decellularization methods inevitably cause damage to the ordered arrangement of the matrix layers to varying degrees.

Existing decellularization methods are mainly divided into physical methods, chemical methods, enzymatic methods, and electrophoresis methods. Physical methods such as freezing and thawing, drying, low efficiency of cell removal, residual corneal cell components, and easy to cause immune rejection after implantation. The reagents used in the chemical method mainly include: ionic detergents, nonionic detergents, alkaline reagents, and acidic reagents; although these reagents have high efficiency in removing cells, the structure of the cornea is destroyed while removing cells. Thereby, the transparency of the corneal stroma is significantly reduced.

The enzymatic reagents mainly include: lipases, nucleases and proteases; different enzymes are specific to the cellular components, lipidases degrade the lipid components of the cell membrane, while proteases can degrade the cell membrane, but the corneal extracellular matrix also exists. Significant damage, leading to a decrease in the transparency of the cornea. The use of electrophoretic methods to remove cellular components within the corneal stroma also has a greater impact on corneal transparency.

In addition to physical decellularization methods, other methods of decellularizing the cornea involve the use of decellularizing agents. The residual of the decellularization reagent has an effect on the transparency, epithelial repair and corneal stroma reconstruction function after acellular corneal implantation. The existing decellularization technology can completely maintain the transparency of the acellular corneal stroma while completely removing the cells, or the corneal reconstruction process is long due to excessive residual, which may cause corneal neovascularization and significantly reduce the decellularized corneal stroma. Transparency or failure of the transplant.

It is well known that the rehydration process of the cornea makes the cornea soft, so that it can be easily transplanted. However, the cornea will greatly increase the water content of the cornea during the rehydration process, and its transparency will be greatly reduced. In the clinic, the corneal water content after transplantation through the semi-dehydration mechanism of the human cornea background makes the artificial lamellar cornea gradually transparent after transplantation. Therefore, the clinical data of the existing products show that the corneal recovery transparent period after transplantation is up to 3 months, and the time of corneal transparency in each case is also uncontrollable.

In addition, as a corneal product with mass production characteristics, the corneal product should have the product attributes of convenient storage, transportation and use under the requirements of product quality identity. At present, the replacement of human cornea by acellular porcine cornea has entered a stage of rapid development. The quality identity and product attributes of corneal products are the key to solving the lack of corneal transplant donors in China. Therefore, it is necessary to find an optimal combination of the necessary characteristics of the comprehensive biology, physics and product morphology of the transplanted corneal products, without relying on the corneal products of the corneal preparation method, in order to realize the marketization of the corneal products. .

Summary of the invention

The object of the present invention is to provide a decellularized pig layer dry cornea, from the perspective of the most basic physiological function "transparency" of the cornea, based on the use of porcine cornea as a raw material, comprehensive consideration of the biology of the cornea for transplantation, The necessary characteristics of physics and its product form, the best solution for the characteristics of acellular corneal products is proposed, and the quality identity of the dried corneal products of the acellular pig layer is realized. Therefore, the defects of the corneal products in the prior art are different due to different preparation methods, and the technical requirements for mass production are achieved, so that the decellularized lamellar cornea can be used as a substitute for the human cornea to promote the development of the ophthalmic medical level in China. It solves the problem that many patients cannot be effectively treated because of the lack of corneal donors.

Another object of the present invention is to provide a decellularized pig layer dry cornea, which has the form of a product convenient for storage, transportation and use, and improves the product properties of the artificial cornea suitable for the market, and meets the requirements for large-scale promotion and use of artificial corneal products. .

A further object of the present invention is to provide a decellularized pig layer dry cornea, which is easy to standardize and simplify The operation of corneal treatment during clinical transplantation makes it convenient for clinical application.

The object of the present invention is achieved by the present invention, which provides a decellularized pig layer dry cornea composed of a front elastic layer and a matrix layer of porcine cornea; the matrix layer maintains a regular arrangement of collagen fibers; Within the range, the dry cornea has a light transmittance of not less than 70%; and the dried cornea has a water content of not more than 20%.

The acellular cell layer of the present invention is used to dry the cornea, and the DNA residue in the matrix layer after decellularization is not more than 100 ng/mg.

In an alternative embodiment of the invention, the light transmission is a post-sterilization detection value, which is the product state of the dried cornea.

In an alternative embodiment of the present invention, the dry cornea is provided with a sealed package which can serve as a support, which can effectively maintain the product state of the cornea after sterilization of the terminal, and avoid pollution and damage to the corneal product during transportation and preservation. .

In an alternative embodiment of the invention, the sealed package is placed before or after sterilization of the dried corneal terminal.

The acellular cell layer of the present invention has a dried cornea, and the matrix layer maintains a regular arrangement of collagen fibers; it means that the average spacing of type I collagen in the dried corneal extracellular matrix is 25 nm ± 10 nm under transmission electron microscope. In particular, the regular arrangement of the collagen fibers in the corneal stroma layer means that the type I collagen in the extracellular matrix of the cornea is clear and the cross-sectional size is uniform under the transmission electron microscope. The collagen fiber structure basically maintains the original regular arrangement structure of the porcine corneal stromal layer, so that the porcine cornea can maintain the most basic physiological function of the "transparent" cornea after undergoing a complicated preparation process such as decellularization. Better transplant effect.

The acellular porcine layer of the present invention has a dried cornea, and the extracted corneal layer has a thickness of 300 um to 700 um.

In the present invention, the plated dry corneal human light transmittance is selected to determine the absorbance value A of two or more different wavelengths, and is calculated according to the formula "T=1/10A×100%".

In a preferred embodiment of the present invention, the light transmittance selection measurement wavelength ranges from 380 to 780 nm, and the measurement step length is not more than 10 nm.

In the present invention, the dried cornea has at least a smooth surface of the front elastic layer, and has no macroscopically visible ridges or fine pleats, which makes it more favorable for the growth of epithelial cells.

The method for using the acellular cell layer dried cornea of the present invention, opening the sealed package before surgery; taking out Dry the cornea and place it in physiological saline for 15 to 30 minutes before transplanting.

In the method of using the dried cornea of the present invention, the optimal time for rehydration of the cornea is 15 to 30 minutes.

The acellular porcine layer dried cornea of the present invention is applied in corneal transplantation.

The acellular porcine lamellar dry cornea of the present invention is applied in a refractive correction surgery.

When the acellular porcine lamellar dry cornea of the present invention is applied to refractive correction, the dry cornea adapted to the diopter may be selected according to the refractive requirement before the refractive correction surgery, or the dry cornea may be precisely processed according to the diopter requirement before surgery. Meet the required diopter requirements.

The technical effect of the present invention is remarkable: First, the inventors based on the statistical analysis results of a large amount of experimental data obtained in the study of the decellularized lamellar cornea using porcine cornea for more than ten years, comprehensive clinical transplantation Based on the necessary characteristics of the cornea and its effect on the transplantation effect, a decellularized pig layer dried cornea was proposed based on the basic requirements of clinical corneal products and a large number of experiments using corneas prepared by different methods. The dried cornea of the invention can achieve an optimal combination scheme in the necessary characteristics of the cornea transplantation such as comprehensive biology, physics and product morphology, and achieve uniform quality of the decellularized pig dried corneal product, thereby overcoming the prior art. Medium corneal products have the defects of different quality or different product properties due to their different preparation methods. This defect makes it difficult to standardize clinical operations, and at the same time does not achieve a predictable transplantation effect, which hinders the large-scale clinical application of pig acellular cornea.

Throughout the course of more than ten years of research, it was initially believed that replacing the human cornea with porcine cornea as a medical material for transplantation, the first problem to be solved is to solve the immunogenicity of heterogeneous materials on the human body, and refer to other heterogeneous alternative materials ( Decellularization methods such as skin, bone, lung and other organs have proposed a number of decellularization methods and different combinations thereof. As mentioned above, the existing decellularization technology in China can effectively remove the cells of the corneal stroma layer, that is, the standard of DNA residue is not more than 100 ng/mg. However, the cornea has distinct characteristics that are different from all other tissues: transparency, which is the most important basis for the cornea to perform its physiological functions. The transparency of the cornea is derived from a highly regular collagen arrangement of the matrix layer. However, a large number of facts prove that the existing decellularization method, in pursuit of high-level decellularization effect, will destroy the highly regular arrangement of the ultrastructure of the corneal matrix layer to varying degrees, thus neglecting the "transparency" of the cornea relative to the cornea. The most important optical performance. Accordingly, the applicant first proposed a decellularized pig layer dry cornea from the most basic and most important performance of the most important feature of the corneal product, "transparency". The dried cornea of the present invention can achieve an optimal combination of the necessary characteristics required for corneal transplantation, such as comprehensive biology, physics, and its product morphology, to achieve corneal product quality consistency and traceability. . Artificial cornea On the basis of ensuring the immunogenicity of the cornea of different materials, the product meets the "transparency" requirements necessary for transplantation. ,. Achieve mass production to meet a large number of clinical needs. The current clinical case of the applicant proves that the corneal product provided by the present invention can be used as a substitute for human cornea.

Secondly, the applicant believes that as a corneal product that can be used in clinical applications, the dried cornea is undoubtedly the best product state of the corneal product, which is convenient for preservation and transportation. Compared with the glycerin preservation method, the dry state of the corneal product can maintain the performance of the dry corneal product quality in the market of preservation and transportation. At the same time, the dried cornea described in the present application has a water content of 5 to 20%, and has a certain toughness while maintaining its intrinsic shape, thereby reducing the defect that the corneal product is brittle due to excessive dryness. The dried cornea of the present invention greatly extends the shelf life of the product relative to the cornea preserved cornea.

Another important technical effect of the dried cornea of the present invention is that the rehydration operation before the 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 recovery time. The invention can completely standardize the rehydration operation before the operation, and achieve the problem of effectively controlling the water content after rehydration of the cornea. However, all current clinical cases of the applicant can prove that in the transplantation of the dried cornea of the present invention after rehydration, the time for the cornea to recover after transplantation can be controlled within 3 days, of which 50% of the cases are in the course of surgery. The cornea is gradually restored to a transparent state, and the surgical effect is achieved after at least 3 months of postoperative operation (see the clinical report of Wuhan Union Medical College). In addition, the simple rehydration operation can accurately control the water content after rehydration of the cornea before surgery, and thus greatly reduce the difficulty of surgical operation.

Thirdly, it has been experimentally and clinically proven that the dry cornea of the present invention has a product having a high surface flatness and is advantageous for attachment and proliferation of epithelial cells. In particular, the flatness of the pre-corneal elastic layer is extremely beneficial to increase the growth rate and growth quality of epithelial cells after corneal implantation.

Fourth, the bioartificial corneas that have entered the clinic on the market and use porcine cornea as materials have not specified DNA residue standards. The applicant has found that the cornea is different from other organs, and its stromal cells are few, about 3% to 10% of the total volume, which is called by some scientists as relatively acellular tissue. Therefore, this indicator is usually ignored in allogeneic transplantation, ie, human corneal transplantation. Therefore, excessive pursuit of corneal DNA residue standards, in the process of corneal preparation, will increase the strength of corneal decellularization, and inevitably destroy the original collagen regular arrangement in the corneal stroma. Applicants believe that due to the absence of vascular structure in the cornea, it has immunogenicity and low biological characteristics, so the appropriate choice of this biological index value of the cornea is Often necessary. Accordingly, the applicant conducted a large number of animal experiments, based on the results of the study, taking into account the special optical characteristics required for corneal products, the DNA residue for dry corneal products is not more than 100 ng / mg, both to achieve cell depletion in corneal preparation The purpose is to maintain the ultrastructure of the regular arrangement of collagen in the corneal stroma layer to meet the basic requirements of corneal immunogenicity, so that the artificial corneal product has a "transparency" that can ensure the transplantation effect, as shown in FIG. .

In summary, the present invention provides a decellularized layer of porcine cornea based on the optimal combination of the necessary characteristics of the integrated biology, physics and product morphology of the transplanted corneal product. cornea. The acellular porcine cornea of the invention can be used as a substitute for the human cornea. The invention solves the problem that the quality of the corneal product is different due to the preparation method and the quality is not uniform, and provides guarantee for the mass production and clinical application of the corneal product.

DRAWINGS

The invention and its specific embodiments and their effects are briefly described below with reference to the accompanying drawings, and the following drawings are merely illustrative of some specific experimental examples of the invention, rather than all of the invention.

Figure 1A is a cross-sectional arrangement of a collagen arrangement of a human cornea;

Figure 1B cross-sectional arrangement of porcine corneal collagen without decellularization;

1C is a cross-sectional arrangement of collagen of a porcine cornea after decellularization treatment of the present invention;

Figure 2 is a photograph of the corneal product of the decellularized and dried lamellar layer of the pig of the present invention.

Figure 3 is a photograph of HE staining of the corneal decellularized plate of the present invention.

Figure 4 is a photograph of the post-transplantation of the corneal decellularized lamellar cornea of the present invention to the New Zealand white rabbit.

Figure 5A is a pre-operative photo of a clinical transplant of the present invention;

Figure 5B is a photograph of the 3 day after clinical transplantation of Figure 5A;

Figure 5C is a photograph of Figure 2A after 2 months of clinical transplantation;

Figure 5D is a photograph of the 6 months after the clinical transplantation of Figure 5A;

Figure 5E is a photograph of Figure 1A after 1 year of clinical transplantation.

detailed description

The technical solutions of the present invention will be clearly and completely described in the following with reference to the embodiments of the present invention. It is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the invention All other embodiments obtained by those skilled in the art without creative efforts are within the scope of the present invention.

Example 1

In the present embodiment, the acellular cell layer dried cornea is made of fresh porcine cornea, and is composed only of the anterior elastic layer and the stromal layer of the porcine cornea, and does not include the epithelial layer, the posterior elastic layer and the endothelial layer. The matrix layer maintains a regular arrangement of collagen fibers; in the visible range, the dried cornea has a light transmittance of not less than 70%; and the dried cornea has a water content of not more than 20%.

Specifically, in the first embodiment, the light transmittance measured by the dried lamellar film having a water content of 15% ± 2% before the terminal sterilization treatment was 86 ± 5%. Transmission electron microscopic observation of the lamellar dry cornea given in this example, as shown in FIG. 1C, the interstitial space of the type I collagen in the extracellular matrix of the cornea is uniform, the boundary of the type I collagen in the extracellular matrix of the cornea is clear, and the cross-sectional size is uniform. The average spacing is approximately 25 ± 10 nm. The structure of this collagen arrangement is very similar to the human cornea in Fig. 1A and the porcine cornea in Fig. 1B which has not been subjected to decellularization. A large number of animal experiments have proved that the structure of the lamellar cornea provided by the present invention can induce the orderly and uniform growth of the recipient corneal stromal cells after implantation, so that the cornea can return to a transparent state soon after transplantation.

It is well known that the regular arrangement of the corneal stroma collagen fibers and the "semi-dehydrated state" together maintain the transparency of the cornea: the regular arrangement of the corneal stroma layer acts as a diffraction grating, reducing the scattering by disrupting the interference; In relation to the relative water content of the cornea, an increase in water content in the cornea results in a decrease in corneal transparency. Therefore, proper selection of the "transparency" and "water content" parameters of corneal products is an important part of the standardization of corneal product quality.

The inventor has studied for more than ten years, and through the statistical analysis of a large number of experimental data, from the perspective of the clinical requirements for corneal products, it is considered that for the lamellar dry corneal products that can be applied to the clinic, the cornea is Transparency and the moisture content of the cornea are important and essential essential characteristics, otherwise the corneal product does not have the value of clinical application at all. The present invention is based on the physiological functions necessary for the cornea, proposes the most basic corneal physics characteristics, achieves an optimal combination scheme, and achieves the quality consistency of the decellularized pig dried corneal products. It cannot be denied that any of the corneal preparation methods currently used, different methods will have different effects on certain characteristics of the cornea, which is the main reason for the current inconsistent quality of corneal products. However, the Applicant believes that as a corneal product that can be used in a large number of clinical applications, it is necessary to ensure uniformity of product quality or identity of product performance. Therefore, in selecting a corneal preparation method, the most basic requirements of the corneal product proposed by the present invention should first be considered.

In an embodiment of the first embodiment, in the preparation of the lamellar cornea, the epithelial layer, the posterior elastic layer and the endothelial layer are removed, and the pre-elastic layer is retained, and the thickness of the matrix layer for preparing the fresh cornea is 500±50 um. Lamellar cornea.

In a preferred embodiment of the first embodiment, the surface of the dry corneal front elastic layer is smooth, and there are no macroscopic folds or fine folds visible to the naked eye. A large number of animal experiments and clinical trials have shown that the smooth corneal elastic layer surface is more conducive to the growth of epithelial cells. As shown in FIGS. 5A to 5E, the dried cornea of the present embodiment 1 has an overall surface smoothness. The effect from the transplant shows that the dry cornea with overall surface smoothness has excellent transparency and is beneficial to the recovery of postoperative vision.

In another preferred embodiment of the first embodiment, the residual amount of DNA in the matrix layer after decellularization is not more than 100 ng/mg. Specifically, in the present Example 1, the DNA residue in the matrix layer after decellularization was 47 ng/mg. The replacement of human cornea with porcine cornea as a medical device for transplantation requires the first problem to solve the problem of immunogenicity of heterogeneous materials. At home and abroad, the research on the replacement of cornea for more than ten years at home and abroad shows that the decellularization of heterogeneous cornea is the most important part of all preparation methods. Based on the conclusions of organ transplantation in dissimilar materials in other fields, it is generally believed that the DNA residue index in the stromal layer is one of the important factors determining the effect of heterogeneous corneal transplantation. Therefore, the treatment of corneal decellularization is increased in various ways to achieve a lower level of DNA residue. However, the present inventors believe that while pursuing a lower level of DNA residue, it is necessary to reduce the destruction of the original regular collagen arrangement structure in the corneal stroma layer by focusing on decellularization treatment, ensuring the transparency of the corneal product and ensuring the cornea. After transplantation, it has a good recovery effect.

In a large number of animal experiments, the lamellar cornea proposed in the first embodiment can meet the requirements of the national DNA residue of animal-derived biological materials, and can also meet the requirements of immunological rejection rate of transplantation, and the corneal stromal layer Maintaining the original regular collagen fiber arrangement structure, the product has a better "transparent" effect after transplantation.

In an alternative embodiment of the embodiment 1, the dried cornea is placed in a sealed package that can serve as a support. The dried cornea provided by the invention can greatly prolong the storage time of the product relative to the cornea preserved by the glycerin, and has the advantages of convenient storage and transportation. Since any sterilization method will have a certain impact on the transparency of the corneal product. Therefore, specifically in the present embodiment, the corneal transmittance measured after sterilization by the 25 kgy irradiation terminal is 78%±5%.

The transmittance of the dried lamellar cornea of the present invention is selected to determine the absorbance value A of two or more different wavelengths, Calculated according to the formula "T = 1/10A × 100%". Wherein the selected spectral scanning range is 380-780 nm and the step size is 10 nm. The "light transmittance" of the dried cornea of the present invention is not limited to the method as described above, and other optical methods may be selected for the measurement, but the obtained transmittance value is equivalently converted and the light transmittance of the present invention is proposed. Corresponding.

The method for using the acellular cell layer dried cornea of the invention has the characteristics of being easy to operate, and the sealed package is opened before the operation; the dried cornea is taken out, and the transplanting is performed after being soaked in physiological saline for 15 to 30 minutes. Specifically, in this embodiment, the corneal rehydration time is 15 to 20 minutes.

A specific clinical case of the present embodiment proves that the applicant has proved that all the clinical cases have effects, and the product indexes of the lamellar dry cornea proposed by the invention can achieve better transplantation effects. 5A to 5E are a clinical case of the present invention. FIG. 5A to FIG. 5E are a group of photos of a human corneal transplantation of a porcine acellular cell layer dried cornea according to Example 1 of the present invention, respectively. Fig. 5A is a photograph of a preoperative case, Fig. 5B is 3 days after surgery; Fig. 5C is 2 months after surgery; Fig. 5D is 6 months after surgery; Fig. 5E is 1 year after surgery. Compared with the existing documented clinical effects, the lamellar dry cornea proposed in the first embodiment has been in a transparent state 3 days after the operation, and the corneal epithelium is basically repaired, and no obvious rejection reaction is observed. The cornea was completely restored to transparency, no neovascularization occurred, and no rejection was observed. It was completely transparent 1 year after operation, basically achieving the same effect as human corneal transplantation, and the corrected visual acuity was 1.0.

Tests have shown that the most important thing for "transparent" corneal products is the most basic performance of "transparency". The invention proposes the technical index of the corneal product, so that on the basis of having good transparency, the mass production of the product is ensured to meet the clinical demand.

Example 2

In the present embodiment, the acellular cell layer dried cornea is made of fresh porcine cornea, and is composed only of the anterior elastic layer and the stromal layer of the porcine cornea, and does not include the epithelial layer, the posterior elastic layer and the endothelial layer. Through all the decellularization preparation processes, the regular arrangement of collagen fibers is still maintained in the matrix layer; specifically, in the second embodiment, the dried cornea of the lamellar layer is measured at a moisture content of about 5% before the terminal sterilization treatment. The resulting light transmittance was 83 ± 5%. The transmission electron microscopic observation of the lamellar dry cornea given in Example 2 showed that the interstitial space of the type I collagen in the extracellular matrix of the cornea was uniform, the boundary of the type I collagen in the extracellular matrix of the cornea was clear, the cross-sectional size was basically the same, and the average spacing was about 25±10 nm and this collagen arrangement structure is substantially similar to the collagen arrangement of the human cornea of Fig. 1A and the porcine cornea without decellularization in Fig. 1B. Test results It is shown that in the state 2 of the dried cornea, the brittleness of the cornea slightly increases in a state where the water content is close to 5%. The corneal transmittance measured after sterilization with a 25 kgy irradiation terminal was 73% ± 2%.

In the second embodiment, in the preparation of the lamellar cornea, the epithelial layer, the posterior elastic layer and the endothelial layer are removed, and the pre-retained elastic layer and the matrix layer thickness of the fresh cornea are 600±50 um to form a lamellar corneal structure.

In the second embodiment, the surface of the dry corneal front elastic layer is smooth, and there are no macroscopic folds or fine folds visible to the naked eye.

In the second embodiment, the DNA residue in the matrix layer after decellularization was 86 ng/mg.

In an alternative embodiment of the second embodiment, the dried cornea is provided with a sealed package which can serve as a support, so that the dried lamellar film of the present invention is maintained in a solid product form for storage and transportation.

The method for detecting the light transmittance described in the second embodiment is substantially the same as that of the first embodiment, and therefore will not be described again. The method for using the acellular cell layer dried cornea of the present invention opens the sealed package before surgery; the dried cornea is taken out and immersed in physiological saline for 25 minutes, and then the transplant operation can be performed.

Example 3

In the present embodiment, the acellular cell layer dried cornea is made of fresh porcine cornea, and is composed only of the anterior elastic layer and the stromal layer of the porcine cornea, and does not include the epithelial layer, the posterior elastic layer and the endothelial layer. The regular arrangement structure of the collagen fibers is still maintained in the matrix layer; specifically, in the third embodiment, the light transmittance measured by the dried cornea of the lamellar layer at a moisture content of about 20% before the terminal sterilization treatment is 85%. ±5%. The transmission electron microscopic observation of the lamellar dry cornea given in Example 3 showed that the interstitial space of the type I collagen in the extracellular matrix of the cornea was uniform, the boundary of the type I collagen in the extracellular matrix of the cornea was clear, the cross-sectional size was basically the same, and the average spacing was about The structure of the collagen arrangement of 25±10 nm is substantially similar to that of the human cornea of Fig. 1A and the porcine cornea of the undecellularized porcine cornea of Fig. 1B. The test results show that the lamellar cornea of the present Example 3 has a good toughness in a state where the water content is close to 20%. The corneal transmittance measured after sterilization with a 25 kgy irradiation terminal was 78% ± 2%.

In the third embodiment, in the preparation of the lamellar cornea, the epithelial layer, the posterior elastic layer and the endothelial layer are removed, and the pre-retained elastic layer and the thickness of the matrix layer for preparing the fresh cornea are 600 um ± 50 um to form a lamellar corneal structure.

In the third embodiment, the surface of the dry corneal front elastic layer is smooth, and there are no macroscopic folds or fine folds visible to the naked eye.

In the third embodiment, the DNA residue in the matrix layer after decellularization was 56 ng/mg.

An alternative embodiment of the present embodiment 3 is that the dried cornea is placed in a sealed package that provides support so that the dried lamellar cornea of the present invention remains in a solid product form for ease of storage and transportation.

The method for detecting the light transmittance described in the third embodiment is substantially the same as that of the first embodiment, and therefore will not be described again.

In the present embodiment, the method for using the acellular porcine layer to dry the cornea is to open the sealed package before surgery; the dried cornea is taken out and immersed in physiological saline for 20 to 30 minutes, and then the transplant operation can be performed.

The lamellar dried cornea of the above embodiment of the present invention is obtained by using different preparation methods. From the results of the test and the effect of clinical transplantation, the dried cornea of the present invention can achieve an optimal combination scheme for the necessary characteristics required for corneal transplantation in biology, physics and product morphology, and realizes acellular pig Quality standardization of dried corneal products. It is necessary to properly select or combine different preparation methods under the conditions of ensuring the same quality of the cornea product and the same product performance.

The acellular porcine layer dried cornea of the present invention is suitable for use in corneal transplantation.

The acellular porcine lamellar dry cornea of the present invention is applied in a refractive correction surgery. The dried lamellar cornea of the invention has good processability, and the dried cornea can be precisely processed according to the requirement of diopter to achieve the required diopter requirement before the refractive correction surgery.

The detailed description of the various embodiments described above is intended to be illustrative of the present invention in order to provide a better understanding of the present invention, but these descriptions are not to be construed as limiting the invention, in particular, The various features described in the embodiments can also be arbitrarily combined with each other to form other embodiments, which are to be understood as being applicable to any one embodiment, and are not limited to the described embodiments. the way.

[Correct according to Rule 26 07.03.2017]

Claims

[Correct according to Rule 26 07.03.2017]
A decellularized porcine lamellar dry cornea consisting of a anterior elastic layer and a stromal layer of porcine cornea; characterized in that the matrix layer maintains a regular arrangement of collagen fibers; in the visible range, the dried cornea is permeable The light rate is not less than 70%; the moisture content of the dried cornea is not more than 20%.
[Correct according to Rule 26 07.03.2017]
The acellular porcine lamellar dried cornea according to claim 1, wherein the DNA residue of the dried cornea of the pig layer after decellularization is not more than 100 ng/mg.
The acellular porcine layer dried cornea according to claim 1, wherein the light transmittance is a post-sterilization detection value.
The acellular porcine layer dried cornea according to claim 1, wherein said dried cornea is provided with a sealed package which can serve as a support.
A decellularized pig lamellar dried cornea according to claim 4, wherein said hermetic package is placed before or after sterilization of the dried corneal terminal.
The acellular porcine layer dried cornea according to claim 1, wherein the matrix layer maintains a regular arrangement of collagen fibers; and the average spacing of type I collagen in the extracellular matrix of the dried cornea is 25 nm under transmission electron microscope. ±10 nm.
The acellular porcine lamellar dried cornea according to claim 1 or 6, wherein the matrix layer maintains the regular arrangement of the original collagen fibers, and the cross-section of the type I collagen in the extracellular matrix of the cornea is clear under transmission electron microscopy. The size is the same.
The acellular porcine lamellar dried cornea according to claim 1, wherein said pre-dried corneal lamellar layer has a thickness of from 300 μm to 700 μm.
In the present invention, the plated dry corneal human light transmittance is selected to determine the absorbance value A of two or more different wavelengths, and is calculated according to the formula "T=1/10A×100%".
The acellular porcine layer dried cornea according to claim 9, wherein said light transmittance is selected to be measured in a wavelength range of from 380 to 780 nm, and the measurement step is not more than 10 nm.
The acellular porcine lamellar dried cornea of claim 1 wherein said dried cornea has at least a smooth surface of the front elastic layer and no macroscopically visible ridges or fine pleats.
A method for using acellular cell layer dried cornea according to any one of the preceding claims 1 to 10, characterized in that the sealed package is opened before surgery; the dried cornea is taken out and placed in physiological saline for 15 to 30 minutes after rehydration. The transplant can be performed.
A method of using a dry cornea according to claim 12, wherein the optimal time for rehydration of the cornea is 15 to 20 minutes.
A decellularized pig lamellar dry cornea according to any one of the above 1 to 12 is applied in a corneal transplant operation.
[Correct according to Rule 26 07.03.2017]
A decellularized pig lamellar dry cornea according to any one of the preceding claims 1 to 12 for use in refractive correction surgery.
[Correct according to Rule 26 07.03.2017]
The acellular porcine layer dried cornea according to claim 15, before the refractive correction operation, the dry cornea adapted to the diopter may be selected according to the refractive requirement, or the dried cornea may be processed according to the diopter requirement before surgery to achieve the required diopter requirement. .