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WO2018107486A1 - Procédé de séchage de cornée décellularisée et cornée lamellaire de porc décellularisée séchée - Google Patents

Procédé de séchage de cornée décellularisée et cornée lamellaire de porc décellularisée séchée Download PDF

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Publication number
WO2018107486A1
WO2018107486A1 PCT/CN2016/110464 CN2016110464W WO2018107486A1 WO 2018107486 A1 WO2018107486 A1 WO 2018107486A1 CN 2016110464 W CN2016110464 W CN 2016110464W WO 2018107486 A1 WO2018107486 A1 WO 2018107486A1
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Prior art keywords
drying
cornea
pressure
decellularized
dried
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PCT/CN2016/110464
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English (en)
Chinese (zh)
Inventor
詹晓亮
李洁
李志寒
董晓鸥
刘靖
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Xiamen Dakai Biotechnology Co Ltd
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Xiamen Dakai Biotechnology Co Ltd
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Priority to CN201680077793.4A priority Critical patent/CN109069264B/zh
Priority to PCT/CN2016/110464 priority patent/WO2018107486A1/fr
Publication of WO2018107486A1 publication Critical patent/WO2018107486A1/fr
Anticipated expiration legal-status Critical
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61F—FILTERS IMPLANTABLE INTO BLOOD VESSELS; PROSTHESES; DEVICES PROVIDING PATENCY TO, OR PREVENTING COLLAPSING OF, TUBULAR STRUCTURES OF THE BODY, e.g. STENTS; ORTHOPAEDIC, NURSING OR CONTRACEPTIVE DEVICES; FOMENTATION; TREATMENT OR PROTECTION OF EYES OR EARS; BANDAGES, DRESSINGS OR ABSORBENT PADS; FIRST-AID KITS
    • A61F2/00—Filters implantable into blood vessels; Prostheses, i.e. artificial substitutes or replacements for parts of the body; Appliances for connecting them with the body; Devices providing patency to, or preventing collapsing of, tubular structures of the body, e.g. stents
    • A61F2/02—Prostheses implantable into the body
    • A61F2/14—Eye parts, e.g. lenses or corneal implants; Artificial eyes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells

Definitions

  • 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.
  • 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.
  • 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).
  • unseasoned lamellar scaffold referred to as water content dehydrated to the original cornea
  • dry lamellar scaffold referred to as dry lamellar scaffold and rehydred lamellar scaffold.
  • the test data or clinical effects are not exactly the same.
  • 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.
  • the biggest drawback of non-dried corneas is that they are extremely difficult to store and transport.
  • 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.
  • 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.
  • 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.
  • many of the alternative drying methods currently proposed are conventional drying methods.
  • 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.
  • 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.
  • 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.
  • the highly regular arrangement of collagen fibers in the corneal stroma is also the structural basis of the mechanical elasticity of the cornea.
  • 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.
  • 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.
  • the boiling point drying method is rarely used to dry the cornea.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the liquid preservation state is not conducive to transportation, and glycerin is prone to explosion and other safety hazards during transportation.
  • 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.
  • 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.
  • the higher the transparency the higher the transparency of the recovery after transplantation.
  • 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.
  • 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.
  • 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 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.
  • 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.
  • 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.
  • 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.
  • the decompression range of each gradient is 10 100 kpa; wherein the preferential decompression range is 10 ⁇ 30 kPa.
  • the duration of the pressure gradient for each stage is set to decrease as the pressure decreases.
  • the vacuum adjustment system adjusts the previous stage pressure block to adjust directly to the subsequent stage pressure block as the pressure gradient changes.
  • the vacuum adjustment system adjusts the previous stage pressure differential to gradually decrease to the next stage pressure differential as the pressure gradient changes.
  • the duration of each stage of the reduced pressure gradient is 0.5 12 hours; wherein the priority duration is 0.54 hours.
  • 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.
  • the operation of the reduced pressure can be achieved by means of automatic control.
  • the temperature in the enclosed drying chamber is controlled between 0 ° C and 30 ° C.
  • 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.
  • the pressure regulating range of the closed drying chamber is from atmospheric pressure to a set maximum vacuum.
  • the set maximum vacuum is at a pressure close to the ultimate vacuum.
  • the corneal dryness is set according to the corneal moisture content.
  • 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 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.
  • 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.
  • 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.
  • the cornea 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.
  • 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.
  • 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 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.
  • 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.
  • 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 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.
  • 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.
  • 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.
  • 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.
  • another significant clinical effect is that the postoperative epithelial cell attachment and proliferation rate is fast.
  • 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).
  • 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.
  • 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.
  • 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 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.
  • the vacuum regulating system adjusts the first stage pressure stop to instantaneously decrease to the next stage pressure block.
  • 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. ;
  • the pressure is reduced to the maximum vacuum of the device.
  • the maximum vacuum of the device is 0.5 kPa, and then the artificial cornea is completely dried.
  • the duration is about 1 hour in the maximum vacuum state of the device.
  • 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.
  • 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.
  • 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
  • the gradient is used to reduce the pressure from 60 kPa to the maximum vacuum of 0.3 kpa through two steps.
  • the vacuum adjustment system adjusts the previous stage pressure block to instantaneously adjust to the next stage pressure block.
  • 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 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 decompression curve is shown in Figure 2.
  • the decompression gradient is 60kpa and 30kpa, and the duration is 4h and 4h respectively .
  • 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.
  • 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.
  • the dried cornea 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
  • the pressure is reduced from lOlkpa to a maximum vacuum of 0.3 kpa through nine steps using a gradient decompression method.
  • 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 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;
  • 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.
  • 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.
  • the pressure is reduced from 50 kPa to the maximum vacuum of the device by 0.2 kPa by means of gradient decompression.
  • 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 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 4.
  • the decompression gradient is 50kpa, 30kpa, 15kpa, and the duration is 3h, 2h, lh, respectively.
  • 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.
  • 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.
  • 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.
  • the vacuum regulating system adjusts the pressure of the previous stage to gradually decrease to the pressure of the latter stage.
  • 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 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;
  • 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.
  • the pressure is reduced to the maximum vacuum of the apparatus for a period of time by means of continuous decompression.
  • 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 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.
  • 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.
  • this embodiment is another embodiment of continuous decompression.
  • 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 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.
  • this embodiment is a third embodiment of continuous decompression.
  • 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 is to continuously decompress the pressure in the closed and dry container at different speeds.
  • 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.
  • the present invention can satisfy corneal conditions in large quantities.
  • 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.
  • 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%.
  • the biomatrix cornea of the present invention has incomparable biocompatibility with respect to chemical contact lenses.

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Abstract

L'invention concerne un procédé de séchage d'une cornée lamellaire décellularisée et une cornée lamellaire de porc décellularisée séchée obtenue par ce procédé. Le procédé de séchage comprend le placement initial de la cornée dans une chambre de séchage sous vide ; la réduction progressive d'une pression à l'intérieur de la chambre de séchage fermée ; et la réduction progressive de la pression à une pression d'un degré de vide maximal défini sur une période donnée jusqu'à ce qu'une sécheresse cornéenne exigée soit atteinte. Le procédé de séchage permet un processus de séchage cornéen plus doux et réduit au minimum les dommages causés à une structure d'alignement de collagène cornéen durant le séchage sous vide.
PCT/CN2016/110464 2016-12-16 2016-12-16 Procédé de séchage de cornée décellularisée et cornée lamellaire de porc décellularisée séchée Ceased WO2018107486A1 (fr)

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PCT/CN2016/110464 WO2018107486A1 (fr) 2016-12-16 2016-12-16 Procédé de séchage de cornée décellularisée et cornée lamellaire de porc décellularisée séchée

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