CN101947144A - Ply tissue engineering corneal frame and manufacturing method and application thereof - Google Patents

Abstract

A lamellar tissue engineering corneal stent and its manufacturing method and application relate to a biomedical lamellar tissue engineering corneal stent. Compared with the existing materials, it provides a lamellar tissue that has rich sources, high transparency, good biocompatibility, performance close to that of fresh cornea, complete decellularization, and strong safety, which can be accepted by a large number of patients and used in clinical practice for a long time Engineering corneal stent and its manufacturing method and application. The lamellar tissue engineering corneal scaffold is an animal-derived decellularized lamellar corneal stroma sheet, which does not contain cell components, the collagen fibers are arranged neatly, the gaps are regular, the corneal light transmittance is 80% to 95%, and the tensile strength is 2～5N/mm ² . It can be used as a substitute for various donor materials in corneal transplantation, and can be used in the treatment of corneal trauma and chemical burn series of diseases, corneal tumors and proliferative diseases, corneal vascularization and scar series of diseases, corneal immune diseases, corneal transplantation rejection series It is used in corneal diseases such as diseases.

Description

A kind of lamellar tissue engineering corneal scaffold and its production method and application

technical field

The invention relates to a biomedical lamellar tissue engineering corneal scaffold, in particular to a scaffold for treating corneal trauma, tumors, chemical burns (alkali burns), vascularized severe dry eye, limbal stem cell deficiency, and immune diseases Such as Stevens-Johnson syndrome (SJS), ocular pemphigoid, herpes simplex virus leukoplakia, corneal transplant rejection and other blinding eye diseases such as lamellar corneal tissue engineering materials and their preparation methods and applications.

Background technique

According to the report of the World Health Organization, corneal disease is the second major cause of vision loss. Every year, 1.5 million to 2 million new corneal blindness are caused by corneal ulcers and eye trauma. The main cause of childhood blindness is corneal blindness caused by measles. Vitiligo. The only effective treatment for corneal blindness is keratoplasty. However, due to the influence of religious beliefs and customs, very few people donate corneas after death; in addition, human immunodeficiency virus (HIV), hepatitis B virus, rabies virus, Creutzfeldt-Jakob virus can be transmitted through implants, corneal refractive surgery, Due to population aging and other reasons, there is a serious shortage of corneal donor materials, which greatly limits the blindness of corneal blind patients. As people's requirements for visual quality are increasing day by day, corneal transplantation technology has also been continuously improved and updated, but the lack of donor sources, immune rejection, corneal transplantation complications, etc. limit the development of this technology. With the increase in the number of patients with corneal trauma, tumors, chemical burns (alkali burns), severe vascularized dry eye, limbal stem cell deficiency, immune diseases, and corneal transplant rejection, the source of corneal materials has become the current ophthalmologist. A tricky problem exists. How to maintain the function of the lamellar corneal substitute in the eye with high quality for a long time, and how to find a corneal substitute that can achieve an ideal state has become a hot spot in the current ophthalmology field. However, various seed cell scaffold materials developed at home and abroad have some defects in corneal curvature and topography, biocompatibility, strength, degradation rate, transparency, homology, antigenicity and pathogenicity. . Porcine cornea has a wide range of sources and is cheap, and its thickness, topography and refraction are very similar to those of human cornea ([1] Liu W, McLaughlin CR, Fagerholm P, Lagali NS, Heyne B, et al. Collagen-phoshphorylcholine interpenetrating network hydrogels as corneal substitutes.Biomaterials, 2009.p.1551-9; [2]Biomaterials and Culture Methods.Res 2008;63:535-544; [3]recombinant humancollagen-based corneal substitutes for Implantation: performance of type I versus type IIIcollagen. Invest Ophthalmol Vis Sci 2008; 49: 3887-3894; [4] Li F, Lohmann C, Suuronen E, Vascotto S, Kobuch K, et al. Cellular and nerve regeneration within a biosynthetic extracellular matrix S for corneal transplantation. USA 2003; 100: 15346-15351; [5] Gilbert TW, BadylakSF. Decellualrization of tissues and organs. Biomaterials 2006; 27: 3675-3683), more and more attention of scientists, while other organs of pigs have been used for Xenotransplantation ([6] Sykes M, Sandrin M. Position paper of the Ethics Committee of the International Xenotransplantation Association. Transplantation 2004; 78: 1101-1107; [7] Kampmeier J, Birngruber R, Brinkmann R. Thermal and biomechanical parameters of ne .2000; 19 : 355-363).

Contents of the invention

The purpose of the present invention is to provide a cornea with abundant sources, high transparency, good biocompatibility, performance close to that of fresh cornea, complete decellularization and strong safety compared with the existing materials, which can be accepted by the majority of patients and used for a long time. Clinical lamellar tissue engineering corneal stent and its production method and application.

The lamellar tissue engineering corneal support can be used as a substitute for various donor materials for corneal transplantation.

The lamellar tissue engineering corneal scaffold is an animal-derived decellularized lamellar corneal stroma sheet, which does not contain cell components, the collagen fibers are arranged neatly, the gaps are regular, the corneal light transmittance is 80% to 95%, and the tensile strength is 2～5N/mm ² .

The lamellar tissue engineered corneal scaffold may include an undried lamellar corneal scaffold, a dried lamellar corneal scaffold, or a rehydrated lamellar corneal scaffold.

The water content and expansion rate of the undried lamellar corneal stent are 75%-90%.

The dry lamellar corneal stent is based on the undried lamellar corneal stent, and the lamellar corneal stent has been dried. The drying process can be vacuum freeze-drying, vacuum drying, dry anhydrous _CaCl2 vacuum drying, natural At least one of air drying, blow drying, and drying in a 30-60°C thermostat.

The rehydrated lamellar corneal stent is based on a dry lamellar corneal stent and processed through a rehydration process. The rehydration agent used in the rehydration process is selected from Ringer's solution, balance solution, At least one of 1×PBS solution, DMEM culture solution, short-term and medium-term corneal preservation solutions, etc., with a water content and expansion rate of 75% to 90%.

The preparation method of described lamellar tissue engineering corneal stent comprises the following steps:

1) Soak fresh animal eyeballs in iodophor, or in PBS containing antibiotics, and rinse;

2) After disinfection, cover the surface of the cornea with filter paper or soak it directly, and then wipe off the epithelial cell layer;

3) Make a limbal incision under the operating microscope, insert the iris restorer to separate the anterior cornea, cut off the anterior cornea with corneal scissors after the separation is complete, or directly separate the anterior lamellar cornea with a lamellar keratectomy knife;

4) Place the separated anterior lamellar cornea on the corneal occipital, with the anterior elastic layer facing down, and then use a corneal ring to drill a lamellar corneal stroma piece with a diameter of 3-10 mm (mark the epithelial surface with 10-0 nylon thread); The fresh lamellar corneal stroma slices were placed directly in airtight sterile cryopreservation tubes and subjected to repeated freeze-thaw rewarming treatment. The freezing time was 1-5 minutes, the rewarming time was 3-15 minutes, and the number of cycles was 1-5 times;

5) Soak the frozen-thawed lamellar corneal stroma sheet in hypotonic solution for 20-120 minutes;

6) Put the lamellar corneal stromal slices treated in step 5) in an incubator at 30-40°C, incubate successively with DNase for 60-180 minutes, and incubate with RNase for 60-180 minutes. At the same time, add 0.5-2.5ug/ml protease inhibitor, 1.10±0.25μg DNA per milligram dry weight of corneal stroma, corneal stroma water content is 50%-80%;

7) Place the enzymatically digested lamellar corneal stroma piece in the electrophoresis solution TA (mark the electrophoresis direction of the cornea), then put the entire electrophoresis tank in a closed disinfection box, and freeze it in a constant temperature refrigerator at 0-10°C. bath electrophoresis;

8) Gradient dehydration is performed on the lamellar corneal stroma sheet after electrophoresis to obtain an undried lamellar tissue engineered corneal scaffold, which is sterilized with ethylene oxide and stored for later use;

9) putting the undried corneal stroma sheet in a 24-well plate for drying treatment to obtain a dry lamellar tissue engineered corneal scaffold;

10) Sterilize the dry lamellar tissue engineered corneal stent with cobalt 60 and perform rehydration treatment to obtain the rehydrated lamellar corneal stent.

In step 1), the povidone iodine can be 0.5% to 2% povidone iodide in mass percent concentration, and the soaking time of the povidone iodine can be 2 to 5 minutes; the soaking time with PBS containing antibiotics It can be 5-10 minutes, and the PBS washing can be washed 3 times, and the time of each washing can be 5 minutes; the antibiotics include 50,000 U/L penicillin, 80,000 U/L tobramycin or 100 mg/L chain Mycin etc.

In step 2), the filter paper is preferably a filter paper dipped in 15% to 30% alcohol by mass percentage; the time for the direct soaking can be 1 to 5 minutes; the erasing of the epithelial cell layer can be Use a cotton swab or a corneal epidermotomy to scrape off the epithelial layer.

In step 3), the limbal incision is made under the operating microscope, and a gem knife can be used to make a limbal incision with a depth of 3 to 4 mm, a thickness of 0.8 to 1.2 mm, and a length of 2.5 to 3.5 mm; The depth of the anterior lamellar cornea separated by the lamellar keratectomy knife may be 3-4 mm, and the thickness may be 0.8-1.2 mm.

In step 5), the hypotonic solution can be selected from double distilled water or triple distilled water.

In step 6), the volume ratio of the enzyme to the buffer can be 1:50, and the volume ratio of the DNase to the RNase can be 10:1; the protease inhibitor can use the product of Roche Company, without EDTA , can inhibit Protease, Calpain II, Cathepsin B, Elastase, leukocyte, Elastase, pancreatic, Papain, Plasmin, Thermolysin, Trypsin and other proteases.

In step 7), the time of the ice-bath electrophoresis can be 1-4 hours, the electrophoresis can be single-phase horizontal electrophoresis or biphasic electrophoresis, the condition of electrophoresis is 100-180V/cm, 1×TA solution configuration method: Dissolve 4.84g TrisBase in 100ml double distilled water, add 1.142ml Glacial Acetic Acid dropwise, then add double distilled water to 1L;

In step 9), the drying process can be at least one of vacuum freeze-drying, vacuum drying, drying anhydrous _Cacl2 vacuum drying, natural drying, drying, drying in a 30-60°C thermostat, etc. , the drying time may be 6-24 hours.

In step 10), the rehydration agent used in the rehydration treatment can be selected from at least one of Ringer's solution, equilibrium solution, 1×PBS solution, DMEM culture solution, short-term corneal preservation solution, medium-term corneal preservation solution, etc. A sort of.

The lamellar tissue engineering corneal stent described in the present invention can be used as a substitute for various donor materials for corneal transplantation, and can be used in the treatment of corneal trauma and chemical burn series of diseases, corneal tumors and proliferative series of diseases, corneal vascularization and scar series Diseases, corneal immune diseases, corneal transplant rejection series of diseases and other corneal diseases.

The present invention starts from a detailed analysis of the commonly used decellularization methods at present, and conducts strict screening and effective combination according to their respective advantages and disadvantages. Breaking the nuclear membrane, using DNA/RNA enzymes that have no effect on the collagen fibrous protein scaffold to destroy and digest residual nucleic acids, and adding protease inhibitors to the buffer, reducing the extracellular matrix of the corneal stroma skeleton (mainly glycosaminoglycans) The destruction of cell residues and charged components is removed by single-phase/two-phase electrophoresis to achieve the purpose of completely removing the antigenic components of the scaffold material, and the remaining nucleic acid components are removed by complete electrophoresis and Co60 disinfection. Pathogenicity of scaffold materials. In order to meet the requirements of product development, all aspects of decellularization treatment, such as time, temperature, voltage, how to ensure sterility and electrolyte configuration, have been carefully designed and repeatedly rigorously explored; through a series of production processes Repeated improvement and optimization make the present invention have the following advantages:

1) Adopting the technical solution of the present invention, the production process is simple and reliable, the time is short and easy to implement;

2) The lamellar tissue engineered corneal stent has a wide range of sources, low cost and convenient use;

3) Adopting the technical scheme of the present invention, the decellularization is thorough, the destruction of the extracellular matrix of the corneal stromal skeleton is minimized, the collagen is arranged regularly, the gap is uniform, no nucleic acid substance and soluble protein components are left, and the surface is composed of type IV collagen The pre-bowman's layer and basement membrane are preserved, which improves the biocompatibility of the scaffold material and facilitates the proliferation and crawling of autologous cells, while the application of protease inhibitors maximizes the protection of the collagen in the matrix;

4) The lamellar tissue engineered corneal stent produced by the technical solution of the present invention was tested for biological properties (transparency, water content, expansion rate, maximum stretching length), ultrastructure observation and optometry detection. Human corneal stroma is very similar;

5) The invention not only helps to optimize the decellularization conditions of animal corneal stroma, but also provides important technical support for the industrialization of tissue engineering cornea.

Description of drawings

Fig. 1 is the DAPI staining result of the undried decellularized porcine lamellar corneal stromal sheet made by the method of the present invention.

Fig. 2 is the DNAGel result of the undried decellularized porcine lamellar corneal stromal sheet made by the method of the present invention.

Fig. 3 is the transparency of the acellular porcine lamellar corneal stromal sheet made by the method of the present invention.

Fig. 4 is the result of the ultrastructure under the transmission electron microscope of the undried acellular porcine lamellar corneal stromal sheet made by the method of the present invention.

Fig. 5 is a slit-lamp photograph of an undried porcine lamellar corneal stromal sheet (with a diameter of 9 cm) implanted in the back of the rabbit neck 8 months after implantation by the method of the present invention.

Fig. 6 is a slit-lamp photograph of the non-dried porcine lamellar corneal stromal sheet implanted in the anterior chamber of the rabbit for two months after being made by the method of the present invention.

Fig. 7 is a slit-lamp photograph of the undried porcine lamellar corneal stromal sheet made by the method of the present invention after 3 months after tiling of the central pouch of the rabbit cornea.

Fig. 8 is a slit-lamp photograph of undried porcine lamellar corneal stromal slices made by the method of the present invention used for rabbit corneal transplantation for 10 days.

Fig. 9 is a comparison curve of corneal epithelial repair within 12 days of rabbit corneal transplantation using undried porcine lamellar tissue engineered corneal scaffold and fresh pig lamellar tissue engineered corneal scaffold prepared by the present invention. In Figure 9, the abscissa is the repair time time (day), and the ordinate is the proportion of corneal epithelium repaired Accumulation repair; ▲ is the untreated group, and ■ is the experimental group.

Detailed ways

Example 1

The lamellar tissue engineering corneal stent described in the present invention, taking the dry and rehydrated lamellar corneal stent as an example, comprises the following steps: after the fresh animal eyeball is disinfected with iodine, it is treated with alcohol and scraped to remove the epithelial cells, and the Lamellar cornea, remove the lamellar corneal stroma, put it on the corneal pillow and use a corneal trephine to remove the lamellar corneal slices of different diameters, through repeated regular airtight freeze-thaw, hypotonic swelling, DNA, RNase digestion, airtight Ice bath electrophoresis in a sterilization box, drying technology in a 24-well plate, cobalt 60 disinfection technology, etc., and finally the lamellar corneal stroma sheet that was not dried, dried, and rehydrated after drying was obtained to obtain the lamellar tissue engineered corneal stroma scaffold. Save it for later use, and add the required growth factors before use.

Preparation method 1 includes the following steps: soak fresh animal eyeballs in 2% povidone iodine for 5 minutes, after disinfection, cover the corneal surface with filter paper dipped in 20% alcohol for 3 minutes, and then gently wipe the epithelial cells with a cotton swab/corneal epidermal knife Under the operating microscope, use a jewel knife to make a limbal incision about 3/4 deep (about 0.8-1.2mm thick) and about 3mm long, and extend into the iris restorer to separate the anterior cornea with a thickness of about 0.8-1.2mm. After the separation is complete, use corneal scissors to cut off the anterior cornea; put the separated anterior lamellar cornea on the corneal pillow (the anterior elastic layer faces down), and then use a corneal trephine drill to obtain a lamellar corneal stroma sheet with a diameter of 3 to 10 mm (10 to 10 mm). 0 nylon thread to mark the epithelial surface). The fresh lamellar corneal stromal slices after de-epithelialization were directly placed in a closed sterile 2ml cryopreservation tube and subjected to repeated freeze-thaw rewarming treatment, the freezing time was 2 minutes, the rewarming time was 5 minutes, and the number of cycles was 3 times; then the frozen The melted lamellar corneal stroma piece was placed in triple distilled water and soaked for 60 minutes. Then put the above-mentioned substrate slices in an incubator at 37°C, and incubate in DNase for 120 minutes and RNase for 120 minutes, and add 1 ug/ml of protease inhibitors to the respective buffers, wherein the ratio of enzyme to buffer 1:50, the ratio of DNase to RNase is 10:1, about 1.10±0.25 μg DNA per milligram of dry weight of corneal stroma, water content of corneal stroma is 50% to 80%, protease inhibitor is a product of Roche Company , does not contain EDTA, can inhibit Protease, Calpain II, Cathepsin B, Elastase, leukocyte, Elastase, pancreatic, Papain, Plasmin, Thermolysin, Trypsin and other proteases. Then place the enzymatically digested lamellar corneal stroma in a special electrophoresis solution TA solution (mark the electrophoresis direction of the cornea), then put the entire single-phase horizontal electrophoresis tank in a closed disinfection box, and conduct ice bath electrophoresis in a constant temperature refrigerator at 4°C for 1-4 hours. The electrophoresis condition is 100-180V/cm Gradient dehydration of the lamellar corneal stroma sheet after electrophoresis to obtain the undried lamellar tissue engineering corneal scaffold, which is sterilized with ethylene oxide and stored for later use; then the undried corneal stroma sheet is placed in a 24-well plate Carry out vacuum drying with anhydrous Cacl ₂ for 12 hours to obtain dry lamellar tissue engineered corneal scaffolds; the dried samples are sterilized with cobalt 60 and rehydrated with Ringer's solution when used. Finally, the required undried, dried and rehydrated lamellar corneal stroma sheet is obtained, which is the lamellar tissue engineered corneal stroma scaffold. Save it for later use, and add the required growth factors before use.

Preparation method 2 includes the following steps: soak fresh animal eyeballs in 2% iodophor for 5 minutes, after disinfection, cover the corneal surface with filter paper dipped in 20% alcohol for 3 minutes, and then gently wipe the epithelial cells with a cotton swab/corneal epidermal knife directly use a lamellar keratectomy knife to separate the anterior lamellar cornea about 3/4 deep (about 0.8-1.2mm thick); put the separated anterior lamellar cornea on the corneal pillow (the anterior elastic layer faces down) ), and then use a corneal ring drill to get a lamellar corneal stromal piece with a diameter of 3 to 10 mm (10 to 0 nylon thread marks the epithelial surface). The fresh lamellar corneal stromal slices after de-epithelialization were directly placed in a closed sterile 2ml cryopreservation tube and subjected to repeated freeze-thaw rewarming treatment, the freezing time was 2 minutes, the rewarming time was 5 minutes, and the number of cycles was 3 times; then the frozen The melted lamellar corneal stroma piece was placed in triple distilled water and soaked for 60 minutes. Then put the above-treated matrix slices in a 37°C incubator, incubate in DNase for 120 minutes, and incubate in RNase for 120 minutes. At the same time, add 1ug/ml of protease inhibitors to the respective buffers, wherein the ratio of enzyme to buffer The dosage ratio of DNase and RNase is 1:50, the dosage ratio of DNase and RNase is 10:1, the dry weight of corneal stroma per mg contains about 1.10±0.25μg DNA, the water content of corneal stroma is 50%-80%, and the protease inhibitor is from Roche Company The product does not contain EDTA and can inhibit Protease, Calpain II, Cathepsin B, Elastase, leukocyte, Elastase, pancreatic, Papain, Plasmin, Thermolysin, Trypsin and other proteases. Then the enzymatically digested lamellar corneal stroma sheet is placed in a special electrophoresis TA solution (mark the electrophoresis direction of the cornea), then put the entire single-phase horizontal electrophoresis tank in a closed disinfection box, and conduct ice bath electrophoresis in a constant temperature refrigerator at 4°C for 1-4 hours. The electrophoresis condition is 100-180V/ cm; dehydrate the lamellar corneal stroma sheet after electrophoresis to obtain the undried lamellar tissue engineering corneal scaffold, sterilize it with ethylene oxide, and save it for later use; then put the undried corneal stroma sheet in a 24-well plate Vacuum-dried with anhydrous CaCl ₂ for 12 hours to obtain dry lamellar tissue engineered corneal scaffolds; the dried samples were sterilized with cobalt 60, and rehydrated with Ringer's solution when used. Finally, the required undried, dried and rehydrated lamellar corneal stroma sheet is obtained, which is the lamellar tissue engineered corneal stroma scaffold. Save it for later use, and add the required growth factors before use.

Example 2

The lamellar tissue engineered corneal stent described in the present invention, taking the dry and rehydrated lamellar corneal stent as an example, includes the following steps: after the fresh animal eyeball is sterilized with antibiotics, it is treated with alcohol and scraped to remove the epithelial cells, and the lamellar tissue is separated. Cornea, remove the lamellar corneal stroma sheet, put it on the corneal pillow and use the corneal trephine to remove the lamellar corneal sheet of different diameters, through repeated regular sealing freeze-thawing, hypotonic swelling, DNA, RNase digestion, airtight disinfection In-box ice-bath electrophoresis, 24-well plate drying technology, cobalt 60 disinfection technology, etc., finally obtained undried, dried and rehydrated lamellar corneal stroma sheets to obtain the lamellar tissue engineered corneal stroma scaffold. Save it for later use, and add the required growth factors before use.

The first preparation method includes the following steps: soak fresh animal eyeballs in PBS containing antibiotics for 10 minutes, and then rinse with PBS for 3 times×5 minutes. The antibiotics used include 50,000 U/L penicillin, 80,000 U/L tobramycin, and 100 mg/L streptomycin; after disinfection, cover the corneal surface with filter paper dipped in 20% alcohol for 3 minutes, and then use a cotton swab/corneal epithelial Gently wipe off the epithelial cell layer with a knife; under the operating microscope, use a gemstone knife to make a limbal incision about 3/4 deep (0.8-1.2mm thick) and 3mm long, and insert the iris restorer to separate the anterior cornea. The thickness is about 0.8-1.2 mm. After the separation is complete, use corneal scissors to cut off the anterior cornea; place the separated anterior lamellar cornea on the corneal pillow (the anterior elastic layer faces down), and then use a corneal ring to drill a 3-10 mm diameter Lamellar corneal stroma sheet (10-0 nylon thread marks the epithelial side). The fresh lamellar corneal stromal slices after de-epithelialization were directly placed in a closed sterile 2ml cryopreservation tube and subjected to repeated freeze-thaw rewarming treatment, the freezing time was 2 minutes, the rewarming time was 5 minutes, and the number of cycles was 3 times; then the frozen The melted lamellar corneal stroma piece was placed in triple distilled water and soaked for 60 minutes. Then put the above-treated matrix slices in a 37°C incubator, incubate in DNase for 120 minutes, and incubate in RNase for 120 minutes. At the same time, add 1ug/ml of protease inhibitors to the respective buffers, wherein the ratio of enzyme to buffer The dosage ratio of DNase and RNase is 1:50, the ratio of DNase and RNase is 10:1, about 1.10±0.25 μg DNA is contained in each milligram of dry weight of corneal stroma, and the water content of corneal stroma is between 50-80%. The protease inhibitor is from Roche Company The product does not contain EDTA and can inhibit Protease, Calpain II, Cathepsin B, Elastase, leukocyte, Elastase, pancreatic, Papain, Plasmin, Thermolysin, Trypsin and other proteases. Then the enzymatically digested lamellar corneal stroma sheet is placed in a special Put the electrophoresis solution in TA solution (mark the electrophoresis direction of the cornea), then put the entire single-phase horizontal electrophoresis tank in a closed disinfection box, and conduct ice bath electrophoresis in a constant temperature refrigerator at 4°C for 1-4 hours. The electrophoresis condition is 100-180V /cm; Gradient dehydration of the lamellar corneal stroma sheet after electrophoresis is performed to obtain the undried lamellar tissue engineering corneal scaffold, which is sterilized with ethylene oxide and stored for later use; then the undried corneal stroma sheet is placed in 24 wells Vacuum drying with anhydrous CaCl ₂ in the plate for 12 hours to obtain dry lamellar tissue engineered corneal scaffolds; the dried samples were sterilized with cobalt 60, and rehydrated with Ringer's solution when used. Finally, the required undried, dried and rehydrated lamellar corneal stroma sheet is obtained, which is the lamellar tissue engineered corneal stroma scaffold, which is stored for future use.

The second preparation method includes the following steps: soak fresh animal eyeballs in PBS containing antibiotics for 10 minutes, and rinse with PBS for 3 times×5 minutes. The antibiotics used include 50,000 U/L penicillin, 80,000 U/L tobramycin, and 100 mg/L streptomycin; after disinfection, cover the corneal surface with filter paper dipped in 20% alcohol for 3 minutes, and then use a cotton swab/corneal epithelial Gently wipe the epithelial cell layer with the knife; directly use the lamellar keratotomy knife to separate the anterior lamellar cornea about 3/4 deep (about 0.8-1.2mm thick) under the operating microscope; put the separated anterior lamellar cornea on Put the cornea on the occipital (the Bowman's layer facing down), and then drill a 3-10mm diameter lamellar corneal stromal piece (the epithelial surface is marked with 10-0 nylon thread) with a corneal ring drill. The fresh lamellar corneal stromal slices after de-epithelialization were directly placed in a closed sterile 2ml cryopreservation tube and subjected to repeated freeze-thaw rewarming treatment, the freezing time was 2 minutes, the rewarming time was 5 minutes, and the number of cycles was 3 times; then the frozen The melted lamellar corneal stroma piece was placed in triple distilled water and soaked for 60 minutes. Then put the above-treated matrix slices in a 37°C incubator, incubate in DNase for 120 minutes, and incubate in RNase for 120 minutes. At the same time, add 1ug/ml of protease inhibitors to the respective buffers, wherein the ratio of enzyme to buffer 1:50, the dosage ratio of DNase and RNase is 10:1, about 1.10 ± 0.25 micrograms of DNA per milligram of dry weight of corneal stroma, the water content of corneal stroma is between 50-80%, and the protease inhibitor is Roche company The product does not contain EDTA and can inhibit Protease, Calpain II, Cathepsin B, Elastase, leukocyte, Elastase, pancreatic, Papain, Plasmin, Thermolysin, Trypsin and other proteases. Then the enzymatically digested lamellar corneal stroma sheet is placed in a special Put the electrophoresis solution in TA solution (mark the electrophoresis direction of the cornea), then put the entire single-phase horizontal electrophoresis tank in a closed disinfection box, and conduct ice bath electrophoresis in a constant temperature refrigerator at 4°C for 1-4 hours. The electrophoresis condition is 100-180V /cm; Gradient dehydration of the lamellar corneal stroma sheet after electrophoresis is performed to obtain the undried lamellar tissue engineering corneal scaffold, which is sterilized with ethylene oxide and stored for later use; then the undried corneal stroma sheet is placed in 24 wells Vacuum drying with anhydrous CaCl ₂ in the plate for 12 hours to obtain dry lamellar tissue engineered corneal scaffolds; the dried samples were sterilized with cobalt 60, and rehydrated with Ringer's solution when used. Finally, the required undried, dried and rehydrated lamellar corneal stroma sheet is obtained, which is the lamellar tissue engineered corneal stroma scaffold. Save it for later use, and add the required growth factors before use.

The present invention analyzes in detail the commonly used decellularization methods at present, and conducts strict screening and effective combination according to their respective advantages and disadvantages, and finally selects the freezing-thawing method that has less influence on the tissue structure to destroy and disintegrate the cell membrane, and uses the hypotonic method to burst the nucleus Membrane, the use of collagen fibrin scaffolds has no effect on DNA/RNase damage and digested residual nucleoli, and protein inhibitors are added to the buffer to reduce the damage to the ECM of the corneal stroma skeleton, using single-phase/bi-phase at low temperature The method of electrophoresis removes the residual debris and charged components of the cells, and achieves the purpose of completely removing the antigenic components of the scaffold material. The pathogenicity of the scaffold material is removed by completely electrophoresis to remove the residual nucleic acid components and Co60 disinfection. In order to meet the requirements of product development, all aspects of decellularization treatment, such as time, temperature, voltage, how to ensure sterility and electrolyte configuration, have been carefully designed and repeatedly rigorously explored; through a series of production processes The repeated improvement and optimization of the present invention has the following advantages: (1) adopting the technical scheme of the present invention, the production process is simple and reliable, the time is short and easy to implement; (2) the lamellar tissue engineering corneal scaffold has a wide range of sources, low cost, and and easy to use; (3) through DAPI staining and DNA Gel results show (Fig. 1 and 2), the porcine lamellar corneal stroma that the inventive method makes, decellularization is thorough, the minimum reduces the destruction of corneal stroma ECM, does not stay any Nucleic acid components and soluble protein, the pre-blastoma and basement membrane formed by type IV collagen on the surface are preserved, which improves the biocompatibility of the scaffold material and is conducive to autologous cell proliferation and crawling; (4) pigs made by adopting the technical scheme of the present invention Lamellar tissue engineered corneal stent, with high transparency (see Figure 3), biological property testing includes measurement of transparency, water content, expansion rate, maximum stretching length, etc. (see Table 1), ultrastructural observation (see Figure 4) and As a result of optical inspection, it was found that this material is very similar to human corneal stroma, the collagen is arranged regularly, and the gaps are even; (5) the non-dried porcine lamellar tissue engineering corneal scaffold made by the technical scheme of the present invention is implanted in 8 rabbit subcutaneous (seeing Fig. 5), in the anterior chamber of the rabbit (seeing Fig. 6) 6 months all show that it has good histocompatibility, do not see obvious inflammatory reaction and rejection; Porcine lamellar tissue-engineered corneal scaffolds were used for 3 months after the central tiling of rabbit corneas (see Figure 7). It was found that the cornea was relatively transparent without affecting its transparency, and no obvious inflammatory reaction and rejection reaction were seen; (7) using this method The porcine lamellar tissue engineering corneal scaffold made by the technical scheme of the invention was used for 10 days after corneal transplantation in rabbits (see Figure 8), and fluorescein staining found that the corneal epithelium grew intact on its surface 10 days after the operation (see Figure 9), and the cornea Gradually transparent, no obvious inflammatory reaction and rejection reaction; (7) The present invention not only helps to optimize the decellularization conditions of animal corneal stroma, but also provides important technical support for the industrialization of tissue engineering cornea.

Table 1

Note: The unit of tensile strength is N/mm ² , and the units of water content, expansion rate and light transmittance are %.

The decrease of corneal transparency caused by various diseases, irreversible turbidity of corneal stroma is the main cause of vision loss, ranking second in the cause of blindness, second only to cataract. The most effective treatment for corneal blindness is penetrating keratoplasty, but insufficient donor sources, immune rejection, and complications of corneal transplantation limit the development of this technique. Therefore, it is particularly important to find a new corneal substitute material. Corneal substitutes are mainly divided into two categories: artificial corneas and tissue engineered corneal equivalents (TECE). TECE is a cell-based active substitute, mainly composed of three types of cells and collagen components of the natural cornea. The key to constructing a tissue-engineered cornea equivalent is a scaffold material that supports the three-dimensional growth of cells. Collagen-based natural biopolymer glue exhibits excellent biological properties and supports three-dimensional growth of cells, and has been widely used in tissue engineering technology. At present, there is no report on the clinical application of TECE constructed with this method, mainly because the collagen has poor toughness and cannot tolerate sutures. The main components of decellularized xenogeneic tissues are collagen, elastic fibers, etc., with little change in shape and toughness. Moreover, decellularization treatment can reduce the inflammatory response and immune rejection of heterogeneous tissues, and expand the source of tissues. In tissue engineering technology and regenerative medicine research, decellularized bioscaffold materials such as heart valves, blood vessels, skin, nerves, skeletal muscles, aponeurosis, ligaments, small intestinal submucosal tissue, bladder and liver have entered preclinical animal experiments or clinical trials. treatment phase. However, in current studies, physical, chemical and detergent methods are often used to remove stromal cells. Although the cellular components are removed as much as possible, the corneal stromal skeleton and ECM are greatly damaged, which seriously affects the transparency and toughness of the stromal sheet.

The key technology of tissue engineering to construct cornea is to form a three-dimensional complex of corneal epithelial cells, stromal cells, endothelial cells and biological scaffolds. Scaffold material is the main content of tissue engineering research and one of the basic elements of tissue engineering cornea. The characteristics of the scaffold material directly affect whether the composite seed cells can adhere, grow and differentiate normally.

An ideal TECE scaffold material should have the following characteristics:

1) Good biocompatibility, various cells of the cornea can grow well on or in it.

2) Having a three-dimensional structure and carrying a variety of biologically inducible chemical signals can induce seed cells to grow along the scaffold material and differentiate into corneal cells.

3) The degradation rate of the scaffold material matches the rate of the tissue formed by the implanted seed cells, and can be gradually degraded and absorbed or become an integral part of the fusion with the new tissue, and can be molded according to the defect of the diseased cornea to achieve Perfect shape restoration.

4) It has certain strength and toughness, and can resist intraocular pressure.

5) Can gradually become transparent. The ideal method needs to be gentle on the corneal stroma (reduce steps, operate as little as possible, time as short as possible, and temperature as low as possible), remove cells as much as possible without damaging the stroma skeleton and extracellular matrix, and keep the corneal stroma as it is. Biological and mechanical properties without affecting the transparency of the tissue. In order to meet most of the above requirements, we analyzed in detail the currently commonly used decellularization methods, and conducted strict screening and effective combination according to their respective advantages and disadvantages, and finally invented the above production process.

The undried porcine lamellar tissue engineering corneal stent made by the present invention was implanted under the rabbit skin for 8 months, and the anterior chamber of the rabbit for 6 months showed that it had good histocompatibility, and no obvious inflammatory reaction and rejection reaction were seen. The implantation results of the untreated porcine lamellar corneal stromal sheet were obviously superior. At the same time, it was used in the central and peripheral pockets of the rabbit cornea to tile the cornea 3 months after operation and 10 days after keratoplasty. The cornea was gradually transparent, and no Obvious inflammatory reaction and rejection reaction were obviously superior to those of untreated porcine lamellar corneal stromal sheet implantation. Fluorescein staining showed that the corneal epithelium grew intact on the surface 10 days after operation.

Claims (10)

1. a flaggy tissue engineering comea support is characterized in that not containing cell component for the animal derived cell plate layer corneal stroma sheet that takes off, and arrangement of collagen fibers is neat, the gap rule, and the cornea light transmittance is 80%～95%, hot strength is 2～5N/mm ²

2. a kind of flaggy tissue engineering comea support as claimed in claim 1 is characterized in that described flaggy tissue engineering comea support comprises not dry flaggy CF, dry flaggy CF or rehydration lamellar cornea support; The water content of described not dry flaggy CF and expansion rate are 75%～90%; Described dry flaggy CF is based on not dry flaggy CF, and through the lamellar cornea support of dried, described dried is a vacuum freeze-drying, and vacuum is dried, dry anhydrous CaCl ₂Vacuum drying dries naturally, dries up at least a in 30～60 ℃ of calorstats in the oven dry; Described rehydration lamellar cornea support is based on dry flaggy CF, lamellar cornea support through the reconstitution process processing, described reconstitution process is handled used rehydration agent and is selected from ringer's solution, balance liquid, 1 * PBS solution, at least a in the DMEM culture fluid, short, mid-term corneal storage medium, water content and expansion rate are 75%～90%.

3. the manufacture method of a kind of flaggy tissue engineering comea support as claimed in claim 1 is characterized in that may further comprise the steps:

1) the fresh animal eyeball is soaked through povidone iodine, or with after the PBS immersion that contains antibiotics, flushing;

2) the sterilization back is covered in anterior corneal surface with filter paper or directly soaks, and wipes epithelium layer then;

3) under operating microscope, make cut-out of limbus corneae, stretch into iris repositor and separate the anterior layer cornea, separate complete back and cut the anterior layer cornea with corneal scissors, or lamellar cornea before directly separating with the lamellar cornea separating knife;

4) lamellar cornea before isolating is put on the cornea pillow, bowman's lamina faces down, and drills through the lamellar cornea substrate tablet of 3～10mm diameter then with corneal ring; Handle going fresh lamellar cornea substrate tablet behind the epithelium directly to be put in to give the multigelation rewarming in the airtight aseptic frozen pipe, cooling time is 1～5min, and rewarming time is 3～15min, and cycle-index is 1～5 time;

5) the lamellar cornea substrate tablet of freeze thawing is positioned over soaks 20～120min in the hypotonic medium;

6) will be put in 30～40 ℃ of calorstats through the lamellar cornea substrate tablet that step 5) was handled, successively hatch 60～180min in the DNA enzyme, hatch 60～180min in the RNA enzyme, the protease inhibitor that in buffer separately, adds simultaneously 0.5～2.5ug/ml, every milligram of corneal stroma dry weight contains 1.10 ± 0.25 μ g DNA, and the corneal stroma water content is 50%～80%;

7) will be positioned over through the lamellar cornea substrate tablet of enzymic digestion in the electrophoresis liquid TA liquid and (perform the electrophoresis bearing mark of cornea), then whole electrophoresis tank will be put in the airtight disinfection box, carry out the ice bath electrophoresis in 0～10 ℃ of constant temperature refrigerator;

8) the lamellar cornea substrate tablet that electrophoresis is finished carries out the gradient dehydration, promptly obtains not drying plate layer tissue engineering cornea support, adopts ethylene oxide sterilization processes, preserves standby;

9) not dry corneal stroma sheet is put in carries out dried in 24 orifice plates, promptly obtain drying plate layer tissue engineering cornea support;

10) drying plate layer tissue engineering cornea support is carried out cobalt 60 sterilizations, carry out rehydration and handle, get rehydration lamellar cornea support.

4. the manufacture method of a kind of flaggy tissue engineering comea support as claimed in claim 3 is characterized in that in step 1), and described povidone iodine is that the employing mass percentage concentration is 0.5%～2% povidone iodine, and the time that described povidone iodine soaks is 2～5min; The time that described usefulness contains the PBS immersion of antibiotics is 5～10min, and described PBS flushing is flushing 3 times, and the time of each flushing is 5min; Described antibiotics comprises 50,000 U/L penicillins, 80,000 U/L tobramycins or 100mg/L streptomycin.

5. the manufacture method of a kind of flaggy tissue engineering comea support as claimed in claim 3 is characterized in that in step 2) in, described filter paper is to dip in the filter paper that mass percentage concentration is 15%～30% ethanol; The time of described direct immersion is 1～5min; The described epithelium layer of wiping is to adopt cotton swab or epi-microkerotome to wipe epithelium layer.

6. the manufacture method of a kind of flaggy tissue engineering comea support as claimed in claim 3, it is characterized in that in step 3), describedly make cut-out of limbus corneae under operating microscope, is that to adopt the gem cutter be 3～4mm as the degree of depth, thickness is 0.8～1.2mm, and length is the cut-out of limbus corneae of 2.5～3.5mm; The described direct degree of depth with the preceding lamellar cornea of lamellar cornea before the separation of lamellar cornea separating knife is 3～4mm, and thickness is 0.8～1.2mm.

7. the manufacture method of a kind of flaggy tissue engineering comea support as claimed in claim 3 is characterized in that in step 5), and described hypotonic medium is selected from distilled water or tri-distilled water.

8. the manufacture method of a kind of flaggy tissue engineering comea support as claimed in claim 3 is characterized in that in step 6), and the volume ratio of described enzyme and buffer is 1: 50, and the volume ratio of DNA enzyme and RNA enzyme is 10: 1; Described protease inhibitor adopts the product of roche company, does not contain EDTA;

In step 7), the electrophoretic time of described ice bath is 1～4h, described electrophoresis is single-phase horizontal strip electrophoresis or two-phase electrophoresis, electrophoretic condition is 100～180V/cm, 1 * TA solution allocation method: 4.84g TrisBase is dissolved in the 100ml distilled water, drip 1.142ml Glacial Acetic Acid, add distilled water then to 1L.

9. the manufacture method of a kind of flaggy tissue engineering comea support as claimed in claim 3 is characterized in that in step 9) described dried is a vacuum freeze-drying, and vacuum is dried, dry anhydrous Cacl ₂Vacuum drying dries naturally, dries up, and at least a in 30～60 ℃ of calorstats in the oven dry, the time of described dried is 6～24h; In step 10), described rehydration is handled the rehydration agent of being adopted and is selected from ringer's solution, balance liquid, 1 * PBS solution, DMEM culture fluid, at least a in the short-term corneal storage medium, mid-term corneal storage medium.

10. a kind of according to claim 1 flaggy tissue engineering comea support is used for the substitute of the various donor materials of corneal transplantation, outside the treatment cornea, injure chemical burn series disease, corneal tumor and hypertrophy series disease, vascularization of cornea and cicatrix series disease, the cornea immune disease is used in the corneal allograft rejection series disease.

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