CN109069264B - Drying method of acellular cornea and acellular pig lamina layer dried cornea - Google Patents
Drying method of acellular cornea and acellular pig lamina layer dried cornea Download PDFInfo
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Abstract
A drying method of acellular lamellar cornea and an acellular porcine lamellar dry cornea thereof. The drying method comprises the steps of firstly putting the cornea into a vacuum drying chamber, gradually reducing the pressure in the closed drying chamber, and gradually reducing the pressure to the set maximum vacuum degree within a period of time until the requirement of the dryness of the cornea is met. The drying method of the invention makes the drying process of the cornea milder, and minimizes the damage to the collagen arrangement structure of the cornea in the vacuum drying link.
Description
Technical Field
The invention relates to a preparation method of an artificial cornea for treating eye blindness caused by a cornea, in particular to a method for drying an acellular cornea and an acellular lamellar dry cornea taking a pig cornea as a donor source.
Background
The cornea transplantation is the only effective method for treating the corneal blindness at present, the wide-range popularization of the operation is greatly influenced due to the serious shortage of the current situation of the corneal donor in China, but the research of China in the field of xenogenic cornea replacement is promoted to obtain the achievement of drawing attention, and particularly the application result of the acellular pig lamellar cornea taking the pig cornea as the donor source is praised by the foreign scientific community as one of the five major innovations in the contemporary China.
The last decade is a well-established conclusion that corneal substitutes have been the best choice based on porcine cornea having highly similar tissue structure, biophysical properties, and optical properties to human cornea. The ocular academicians in China successfully realize the direct application of the porcine lamellar cornea obtained by cell-free treatment to human body transplantation and obtain certain clinical effect. Compared with the artificial cornea made of other materials, the artificial cornea made of the pig cornea has the advantages of wide material source, low cost, simple processing method, good clinical effect and the like. Two companies have developed artificial cornea products taking pig cornea as donor source in China, and the artificial cornea products are applied to clinic through market admission approval of relevant supervision departments in China, bring good news to patients with keratopathy and bring a huge market demand.
Lamellar corneas are corneas of lamellar structure (scaffolds) comprising only a pre-elastic layer and a stromal layer, as opposed to full-lamellar corneas. The method comprises three forms: undried lamellar supports (meaning dehydrated to the moisture content of the original cornea), dried lamellar supports, and rehydrated lamellar supports. (see the inventor's prior application No. CN 10197144). A great number of methods for preparing acellular porcine cornea laminae disclosed in the patent literature and the related literature at present, and the detection data, animal test data and clinical effects obtained by the preparation method adopted by the methods. Most of them were obtained in a state of non-dried cornea after completion of decellularization treatment in a laboratory. And because of different preparation methods, the test data or clinical effects are not completely the same. Needless to say, in the prior art disclosed at present, the cornea product and its application effect are only established in the state of test data of a non-dry cornea, and the conditions for popularization and use in the market, that is, the identity and performance stability of the product quality are guaranteed in each step of market operation such as production, storage, transportation and use, are not yet provided.
The biggest drawback of non-dry corneas in particular is their extreme ease of storage and transport. During storage and transport, the quality of the lamellar corneas and their biophysical properties in the non-dry state change with changes in storage and transport conditions, which are unpredictable. Therefore, the doctor can not judge the postoperative effect of the artificial cornea used by the doctor in clinic. According to published data, the clinical effect statistics of the artificial cornea transplantation on the market at present can show that even if the operation is successful, the postoperative recovery time is 3 to 6 months long. Not only is the result of such transplantation far from that of human cornea, but it is also difficult for the doctor to make an accurate judgment as to whether the transplantation is successful and the postoperative recovery effect in as short a time as possible, which is disadvantageous for taking as early a remedy as possible for the result after the transplantation, which is failed or has a poor recovery effect.
To solve the above problems, researchers have proposed a method of obtaining a dried cornea which is easy to store and transport by subjecting the cornea after decellularization to a drying treatment as necessary. However, many alternative drying methods proposed so far are conventional drying methods. For example, the methods for drying the cornea disclosed so far mainly include freeze drying, glycerin dehydration, modified silica gel dehydration, phosphorus pentoxide dehydration, anhydrous calcium chloride dehydration, and the like, which are widely used in the conventional drying techniques for medical devices. The applicant has exhausted the current conventional drying methods to perform a number of drying treatment trials on keratoprostheses. Comparing all biophysics of the artificial cornea before and after drying, it was found that any conventional drying process may have some adverse effect on the biophysical properties of the artificial cornea. The applicant has found, after conducting research statistical analysis on a large amount of test data obtained during the drying process for each drying method, that the reason why the conventional drying method described above adversely affects the artificial cornea is that the drying process is excessively severe. In particular, after the artificial cornea is decellularized, the collagen scaffold becomes very loose due to the large amount of water contained in the cornea and the gaps existing after the cells are removed, and simultaneously the original supporting force of collagen arrangement in the stromal layer is lost. In this state, the severe drying process inevitably causes irregular changes in the arrangement of collagen tissues, thereby destroying the highly regular arrangement of collagen fibers in the original corneal stroma.
The cornea has distinct characteristics from all other tissues: and (4) transparent. This is also the most important basis for the cornea to perform its physiological functions. While corneal transparency is due to the highly regular arrangement of collagen fibril structures in the stromal layer. Meanwhile, the highly regular arrangement of collagen fiber structures of the corneal stroma layer is also the structural basis of the mechanical elasticity of the cornea. Numerous experiments have demonstrated that any of the above-described conventional drying methods can disrupt the regular arrangement of collagen fibers in the corneal stroma to varying degrees, adversely affecting biophysical properties of the artificial cornea product, such as transparency, mechanical elasticity, and the like. For example, in patent No. CN104645415, all conventional drying methods are disclosed, including vacuum freeze-drying, vacuum air-drying, natural air-drying, vacuum drying of dry anhydrous calcium chloride, oven-drying at 30-60 ℃ and the like. All the conventional drying methods except natural airing achieve the purposes of shortening the drying time and ensuring the same drying effect through the interference of unnatural factors.
In research practice, the drying treatment of biological materials is also carried out by using a vacuum drying method. Because the vacuum drying has the characteristics of low temperature, no overheating phenomenon, easy evaporation of water, short drying time, reduction of the contact chance of materials and air, avoidance of pollution and the like, the method is the most widely used drying method at present. However, vacuum drying is to reduce the boiling point (boiling point drying) or melting point (freeze drying) of water by reducing pressure in a closed environment, so as to accelerate the vaporization or sublimation of water in an object, thereby achieving the purpose of rapid drying. According to the principle of vacuum drying, boiling point drying belongs to a high-temperature drying process. The evaporation and boiling of the water in the material during the drying process are carried out simultaneously with sufficient heat, and although the boiling point of the water is reduced under the low pressure condition, a certain amount of heat is required to ensure that the water in the drying object reaches the evaporation and boiling conditions, which is obviously violent. Meanwhile, when the vaporized steam is pumped out quickly by vacuum pumping, a negative pressure state is formed around the material, and a larger humidity gradient is formed between the inner layer and the outer layer of the material, so that the boiling point drying is also extremely uneven drying, and the direct application of the material to the drying of fresh cornea can cause certain damage to the arrangement of corneal collagen fibers. Therefore, boiling point drying methods are rarely used for drying the cornea in scientific research and production practice.
In the sublimation drying (freeze-drying) method, the moisture of the material is first frozen into a solid state, and then the solid moisture therein is sublimated directly into a gaseous state under a high vacuum to be removed. The method has no high-temperature violent reaction such as evaporation and boiling in boiling point drying, and is a drying method which is applied to a large number of biological materials. However, since the principle of this drying method is to lower 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 porous structure. The experimental conclusion presented in the paper "comparison of dried and lyophilized acellular porcine corneal stroma as a tissue engineering corneal scaffold material" published by the fourth university of military medical science shows that the collagen arrangement of the lyophilized (freeze-dried) porcine corneal stroma is disordered and has a porous structure under TEM observation, and the front elastic surface has ridge-like protrusions and loses the regular lamellar structure. Numerous experiments have demonstrated that during corneal acellular processes, when the collagen tissue is in a relaxed state with an increased amount of water in the corneal stroma, the interplanar collagen and intralamellar collagen distances are increased and completely dominated by water. When the flowing water distribution is not uniform, the distance between the collagen fibers in the corneal lamellae is also not uniform, the arrangement structure of the collagen fibers in the corneal lamellae is also in an unstable state, and when the water is solidified into ice crystals at a low temperature, the original high regularity of the arrangement of the collagen fibers in the corneal lamellae is difficult to ensure. It is readily evident that the vacuum freeze-drying method has a considerable effect on the transparency of the artificial cornea.
The patent No. CN 104188957 proposes basically the same natural drying method without influence of unnatural factors by sterile air drying, namely, the acellular corneal stroma is paved on a 24-hole plate culture plate and is placed on an ultra-clean bench for sterile air drying for 48-72 hours. The natural drying method has the defect that the drying time is too long, so that collagen of the cornea is easily denatured, and the transparency of the cornea is affected. And the difficulty of maintaining the sterility of the environment throughout all drying times is quite high. In addition, other conditions of natural drying such as temperature control, wind power adjustment, etc. are difficult to control, and the degree of drying and the drying time are difficult to control. The effect of other uncontrollable factors in the natural environment can cause different drying effects, and even the drying effect of the same batch of corneas is different due to uneven drying, so that the drying effect of all batches can not be ensured to be the same. Therefore, the current natural drying method is only suitable for laboratory or small-batch production conditions, and basically cannot be applied to large-batch industrial scale production. This is also why there is currently substantially no adoption in the field of medical device industrialization.
Based on the preservation experience of human cornea, some documents propose to use more glycerol preservation method. The method comprises placing non-dried cornea in glycerol, and sealing for storage. This method is mainly a gradual dehydration process in which the cornea is preserved in glycerol liquid and is performed during the preservation process. Firstly, the liquid storage state is not beneficial to transportation, and potential safety hazards such as explosion and the like are easily generated in the transportation process of the glycerol; secondly, the quality identity of the cornea is difficult to ensure in the gradual dehydration process when the cornea is in a clinical use state, and the cornea transplantation can not be standardized, so that doctors need to make adaptive changes according to the current preservation state of the cornea in clinic; thirdly, cleaning is needed before operation to remove glycerin stuck on the cornea, and if the cleaning is not clean, residues are easily formed on the cornea to seriously affect the operation effect. Fourth, non-dry corneas are incapable of shape modification. For example, the corneal shape is modified according to the diopter requirements of the transplanted cornea.
In a large number of animal experiments and partial clinical experiments on the dry cornea, the applicant found that when the dry cornea is used for corneal transplantation, the transparency of the dry cornea and the shape of the cornea, particularly the flatness of the cornea are very important for the postoperative effect of corneal transplantation. First, the higher the transparency, the higher the clarity of the graft post-surgery vision; secondly, the drying cornea with basically the same water content can standardize the rehydration operation of the transplantation, can realize the effective control of the rehydration saturation of the cornea after rehydration, and is beneficial to shortening the postoperative transparent time of the cornea; thirdly, the higher the flatness of the cornea, especially the elastic layer before the cornea, is, the more favorable the adhesion and proliferation of epithelial layer cells are. A dry cornea that is uneven or has a visually apparent ridged protrusion or fine fold on its surface may affect the post-transplant vision recovery to varying degrees.
With the increasingly mature and development of technology for treating corneal blindness and blindness by replacing human cornea with the porcine acellular lamellar cornea, the demand of the porcine acellular lamellar cornea in the market is greatly improved. Therefore, there is a need for a method for preparing a dried cornea that maximizes the quality consistency and performance stability of the dried cornea product while meeting the market attributes of being convenient for storage and transportation.
Disclosure of Invention
The invention aims to provide a method for drying a decellularized cornea, which ensures that the drying process of the cornea is milder, minimizes the damage to the arrangement structure of collagen fibers of the cornea in the drying link, and ensures that the cornea after drying maintains the physical characteristics which are basically the same as those of the cornea before drying to the maximum extent.
Another object of the present invention is to provide a method for drying a decellularized cornea and a dried cornea thereof, which can improve the transparency of the dried cornea to shorten the time for restoring the transparency after the corneal implantation.
Still another object of the present invention is to provide a method for drying a decellularized cornea and a dried cornea thereof, which can improve the flatness of the appearance of the dried cornea, particularly the flatness of the elastic layer before the cornea, so as to improve the growth rate and growth quality of epithelial cells after the cornea is implanted.
The fourth purpose of the present invention is to provide a method for drying a decellularized cornea and a dried cornea thereof, which are convenient for storage and transportation and which make an artificial cornea have the attributes of products that are distributed in the market. The standardization, industrialization and marketization development of the artificial cornea product are promoted, and the huge market demand of the artificial cornea is met.
The fifth objective of the present invention is to provide a method for drying a decellularized cornea and a dried cornea thereof, so that the cornea can be conveniently and precisely modified and processed according to the requirements, particularly the corneal diopter can be processed, and the application range of the artificial cornea in the refractive correction can be expanded.
The invention aims to realize the method, the cornea to be dried is placed on a supporting device and is placed in a closed drying chamber with adjustable vacuum degree; reducing the pressure in the closed drying chamber to be lower than the atmospheric pressure by a vacuum regulating system; the method is characterized in that the pressure of the closed drying chamber is regulated to be gradually reduced to the pressure when the set maximum vacuum degree is reached in a certain time period, and the pressure is continued to reach the requirement of the corneal dryness under the set maximum vacuum degree state.
The working principle of the invention is that after the cornea to be dried is put into the closed drying chamber with adjustable vacuum degree, the pressure in the vacuum drying chamber is set to be lower than the atmospheric pressure but higher than the maximum vacuum degree of the drying chamber, and the pressure in the closed drying chamber is gradually adjusted to the set maximum vacuum degree through the adjusting system until the cornea reaches the set dryness requirement. Compared with the existing vacuum drying method, the vacuum drying process of the invention becomes milder, thereby overcoming the defect that the drying process of the existing drying method is too violent. Therefore, the invention minimizes the damage degree of the regular arrangement of the collagen of the stroma during the corneal desiccation process. The dried cornea obtained using the drying method of the present invention has a stroma layer collagen arrangement that is substantially unchanged from that before drying without further destructive alteration under the same conditions of the decellularization process (i.e., excluding the effects of other preparative processes on the cornea). The drying method is suitable for drying treatment of 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-stage pressure reduction from high to low gradient, and the pressure gradient of each stage lasts for a period of time; and reducing the final stage of gradient pressure to a set maximum vacuum degree state. The preferred embodiment is more convenient to operate and easy to control. Wherein:
in an optional example of the invention, the decompression range of each stage of gradient is 10-100 kpa; wherein the preferential pressure reduction range is 10 to 30 kpa.
In an alternative embodiment of the invention, the duration of the pressure gradient at each stage is set to decrease with decreasing pressure.
In an alternative embodiment of the invention, the vacuum regulation system regulates the pressure range of the previous stage directly to the pressure range of the subsequent stage when the pressure gradient changes.
In another alternative example of the present invention, the vacuum regulating system regulates the pressure of the previous stage to gradually decrease to the pressure of the next stage when the pressure gradient changes.
In another alternative example of the present invention, the duration of each stage of the decompression gradient is 0.5 to 12 hours; wherein the preferential duration time is 0.5-4 hours.
In another embodiment of the present invention, the method of controlling the pressure reduction in the sealed drying chamber is a method of continuously reducing the pressure to a predetermined maximum vacuum state for a certain period of time. In the continuous pressure reduction embodiment, the pressure reduction operation may be automatically controlled.
In an alternative embodiment of the present invention, the temperature in the closed drying chamber is controlled to be within 0 ℃ to 30 ℃.
In an optional example of the invention, the vacuum regulation system regulates the pressure of the closed drying chamber when the pressure is reduced to the set maximum vacuum degree in not more than 24 hours; wherein the pressure is preferably reduced to a predetermined maximum vacuum degree within 6 to 11 hours.
In an alternative example of the present invention, the pressure of the sealed drying chamber is adjusted from normal pressure to a set maximum vacuum.
In an alternative embodiment of the invention, the pressure at the set maximum vacuum level is close to the ultimate vacuum.
In an alternative embodiment of the present invention, the corneal dryness is set according to the corneal moisture content.
In a preferred embodiment of the present invention, the dryness of the cornea is optimally set to a moisture content of not more than 20%.
The invention provides a decellularized pig dried cornea, which consists of a porcine cornea front elastic layer and a stroma layer which are subjected to decellularization treatment, and is obtained by any one of the 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 dried corneal surface was smooth with no visible ridges or fine folds.
Compared with the current conventional drying technology, the invention has quite remarkable effect. Firstly, the invention is based on the principle of vacuum drying, thus having the advantages of low vacuum drying temperature, no overheating phenomenon, easy evaporation of water and short drying time. However, the invention effectively overcomes the defect of over-violent drying in the existing vacuum drying method by gradually reducing the pressure, so that the vacuum drying process becomes milder. And thus minimize the disruption of the regular arrangement of collagen fibers in the stromal layer during corneal desiccation. The dried cornea obtained by the drying method of the present invention has a stroma layer collagen fibril arrangement that is not substantially further destructively altered than before drying, under the same conditions of the decellularization process (i.e., excluding the effects of other preparative processes on the cornea). The cornea after desiccation retains to the maximum extent the biophysical properties of the cornea substantially the same as those of the cornea before desiccation. The test results show that the dried cornea obtained by the drying method has higher transparency, smooth surface and excellent flatness visible to the naked eye compared with the dried cornea obtained by the conventional drying method.
The clinical effect of the dry cornea of the invention is more obvious: first, the dry cornea of the present invention begins to become transparent during the corneal transplant procedure. Compared with the existing cornea drying process which requires at least 3 months of recovery period, the drying method of the invention can greatly shorten the time for restoring the transparency of the cornea after implantation. Secondly, the flatness of the dried cornea is extremely high based on the invention, and another remarkable clinical effect is that the epithelial cells are fast in adhesion and proliferation speed after operation and good in effect.
The other important technical effect of the invention is that the whole drying process of the invention is carried out in a vacuum closed environment, thereby greatly reducing the contact chance between the cornea and the air and effectively avoiding pollution compared with natural drying. Therefore, the invention can meet the requirement of preparing the cornea in large batch. Particularly, in the present invention, controllability of all the influencing factors such as temperature, decompression curve, drying time and the like can be used to achieve the same quality of corneal drying in the same batch or even multiple batches by adopting the optimal combination of various decompression modes most suitable for the cornea according to the pre-drying state of the cornea obtained by different acellular methods.
Compared with non-dried cornea, the dried cornea obtained by the drying method has the characteristic of stable performance. The quality and the characteristics of the cornea can not be changed due to the length of the preservation time or other factors through the simplest low-temperature preservation method (for example, the low-temperature preservation at 0-8 ℃ in a refrigerator), the quality identity of the dried cornea can still be ensured when the dried cornea enters a clinical use state after preservation and transportation, and the purposes of convenient preservation and transportation are realized. So that the dry cornea has the necessary conditions of industrialization and marketization as a product, and is beneficial to the market popularization of the artificial cornea.
Experiments prove that the dried cornea obtained by the drying method has the machining performance similar to that of a chemical contact lens when the water content is lower than 10-15%. Can conveniently and accurately process the shape of the cornea according to the requirement, thereby meeting the special requirement of corneal transplantation. In particular to the processing of dry cornea diopter, thereby realizing the application of the artificial cornea in the aspect of refraction correction. The bio-stromal cornea of the present invention is not comparable to chemical contact lenses in biocompatibility.
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The present invention, its embodiments and effects will be described briefly with reference to the accompanying drawings, which are only illustrative of some specific test examples selected for the present invention and are not all the present invention.
FIG. 1 is a first embodiment of the gradient depressurization of the present invention;
FIG. 2 is a second embodiment of the gradient depressurization of the present invention;
FIG. 3 is a third embodiment of the gradient depressurization of the present invention;
FIG. 4 is a fourth embodiment of the gradient depressurization of the present invention;
FIG. 5 is a fifth embodiment of the gradient depressurization of the present invention;
FIG. 6 shows a first embodiment of the present invention with continuous pressure reduction;
FIG. 7 is a second embodiment of the present invention for continuous pressure reduction;
FIG. 8 is a third embodiment of the present invention for continuous pressure reduction;
FIG. 9 shows an electron microscopic microstructure of a lamellar dried cornea of the present invention;
FIG. 10 is an ultra-microstructure of a lamellar desiccation cornea of the present invention under an electron microscope;
FIG. 11 is a photograph of the transparency of a lamellar dried cornea of the invention;
FIG. 12 is a photograph of a lamellar dry keratoplasty of the invention taken 3 days after surgery;
FIG. 13 is a photograph of a lamina dry cornea of the present invention taken 2 months after transplantation.
Detailed Description
The invention provides a drying method of a decellularized cornea, which comprises the steps of placing a cornea to be dried on a supporting device, and placing the cornea to be dried in a closed drying chamber with adjustable vacuum degree; as shown in fig. 1 to 7, the pressure in the sealed drying chamber is reduced to the pressure at the set maximum vacuum degree in a period of time by the vacuum regulation system, and is continued to reach the requirement of corneal dryness under the set maximum vacuum degree state, so as to obtain the dry cornea.
The drying method is based on the principle of vacuum drying, so that the method has the advantages of low vacuum drying temperature, no overheating phenomenon, easy evaporation of water and short drying time. Meanwhile, the invention adopts a mode of gradually reducing pressure within a time period, thereby effectively overcoming the defect that the drying process is too violent as the pressure is directly adjusted to the maximum vacuum degree in the existing vacuum drying. The present invention makes the vacuum drying process milder and thus minimizes the damage to the regular arrangement of collagen fibers in the stromal layer during corneal drying. Under the same conditions of the decellularization method (i.e., excluding the influence of other preparation processes on the cornea), the dried cornea obtained by the drying method of the present invention has the collagen fiber arrangement of the stroma layer not changed substantially destructively with that before drying, and the dried cornea maintains the biophysical characteristics substantially similar to that of a fresh cornea before drying to the maximum extent.
The water content of the dried cornea obtained by the drying method is not higher than 20%. The light transmittance is not less than 70%. The dried corneal surface was smooth with no visible ridges or fine folds.
The clinical effect of the dried cornea obtained by the drying method is obvious: first, the dry cornea begins to become transparent during the corneal transplant procedure. Compared with the prior artificial cornea which needs at least 3 months of transparency, the dry cornea obtained by the drying method greatly shortens the time for realizing transparency after the cornea is implanted. Secondly, the flatness of the dried cornea is extremely high based on the invention, and another remarkable clinical effect is that the postoperative epithelial cell attaching and proliferation speed is high and the effect is good.
As shown in fig. 9 to 10, the ultra-fine structure and the appearance picture of the dried artificial cornea obtained by the drying method of the present invention are shown. As can be seen from the dried cornea structures under electron microscope shown in FIGS. 9 to 10, the cornea of the present invention after drying maintains the collagen microstructure with regular arrangement to the maximum extent, with little structural damage and uniform average gaps (25. + -.10 nm) between collagen fibers.
As shown in FIG. 11, the dried cornea of the present invention was extremely transparent, smooth in surface, and had excellent flatness visible to the naked eye. Animal experiments show that the performance of the cornea-based cornea-.
Fig. 12 and 13 are photographs showing the dried cornea of example 1 at 3 days and 2 months after the human corneal transplantation. As can be seen from the photographs of the effects after the operations of fig. 11 and fig. 12, the dry cornea of the invention is already in a transparent state 3 days after the operations, the corneal epithelium is basically repaired, no obvious rejection reaction is seen, the cornea is completely recovered to be transparent after 2 months after the operations, no new blood vessel grows in, and no rejection reaction is seen.
In the invention, the temperature in the closed drying chamber is controlled within 0-30 ℃. The pressure range in the closed drying chamber is from normal pressure to the set maximum vacuum degree. The set maximum vacuum level may be the set maximum vacuum level. Because each closed drying chamber has different equipment, the maximum vacuum degree which can be reached by the equipment also has difference, and in the invention, the pressure when the set maximum vacuum degree is close to the limit vacuum.
In the invention, the pressure refers to a pressure value which needs to be reached by a vacuum adjusting system to adjust the closed drying chamber, and is not an actually measured pressure value in the closed drying chamber.
The invention is illustrated in more detail below by means of several specific examples.
Example 1
In this embodiment, as shown in fig. 1, the pressure reduction mode in the closed drying chamber is to reduce the pressure from high to low in at least two stages, and the pressure gradient at each stage lasts for a period of time; and reducing the pressure of the last stage of gradient to the maximum vacuum state.
The decompression value of each grade of gradient is 10-50 kpa. The duration of each step of reduced pressure gradient is between 30 minutes and 4 hours.
As shown in fig. 1, in the present embodiment, the vacuum regulation system regulates the pressure level of the previous stage to decrease to the pressure level of the next stage instantaneously when the pressure gradient changes.
The drying method of the embodiment specifically comprises the following steps:
firstly, placing an artificial cornea to be dried on a supporting device, and placing the artificial cornea in a vacuum drying oven at the temperature of 0-30 ℃;
secondly, controlling a vacuum adjusting system, and starting gradient pressure reduction operation, wherein the pressure reduction curve of pressure control is shown in figure 1, the pressure reduction gradient is 80kpa, 60kpa, 40kpa and 20kpa, and the duration time is 2h, 2h and 1h respectively;
thirdly, the decompression is continued to the maximum vacuum of the apparatus, which in one specific example is 0.5kpa, and then is maintained until the artificial cornea is completely dry. In the specific example shown in fig. 1, the duration is about 1 hour in the state of the maximum degree of vacuum of the apparatus.
In this example, the pressure is gradually reduced to about 7 hours or so through a plurality of gradient levels to the maximum vacuum of the apparatus, and then the corneal desiccation requirement is achieved after about 1 hour of maximum vacuum. Compared with the existing method for drying in the maximum vacuum environment, the multi-gradient decompression mode of the embodiment has a milder drying process, so that the damage to the regular arrangement of the collagen fibers of the stroma layer in the cornea drying process is reduced to the maximum extent, and the dried cornea product can keep better transparency and appearance smoothness. In addition, compared with natural drying, the low-temperature gradient vacuum drying method has the advantages that the time is greatly shortened, the corneal protein denaturation is effectively reduced, and the consistency of corneal products is guaranteed while the corneal transparency is maintained.
The water content of the dried cornea obtained by the gradient decompression method of the embodiment is 10 +/-2%, and the light transmittance is 90 +/-2%. The dried corneal surface was smooth with no visible ridges or fine folds as shown in fig. 11.
Example 2
In the present embodiment, as shown in fig. 2, a gradient pressure reduction method is adopted, and the pressure is reduced from 60kpa to 0.3kpa of the maximum vacuum degree of the device through two steps. As shown in fig. 2, the vacuum regulating system adjusts the pressure level of the previous stage to the pressure level of the next stage instantaneously when the pressure gradient changes.
In this embodiment, the duration over the pressure gradient of each stage is set to decrease with decreasing pressure.
The drying procedure for this example is as follows:
firstly, placing an artificial cornea to be dried on a supporting device, and placing the artificial cornea in a vacuum drying oven at the temperature of 0-30 ℃;
secondly, controlling a vacuum regulating system, and starting gradient decompression operation, wherein decompression curves are shown in figure 2, decompression gradients are 60kpa and 30kpa, and duration time is 4h and 4h respectively;
and thirdly, continuously reducing the pressure to the maximum vacuum degree of the device of 0.3kpa, and then keeping the pressure until the artificial cornea is completely dried. The duration is approximately 1 hour.
In this example, the pressure is gradually reduced to about 8 hours after 2 gradient steps to the maximum vacuum of the apparatus, and then the corneal desiccation requirement is achieved after about 1 hour of maximum vacuum.
The water content of the dried cornea obtained by the gradient decompression method of this example was 18. + -. 2% and the light transmittance was 82. + -. 2%. The dried corneal surface was smooth with no visible ridges or fine folds.
Example 3
In the present embodiment, as shown in fig. 3, the pressure is reduced from 101kpa to 0.3kpa at the maximum vacuum degree of the equipment by 9 steps in a gradient pressure reduction manner. As shown in fig. 3, the vacuum regulating system regulates the pressure level of the previous stage to decrease to the pressure level of the next stage instantaneously when the pressure gradient changes. The pressure gradient of each stage of pressure reduction is not equally distributed.
The drying procedure for this example is as follows:
firstly, placing an artificial cornea to be dried on a supporting device, and placing the artificial cornea in a drying oven at the temperature of 0-30 ℃;
secondly, controlling a vacuum adjusting system, and starting gradient decompression operation, wherein a decompression curve is shown in fig. 3, decompression gradients are 85kpa, 70kpa, 60kpa, 45kpa, 35kpa, 25kpa and 15kpa, and duration time is 2h, 1.5h, 1h and 1h respectively;
and thirdly, continuously reducing the pressure to the maximum vacuum degree of the device of 0.3kpa, and then keeping the pressure until the artificial cornea is completely dried. The duration is approximately 1 hour.
In this example, the pressure was gradually reduced to about 11 hours to the maximum vacuum of the apparatus over 7 gradient steps, and then the corneal desiccation requirement was achieved after about 1 hour at maximum vacuum.
The water content of the dried cornea obtained by the gradient decompression method of this example was 5. + -. 0.5%, and the light transmittance was 85. + -. 2%. The dried corneal surface was smooth with no visible ridges or fine folds.
Example 4
As shown in FIG. 4, in the present embodiment, the pressure is reduced from 50kpa to 0.2kpa at the maximum vacuum degree of the apparatus by 4 steps in a gradient decompression manner. As shown in fig. 4, the vacuum regulating system regulates the pressure level of the previous stage to decrease to the pressure level of the next stage instantaneously when the pressure gradient changes. The pressure gradient of each stage of pressure reduction is not equally distributed.
The drying procedure for this example is as follows:
firstly, placing an artificial cornea to be dried on a supporting device, and placing the artificial cornea in a drying oven at the temperature of 0-30 ℃;
secondly, controlling a vacuum regulating system, starting gradient decompression operation, wherein the decompression curve is shown in figure 4, the decompression gradient is 50kpa, 30kpa and 15kpa, the duration time is 3h, 2h and 1h respectively,
and thirdly, continuously reducing the pressure to the maximum vacuum degree of the device of 0.2kpa, and then keeping the pressure until the artificial cornea is completely dried. The duration is approximately 1 hour.
In this example, the pressure is gradually reduced to about 6 hours after 3 gradient steps to the maximum vacuum of the apparatus, and then the corneal desiccation requirement is achieved after about 1 hour of maximum vacuum.
The moisture content of the dried cornea obtained by the gradient decompression method of this example was 18. + -. 2% and the light transmittance was 84. + -. 2%. The dried corneal surface was smooth with no visible ridges or fine folds.
Example 5
As shown in FIG. 5, in the present embodiment, the pressure is reduced from 80kpa to 0.5kpa at the maximum vacuum degree of the apparatus by 5 steps in a gradient decompression manner.
In the present embodiment, as shown in fig. 5, the vacuum regulating system regulates the pressure level of the previous stage to gradually decrease to the pressure level of the next stage when the pressure gradient changes.
The drying procedure for this example is as follows:
firstly, placing an artificial cornea to be dried on a supporting device, and placing the artificial cornea in a drying oven at the temperature of 0-30 ℃;
and secondly, controlling a vacuum regulation system, and starting gradient decompression operation, wherein a decompression curve is shown in figure 5, and a decompression gradient is as follows: hold at 80kpa for 2 h;
gradually reducing the pressure of 80kpa to 60kpa for 1h, and keeping the pressure at 60kpa for 1 h;
gradually reducing the pressure of 60kpa to 40kpa for 1h, and keeping the pressure at 40kpa for 1 h;
gradually reducing the pressure of 40kpa to 20kpa for 1h, and keeping the pressure at 20kpa for 1 h;
thirdly, gradually reducing the pressure of 20kpa to the maximum vacuum degree of 0.5kpa of the equipment after 1 hour; and then held at 0.5kpa until the artificial cornea is completely dry. The duration is approximately 1 hour.
The water content of the dried cornea obtained by the gradient decompression method of this example was 10. + -. 2% and the light transmittance was 84. + -. 1%. The dried corneal surface was smooth with no visible ridges or fine folds.
Example 6
As shown in fig. 6, in the present embodiment, the pressure is reduced to the maximum vacuum degree of the apparatus in a time period by continuously reducing the pressure.
The drying procedure for this example is as follows:
firstly, placing an artificial cornea to be dried on a supporting device, and placing the artificial cornea in a drying oven at the temperature of 0-30 ℃;
secondly, controlling the vacuum regulating system to start continuous decompression operation, wherein the decompression curve is shown in figure 6,
continuously reducing the pressure in the closed drying container to 0.5kpa of the maximum vacuum degree of the equipment at a constant speed within 12 hours;
and thirdly, drying the cornea under the state of 0.5kpa until the water content of the cornea is reached, wherein the time is about 1 hour.
The water content of the dried cornea obtained by the gradient decompression method of this example was 5. + -. 1%, and the light transmittance was 85. + -. 2%. The dried corneal surface was smooth with no visible ridges or fine folds.
Example 7
As shown in fig. 7, this embodiment is another embodiment of continuous depressurization, and in this embodiment, the depressurization is performed by continuously depressurizing the pressure in the sealed drying container to the maximum vacuum pressure of the apparatus at different speeds over a period of time.
The drying procedure for this example is as follows:
firstly, placing an artificial cornea to be dried on a supporting device, and placing the artificial cornea in a drying oven at the temperature of 0-30 ℃;
secondly, controlling a vacuum regulation system to start continuous decompression operation, wherein a decompression curve is shown in FIG. 7, and firstly, the pressure is reduced from 101kpa to 70kpa in 4 hours; reducing the pressure from 70kpa to 20kpa over a further 3 hours; then the pressure was reduced from 20kpa to 0.5kpa for 3 hours;
and thirdly, drying the cornea under the state of 0.5kpa until the water content of the cornea is reached, and about 2 hours.
The water content of the dried cornea obtained by the gradient decompression method of this example was 12. + -. 1%, and the light transmittance was 85. + -. 3%. The dried corneal surface was smooth with no visible ridges or fine folds.
Example 8
As shown in fig. 8, this example is a third embodiment of continuous depressurization, and in this example, the pressure in the sealed drying container is continuously depressurized to the maximum vacuum pressure of the apparatus at different speeds over a period of time.
In this example 8, a vacuum regulation system was controlled to continuously reduce the pressure at a uniform rate, and the pressure was continuously reduced from 95kpa to 0.3kpa, which is the ultimate system pressure, within 24 hours, as shown in fig. 8;
the water content of the dried cornea obtained by the gradient decompression method of this example was 5. + -. 1%, and the light transmittance was 79. + -. 3%. The dried corneal surface was smooth with no visible ridges or fine folds.
By adopting the drying method of the invention, as the whole drying process is carried out in a vacuum closed environment, the contact chance between the cornea and the air is greatly reduced, and compared with natural drying, the pollution can be effectively avoided. Therefore, the invention can meet the requirement of preparing the cornea in large batch. Particularly, in the present invention, controllability of all the influencing factors such as temperature, decompression curve, drying time and the like can be used to achieve the same quality of corneal drying in the same batch or even multiple batches by adopting the optimal combination of various decompression modes most suitable for the cornea according to the pre-drying state of the cornea obtained by different acellular methods.
Compared with non-dried cornea, the dried cornea obtained by the drying method has the characteristic of stable performance. The quality and the characteristics of the cornea can not be changed due to the length of the preservation time or other factors through the simplest low-temperature preservation method (for example, the low-temperature preservation at 0-8 ℃ in a refrigerator), the quality identity of the dried cornea can still be ensured when the dried cornea enters a clinical use state after preservation and transportation, and the purposes of convenient preservation and transportation are realized. So that the dry cornea has the necessary conditions of industrialization and marketization as a product, and is beneficial to the market popularization of the artificial cornea.
In addition, experiments prove that the dried cornea obtained by the drying method has the mechanical processing performance similar to that of a chemical contact lens when the water content is lower than 10-15%. Can conveniently and accurately process the shape of the cornea according to the requirement, thereby meeting the special requirement of corneal transplantation. In particular to the processing of dry cornea diopter, thereby realizing the application of the artificial cornea in the aspect of refraction correction. The bio-stromal cornea of the present invention is not comparable to chemical contact lenses in biocompatibility.
The present invention is not limited to the above embodiments, and in particular, various features described in different embodiments can be arbitrarily combined with each other to form other embodiments, and the features are understood to be applicable to any embodiment except the explicitly opposite descriptions, and are not limited to the described embodiments.
Claims (21)
1. A method for drying a decellularized cornea comprises the steps of placing a cornea to be dried on a supporting device, and placing the cornea to be dried in a closed drying chamber with adjustable vacuum degree; reducing the pressure in the closed drying chamber to be lower than the atmospheric pressure by a vacuum regulating system; the method is characterized in that the pressure of the closed drying chamber is regulated to be gradually reduced to the pressure when the set maximum vacuum degree is reached in a certain time period, and the pressure is continued to reach the requirement of the corneal dryness under the set maximum vacuum degree state.
2. The method for drying a decellularized cornea of claim 1, wherein said closed drying chamber is depressurized in at least two stages with a gradient from high to low for a period of time at each stage of pressure gradient; and reducing the final stage of gradient pressure to a set maximum vacuum degree state.
3. The method of claim 1, wherein the sealed drying chamber is continuously depressurized to a predetermined maximum vacuum level for a period of time.
4. The method for drying a decellularized cornea of claim 1, wherein the temperature in said closed drying chamber is controlled within a range of 0 ℃ to 30 ℃.
5. The method for drying a decellularized cornea as claimed in claim 1, wherein said vacuum regulation system regulates the pressure of the sealed drying chamber to a pressure at which a set maximum vacuum degree is obtained within 24 hours.
6. The method for drying a decellularized cornea as claimed in claim 5, wherein the preferable decompression time when the pressure in the closed drying chamber is reduced to the set maximum degree of vacuum is adjusted to 6 to 12 hours.
7. The method for drying a decellularized cornea as claimed in claim 1, wherein the pressure regulation range of said sealed drying chamber is from atmospheric pressure to a set maximum vacuum degree.
8. The method of claim 1, wherein the pressure at the set maximum vacuum level is near ultimate vacuum.
9. The method of claim 2, wherein the reduced pressure is in the range of 10 to 100kpa per step.
10. The method of claim 9, wherein the preferred reduced pressure range for each step of the gradient is 10 to 30 kpa.
11. The method of claim 8, wherein the duration of each step of the pressure gradient is set to vary with the pressure.
12. The method of claim 2, wherein the vacuum adjustment system adjusts the pressure from a previous pressure level to a subsequent pressure level directly as the pressure gradient changes.
13. The method of claim 2, wherein the vacuum adjustment system adjusts the pressure level of the previous stage to gradually decrease to the pressure level of the next stage when the pressure gradient changes.
14. The method of claim 2, wherein each step of the pressure gradient has a duration of 0.5 to 12 hours.
15. The method of drying a decellularized cornea of claim 14, wherein said pressure gradient of each stage has a preferred duration of 0.5 to 4 hours.
16. The method of claim 1, wherein the degree of corneal dryness is set according to the percentage of corneal water.
17. The method of drying a decellularized cornea of claim 15, wherein the degree of corneal dryness is set to a moisture content of not more than 20%.
18. An acellular porcine lamellar dried cornea, consisting of an acellular porcine cornea pro-elastic layer and a stromal layer, characterized in that the dried cornea is obtained by the drying method according to any one of the claims 1 to 16.
19. The decellularized porcine lamellar dried cornea of claim 18, wherein the water content of the dried cornea is greater than 0% and not greater than 20%.
20. The decellularized porcine lamellar dried cornea of claim 18, wherein the light transmittance of the dried cornea is not less than 70%.
21. The decellularized porcine lamellar dried cornea of claim 18, wherein the dried cornea is flat in surface, free of macroscopic ridges or fine folds.
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| PCT/CN2016/110464 WO2018107486A1 (en) | 2016-12-16 | 2016-12-16 | Method of drying decellularized cornea and dried decellularized pig lamellar cornea |
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| GB201916506D0 (en) * | 2019-11-13 | 2019-12-25 | Univ Nottingham | Corneal tissue |
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| CN101947144A (en) * | 2010-09-29 | 2011-01-19 | 厦门大学 | Ply tissue engineering corneal frame and manufacturing method and application thereof |
| CN101985051A (en) * | 2010-10-21 | 2011-03-16 | 暨南大学 | Acellular cornea or acellular corneal stroma, preparation method and application thereof |
| WO2011154687A1 (en) * | 2010-06-11 | 2011-12-15 | Ucl Business Plc | Biomimetic corneal tissue |
| CN102599991A (en) * | 2012-03-20 | 2012-07-25 | 中山大学中山眼科中心 | Application of beautifying cornea lens |
| CN104001214A (en) * | 2014-05-28 | 2014-08-27 | 青岛中皓生物工程有限公司 | Lamellar corneal stroma bracket as well as preparation method and application thereof |
| CN104645415A (en) * | 2014-11-28 | 2015-05-27 | 南昌大学第一附属医院 | Method for preparing decellularized lamellar cornea matrix sheet |
| CN104665956A (en) * | 2015-02-12 | 2015-06-03 | 厦门大开医疗器械有限公司 | Preparation method for artificial corneas |
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| US20110212524A1 (en) * | 2006-12-04 | 2011-09-01 | Body Organ Biomedical Corporation | Biomaterial and preparation method thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2011154687A1 (en) * | 2010-06-11 | 2011-12-15 | Ucl Business Plc | Biomimetic corneal tissue |
| CN101947144A (en) * | 2010-09-29 | 2011-01-19 | 厦门大学 | Ply tissue engineering corneal frame and manufacturing method and application thereof |
| CN101985051A (en) * | 2010-10-21 | 2011-03-16 | 暨南大学 | Acellular cornea or acellular corneal stroma, preparation method and application thereof |
| CN102599991A (en) * | 2012-03-20 | 2012-07-25 | 中山大学中山眼科中心 | Application of beautifying cornea lens |
| CN104001214A (en) * | 2014-05-28 | 2014-08-27 | 青岛中皓生物工程有限公司 | Lamellar corneal stroma bracket as well as preparation method and application thereof |
| CN104645415A (en) * | 2014-11-28 | 2015-05-27 | 南昌大学第一附属医院 | Method for preparing decellularized lamellar cornea matrix sheet |
| CN104665956A (en) * | 2015-02-12 | 2015-06-03 | 厦门大开医疗器械有限公司 | Preparation method for artificial corneas |
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