WO2018107486A1

WO2018107486A1 - Method of drying decellularized cornea and dried decellularized pig lamellar cornea

Info

Publication number: WO2018107486A1
Application number: PCT/CN2016/110464
Authority: WO
Inventors: 詹晓亮; 李洁; 李志寒; 董晓鸥; 刘靖
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: CN109069264A; CN109069264B

Abstract

A method of drying a decellularized lamellar cornea and a dried decellularized pig lamellar cornea obtained thereby. The drying method comprises first placing the cornea into a vacuum drying chamber; gradually reducing a pressure within the closed drying chamber; and gradually reducing the pressure to a pressure at a set maximum vacuum degree over a time period until a required corneal dryness is reached. The drying method allows a milder corneal drying process, and minimizes damage to a corneal collagen alignment structure during the vacuum drying.

Description

Method for drying decellularized cornea and dried cell of acellular pig layer

The present invention relates to a method for preparing an artificial cornea for treating corneal blinding eye diseases, in particular to a method for drying a decellularized cornea and a decellularized lamellar dried cornea using a porcine cornea as a donor source. Background technique

Corneal transplantation is currently the only effective method for treating corneal blindness. Due to the serious lack of status of corneal donors in China, it has greatly affected the wide-scale promotion of this operation, but it has also made China's research in the field of heterogeneous corneal replacement a world-famous The achievements, especially the application of acellular porcine lamellar cornea with porcine cornea as donor source, have been praised by foreign scientific circles as one of the five major innovations in contemporary China.

In the last decade, the porcine cornea has a tissue structure, biophysical properties and optical properties similar to human cornea height, which is the accepted conclusion of the best choice for corneal substitutes. The domestic ophthalmologists have successfully realized the transplantation of the porcine lamellar cornea directly into the human body, and achieved certain clinical effects. The artificial cornea with porcine cornea as the material has many advantages such as wide material source, low cost, simple treatment method and good clinical effect compared with other artificial cornea techniques. Two domestic companies have developed artificial corneal products with porcine cornea as donor source, which has been applied to the clinic through market access approval of relevant Chinese regulatory authorities, bringing good news to patients with corneal diseases and bringing a huge market demand. .

The lamellar cornea is a cornea (scaffold) comprising only the lamella structure of the front elastic layer and the matrix layer with respect to the cornea of the full-layer structure. It is available in three forms: unseasoned lamellar scaffold (referred to as water content dehydrated to the original cornea), dry lamellar scaffold and rehydred lamellar scaffold. (See the applicant's prior application No. CN10197144). A large number of patent documents and methods for preparing a variety of acellular porcine corneal lamellar corneas disclosed in the related literature, as well as test data, animal test data, and clinical effects obtained by the preparation method employed therefor. Most of them are obtained according to the non-drying corneal state after completion of the decellularization treatment in the laboratory. And because of the different preparation methods, the test data or clinical effects are not exactly the same. Undoubtedly, in the currently disclosed prior art, the so-called corneal products and their application effects are only based on the test data state of the non-dried cornea, and there is no condition that can be promoted and used in the market, that is, in production and preservation. , product quality and performance stability at every stage of market operations such as transportation and use.

In particular, the biggest drawback of non-dried corneas is that they are extremely difficult to store and transport. During storage and transportation, the quality and biophysical properties of the non-drying lamellar cornea will be preserved and transported. The change in the conditions of the change changes with it, and this change is unpredictable. Therefore, the doctor cannot judge the postoperative effect that the artificial cornea used can be achieved in the clinic. According to the published data, the clinical effect of using such artificial corneal transplantation on the market is that, even if the operation is successful, the time for postoperative recovery needs to be as long as 3 months to 6 months. This transplantation result is not only far from the transplantation effect of human cornea, but also it is difficult for doctors to make accurate judgments on the success of transplantation and the effect of postoperative recovery in the shortest possible time, which is not conducive to failure or recovery. Poor results After transplantation, the results were taken as soon as possible.

In order to solve the above problems, the current researchers have proposed a method of drying the cornea after decellularization to obtain a dry cornea which is easy to store and transport. However, many of the alternative drying methods currently proposed are conventional drying methods. For example, the currently disclosed methods of drying the cornea mainly include freeze-drying, dehydration of glycerin, dehydration of denaturing silica, dehydration of phosphorus pentoxide and dehydration of anhydrous calcium chloride, and are widely used in conventional drying techniques in the field of medical devices. The Applicant has exhausted all of the conventional drying methods to perform extensive drying treatment tests on artificial corneas. By comparing all biophysical properties of the artificial cornea before and after drying, it was found that any conventional drying process would have a certain degree of adverse effect on the biophysical properties of the artificial cornea. The present inventors have conducted research and statistical analysis on a large amount of test data obtained by each drying method in the drying process, and found that the above-mentioned conventional drying method has an adverse effect on the artificial cornea because the drying process is too severe. In particular, after the decellularized treatment of the artificial cornea, the collagen scaffold becomes very loose due to the large amount of water contained in the cornea and the gap existing after the removal of the cells, and at the same time, the supporting force of the collagen arrangement in the original matrix layer is lost. In this state, the violent drying process inevitably causes an irregular change in the arrangement of the collagen tissue, thereby destroying the highly regular arrangement of the collagen fibers in the original corneal stroma.

The cornea has distinct characteristics that are different from all other tissues: transparent. This is also the most important basis for the cornea to perform its physiological functions. The transparency of the cornea is derived from the highly regular arrangement of collagen fibers in the matrix layer. At the same time, the highly regular arrangement of collagen fibers in the corneal stroma is also the structural basis of the mechanical elasticity of the cornea. A large number of experiments have proved that any of the conventional drying methods described above can damage the regular arrangement of collagen fibers in the corneal stroma layer to a certain extent, and have a certain degree of adverse effect on the biophysical properties of artificial corneal products such as transparency and mechanical elasticity. . For example, all conventional drying methods are disclosed in the patent number CN104645415, including vacuum lyophilization, vacuum drying, natural drying, dry anhydrous calcium chloride vacuum drying, drying in a 30-60 ° C incubator, and the like. In addition to naturally drying in all of these conventional drying methods, other methods achieve the same drying time and the same drying effect by interference of unnatural factors. At present, in the research practice, the biological material is dried by using a vacuum drying method. Because vacuum drying has low temperature, no overheating, easy evaporation of water, short drying time, and reduced contact with materials and air, it can avoid pollution and is the most widely used drying method. However, since vacuum drying is achieved by lowering the pressure by lowering the pressure (dry boiling point) or melting point (freeze drying) in a closed environment, the vaporization or sublimation of moisture in the object is accelerated, thereby achieving rapid drying. According to the principle of vacuum drying, boiling point drying is a process of high temperature drying. During the drying process, the moisture in the material is simultaneously evaporated and boiled under sufficient heat. Although the boiling point of the water is lowered in the low pressure state, a certain amount of heat is required to ensure that the water in the dried object reaches the evaporation and boiling state. It is violent. At the same time, when the vaporized steam is quickly extracted, a negative pressure state is formed around the material, and a large humidity gradient is formed between the inner and outer layers of the material, so the boiling point is also dried very unevenly, and is directly used for drying the fresh cornea. It will cause some damage to the corneal collagen fiber arrangement. Therefore, in the scientific research and production practice, the boiling point drying method is rarely used to dry the cornea.

In the sublimation drying (freeze drying) method, the moisture of the material is first frozen to a solid state, and then the solid water is directly sublimated into a gaseous state under a high vacuum to be removed. This method does not have a high temperature and violent reaction such as evaporation and boiling in boiling point drying, and is a drying method which is widely used in biological materials. However, since the drying method is based on lowering the boiling point of water under low pressure conditions, the ice crystals in the tissue are sublimated into a vapor state and removed, and the volume occupied by the ice crystals forms a pore-like structure. The conclusions given in the paper "Comparison of dried and lyophilized decellularized porcine corneal stroma as tissue engineering corneal scaffolds" published by the Fourth Military Medical University show that lyophilized (lyophilized) porcine corneal stroma is observed under TEM observation. The collagen is arranged in a disorderly structure with a porous structure, and the front elastic surface has a ridge-like protrusion, which loses the regular slab structure. A large number of experiments have shown that during the process of corneal decellularization, when the water content in the corneal stroma increases and the collagen tissue is released, the distance between the collagen and the collagen in the lamellar layer increases and is completely occupied by water. . When the water distribution in the flowing state is uneven, the distance between the collagen layers in the lamellar layer is also uneven, and the collagen arrangement structure of the corneal lamellar layer is also unstable. When the water is consolidated into ice crystals at a low temperature, it is very It is difficult to ensure that the collagen fiber arrangement of the corneal layer remains the original high degree of regularity. It is not difficult to prove that the vacuum freeze-drying method has a considerable influence on the transparency of the artificial cornea.

Patent No. CN 104188957 proposes a natural drying method which is basically the same as that of a non-natural factor that is sterile and air-dried, that is, the acellular corneal stroma is placed in a 24-well plate culture plate and placed in a clean bench for 48°. 72 hours. The drawback of this natural drying method is that the drying time is too long, which tends to cause collagen denaturation of the cornea, thereby affecting the transparency of the cornea. And throughout all drying time, The maintenance of the sterility of the environment is quite difficult. In addition, other conditions such as temperature control, wind adjustment, etc., which are naturally dried, are difficult to control, and the degree of drying and drying time are difficult to control. However, the influence of other uncontrollable factors in the natural environment will result in different drying effects. Even the corneal drying in the same batch will be uneven and the drying effect will be different, and the drying effect of all batches cannot be guaranteed to be the same. Therefore, the current natural drying method is only applicable to the laboratory or small batch production conditions, and basically cannot be applied to large-scale industrial scale production. This is also the reason why there is basically no adoption in the field of medical device industrialization.

Based on the preservation experience of human cornea, some literatures suggest that more glycerin preservation methods are used. The method is to seal the non-dried cornea in glycerin. This method is mainly a process in which the cornea is stored in a glycerin liquid and is gradually dehydrated during storage. The defects are also obvious. First, the liquid preservation state is not conducive to transportation, and glycerin is prone to explosion and other safety hazards during transportation. Second, the gradual dehydration process is difficult to ensure the quality of the cornea in clinical use. It is also impossible to regulate the corneal transplantation. It is necessary for the doctor to make adaptive changes according to the preservation state of the cornea at the time of clinical use. Third, it is necessary to clean before the operation to remove the glycerin sticking to the cornea. If the cleaning is not clean, It is easy to form residues on the cornea and seriously affect the surgical effect. Fourth, the non-dried cornea cannot modify its shape. For example, the shape of the cornea is corrected based on the diopter requirements of the transplanted cornea.

In the large number of animal tests and some clinical trials on dry cornea, the applicant found that the transparency of the cornea and the morphology of the cornea, especially the smoothness of the cornea, are crucial for the postoperative effect of keratoplasty when the cornea is transplanted with dry cornea. important. First, the higher the transparency, the higher the transparency of the recovery after transplantation. Secondly, the water content is basically the same. The dry cornea can regulate the rehydration operation of the transplant, which can effectively control the rehydration of the cornea after rehydration. It is beneficial to shorten the time of transparency after corneal surgery. Thirdly, the flatness of the cornea, especially the higher the level of the pre-corneal elastic layer, is more conducive to the attachment and proliferation of epithelial cells. Uneven or dry corneas with visually visible verrucous protrusions or fine folds can affect the healing effect after transplantation to varying degrees.

With the replacement of human cornea with porcine acellular lamellar cornea for the treatment of corneal blind replication technology, the demand for corneal decellularized lamellar cornea is greatly increased. Therefore, it is necessary to propose a method for preparing a dry cornea, which satisfies the market property of maximizing the quality identity and performance stability of the dried corneal product under conditions suitable for preservation and transportation. Summary of the invention

The object of the present invention is to provide a method for drying acellular cornea, which makes the cornea drying process more The mildening minimizes the destruction of the corneal collagen fiber alignment structure during the drying process, so that the cornea after drying has the same physical properties as the cornea before drying.

Another object of the present invention is to provide a method for drying a decellularized cornea and a dry cornea thereof, which improves the transparency of the cornea after drying to facilitate shortening the time for recovery of transparency after corneal implantation.

A further object of the present invention is to provide a method for drying acellular cornea and a dry cornea thereof, which improves the flatness of the appearance of the dried cornea, in particular, the flatness of the pre-corneal elastic layer to facilitate the growth of epithelial cells after corneal implantation. Speed and growth quality.

A fourth object of the present invention is to provide a method for drying a decellularized cornea and a dried cornea, which are convenient for storage and transportation, and which provide artificial corneal with product properties of market circulation. Promote the standardization, industrialization and market development of artificial corneal products to meet the huge market demand for artificial corneas.

The fifth object of the present invention is to provide a method for drying acellular cornea and a dry cornea, which can conveniently perform precise correction processing on the cornea according to requirements, in particular, processing corneal diopter, and expanding artificial cornea in refractive correction. The scope of application. The object of the present invention is achieved by a method for drying a decellularized cornea, which is first placed on a support device and placed in a closed drying chamber with adjustable vacuum; and the vacuum drying system is used to reduce the closed drying chamber. The pressure is lower than the atmospheric pressure; and the pressure in the closed drying chamber is gradually reduced to a set maximum pressure during a period of time, and continues to reach the corneal drying under the set maximum vacuum state. Degree requirements.

The working principle of the invention is that after the cornea to be dried is placed in a closed drying chamber with adjustable vacuum degree, the pressure in the vacuum drying chamber is set at a pressure lower than atmospheric pressure but higher than the maximum vacuum of the drying chamber. And through the adjustment system, the pressure of the closed drying chamber is gradually adjusted to the set maximum vacuum until the cornea reaches its set dryness requirement. Compared with the existing vacuum drying method, the vacuum drying process of the present invention becomes gentler, thereby overcoming the drawback that the drying process of the prior drying method is too severe. Therefore, the present invention minimizes the degree of destruction of collagen regular alignment of the matrix layer during corneal drying. Under the conditions of the same decellularization method (ie, excluding the influence of other preparation processes on the cornea), the dried cornea obtained by the drying method of the present invention has substantially no further destructive collagen arrangement of the matrix layer before drying. change. The drying method of the present invention is suitable for the drying treatment of the full-thickness cornea and lamellar cornea.

In a preferred embodiment of the present invention, the pressure reduction mode in the closed drying chamber is at least two stages from high to low gradient pressure reduction, and the pressure gradient of each stage lasts for a period of time; stress reliever To the set maximum vacuum state. The preferred embodiment is more convenient to operate and easier to control. Wherein: In an optional example of the present invention, the decompression range of each gradient is 10 100 kpa; wherein the preferential decompression range is 10~30 kPa.

In an alternative embodiment of the invention, the duration of the pressure gradient for each stage is set to decrease as the pressure decreases.

In an alternative embodiment of the invention, the vacuum adjustment system adjusts the previous stage pressure block to adjust directly to the subsequent stage pressure block as the pressure gradient changes.

In another alternative embodiment of the invention, the vacuum adjustment system adjusts the previous stage pressure differential to gradually decrease to the next stage pressure differential as the pressure gradient changes.

In another optional embodiment of the invention, the duration of each stage of the reduced pressure gradient is 0.5 12 hours; wherein the priority duration is 0.54 hours.

In another embodiment of the present invention, the method of controlling the pressure reduction in the sealed drying chamber is to continuously reduce the pressure to a set maximum vacuum state for a period of time. In a continuous depressurization embodiment, the operation of the reduced pressure can be achieved by means of automatic control.

In an alternative embodiment of the invention, the temperature in the enclosed drying chamber is controlled between 0 ° C and 30 ° C. In an optional example of the present invention, the vacuum adjustment system adjusts the pressure at which the pressure of the closed drying chamber is reduced to a set maximum vacuum within no more than 24 hours; wherein the pressure is preferentially set to 6 to 11 hours. The maximum vacuum when the pressure.

In an alternative embodiment of the invention, the pressure regulating range of the closed drying chamber is from atmospheric pressure to a set maximum vacuum.

In an alternative example of the invention, the set maximum vacuum is at a pressure close to the ultimate vacuum. In an alternative embodiment of the invention, the corneal dryness is set according to the corneal moisture content.

In a preferred embodiment of the invention, the corneal dryness is optimally set to a moisture content of no greater than 20%. The invention provides a decellularized porcine dry cornea, which comprises a decellularized porcine corneal anterior elastic layer and a matrix layer, and is obtained by any of the above drying methods. The moisture content of the dried cornea is greater than 0% and not greater than 20%. The light transmittance of the dried cornea is not less than 70%. The surface of the dried cornea is flat, with no visible ridges or fine folds visible to the naked eye.

The effect of the present invention is quite significant compared to current conventional drying techniques. First of all, the present invention is based on the principle of vacuum drying, so that the vacuum drying temperature is low, there is no overheating, the water is easy to evaporate, and when dry Short advantage. However, in the present invention, the excessively severe drying defects in the conventional vacuum drying method are effectively overcome by the gradual decompression method, so that the vacuum drying process becomes gentler. Therefore, the degree of regular destruction of collagen fibers in the matrix layer during corneal drying is minimized. Under the conditions of the same decellularization method (ie, excluding the influence of other preparation processes on the cornea), the dried cornea obtained by the drying method of the present invention has substantially no further damage of the collagen fibers of the matrix layer before drying. Sexual change. After drying, the cornea is kept to the maximum of the same biophysical properties as the cornea before drying. The test results show that the dried cornea obtained by the drying method of the method has higher transparency and smooth surface than the dried cornea obtained by the conventional drying method, and has excellent flatness visible to the naked eye.

The clinical effect of the dried cornea of the present invention is more remarkable: First, the dried cornea of the present invention begins to gradually become transparent during the corneal transplantation. The dry cornea obtained by the drying method of the present invention greatly reduces the time for recovery of the cornea after implantation due to the curing period of at least 3 months compared to the existing dry cornea. Secondly, the flatness of the dried cornea based on the present invention is extremely high, and another significant clinical effect is that the postoperative epithelial cells adhere and proliferate quickly and have a good effect.

Another important technical effect of the present invention is that the entire drying process of the present invention is carried out in a vacuum-tight environment, greatly reducing the chance of contact between the cornea and the air, and effectively avoiding contamination as compared with natural drying. Therefore, the present invention can satisfy corneal conditions in large quantities. In particular, in the present invention, all influencing factors such as temperature, decompression curve, and drying time are controllable, and the pre-dry state of the cornea obtained by different decellularization methods can be used, and various decompression methods most suitable for the cornea are used. The best combination of the corneal drying quality identity of the same batch or even batches.

The dried cornea obtained by the drying method of the present invention has characteristics of stable performance with respect to the non-dried cornea. It can be cleaned by the simplest method of cryopreservation (such as 0~8 degree cryopreservation in the refrigerator). Its quality and characteristics will not change its performance due to the length of storage or other factors, ensuring that the dried cornea is preserved and transported. After entering the clinical use state, the quality identity can still be guaranteed, and the purpose of preservation and transportation can be achieved. The dry cornea has the necessary conditions for industrialization and marketization as a product, which is beneficial to the market of artificial cornea.

Tests have shown that the dried cornea obtained by the drying method of the present invention has a mechanical processing property similar to that of a chemical contact lens when the water content is less than 10 to 15%. It is convenient to accurately process the shape of the cornea according to the requirements to achieve the special needs of corneal grafting. In particular, the processing of dry corneal diopter, the application of artificial cornea in refractive correction. The biomatrix cornea of the present invention has incomparable biocompatibility with respect to chemical contact lenses. DRAWINGS

The invention and its specific embodiments and their effects are briefly described below with reference to the accompanying drawings, which are merely illustrative of some specific experimental examples of the invention, and not all of the invention.

Figure 1 shows a first embodiment of the gradient decompression of the present invention;

Figure 2 is a second embodiment of the gradient decompression of the present invention;

Figure 3 is a third embodiment of the gradient decompression of the present invention;

Figure 4 is a fourth embodiment of the gradient decompression of the present invention;

Figure 5 is a fifth embodiment of the gradient decompression of the present invention;

Figure 6 is a first embodiment of continuous decompression of the present invention;

Figure 7 is a second embodiment of the continuous decompression of the present invention;

Figure 8 is a third embodiment of the continuous decompression of the present invention;

Figure 9 Ultrastructural ultrastructure of the lamellar dried cornea of the present invention;

Figure 10 is an ultrastructure of the lamellar dry corneal electron microscope of the present invention;

Figure 11 is a photograph of the dried corneal transparency of the ply of the present invention;

Figure 12 is a photograph of a 3-day postoperative lamellar keratoplasty of the present invention;

Figure 13 Photograph of the 2 months after lamellar keratoplasty of the present invention. detailed description

The invention provides a method for drying a decellularized cornea, which is first placed on a supporting device and placed in a closed drying chamber with adjustable vacuum; as shown in FIG. 1 to FIG. 7, the vacuum regulating system is adopted. The pressure in the closed drying chamber is depressurized to a set maximum vacuum degree for a period of time, and continues to reach the requirement of corneal dryness under the set maximum vacuum state, thereby obtaining a dried cornea.

The drying method of the present invention is based on the principle of vacuum drying, and therefore has the advantages of low vacuum drying temperature, no overheating, easy evaporation of water, and short drying time. At the same time, since the present invention adopts a method of gradually depressurizing by a period of time, the existing vacuum drying is effectively overcome to directly adjust the pressure to the maximum degree of vacuum, thereby causing the drying process to be too severe. The present invention makes the process of vacuum drying more gentle, and thus minimizes the degree of regular destruction of collagen fibers in the matrix layer during corneal drying. Under the conditions of the same decellularization method (ie, excluding the influence of other preparation processes on the cornea), the dried cornea obtained by the drying method of the present invention, the collagen fibers of the matrix layer are arranged and dried substantially before drying. There is no destructive change, and the cornea is kept to a maximum of biophysical properties that are substantially similar to the fresh cornea before drying.

The dried cornea obtained by the drying method of the present invention has a moisture content of the dried cornea of not more than 20%. The light transmittance is not less than 70%. The surface of the dried cornea is flat, with no visible protrusions or fine folds visible to the naked eye.

The clinical effect of the dried cornea obtained by the drying method of the present invention is remarkable: First, the dried cornea begins to gradually become transparent during the corneal transplantation. The dry cornea obtained by the drying method of the present invention greatly shortens the time for achieving transparency after corneal implantation, compared to the existing artificial cornea requiring a clear period of at least 3 months. Secondly, based on the extremely high flatness of the dried cornea of the present invention, another significant clinical effect is that the postoperative epithelial cell attachment and proliferation rate is fast.

As shown in Figs. 9 to 10, the ultrastructure and appearance of the dried artificial cornea obtained by the drying method of the present invention are shown. It can be seen from the dry corneal structure under the electron microscope shown in FIG. 9 to FIG. 10 that the cornea of the present invention retains the fine structure of the collagen tissue at a maximum, and the structural damage is small, and the average gap between the collagen fibers is uniform (25). ± 10nm).

As shown in Fig. 11, the dried cornea of the present invention has an extremely high transparency and a smooth surface, and has excellent flatness which is visually visible to the naked eye. Through animal experiments, its performance is closest to that of human cornea.

Fig. 12 and Fig. 13 are photographs showing the dry cornea shown in Example 1 at 3 and 2 months after human corneal transplantation. It can be seen from the postoperative effect photographs of Fig. 11 and Fig. 12 that the dried cornea of the present invention has been in a transparent state 3 days after the operation, the corneal epithelium is basically repaired, no obvious rejection reaction is observed, and the cornea is completely recovered 2 months after the operation. Transparent, no neovascularization, no rejection.

In the present invention, the temperature in the closed drying chamber is controlled within a range of 0 ° C to 30 ° C. The pressure in the closed drying chamber ranges from atmospheric pressure to the set maximum vacuum. The set maximum vacuum may be the set maximum vacuum. The maximum vacuum that can be achieved by the equipment varies depending on the equipment of each closed drying chamber. In the present invention, the pressure at the set maximum vacuum is close to the ultimate vacuum.

In the present invention, the pressure refers to a pressure value that the vacuum regulating system needs to adjust to adjust the closed drying chamber, and is not the measured pressure value in the closed drying chamber.

The invention will now be described in more detail by way of several specific embodiments.

Example 1

In this embodiment, as shown in FIG. 1, the decompression mode in the closed drying chamber is decompressed from a high to low gradient in at least two stages, and continues for a period of time on the pressure gradient of each stage; Then depressurize to the maximum vacuum state. The decompression value of each gradient is 10~50kpa. The duration of each stage of the decompression gradient is 30 minutes to 4 hours.

As shown in Fig. 1, in the present embodiment, when the pressure gradient changes, the vacuum regulating system adjusts the first stage pressure stop to instantaneously decrease to the next stage pressure block.

The specific steps of the drying method of this embodiment are as follows:

In the first step, the artificial cornea that needs to be dried is placed on the support device and placed in a vacuum drying oven at 0 ° C to 30 ° C;

The second step is to control the vacuum regulation system and start the gradient decompression operation. The pressure control curve is shown in Figure 1. The decompression gradient is 80kpa, 60kpa, 40kpa, 20kpa, and the duration is 2h, 2h, 2h, lh respectively. ;

In the third step, the pressure is reduced to the maximum vacuum of the device. In a specific example, the maximum vacuum of the device is 0.5 kPa, and then the artificial cornea is completely dried. In the specific example shown in Fig. 1, the duration is about 1 hour in the maximum vacuum state of the device.

In this embodiment, the pressure is gradually reduced to a maximum vacuum of the apparatus over a period of about 7 hours through a plurality of gradient levels, and then the corneal drying requirement is reached after a maximum vacuum of about 1 hour. Compared with the existing method of directly entering the maximum vacuum environment, the multi-gradient decompression method of the present embodiment has a milder drying process, thereby minimizing the regular arrangement of the collagen fibers in the matrix layer during the corneal drying process. The damage, so that the dried corneal product after drying can maintain good transparency and appearance flatness. In addition, the method of the low-temperature gradient vacuum drying method of the present embodiment greatly shortens the time compared with the natural drying, effectively reduces the denaturation of the corneal protein, and maintains the transparency of the cornea while ensuring the consistency of the corneal product.

The dried cornea obtained by the gradient decompression method of the present embodiment has a moisture content of 10 ± 2% and a light transmittance of 90 ± 2%. The surface of the dried cornea is flat, with no visible protrusions or fine folds as shown in Figure 11. Example 2

As shown in Fig. 2, in the present embodiment, the gradient is used to reduce the pressure from 60 kPa to the maximum vacuum of 0.3 kpa through two steps. As shown in Figure 2, when the pressure gradient changes, the vacuum adjustment system adjusts the previous stage pressure block to instantaneously adjust to the next stage pressure block.

In the present embodiment, the duration of the pressure gradient for each stage is set to decrease as the pressure decreases. The drying steps of this embodiment are as follows:

The second step is to control the vacuum regulation system and start the gradient decompression operation. The decompression curve is shown in Figure 2. The decompression gradient is 60kpa and 30kpa, and the duration is 4h and 4h respectively _.

In the third step, continue to decompress until the maximum vacuum of the device is 0.3kpa, and then keep until the artificial cornea is completely dry. The duration is approximately 1 hour.

In the present embodiment, after two gradient levels, the pressure is gradually reduced to about the maximum vacuum of the apparatus for about 8 hours, and then the corneal drying requirement is reached after the maximum vacuum for about one hour.

With the dried cornea obtained by the gradient decompression method of this example, the dried cornea has a water content of 18 ± 2% and a light transmittance of 82 ± 2%. The surface of the dried cornea is flat and has no visible protrusions or fine folds. Example 3

As shown in Fig. 3, in the present embodiment, the pressure is reduced from lOlkpa to a maximum vacuum of 0.3 kpa through nine steps using a gradient decompression method. As shown in Figure 3, when the pressure gradient changes, the vacuum adjustment system adjusts the first stage pressure block to instantaneously lower to the next stage pressure block. The pressure gradient of each stage of decompression is not evenly distributed.

The drying steps of this embodiment are as follows:

In the first step, the artificial cornea that needs to be dried is placed on the support device and placed in a drying oven at 0 ° C ~ 30 ° C;

The second step is to control the vacuum regulation system and start the gradient decompression operation. The decompression curve is shown in Figure 3. The decompression gradients are 85kpa, 70kpa, 60kpa, 45kpa, 35kpa, 25kpa, 15kpa, respectively. 2h, 2h, 2h, 1.5h, 1.5h, lh, lh;

In this embodiment, after 7 gradient levels, the pressure is gradually reduced to about the maximum vacuum of the apparatus for about 11 hours, and then the corneal drying requirement is reached after about 1 hour at the maximum vacuum.

The dried cornea obtained by the gradient decompression method of the present embodiment has a moisture content of 5 ± 0.5% and a light transmittance of 85 ± 2%. Dry corneal surface is flat, no macroscopically visible ridges or small Wrinkles. Example 4

As shown in Fig. 4, in the present embodiment, the pressure is reduced from 50 kPa to the maximum vacuum of the device by 0.2 kPa by means of gradient decompression. As shown in Figure 4, when the pressure gradient changes, the vacuum adjustment system adjusts the first stage pressure block to instantaneously lower to the next stage pressure block. The pressure gradient of each stage of decompression is not evenly distributed.

The drying steps of this embodiment are as follows:

The second step is to control the vacuum regulation system and start the gradient decompression operation. The decompression curve is shown in Figure 4. The decompression gradient is 50kpa, 30kpa, 15kpa, and the duration is 3h, 2h, lh, respectively.

In the third step, continue to decompress to a maximum vacuum of 0.2kpa, and then keep the artificial cornea completely dry. The duration is approximately 1 hour.

In the present embodiment, after three gradient levels, the pressure is gradually lowered to about the maximum vacuum of the apparatus for about 6 hours, and then the corneal drying requirement is reached after the maximum vacuum for about one hour.

The dried cornea obtained by the gradient decompression method of the present embodiment has a moisture content of 18 ± 2% and a light transmittance of 84 ± 2%. The surface of the dried cornea is flat and has no visible protrusions or fine folds. Example 5

As shown in Fig. 5, in the present embodiment, the gradient pressure is reduced by 5 steps to reduce the pressure from 80 kPa to the maximum vacuum of the device of 0.5 kPa.

As shown in Fig. 5, in the present embodiment, when the pressure gradient changes, the vacuum regulating system adjusts the pressure of the previous stage to gradually decrease to the pressure of the latter stage.

The drying steps of this embodiment are as follows:

The second step is to control the vacuum regulation system and start the gradient decompression operation. The decompression curve is shown in Figure 5. The decompression gradient is: Maintain 2h at 80kpa;

80kpa gradually reduced pressure to 60kpa after lh, and maintained lh at 60kpa;

60kpa gradually reduced pressure to 40kpa after lh, and maintained lh at 40kpa;

40kpa gradually depressurizes to 20kpa after lh, and maintains lh at 20kpa;

In the third step, 20kpa is gradually decompressed to a maximum vacuum of 0.5kpa after lh _; then it is kept at 0.5kpa until the artificial cornea is completely dry. The duration is approximately 1 hour.

The dried cornea obtained by the gradient decompression method of this example has a water content of 10 ± 2% and a light transmittance of 84 ± 1%. The surface of the dried cornea is flat and has no visible protrusions or fine folds. Example 6

As shown in Fig. 6, in the present embodiment, the pressure is reduced to the maximum vacuum of the apparatus for a period of time by means of continuous decompression.

The drying steps of this embodiment are as follows:

The second step is to control the vacuum regulation system and start the continuous decompression operation. The decompression curve is as shown in Fig. 6. The pressure in the closed and dry vessel is continuously decompressed at a constant speed to the maximum vacuum pressure of the device within 12 hours. Kpa;

The third step is to dry at 0.5kpa until the corneal moisture content is reached, about 1 hour. With the dried cornea obtained by the gradient decompression method of the present embodiment, the dried cornea has a water content of 5 ± 1% and a light transmittance of 85 ± 2%. The surface of the dried cornea is flat and has no visible protrusions or fine folds. Example 7

As shown in FIG. 7, this embodiment is another embodiment of continuous decompression. In this embodiment, the decompression mode is to continuously depressurize the pressure in the closed and dried container at different speeds to a period of time. The maximum vacuum pressure of the equipment.

The drying steps of this embodiment are as follows:

In the first step, the artificial cornea that needs to be dried is placed on the support device, and placed in a drying oven at 0 ° C ~ 30 ° C Inside;

The second step is to control the vacuum regulation system and start continuous decompression operation. The decompression curve is shown in Figure 7. The pressure is reduced from lOlkpa to 70kpa after 4 hours; then the pressure is reduced from 70kpa to 20kpa after 3 hours; Reduce the pressure from 20kpa to 0.5kpa in 3 hours;

The third step is to dry at 0.5kpa to reach the corneal moisture content requirement, about 2 hours. The dried cornea obtained by the gradient decompression method of the present embodiment has a moisture content of 12 g of the dried cornea.

1%, light transmittance 85 ± 3%. The surface of the dried cornea is flat and has no visible protrusions or fine folds. Example 8

As shown in FIG. 8, this embodiment is a third embodiment of continuous decompression. In this embodiment, the decompression mode is to continuously decompress the pressure in the closed and dry container at different speeds to a period of time. The maximum vacuum pressure of the equipment.

In the eighth embodiment, the vacuum regulating system is controlled, and the pressure reducing operation is continuously decompressed in a constant-speed decompression manner. The decompression curve is as shown in FIG. 8 , and the pressure is continuously reduced from 95 kPa to the system limit pressure of 0.3 kPa in 24 hours; The dried cornea obtained by the gradient decompression method of the example had a moisture content of 5 ± 1% and a light transmittance of 79 ± 3%. The surface of the dried cornea is flat and has no visible protrusions or fine folds.

According to the above drying method of the present invention, since the entire drying process is carried out in a vacuum-tight environment, the contact chance of the cornea and the air is greatly reduced, and contamination can be effectively prevented as compared with natural drying. Therefore, the present invention can satisfy corneal conditions in large quantities. In particular, in the present invention, all the influencing factors such as temperature, decompression curve, and drying time are controllable, and the pre-dry state of the cornea can be obtained according to different decellularization methods, and various decompression methods most suitable for the cornea are used. The best combination of the corneal drying quality identity of the same batch or even batches.

The dried cornea obtained by the drying method of the present invention has the characteristics of stable performance with respect to the non-dried cornea. It can be cleaned by the simplest method of cryopreservation (such as 0~8 degree cryopreservation in the refrigerator). Its quality and characteristics will not change its performance due to the length of storage or other factors, ensuring that the dried cornea is preserved and transported. After entering the clinical use state, the quality identity can still be guaranteed, and the purpose of preservation and transportation can be achieved. The dry cornea has the necessary conditions for industrialization and marketization as a product, which is beneficial to the market promotion of artificial cornea. In addition, tests have shown that the dried cornea obtained by the drying method of the present invention has a mechanical processing property similar to that of a chemical contact lens when the water content is less than 10 to 15%. It is convenient to accurately process the shape of the cornea according to the requirements to achieve the special needs of corneal grafting. In particular, the processing of dry corneal diopter, the application of artificial cornea in refractive correction. The biomatrix cornea of the present invention has incomparable biocompatibility with respect to chemical contact lenses.

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 invention, but the description should not be construed as limiting the invention in any way, particularly The various features described in the embodiments can also be arbitrarily combined with each other to form other embodiments, and the features are to be understood as being applicable to any one embodiment, and not limited to the described embodiments. the way.

Claims

Rights request

1. A method for drying a decellularized cornea, first placing the cornea to be dried on a support device, and placing it in a closed drying chamber with adjustable vacuum; and reducing the pressure in the closed drying chamber to below atmospheric pressure by a vacuum regulating system; The method is characterized in that the pressure of the closed drying chamber is gradually reduced to a pressure at a set maximum vacuum for a period of time, and continues to reach the requirement of corneal dryness under the set maximum vacuum state.

The method for drying acellular cornea according to claim 1, wherein the decompression mode in the closed drying chamber is at least two stages from high to low gradient decompression, and continues at a pressure gradient of each stage. A period of time; the final stage gradient pressure is then depressurized to a set maximum vacuum state.

The method for drying a decellularized cornea according to claim 1, wherein the method of controlling the decompression in the closed drying chamber is continuous depressurization to a set maximum vacuum state for a period of time.

The method for drying a decellularized cornea according to claim 1, wherein the temperature in the sealed dry chamber is controlled within a range of 0 ° C to 30 ° C.

The method for drying a decellularized cornea according to claim 1, wherein the true air-conditioning system adjusts the pressure at which the pressure in the closed drying chamber is reduced to a set maximum vacuum within 24 hours.

The method for drying a decellularized cornea according to claim 5, wherein the preferred decompression time for adjusting the pressure in the closed drying chamber to a set maximum vacuum is 6 to 12 hours.

The method for drying an acellular cornea according to claim 1, wherein the pressure-regulating range of the sealed drying chamber is from a normal pressure to a set maximum vacuum.

The method of drying a decellularized cornea according to claim 1, wherein the pressure at the set maximum degree of vacuum is close to an ultimate vacuum.

The method for drying a decellularized cornea according to claim 2, wherein the decompression range of each step is 10 to 100 kPa.

The method for drying a decellularized cornea according to claim 9, wherein the preferred decompression range of each step is 10 to 30 kPa.

11. A method of drying a decellularized cornea according to claim 8, wherein the duration of the pressure gradient for each stage is set to vary with changes in pressure.

12. The method of drying a decellularized cornea according to claim 2, wherein the vacuum regulating system adjusts the first stage pressure block to directly adjust to the second stage pressure block when the pressure gradient changes.

The method for drying a decellularized cornea according to claim 2, wherein the vacuum regulating system adjusts the pressure of the first stage to gradually decrease to the pressure of the latter stage when the pressure gradient changes.

The method for drying a decellularized cornea according to claim 2, wherein the duration of each of the decompression gradients is 0.5 to 12 hours.

The method of drying a decellularized cornea according to claim 14, wherein the preferred duration of each of the reduced pressure gradients is 0.5 to 4 hours.

The method of drying a decellularized cornea according to claim 1, wherein the corneal dryness is set in accordance with a corneal moisture content.

The method for drying acellular cornea according to claim 15, wherein the corneal dryness is set to a moisture content of not more than 20%.

18. A decellularized porcine lamellar dried cornea, the decellularized porcine corneal anterior elastic layer and a matrix layer, characterized in that the dried cornea is obtained by any of the drying methods according to any one of claims 1 to 16.

19. A decellularized pig lamellar dried cornea according to claim 18, wherein the dried cornea has a moisture content of greater than 0% and not greater than 20%.

20. A decellularized pig layer dried cornea according to claim 18, wherein the dried cornea has a light transmittance of not less than 70%.

21. A decellularized porcine lamellar dried cornea according to claim 18, wherein the surface of the dried keratome is flat without macroscopic ridges or fine pleats.