CN107050515B

CN107050515B - Corneal stroma, preparation method and application

Info

Publication number: CN107050515B
Application number: CN201710125099.1A
Authority: CN
Inventors: 赵博; 夏磊磊; 王洪权; 赵延瑞; 李学军; 张晋辉
Original assignee: Beijing Bohui Ruijin Biological Science & Technology Co Ltd
Current assignee: Beijing Bohui Ruijin Biological Science & Technology Co Ltd
Priority date: 2017-03-03
Filing date: 2017-03-03
Publication date: 2020-01-21
Anticipated expiration: 2037-03-03
Also published as: CN107050515A

Abstract

The invention provides a corneal stroma, a preparation method and application, wherein animal cornea is used as a raw material, cell components and DNA components in a corneal epithelial layer and the corneal stroma are removed, a front elastic layer and a partial thickness corneal stroma are reserved, and complete extracellular matrix components are reserved; the corneal stroma is suitable for covering of a diseased cornea, particularly for drug-ineffective, unperforated corneal ulcers, infectious keratitis patients in need of lamellar transplantation therapy, and for temporary covering of corneal perforations.

Description

Corneal stroma, preparation method and application

Technical Field

The invention relates to the technical field of medical biomaterials, in particular to a corneal stroma, a preparation method and application.

Background

The cornea is positioned at the forefront of the eyeball and is directly contacted with the external environment, so that the cornea is easy to be damaged and infected, and if the eye is unsanitary, the cornea is easy to be inflamed and even turbid, and the vision is reduced and even blindness is caused. According to the world health organization reports, keratopathy is the second leading cause of vision loss, second only to cataracts. At present, the only effective means for treating corneal blindness is corneal transplantation, and the cornea used by corneal transplantation mainly has three sources: allogeneic corneal donation, keratoprostheses, and tissue engineered corneas. The donated cornea source is limited, the cornea donor is in severe shortage, the treatment requirements of the existing corneal blindness patients cannot be met, the blindness removal of the corneal blindness patients is greatly limited, and the problem to be solved is urgently needed in all countries in the world. The artificial cornea is made of hard artificial cornea materials, silica gel materials, PHEMA, PMMA materials and the like, the materials have certain effect of relieving keratopathy, but cannot be well combined with the tissues of a patient, and some materials even can cause the necrosis of the eye tissues of the patient to generate complications.

Based on the deep understanding of the interaction between synthetic materials of artificial cornea and cells, scientists begin to research the construction of human cornea equivalent from living extracellular matrix, hope to obtain tissue engineering organs with similar structure and physical and chemical properties to natural cornea, and can be used for clinical treatment of corneal blindness. Human studies on tissue engineering corneas have been made in various attempts, such as culturing bovine corneal epithelial cells on collagen, culturing autologous limbal epithelial cells using amniotic membrane to successfully repair ocular surface damage, culturing autologous oral mucosal cells outside a temperature sensitive culture matrix to repair ocular surface, culturing corneal epithelial cell membranes outside autologous limbal stem cells, etc., which lay a theoretical foundation for the studies on tissue engineering corneas, but still cannot satisfy clinical use requirements, such as poor collagen toughness, inability to resist intraocular pressure and mechanical traction of sutures, and people still need to deeply study the healing mechanism of corneal wounds. Through years of research, an ideal tissue engineering cornea material is found, and the immunogen-removed pig cornea stroma material has a structure very similar to that of a human cornea stroma and has the characteristics of low immunogenicity and rich sources. The main components of the xenogenic tissue removed by the immunogen are collagen, elastic fiber and the like, and the change of the shape and the toughness is small. The immunogen removal can reduce the inflammatory reaction and the immunological rejection reaction of the xenogeneic tissue, has good biocompatibility with a human body, and simultaneously, the growth factor in the stroma can induce the differentiation of human corneal limbus cells into corresponding corneal cells, and the removal of the pig corneal stroma material by the immunogen becomes a poor choice for treating corneal blindness.

For the study of tissue engineered corneas, china went to the frontier of the world. Shenzhen Ainei cornea engineering Co., Ltd, in 2015, 4 and 22, provided the first global bioengineering cornea, the cornea product is prepared from a pig eye cornea through virus inactivation, immunogen removal and other processes, and is an extracellular matrix of the pig cornea, and the main component of the extracellular matrix is collagen. The product has collagen fiber structure of natural cornea, and retains the front elastic layer and partial matrix layer of natural cornea. Patent No. CN201310527550.4 of Guangzhou Youdeqing Biotechnology Co., Ltd, which is held by Guangdong Guanhao Biotechnology Co., Ltd, discloses a preparation method of a cornea material, which takes an animal cornea as a raw material to prepare a heterogeneous cornea material with high transparency, low immunogenicity, good bioactivity and biocompatibility, keeps the three-dimensional structure of collagen close to that of a fresh cornea, and further reduces the anti-degradation property and the immunogenicity of the collagen through collagen crosslinking.

Disclosure of Invention

The invention provides a cornea matrix which is prepared by taking the cornea of an animal as a raw material and utilizing a repeated freeze-thaw method and an enzyme solution immunogen removing method, and has high tensile strength, no immunogenicity and good biocompatibility.

In order to solve the technical problems, the technical scheme adopted by the invention is as follows: a corneal stroma is prepared from animal cornea, by removing cell components and DNA components from corneal epithelium layer and corneal stroma, retaining elastic layer and partial thickness of corneal stroma, and retaining intact extracellular matrix components (tested for its mechanical properties, chemical properties, biological properties and degradation properties).

The animal is a mammal, preferably a pig or a cow.

The partial thickness corneal stroma is a corneal stroma with a certain thickness connected with the front elastic layer.

The extracellular matrix components are collagen and glycosaminoglycan.

The collagen is type I collagen and type IV collagen, and the content of the type I collagen and the type IV collagen is 85-90%.

The glycosaminoglycan is chondroitin sulfate and keratan sulfate.

The corneal stroma is a circular sheet with the diameter of 7-10mm and the thickness of 0.1-0.2 mm.

The removal of cell components and DNA components in the corneal epithelium layer and the corneal stroma is evaluated by cell residual quantity, DNA residual quantity and galactosidase clearance, namely, the biological properties are cell residual quantity, DNA residual quantity and galactose glycolytic enzyme (alpha-Gal) clearance.

The corneal stroma of the invention has a residual cell amount of 0, a residual DNA amount of less than 10ng/mg, and a galactose glanzyme (α -Gal) clearance rate of 99% or more.

The invention also provides a preparation method of the corneal stroma, which is characterized by comprising the following steps: the corneal stroma is prepared by a method of freeze-thaw swelling and immunogen removal.

Specifically, the method for preparing the corneal stroma comprises the following steps:

(1) raw material treatment: taking eyeball of animal, taking cornea (such as using trephine to drill), cleaning, and soaking in purified water;

(2) freeze-thawing swelling: soaking cornea in water, and freezing; taking out, washing with purified water after completely thawing, and soaking; repeating the freezing and thawing process for multiple times to obtain water-swelling cornea;

(3) virus inactivation: soaking cornea with peroxyacetic acid-ethanol solution for virus inactivation;

(4) and (3) cleaning: treating the cornea with PBS solution and purified water in an ultrasonic device respectively;

(5) immunogen removal: the immunogen removing solution is a PBS solution containing trypsin and EDTA, and the immunogen removing process is carried out in a multi-frequency ultrasonic device, wherein the multi-frequency ultrasonic device can provide at least two ultrasonic frequencies;

(6) and (3) cleaning: treating the cornea with PBS solution and purified water in an ultrasonic device respectively;

(7) repeating the steps (5) and (6) for a plurality of times, and then soaking the cornea in purified water;

(8) cutting and material taking: cutting off a pre-corneal elastic layer and a part of stroma by using a corneal lamellar knife under a dissecting mirror, and then soaking the pre-corneal elastic layer and the part of stroma in purified water;

(9) drying: the cornea was flattened onto the support surface and placed in an oven for drying.

The concentration (volume percentage) of the peroxyacetic acid in the peroxyacetic acid-ethanol solution in the step (3) is 0.1-5%, the concentration (volume percentage) of the ethanol is 5-40%, the ratio (volume ratio) of the peroxyacetic acid-ethanol solution to the cornea is (30-60): 1, the inactivation time is 2-4 hours, and the temperature range is 10-40 ℃.

The cleaning solution in the step (4) is PBS solution with pH of 6-8, the temperature of the PBS solution is 10-40 ℃, the ratio (volume ratio) of the PBS solution to the cornea is (30-60): 1, the cleaning is carried out for 2-4 times, each time is 10-30min, then the cleaning is carried out by using injection water with 10-40 ℃, namely purified water, the ratio (volume ratio) of the purified water to the cornea is (30-60): 1, and the operation is terminated when the detected conductivity is below 10 muS/cm; the cleaning process is carried out in an ultrasonic cleaning machine.

The immune source removing solution in the step (5) comprises: PBS solution with pH value of 6-8 and dissolved with trypsin and EDTA; the mass percentage concentration of trypsin in the immune source removing solution is 0.01-0.2%, and the concentration of EDTA is 0.1-1 mmol/L; further: the mass percentage concentration of trypsin in the immune source removing solution is 0.02-0.05%, the concentration of EDTA is 0.4-0.8mmol/L, and the pH value of the immune source removing solution is 7.0-8.0, preferably 7.2-7.5; the volume ratio of the immunity source removing liquid to the cornea is (30-60): 1; the multi-frequency ultrasound at least comprises two ultrasound frequencies, wherein the low-frequency range is 20-40KHz, the high-frequency range is 60-90KHz, the low-frequency treatment lasts for 5-40min, the high-frequency treatment lasts for 5-40min, and the temperature range is 20-35 ℃.

And (3) cleaning the cornea with the cleaning solution in the step (6) at pH6-8 in PBS solution at 10-40 ℃ for 10-30min each time at a PBS solution volume ratio of (30-60): 1, cleaning the cornea for 2-4 times at a time of 10-30min, cleaning the cornea with injection water at 10-40 ℃ at a volume ratio of (30-60): 1, and stopping when the difference between the conductivity of the injection water and the conductivity of the injection water not cleaned is less than 1 muS/cm.

The drying and drying in the step (9) are carried out in a thermal cycle oven: preheating an oven to 25-40 ℃, then placing the material obtained in the step (8) in the oven for drying, and drying the water in the air by hot circulating air for 12-24 hours.

The preparation method also comprises the steps of (10) packaging and (11) sterilizing and resolving, wherein the packaging step specifically comprises the following steps: packaging the drying material with tyvek packaging paper and a plastic uptake box; the sterilization and analysis steps are as follows: firstly, the temperature is kept at 20-40 ℃ for 2-4 hours, the humidity is 30-70%, then ethylene oxide with the concentration of 300-1000mg/L is introduced, and the sterilization is carried out for 4-8 hours; then analyzing, wherein the analyzing process is carried out in a ventilated analyzing chamber, the temperature is controlled between 10 ℃ and 30 ℃, and the time is 14-28 d.

The invention further provides the use of the above-described corneal stroma in an ophthalmic implant suitable for the covering of diseased corneas, in particular for drug-ineffective, not yet perforated corneal ulcers, infectious keratitis patients in need of lamellar transplantation therapy, and for the temporary covering of corneal perforations. Firstly, a diseased cornea part of a patient is completely cut off by using a corneal trephine and a corneal lamellar cutter, the cutting depth accords with the diseased depth, a part of corneal stroma and a rear elastic layer of the patient are reserved, a wound foreign matter is thoroughly removed, cauterization hemostasis is avoided, and normal saline is thoroughly washed. Then opening the external package of the invention, taking out the internal package bag and cutting, taking out the internal package box under aseptic condition, injecting aseptic normal saline into the internal package box to rehydrate the product of the invention for 10-15min, then placing the convex surface (front elastic layer surface) of the product of the invention upwards, flatly placing on the wound surface and sewing and fixing. The operation method, the medication and the postoperative care are the same as those of the conventional lamellar cornea transplantation operation, and corneal epithelium protective medicines can be used after the operation.

Compared with the prior art, the invention has the following remarkable advantages and beneficial effects:

(1) through low-temperature freezing, cells in the cornea can be cracked and broken under the action of ice crystals, meanwhile, the interlayer spacing of a cornea matrix plate can be increased due to the fact that water is converted into ice and the volume is expanded, when the cornea is unfrozen, the ice is converted into water, the volume is reduced, water can be absorbed from the outside, and therefore the cornea swelling and thickness increasing are achieved;

(2 freeze thawing swelling increases the space between corneal stroma layers, is more beneficial to immunogen removal and is beneficial to genetic material cleaning;

(3) using trypsin and EDTA to break the connection between the cells and the extracellular matrix; the cells are crushed by adopting low-frequency ultrasound, and simultaneously, the high-frequency ultrasound acts on the crushed cells and the extracellular matrix, so that the cells are further separated from the extracellular matrix, and the purpose of removing the cells is achieved. In the above manner, the whole process of separating the cells from the matrix is reinforced, so that the cells are completely separated from the matrix. The optimal immunogen removal effect is achieved; an enzyme method cell elution process: the trypsin and EDTA compound solution is adopted, the process of removing cells is mild, the damage to the matrix structure is reduced, and active growth factors in the matrix are reserved;

(4) the high-concentration immunogen removing solution and the pure water are alternated, and the scouring effect is formed between the corneal stroma plates by using the diffusion effect of the high-concentration immunogen removing solution and the low-concentration immunogen removing solution, so that the genetic material cleaning efficiency is improved; the air-dried cornea can reduce the space between the corneal stroma plates, eliminate the influence of the cornea on the microstructure of the corneal stroma plate in the freeze thawing process, reduce the thickness of the cornea, enable the stroma tissue to be more compact and improve the tensile strength of the cornea;

(5) the cornea after air drying is a plane transparent sheet-shaped film, so that the cornea is prevented from being wrinkled and shrunk due to air drying under a free condition;

(6) the corneal stroma can be used for lamellar corneal transplantation.

Drawings

FIG. 1 shows a front SEM image of an elastic layer of a corneal stroma according to an embodiment of the invention;

FIG. 2 is a front SEM image of an elastic layer of an example of the invention before removal of corneal stroma;

FIG. 3 shows a side SEM photograph of a corneal stroma according to an embodiment of the invention;

FIG. 4 shows SEM pictures of the front elastic layer and the side of the corneal stroma according to the embodiment of the invention;

figure 5 is a schematic cross-sectional view of a corneal stroma according to an embodiment of the invention.

Detailed Description

The present invention will be described in further detail with reference to the following examples, but is not limited thereto.

Example 1:

the present embodiment relates to a method for preparing corneal stroma, comprising the following steps:

(1) raw material treatment:

the eyeball of the pig within 4 hours of death is taken, the blood is washed clean by normal saline, the cornea is drilled by a trephine with the diameter of 8.5mm, and the eyeball is soaked in purified water after being washed clean by the purified water.

(2) Freeze-thawing swelling:

soaking cornea with small amount of water, freezing in a freezer at-40 deg.C for 1 hr, taking out, thawing completely, washing with purified water for 15min, soaking in sufficient purified water for 30min, and repeating the freezing and thawing process for 3 times to obtain water-swelling cornea.

(3) Virus inactivation:

the virus inactivation is carried out by soaking cornea with peroxyacetic acid-ethanol solution, and the process can be carried out in a stainless steel barrel. The concentration (volume percentage) of peracetic acid in the peracetic acid-ethanol solution is 1%, the concentration (volume percentage) of ethanol is 24%, the ratio (volume ratio) of the peracetic acid-ethanol solution to the cornea is 40: 1, the inactivation time is 2 hours, and the temperature is 20 ℃.

(4) And (3) cleaning:

cleaning cornea with cleaning solution (PBS solution with pH7, PBS solution temperature is 20 deg.C, PBS solution and cornea ratio (volume ratio) is 40: 1, cleaning for 3 times, each time for 20min, cleaning with purified water, purified water and cornea ratio (volume ratio) is 40: 1, and stopping until detected conductivity is below 10 μ S/cm; the cleaning process is carried out in an ultrasonic cleaning machine.

(5) Immunogen removal:

the immunogen removing liquid is PBS (phosphate buffer solution) with the pH value of 7 and containing 2% by mass of trypsin and 0.3% by mass of EDTA (ethylene diamine tetraacetic acid), the mixing ratio (volume ratio) of the immunogen removing liquid to the cornea is 40: 1, the immunogen removing process is carried out in an ultrasonic cleaning machine, the multi-frequency ultrasonic comprises at least two ultrasonic frequencies, the low-frequency is 30KHz, the high-frequency is 70KHz, the low-frequency treatment lasts for 8min, the high-frequency treatment lasts for 12min, and the temperature is 20 ℃.

(6) And (3) cleaning:

cleaning with cleaning liquid, wherein the cleaning process is carried out in an ultrasonic cleaning machine, and the power of ultrasonic waves is below 3000W. The cleaning solution is PBS solution with pH7, the temperature of PBS solution is 20 deg.C, the ratio (volume ratio) of PBS solution to cornea is 40: 1, cleaning is carried out for 3 times, each time is 20min, then cleaning is carried out by using injection water with 20 deg.C, the volume ratio of injection water to cornea is 40: 1, and the detection is terminated when the difference between the conductivity of the injection water for cleaning and the conductivity of the injection water for not cleaning is less than 1 mus/cm.

(7) Repeating the steps (5) and (6) for 6 times to ensure that the concentration of the solution outside the cornea is repeatedly changed and the permeation direction of the solution is changed to form a scouring effect, repeatedly cleaning the genetic material in the cornea until the genetic material on the cornea is completely removed, and then soaking the cornea in the injection water.

(8) Cutting and material taking:

the pre-corneal elastic layer and a part of the stroma were cut to a thickness of 2mm using a keratome under a dissecting mirror, and then immersed in injection water.

(9) Drying:

and (4) performing in a thermal cycle hundred-grade oven, preheating the oven to 37 ℃, putting the material obtained in the step (8) in the oven for drying, and air-drying the material by thermal cycle wind for 24 hours. During the air drying process, the cornea needs to be flatly laid on the surface of the stainless steel tray, so that the cornea can be kept in a flat state after air drying, otherwise, the cornea can be contracted into an irregular state during the air drying process, even folds and deformation occur, and the product quality is influenced.

The preparation method of the present embodiment may further include:

(10) packaging:

the drying material is packaged by Tyvek packaging paper and a plastic uptake box.

(11) Sterilization and resolution:

adopting ethylene oxide for sterilization, wherein the sterilization conditions are as follows: firstly, preserving the heat for 4 hours at the temperature of 40 ℃ and the humidity of 70 percent, then introducing epoxy ethane with the concentration of 500mg/L, and sterilizing for 6 hours; the analysis was carried out in a ventilated analysis chamber, the temperature was controlled between 20 ℃ and the time was 14 days.

The cornea sample finally obtained, the cross section of which is schematically shown in FIG. 5, comprises a lower stromal layer and an upper anterior elastic layer; the cornea of the present invention can be measured by placing the soaked cornea on a water filtration device such as a stainless steel screen mesh, draining for more than 5 minutes, and then weighing with a weighing instrument.

Example 2:

through observation of the sample in example 1 by a scanning electron microscope, as shown in fig. 1 and 4, it can be observed that after the immunogen removing treatment, the immunogen-removed cornea front elastic layer is intact, and is not affected and damaged by the enzyme solution, which is beneficial to migration, adhesion and proliferation of corneal epithelial cells, rapid and complete epithelialization of cornea, and infection avoidance. The epithelium can normally grow and cover the surface of the corneal stroma, so that the degradation of various hydrolytic enzymes to the collagen components can be effectively isolated, the degradation time is prolonged, and the reconstruction of the corneal stroma is facilitated.

After the pre-elastic layer is removed, as shown in the attached figures 2 and 3, the interior of the cornea is seen to present a three-dimensional network structure, and the three-dimensional network structure forms a biological scaffold, so that the growth of corneal cells of a patient is facilitated, the attachment number of the cells is increased, the exchange of nutrient substances is facilitated, and the reconstruction of removing corneal stroma by immunogen is facilitated.

As can be seen from the SEM photographs of the side surfaces of the attached figures 3 and 4, the lamellar structure of the front elastic layer is complete, the interior of the corneal stroma is in a lamellar structure, and the gaps between collagen plate layers can be seen, and the size of the gaps is 20-150 μm.

Example 3:

the physical property detection of the sample obtained by the preparation method is as follows:

a. stitching tensile strength: 5 samples were prepared according to example 1, with 3-0 nonabsorbent suture at 2mm from one edge of the patch, the suture and the other end of the patch were secured to a tensile tester and stretched at 20mm/min until the seam point was torn, and the maximum force was recorded, which showed an average maximum of 3.2N.

b. Light transmittance: 5 samples were prepared according to example 1, and the light transmittance of the air-dried sample, the swollen sample, and the glycerin-dehydrated sample were measured, respectively. Air-drying sample preparation: directly sticking the swelled sample to the inner wall of the transparent side of the cuvette, and sending the cuvette into a blast oven to air-dry for 24 hours; preparation of swollen sample: directly sticking the swelled sample to the inner wall of the light-transmitting side of the cuvette; glycerol dehydrated sample: and (3) placing the swelled sample in glycerol for dehydration for 12-48 hours, then taking out the sample, cleaning the glycerol on the surface of the sample, and directly sticking the sample to the inner wall of the light-transmitting side of the cuvette. During testing, one side of the sample is placed on the right side, the light transmittance at 800nm, 700nm, 600nm, 500nm, 400nm and 300nm is respectively measured, the reading is recorded, and the average value is calculated. The average value of the light transmittance of the air-dried sample is 86.6 percent, the average value of the light transmittance of the swelling sample is 39.05 percent, and the average value of the light transmittance of the glycerol dehydration sample is 80.0 percent;

c. expansion ratio: preparing 5 samples according to example 1, spreading the cornea on a flat plate, covering a glass slide, placing the flat plate in a 40 ℃ air-blast drying box to air-dry for 24-48 hours, taking out the samples, cutting the samples into regular cuboids by a scalpel, measuring the length, width and height of the samples by a vernier caliper, recording the reading, soaking the samples in water for 24 hours to absorb water for swelling, measuring the length, width and height after swelling by the vernier caliper, recording the reading, respectively calculating the volume V1 and V2 before and after swelling, and calculating the swelling ratio as:

the average value of the calculation results is 88.5 percent;

d. water content: samples were prepared as in example 1, the cornea was laid flat on a plate, covered with a glass slide, air-dried in a forced air drying cabinet at 40 ℃ for 24 hours, and the samples were removed and weighed to give a dry weight W₁. Then the sample is soaked in water for 24 hours to absorb water and swell, and the wet weight W is weighed₂And calculating the water content:

the average value of the calculation results was 95.9%.

e. Degradation performance: each corneal stroma sample having a diameter of 8.5mm and a thickness of 0.15mm was immersed in 1ml of 0.03% by mass proteinase K solution in a 56 ℃ water bath, and the cornea was weighed every 10min to calculate the corneal residual mass.

The test results are shown in table 1:

TABLE 1 degradation Properties of the samples of the examples

Example 4:

the performance of the sample obtained by the preparation method is detected, and the method specifically comprises the following steps:

a. pH value: the sample is prepared according to the method specified in GB/T14233.1-2008 at 5.4.1, and the difference between the pH values of the sample test solution and the blank control solution is not more than 1;

b. heavy metal content: samples were prepared as in example 1, and lead and chromium were tested by atomic absorption spectrophotometer method as specified in GB/T14233.1-2008 at 5.9.1 and mercury and arsenic were tested by atomic fluorescence spectrometry as specified in GB/T14233.1-2008 at 5.9.3, showing that the heavy metal content was less than 0.1. mu.g/g.

c. Residue on ignition: samples were prepared as in example 1, and the ignition residue was 1.0% as determined by the method specified in pharmacopoeia of the people's republic of China (fourth part of 2015) 0841.

d. And (3) testing bacterial endotoxin: samples were prepared according to example 1 and operated as specified in GB/T14233.2-2005 and showed bacterial endotoxins to be less than 2 EU/g.

e. Cell residue examination: taking 3 products to perform HE staining respectively, selecting 3 fields for each section, observing the number of the intact cells divided by 3 under a 400-fold optical microscope, and displaying that the average number of the intact cells in each field is 0.

Residual amount of DNA: samples were prepared according to example 1, and the amount of residual DNA in the samples provided in example 1 was measured by fluorescent staining according to the method for measuring residual DNA in a biological preparation ("chinese pharmacopoeia 2010, appendix IX-B exogenous DNA residual amount measurement method). The results showed that the residual amount of DNA in the sample was less than 4.56. + -. 0.01 ng/mg.

g. Galactosidase (α -Gal) clearance: taking animal-derived biological material Gal positive reference products and Gal antigen negative reference products which are respectively 2mg, adding 1mL of lysate, cracking for 30-90min, preparing Gal standard curve samples of 20, 10, 5, 2.5, 1.25 and 0.625 mu g, respectively taking 50mg of test products before and after test immunogen removal, adding 2mL of lysate, and cracking for 30-90 min; taking supernatant after reaction of lysate and M86 antibodyAdding 96-well plate, adding secondary antibody and color developing agent, detecting absorbance value by ELISA method at 450nm, calculating Gal value of sample according to standard curve, and Gal value of material before immunogen removing treatment is 25.69 + -2.72 × 10¹⁴In mg, the Gal values of the samples in example 1 are 0.12. + -. 0.01X 10¹⁴The clearance rate of galactosidase (alpha-Gal) is more than 99.52 percent per mg.

h. And (3) identifying collagen subtypes: detecting type I and type IV collagen by immunohistochemical staining method, continuously slicing at 3 μm thickness, dewaxing with xylene, and dehydrating with gradient ethanol. Transferring the slices into water bath of electric cooker (containing 0.01mol/L trisodium citrate buffer solution with pH of 6.0), maintaining the temperature at 95-100 deg.C, decocting for 20min for antigen retrieval, taking out, and naturally cooling at room temperature. Phosphate Buffered Saline (PBS) wash, 5min × 3 times. Immunohistochemistry by a two-step method: respectively dropwise adding type I and type IV collagen monoclonal antibody primary antibodies, wherein the concentration is 1: incubate 100, 4 ℃ refrigerator overnight at room temperature for 60min, and wash 3 times with PBS. The Envision reaction solution was added dropwise and incubated at room temperature for 30 min. PBS was washed 3 times. 0.05% of 3, 3-diaminobenzidine and 0.03% of H2O2 are used for developing the color for 5-10 min. Washing with running water, and lining-dyeing with hematoxylin. And (4) performing ethanol dehydration by an increasing gradient, and performing xylene transparency and conventional resin sealing. The results show that the four staining specimens observed under the microscope can be stained in brown and yellow, and the specimens are positive, which indicates that the collagen I and the collagen IV can be detected in the samples.

i. Detection of glycosaminoglycan content: taking 10 samples, sampling, leaching, and testing the content of chondroitin sulfate and keratan sulfate by using a Biocolor chondroitin sulfate and keratan sulfate detection kit, wherein the average value of the content of chondroitin sulfate in the samples is 573 +/-83 mu g/g; the mean value of the content of the chondroitin sulfate is 132 +/-13 mu g/g.

Example 5:

the sample obtained by the preparation method is biologically detected, and the detection items comprise: pyrogen, cytotoxicity, delayed type hypersensitivity, intradermal reaction, acute systemic toxicity, Ames test, mouse lymphoma cell mutation test, chromosomal aberration, implantation, sub-chronic toxicity.

1) Pyrogen

According to 6cm²The proportion of the sample added to 1ml of leaching medium,preparing test solution at 37 +/-1 ℃ for 72 +/-2 hr, extracting medium: physiological saline. The method is carried out according to the method specified in GB/T14233.2-2005, and the product has no pyrogen reaction.

2) Cytotoxicity

According to 6cm²The sample is added with 1ml of leaching medium, and test solution is prepared at 37 +/-1 ℃ for 24 +/-2 hr, wherein the leaching medium: serum-containing MEM medium. The test solution is taken to carry out the test according to the test method specified in GB/T16886.5-2003, and the result shows that the cytotoxicity reaction of the product is not more than grade 1.

3) Delayed type hypersensitivity reaction

According to 6cm²The sample is added with 1ml of leaching medium, and test solution is prepared at 37 +/-1 ℃ for 72 +/-2 hr, wherein the ratio of leaching medium: normal saline and cottonseed oil. According to GB/T16886.10-2005 part 10: the irritation and delayed type hypersensitivity test method provides that the test is carried out, and the result is that the product has no delayed type hypersensitivity.

4) Intradermal reaction

According to 6cm²The sample is added with 1ml of leaching medium, and test solution is prepared at 37 +/-1 ℃ for 72 +/-2 hr, wherein the ratio of leaching medium: normal saline and cottonseed oil. According to GB/T16886.10-2005 part 10: stimulation and delayed type hypersensitivity test methods the test is specified to be carried out with the results: the difference between the mean scores of the test sample and the solvent control was less than 1.0.

5) Acute systemic toxicity

According to 6cm²The sample is added with 1ml of leaching medium, and test solution is prepared at 37 +/-1 ℃ for 72 +/-2 hr, wherein the ratio of leaching medium: normal saline and cottonseed oil. The test solution was taken and tested according to the test method specified in GB/T16886.11-2011, and the results were: the product has no acute systemic toxicity reaction.

6) Ames test

According to 6cm²The sample is added with 1ml of leaching medium, and test solution is prepared at 37 +/-1 ℃ for 72 +/-2 hr, wherein the ratio of leaching medium: saline and DMSO. The method is carried out according to the method specified in GB/T16886.3-2008, and the result is that: the product was negative to the Ames test.

7) Mouse lymphoma cell mutation assay

According to 6cm²Adding 1ml of leaching medium into the sample, and processing at 37 + -1 deg.C for 72 + -2 hrPreparing test solution, leaching medium: saline and DMSO. The method is carried out according to the method specified in GB/T16886.3-2008, and the result is that: the mouse lymphoma cell mutation test of the product is a negative result.

8) Chromosome aberration test

According to 6cm²The sample is added with 1ml of leaching medium, and test solution is prepared at 37 +/-1 ℃ for 72 +/-2 hr, wherein the ratio of leaching medium: physiological saline and DMSO were performed according to the method specified in GB/T16886.3-2008, and the results were as follows: the product was negative in the chromosome aberration test.

9) Implant

According to the method specified in GB/T16886.6-1997, the results are as follows: muscle implantation for 1 week: neutrophil, lymphocyte and macrophage infiltration can be seen around the sample, and no cyst cavity is formed; muscle implantation for 4 weeks: a small amount of macrophages and lymphocytes, collagen fibers and fibroblasts are proliferated and a fibrous capsule cavity is formed around the sample; muscle implantation for 12 weeks: a small amount of lymphocytes, collagen fibers and fibrous capsule cavities are more compact and regular around the sample.

10) Sub-chronic toxicity

The method is carried out according to the method specified in GB/T16886.11, and the result is that: the material is evaluated to have sub-slow toxicity without sub-chronic toxic reaction.

Example 6:

the sample obtained by the preparation method of the invention is subjected to animal experiments, and the details are as follows:

the samples from example 1 were mixed with 1: the cornea is cleaned by 4000 gentamicin normal saline for 15min to be rehydrated, and the cornea implant with the diameter of 7mm is manufactured by trephine. 12 rabbits, 1-2kg body weight, left eye surgery, right eye blank control. 1% of sodium amobarbital is injected into the abdominal cavity according to 30mg/kg of body weight, 0.5% of cocaine is subjected to surface anesthesia, 2% of novocaine is injected into the 1mL of the retrobulbar cavity, eyeballs are massaged, intraocular pressure is reduced, and eye drops of 1% of neoforline, 1% of atropine and tropicamide are dripped 30min before operation to ensure that the pupils are fully dispersed. The eye is covered by a conventional sterilized cloth, the eyelashes around the eyes are cut off, four rectus muscle index sutures are made, the eyeballs are exposed and fixed, a 6.5 trephine is used for placing in the center of the cornea, the rotation is slightly pressed downwards, when aqueous humor flows out, the rotation is stopped, the cornea which is not drilled through is completed by a corneal scissors, then the cornea which is rehydrated in the embodiment 1 is transplanted in the defect, 9/0 nylon thread is used for four-point intermittent fixation, and then continuous suture fixation is carried out. After operation, 0.4% gentamicin, 0.5% codiethasone and 1% atropine solution are added dropwise. 12 animals were implanted with 3 transparent animals for 1 week, 5 transparent animals for 2 weeks, 1 dead animal, and 3 transparent animals for 3 weeks. In 11 animals, 3 animals were transparent for 43 days, 56 days and 58 days, and the rest 8 animals were transparent for more than 90 days. The cornea of the eye keeps transparent, the cornea has no edema and infection, and the cornea has no new blood vessels. The thickness of the transplanted cornea is 330 +/-20 mu m and the thickness of the normal cornea is 304 +/-11 mu m, and the thickness of the transplanted cornea and the normal cornea are not statistically different. The eye pressure after operation is 11 +/-1 mmHg, the normal eye pressure is 10 +/-1 mmHg, and the eye pressure and the normal eye pressure are not statistically different.

In summary, the corneal stroma and the preparation method thereof have the following advantages:

(1) the three-dimensional space network structure of collagen fibers in the corneal extracellular matrix is reserved;

(2) retaining active growth factors in the corneal extracellular matrix;

(3) through low-temperature freezing, cells in the cornea can be cracked and broken under the action of ice crystals, meanwhile, the interlayer spacing of a cornea matrix plate can be increased due to the fact that water is converted into ice and the volume is expanded, when the cornea is unfrozen, the ice is converted into water, the volume is reduced, water can be absorbed from the outside, and therefore the cornea swelling and thickness increasing are achieved;

(4) the space between the corneal stroma layers is increased, which is more beneficial to the removal of immunogen and the cleaning of genetic material;

(5) the high-concentration immunogen removing solution and the pure water are alternated, and the scouring effect is formed between the corneal stroma plates by using the diffusion effect of the high-concentration immunogen removing solution and the low-concentration immunogen removing solution, so that the genetic material cleaning efficiency is improved;

(6) DNA residue can reach below 10ng/mg, and the removal rate of galactosidase is high and can reach more than 99%;

a molding part:

(1) the air-dried cornea can reduce the space between the corneal stroma plates, eliminate the influence of the cornea on the microstructure of the corneal stroma plate in the freeze thawing process, reduce the thickness of the cornea, enable the stroma tissue to be more compact and improve the tensile strength of the cornea;

(2) the cornea after air-drying is a plane transparent sheet-shaped film, so that the phenomenon that the cornea is folded and shrunk due to air-drying under a free condition is avoided, and the cornea obtained by the method has certain internal stress, can quickly absorb moisture in the rehydration process, and quickly recovers elasticity;

the sterilization process comprises the following steps:

by controlling the sterilization process, the ethylene oxide gas can be effectively permeated into the cornea, so that the sterilization effect is achieved.

The above-described embodiments of the present invention are illustrative of the present invention and are not intended to be limiting, and any changes within the meaning and scope equivalent to the claims of the present invention are intended to be included within the scope of the claims.

Claims

1. A corneal stroma, characterized by: the method comprises the following steps of (1) removing cell components and DNA components in a corneal epithelium layer and a corneal stroma by using an animal cornea as a raw material, reserving a front elastic layer and a partial thickness corneal stroma, and reserving complete extracellular matrix components; the corneal stroma is prepared by the following steps:

(1) raw material treatment: taking eyeball of animal, taking cornea, cleaning and soaking in purified water;

(2) freeze-thawing swelling: soaking cornea with water, and freezing; taking out after freezing, washing with purified water after completely thawing, and then soaking; repeating the freezing and thawing processes for multiple times to obtain a water-swelling cornea;

(4) and (3) cleaning: treating the cornea with PBS solution and purified water in an ultrasonic device respectively; wherein the PBS solution has pH6-8, the solution temperature is 10-40 deg.C, the volume ratio of PBS solution to cornea is (30-60): 1, and cleaning is carried out for 2-4 times, each time for 10-30 min; cleaning with water for injection at 10-40 deg.C, wherein the volume ratio of water for injection to cornea is (30-60): 1, and stopping until the detected conductivity is below 10 μ s/cm;

(5) immunogen removal: the immunogen removing solution is PBS solution, the PBS solution contains trypsin and EDTA, and the immunogen removing process of the cornea is carried out in a multi-frequency ultrasonic device; wherein, the mass percentage concentration of trypsin in the immunogen removing solution is 0.01-0.2%, the concentration of EDTA is 0.1-1mmol/L, and the pH value of the immunogen removing solution is 7-8; the multi-frequency ultrasonic device provides at least two ultrasonic frequencies, wherein the low-frequency range is 20-40KHz, the high-frequency range is 60-90KHz, the low-frequency treatment is 5-40min, the high-frequency treatment is 5-40min, and the temperature range is 20-35 ℃;

(8) cutting and material taking: cutting a pre-corneal elastic layer and a part of stroma by using a corneal lamellar knife under a dissecting mirror, and then soaking the pre-corneal elastic layer and the part of stroma in purified water;

(9) drying: the cornea was flattened on the surface of the support and placed in an oven for drying to obtain a corneal stroma.

2. The corneal stroma of claim 1, wherein: the animal is a mammal.

3. The corneal stroma of claim 2, wherein: the mammal is pig or cattle.

4. The corneal stroma of claim 1, wherein: the partial thickness corneal stroma is a corneal stroma with a certain thickness connected with a front elastic layer, and the extracellular matrix comprises collagen and glycosaminoglycan.

5. The corneal stroma of claim 4, wherein:

the collagen is type I collagen and type IV collagen, and the content of the type I collagen and the type IV collagen is 85 to 90 percent;

the glycosaminoglycan is chondroitin sulfate and keratan sulfate;

the corneal stroma is a circular sheet with the diameter of 7-10mm and the thickness of 0.1-0.2 mm;

the removal of cell components and DNA components in the corneal epithelium layer and the corneal stroma means that the cell residue is 0, the DNA residue is less than 10ng/mg, and the galactose glycolase clearance rate is more than 99%.

6. A method for preparing a corneal stroma, which is characterized by comprising the following steps: the preparation method comprises the following steps:

(5) immunogen removal: the immunogen removing solution is PBS solution, the PBS solution contains trypsin and EDTA, and the immunogen removing process of the cornea is carried out in a multi-frequency ultrasonic device; the mass percentage concentration of trypsin in the immunogen removing solution is 0.01-0.2%, the concentration of EDTA is 0.1-1mmol/L, and the pH value of the immunogen removing solution is 7-8; the multi-frequency ultrasonic device provides at least two ultrasonic frequencies, wherein the low-frequency range is 20-40KHz, the high-frequency range is 60-90KHz, the low-frequency treatment is 5-40min, the high-frequency treatment is 5-40min, and the temperature range is 20-35 ℃;

7. The method for preparing a corneal stroma according to claim 6, wherein:

step (3) the peroxyacetic acid-ethanol solution contains 0.1-5% of peroxyacetic acid by volume, 5-40% of ethanol by volume, the volume ratio of the peroxyacetic acid-ethanol solution to the cornea is (30-60): 1, the inactivation time is 2-4h, and the temperature range is 10-40 ℃;

washing for 2-4 times (10-30 min each time) at the pH6-8 of the PBS solution and the temperature of 10-40 ℃ and the volume ratio of the PBS solution to the cornea of (30-60): 1, then washing with water for injection at the temperature of 10-40 ℃, the volume ratio of the water for injection to the cornea of (30-60): 1, and stopping when the difference between the conductivity of the water for injection for washing and the conductivity of the water for injection for not washing is less than 1 mu s/cm;

and (4) drying and drying in the step (9) are carried out in a thermal cycle oven, the oven is preheated to 25-40 ℃, the material obtained in the step (8) is placed in the oven for drying, and the moisture is dried in the air through thermal cycle air for 12-24 hours.

8. The method for preparing a corneal stroma according to claim 7, wherein: the mass percentage concentration of trypsin in the immunogen removing solution in the step (5) is 0.02-0.05%, the concentration of EDTA is 0.4-0.8mmol/L, and the pH value of the immunogen removing solution is 7.2-7.5; the volume ratio of the immunogen removing liquid to the cornea is (30-60): 1.

9. The method for preparing a corneal stroma according to claim 6, wherein: the preparation steps further comprise the steps of (10) packaging and (11) sterilizing and resolving.

10. The method for preparing a corneal stroma according to claim 9, wherein: the packaging steps are as follows: packaging the drying material with tyvek packaging paper and a plastic uptake box; the sterilization and analysis steps are as follows: firstly, the temperature is kept at 20-40 ℃ for 2-4 hours, the humidity is 30-70%, then ethylene oxide with the concentration of 300-1000mg/L is introduced, and the sterilization is carried out for 4-8 hours; then analyzing, wherein the analyzing process is carried out in a ventilated analyzing chamber, the temperature is controlled between 10 ℃ and 30 ℃, and the time is 14-28 d.

11. Use of a corneal stroma according to any one of claims 1-5, for the preparation of an ophthalmic implant, characterized in that: the ocular implant is used for temporary covering of drug-ineffective, not yet perforated, ulcerated corneas, infectious keratitis corneas, and perforated corneas.