CN117919509A - Surface anti-adhesion cell biological cornea and preparation method and application thereof - Google Patents
Surface anti-adhesion cell biological cornea and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a surface anti-adhesion cell biological cornea, a preparation method and application thereof. A decellularized biological cornea, said cornea being chemically modified on one side surface with a zwitterionic polymer that forms a polymer molecular brush on the cornea surface; the polymer molecular brush is prepared by reacting a zwitterionic monomer or a polymer with a zwitterionic group on the surface of the decellularized biological cornea. The invention modifies the zwitterionic polymer on the inner surface of the decellularized cornea, the zwitterionic polymer forms a super-hydrophilic polymer brush on the inner surface of the cornea, and the polymer brush can form a hydration layer on the inner surface of the cornea through strong electrostatic interaction, thereby resisting protein adhesion and biomembrane formation in tears and aqueous humor, maintaining high transparency of the cornea, preventing transparency reduction, biological pollution and the like caused by protein adsorption in vivo after the cornea is implanted for a long time, and solving the problem that the decellularized cornea matrix can only be applied to lamellar cornea implantation at present.
Description
Technical Field
The invention relates to the field of biological cornea preparation, in particular to a surface anti-adhesion cell-free biological cornea, and a preparation method and application thereof.
Background
The cornea is a highly transparent tissue that takes over two thirds of the refractive function of the eyeball and serves as a barrier to protect the eyeball from mechanical and ultraviolet damage, blocking foreign objects from the outside. Currently, corneal transplantation is the only protocol for clinical treatment of severe corneal lesions. Cornea transplants mainly include penetrating cornea implants that replace the whole layer and lamellar cornea implants that replace only the epithelial and stromal layers. Penetrating corneal transplantation is the primary mode of current corneal transplantation, and adult corneal endothelial cells are not renewable, so that grafts for penetrating corneal transplantation must have a healthy endothelial layer or function similar to an endothelial layer to isolate aqueous humor and maintain corneal transparency. The cornea donors currently available for penetrating cornea transplants are severely in shortage, and therefore, clinically artificial cornea materials are beginning to be used instead of allogenic cornea of human origin for transplants. Cornea equivalents that have been commercialized are mainly classified into artificial corneas using synthetic polymers as a main raw material and biological corneas using decellularized porcine cornea stroma as a main raw material. The artificial cornea comprises Boston type artificial cornea, bone tooth type artificial cornea, alphaCor artificial cornea and the like, and the main material is polymethyl methacrylate (PMMA) or polyhydroxyethyl methacrylate (PHEMA). These materials are bioinert and do not allow for biological integration of host tissues, resulting in post-implantation herniation or complications. And the artificial cornea transplanting process is complex, and the artificial cornea transplanting cases implemented each year are very limited. The cornea substitute commercialized in China is mainly biological cornea taking decellularized cornea stroma as raw material. The decellularized cornea stroma is derived from porcine cornea, immunogenicity is removed by a certain decellularizing method, original fine structure and active substances of cornea are reserved, and the method has the advantages of wide sources, high bioactivity and surgical thread suture resistance, and has wide application prospect in the field of cornea transplantation. Several methods of decellularization are currently in common use including the use of one or more of enzymatic reagents, detergents, and hypotonic solutions, but these methods all suffer from certain drawbacks. Most cells can be removed by using an enzyme reagent, but the enzyme can degrade the cornea stroma collagen, so that the ordered microstructure of collagen fibers is destroyed, and the cornea transparency is low; the high-low permeability solution method is used for decellularizing, so that cornea is easy to swell and the damage to the cornea matrix structure is large. The problems of tissue infection, transparency reduction caused by edema and the like due to surface protein adhesion can occur after the conventional biological cornea is implanted for a long time, and the vision recovery of a patient is poor after the implantation. Importantly, due to the lack of an endothelial layer, the decellularized porcine cornea stroma can only be used for lamellar cornea transplantation, and cannot treat severe cornea damage that trails the full layer.
Chinese patent application 202310156644.9 discloses an artificial lens and a preparation method thereof, wherein after an artificial lens material such as polymethyl methacrylate, polyhydroxyethyl methacrylate, hydrophobic acrylic ester and the like is subjected to plasma treatment, a hydrophilic anti-fouling polymer or polypeptide is covalently bonded to the surface of the artificial lens, so that the artificial lens has good hydrophilicity and anti-fouling property, long-acting resistance to adhesion of pollutants such as protein, bacteria and the like on the surface of the lens, and stability and effectiveness of artificial lens implantation are improved. The artificial lens material used in the patent is a synthetic polymer material, has no bioactivity, is only used for artificial lens implantation, and is not suitable for the field of cornea implantation. The prior art generally has the following disadvantages: commercial artificial cornea mainly uses synthetic materials, has poor biocompatibility, is easy to cause complications such as glaucoma, anterior chamber leakage, endophthalmitis, retinal detachment, intraocular pressure increase, cornea dissolution and the like after implantation, and is difficult for the vision of a patient to recover; the commercial artificial cornea has complicated preparation flow, complex operation flow and difficult application and popularization; the commercialized biological cornea takes decellularized porcine cornea stroma as a raw material, and after the inner surface of the cornea is contacted with aqueous humor, proteins are easy to adsorb, and meanwhile, swelling occurs to cause corneal edema, so that transparency is reduced, and the cornea cannot be directly contacted with the aqueous humor.
Compared with the prior art, the decellularized cornea provided by the invention can maintain the microstructure of a natural cornea, retains most of active substances, is more beneficial to cell adhesion and proliferation, has low immunogenicity, high transparency and good protein adhesion resistance effect, and has outstanding advantages in the aspect of penetrating cornea transplantation.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a single-sided anti-protein adhesion and anti-biofilm decellularized biological cornea subjected to chemical modification treatment and a preparation method thereof. The decellularized biological cornea is derived from the eyeballs of mammals, and has wide sources and easy acquisition. The decellularization method is mild, can largely retain the natural cornea microstructure, and can maintain the cornea high transparency. Compared with an artificial cornea prepared from a synthetic polymer material, the decellularized porcine cornea retains active substances such as proteins, polysaccharides and growth factors in the eyeball, can better simulate a natural cornea microenvironment, provides more attachment sites for cornea epithelial cells, stromal cells and the like, ensures cell adhesion, growth and reproduction, and promotes regeneration and recovery of tissues.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
The first aspect of the invention provides a decellularized biological cornea, wherein a zwitterionic polymer is chemically modified on one side surface of the cornea, and forms a polymer molecular brush on the surface of the cornea; the polymer molecular brush is prepared by reacting a zwitterionic monomer or a polymer with a zwitterionic group on the surface of the decellularized biological cornea;
The zwitterionic monomer includes at least one of methacryloxyethyl sulfobetaine (CAS number: 3637-26-1), methacryloxyethyl carboxylic betaine (CAS number: 24249-95-4), 2-methacryloxyethyl phosphorylcholine (CAS number: 67881-98-5), trimethylammonium, and a sulfonate mixture.
Preferably, the polymer molecular brush is prepared by reacting a zwitterionic monomer on the surface of the decellularized biological cornea; the zwitterionic monomer is 2-methacryloyloxyethyl phosphorylcholine.
The second aspect of the invention provides a preparation method of the decellularized biological cornea, which comprises the following steps:
immersing the decellularized cornea matrix into an acrylic anhydride compound solution, activating active reactive groups in the decellularized cornea to react with the acrylic anhydride compound, washing after the reaction, covering one side surface by using a mask, immersing in a reaction solution containing zwitterionic groups to initiate grafting reaction on the other side surface of the decellularized cornea matrix, washing after the reaction, and immersing in deionized water to obtain the decellularized biological cornea;
the acrylic anhydride compound solution comprises an aqueous solution of methacrylic anhydride;
The reaction solution containing the zwitterionic groups contains a zwitterionic monomer or a polymer with the zwitterionic groups, and the reaction solution containing the zwitterionic groups also contains an initiator.
The zwitterionic polymer is modified on the inner surface of the decellularized cornea by a one-step method of modification and crosslinking, and forms a super-hydrophilic polymer brush on the inner surface of the cornea, and the polymer brush can form a hydration layer on the inner surface of the cornea through strong electrostatic interaction, so that the adhesion of proteins in tears and aqueous humor and the formation of a biological film are resisted, the high transparency of the cornea is maintained, the transparency reduction and biological pollution caused by the absorption of proteins in vivo after the cornea is implanted for a long time are prevented, and the problem that the decellularized cornea matrix can only be applied to lamellar cornea transplantation at present is solved. In addition, the preparation method not only modifies the decellularized cornea on the inner surface, but also crosslinks the cornea matrix structure, so that the collagen fiber structure is more compact, the mechanical property of the decellularized biological cornea is enhanced, and meanwhile, the decellularized biological cornea is not easy to absorb water and expand, thereby being beneficial to maintaining the light transmittance of the decellularized biological cornea. The preparation method of the decellularized cornea is simple and convenient, is easy to operate, and the prepared decellularized cornea has the advantages of high transparency, low immunogenicity, good biocompatibility and surgical thread suture resistance, can be directly contacted with aqueous humor, and is expected to be used for penetrating cornea transplantation.
Preferably, the decellularized corneal stroma is prepared by a preparation method comprising the steps of: taking an animal eyeball to be washed by using a washing liquid; peeling the obtained cornea slice by using a keratome; and (3) performing decellularization treatment on the cornea slice to obtain a decellularized cornea stroma. Further preferably, the decellularization treatment comprises the steps of: immersing the cornea slice in sterile PBS solution, washing by shaking, then immersing in ultrapure water, and washing by shaking; spreading the cornea slice, attaching the cornea slice on the wall of a centrifuge tube, freezing, re-warming at room temperature, and repeating for 2-8 times; placing the cornea slice into a deoxycholate sodium solution, and oscillating and washing in a shaking table; the cornea slice is placed in sterile PBS solution, and is washed by shaking, thus obtaining decellularized cornea stroma.
Preferably, the animal eyeball is from a mammal, and the mammal comprises one of a human, a pig, a sheep, a cow and a dog; further preferably, the animal eyeball is a pig eyeball.
Preferably, the concentration of the acrylic anhydride compound solution is 0.5-10 (v/v)%.
Preferably, the pH of the acrylic anhydride compound solution is in the range of 5-9; further preferably, the pH of the acrylic anhydride compound solution is in the range of 7 to 8.
Preferably, the acrylic anhydride compound solution is a deionized water solution of methacrylic anhydride.
Preferably, the reaction solution containing the zwitterionic group contains a zwitterionic monomer or a polymer with a zwitterionic group; the concentration of the zwitterionic monomer is 0.1-1mol/L.
Preferably, the initiator is one of a redox initiator, a thermal initiator and a photoinitiator; the redox initiator comprises one of a mixture of dibenzoyl peroxide and N-N-dimethylaniline (CAS number: 99-98-9), a mixture of dibenzoyl peroxide (CAS number: 94-36-0) and N-N-dimethyl-p-toluidine (CAS number: 99-97-8), ammonium persulfate and sodium bisulfite; the thermal initiator concentration comprises potassium persulfate and/or azo diisobutyl amidine hydrochloride; the photoinitiator comprises 2-hydroxy-4' - (2-hydroxyethoxy) -2-methylpropionacetone.
Preferably, when the initiator is a redox initiator, the concentration of the redox initiator in the reaction solution containing the zwitterionic group is 0.01-0.5mol/L; when the initiator is a thermal initiator, the concentration of the thermal initiator in the reaction solution containing the zwitterionic groups is 0.05-0.1mol/L; when the initiator is a photoinitiator, the concentration of the photoinitiator in the reaction solution containing the zwitterionic groups is 0.05-0.1 (v/v)%.
Preferably, when the initiator is a redox initiator, the grafting reaction temperature is 10-30 ℃ and the reaction time is 0.5-24h; when the initiator is a thermal initiator, the grafting reaction temperature is 30-40 ℃ and the reaction time is 0.2-2h; when the initiator is a photoinitiator, the illumination wavelength is 290-450 nm, the illumination intensity is 2-10mW/cm 2, and the illumination time is 40-120s.
Compared with the prior art, the invention has the beneficial effects that:
1. The acellular cornea matrix used by the invention has wide sources, is easy to obtain, and can greatly meet the use requirements.
2. The decellularized biological cornea of the invention has high transparency and is suitable for cornea transplantation.
3. The modification method of the invention is not easy to damage the microstructure of the cornea matrix.
4. The decellularized biological cornea in the invention has good mechanical property, is resistant to suture of an operation line, and is easy to operate during operation.
5. The inner surface of the decellularized cornea can resist the adhesion of tears and proteins in aqueous humor and the formation of a biological film, is directly contacted with the aqueous humor, and is expected to be applied to penetrable cornea transplantation.
6. The preparation method is simple and convenient and is easy to operate.
7. The decellularization method used by the invention is mild, and can largely retain the microstructure and optical properties of the natural cornea.
8. The preparation method of the invention can completely remove the cellular components in the natural cornea, and has low immunogenicity.
Drawings
FIG. 1 is a physical view of a decellularized biological cornea of an embodiment;
FIG. 2 is a graph of H & E staining of the decellularized biological cornea before and after decellularization of an example;
FIG. 3 is a graph showing adhesion of fluorescent proteins before and after modification of the surface of the cornea of an decellularized organism;
FIG. 4 is a graph showing the adhesion of bovine serum albumin before and after modifying the surface of the cornea of a decellularized organism in the example;
FIG. 5 shows the change in hydrophilicity of the front and back surfaces of the decellularized biological cornea-modified polyzwitterionic of the examples;
FIG. 6 is a graph showing the results of the light transmittance test of the modified polyzwitterionic on the surface of the decellularized biological cornea of the example;
FIG. 7 shows the results of the mechanical properties of the modified polyampholytics on the surface of the decellularized biological cornea of the example.
Detailed Description
The following describes the invention in more detail. The description of these embodiments is provided to assist understanding of the present invention, but is not intended to limit the present invention. In addition, the technical features of the embodiments of the present invention described below may be combined with each other as long as they do not collide with each other.
The experimental methods in the following examples, unless otherwise specified, are conventional, and the experimental materials used in the following examples, unless otherwise specified, are commercially available.
The single-sided modified zwitterionic polymer decellularized cornea (pDCSM-PMPC) is prepared by the following steps:
(1) Preparation of corneal sections (NPC)
Flushing the obtained pig eyeballs with a sterile PBS solution; the cornea was peeled off partially using a keratome, the thickness of the cornea peeled off was measured with a keratometer, the thickness of the cornea was determined, and the cornea was peeled off along the curvature of the cornea from the opening. And cutting off the stripped cornea from the opening along the boundary between the cornea and the sclera to obtain a cornea slice.
(2) Preparation of decellularized corneal stroma (pDCSM)
The cornea slice is soaked in sterile PBS solution, washed by shaking, then soaked in ultrapure water, and washed by shaking.
The cornea slice is tiled and stuck on the wall of a centrifuge tube, frozen, re-warmed at room temperature and repeated for 2-8 times.
The corneal sections were placed in sodium deoxycholate solution and washed with shaking in a shaker.
The cornea slice is placed in sterile PBS solution, and is washed by shaking, thus obtaining decellularized cornea stroma.
(3) Preparation of Single-sided modified polyzwitterionic decellularized biological cornea (pDCSM-PMPC)
Immersing the prepared decellularized cornea matrix in methacrylic anhydride/deionized water solution for reaction, adjusting the pH value to be 5-9 by using sodium hydroxide solution, and washing by using deionized water after the reaction to obtain methacryloylated decellularized cornea matrix (pDCSM-MA); covering the outer surface of the washed decellularized cornea matrix, and vacuum drying.
Adding 2-methacryloyloxyethyl phosphorylcholine solution at the bottom of a sample bottle, placing the dried decellularized cornea matrix into the sample bottle, adding ammonium persulfate solution and sodium bisulphite solution, performing oscillation reaction, and washing with deionized water after the reaction.
Immersing and rehydrating the sample in deionized water to obtain the decellularized cornea (pDCSM-PMPC) of the single-sided modified zwitterionic polymer.
According to the invention, the decellularized cornea matrix is derived from natural tissues, so that immunogenic substances are removed, and active substances such as proteins, polysaccharides, growth factors and the like and microstructures peculiar to the natural cornea are reserved, so that the cornea cell growth microenvironment can be better simulated, cell adhesion, growth and reproduction are facilitated, secretion and accumulation of extracellular matrixes are promoted, and the decellularized cornea matrix has excellent cell compatibility, tissue compatibility and surgical suture resistance.
Methacrylic anhydride is an organic compound containing carbonyl groups and has a double bond structure. In a weak alkaline environment, the carbonyl carbon atom of the anhydride and the nitrogen atom of the amino on the acellular cornea stroma generate nucleophilic addition reaction to generate an intermediate amidate, then the intermediate amidate is hydrolyzed to generate an amide compound, and double bonds are introduced into the acellular cornea stroma. The double bond serves as an initiation site, and the molecular brush is formed by subsequent polymerization of the double bond and 2-methacryloyloxyethyl phosphorylcholine through free radical polymerization.
The 2-methacryloyloxyethyl phosphorylcholine is a zwitterionic monomer, has a molecular structure of combination of cations and anionic groups, is electrically neutral and highly hydrophilic as a whole, can combine a plurality of water molecules through strong electrostatic interaction to form a hydration layer, and has remarkable advantages in the aspects of preventing nonspecific protein adsorption, interface lubrication and reducing bacterial adhesion. In addition, the molecular structure of the 2-methacryloyloxyethyl phosphorylcholine is similar to that of a natural phospholipid bilayer, and the biocompatibility is good.
Ammonium persulfate and sodium bisulphite are inorganic salts, form a redox initiation system, generate free radicals, initiate free radical polymerization reaction, and enable the polyamphogen to be modified on the inner surface of the acellular cornea matrix.
The implementation of the invention is illustrated below by means of specific examples.
Example 1
The obtained porcine eyeball was rinsed with a sterile PBS solution. A keratome was used to make an incision at the boundary between the cornea and sclera of the porcine eyeball to separate portions of the cornea. The thickness of the peeled cornea was measured with a keratometer and was determined to be between 300 and 400 μm. The cornea is peeled from the opening along the corneal curvature until the lamellar cornea is completely separated. The thickness of the stripped cornea was determined by a repeated measurement with a keratometer. The peeled cornea is sheared off by surgical scissors.
The corneal sections were immersed in sterile PBS solution and washed with shaking in a shaker for 20min. The corneal sections were immersed in ultrapure water and washed with shaking in a shaker. The cornea slices are tiled and stuck on the wall of a centrifuge tube, and are frozen in a refrigerator at the temperature of minus 40 ℃ for 2 hours. The centrifuge tube was removed and placed in a 25℃water bath for 2 hours of rewarming. Repeated 3 times. The corneal sections were placed in sodium deoxycholate solution and washed with shaking in a shaker for 4 hours. The corneal sections were placed in sterile PBS solution and washed with shaking in a shaker for 30 minutes. Obtaining the decellularized cornea stroma.
A physical view of the decellularized corneal stroma (pDCSM) of this example is shown in FIG. 1.
The results of H & E staining before and after corneal decellularization are shown in FIG. 2, and the results of FIG. 2 show that the decellularized biological cornea of the invention is completely removed from the cellular components after decellularization treatment, but the collagen fiber lamellar structure of the original natural cornea is still maintained.
Example 2
3 Pieces of the decellularized cornea stroma prepared in example 1 were taken, immersed in a 2.5% methacrylic anhydride/deionized water solution (v/v), and reacted with stirring for 1 hour while maintaining the pH between 7 and 8 with a 1M sodium hydroxide solution. After the reaction, the sample was washed 5 times with deionized water to obtain methacryloylated decellularized corneal stroma (pDCSM-MA). The outer surface of the decellularized cornea stroma was covered with clear tape and dried under vacuum for 30 minutes. A0.5M solution of 2-methacryloyloxyethyl phosphorylcholine was added to the bottom of the sample bottle, and the dried decellularized corneal stroma was placed in the sample bottle with its inner surface in contact with the solution. To the sample bottle were added a 0.2M ammonium persulfate solution and a 0.2M sodium bisulfite solution. Placing in a shaking table for shaking reaction for 1 hour. The reacted sample was washed 3 times with deionized water. Immersing the sample in deionized water for 6 hours for rehydration to obtain the decellularized cornea (pDCSM-PMPC) of the single-sided modified zwitterionic polymer.
Example 3
3 Pieces of the decellularized cornea stroma prepared in example 1 were taken, immersed in a 2.5% methacrylic anhydride/deionized water solution (v/v), and reacted with stirring for 1 hour while maintaining the pH between 7 and 8 with a 1M sodium hydroxide solution. After the reaction, the sample was washed 5 times with deionized water to obtain methacryloylated decellularized corneal stroma (pDCSM-MA). The outer surface of the decellularized cornea stroma was covered with clear tape and dried under vacuum for 30 minutes. A0.5M solution of 2-methacryloyloxyethyl phosphorylcholine was added to the bottom of the sample bottle, and the dried decellularized corneal stroma was placed in the sample bottle with its inner surface in contact with the solution. To the sample bottle were added a 0.2M ammonium persulfate solution and a 0.2M sodium bisulfite solution. Placing in a shaking table for shaking reaction for 2 hours. The reacted sample was washed 3 times with deionized water. Immersing the sample in deionized water for 6 hours for rehydration to obtain the decellularized cornea (pDCSM-PMPC) of the single-sided modified zwitterionic polymer.
Example 4
3 Pieces of the decellularized cornea stroma prepared in example 1 were taken, immersed in a 2.5% methacrylic anhydride/deionized water solution (v/v), and reacted with stirring for 1 hour while maintaining the pH between 7 and 8 with a 1M sodium hydroxide solution. After the reaction, the sample was washed 5 times with deionized water to obtain methacryloylated decellularized corneal stroma (pDCSM-MA). The outer surface of the decellularized cornea stroma was covered with clear tape and dried under vacuum for 30 minutes. A0.5M solution of 2-methacryloyloxyethyl phosphorylcholine was added to the bottom of the sample bottle, and the dried decellularized corneal stroma was placed in the sample bottle with its inner surface in contact with the solution. To the sample bottle were added a 0.2M ammonium persulfate solution and a 0.2M sodium bisulfite solution. Placing in a shaking table for shaking reaction for 3 hours. The reacted sample was washed 3 times with deionized water. And soaking the sample in deionized water for 6 hours for rehydration to obtain the single-sided modified polyamphogen decellularized cornea (pDCSM-PMPC).
The single-sided modified zwitterionic polymer decellularized cornea (pDCSM-PMPC) physical diagram of the embodiment is shown in figure 1, and the result of figure 1 shows that the decellularized biological cornea of the invention can keep good light transmission performance before and after chemical modification.
Example 5
And (5) testing protein adhesion resistance.
The pDSM, pDSM-MA and pDCSM-PMPC prepared in example 3 were cut into disc-like samples with a diameter of 5mm, and immersed in PBS solution for equilibration for 20 minutes. Samples were placed in 48-well cell culture dishes, and 500. Mu.L of fluorescein isothiocyanate-labeled bovine serum albumin (FITC-BSA, 0.5 mg/mL) was added to each well. The 48-well cell culture dish was placed in a constant temperature shaker and incubated with shaking for 2 hours. The sample was removed, washed 3 times with PBS solution under dark conditions, and the FITC-BSA physically attached to the surface of the sample was removed, and then placed in new wells, each well being filled with 1mLPBS solution. The fluorescent emission of the sample was recorded by observation with an inverted fluorescent microscope and photographed.
The results are shown in fig. 3, and the results in fig. 3 show that after the surface of the inner surface of the decellularized biological cornea is modified by the zwitterionic polymer, the amount of fluorescent protein adhered to the surface is obviously reduced, and the surface has good protein adhesion resistance.
Example 6
And (5) testing protein adhesion resistance.
The pDSM, pDSM-MA and pDCSM-PMPC prepared in example 3 were cut into disc-like samples with a diameter of 5mm, and immersed in PBS solution for equilibration for 20 minutes. Samples were placed in 24-well cell culture dishes and 1mL of a 2mg/mL bovine serum albumin solution was added to each well. The 24-hole cell culture dish is placed in a constant temperature shaking table, and after shaking incubation for 4 hours, the sample is taken out. Samples were washed 3 times with PBS and placed in new wells. 1mL of sodium dodecyl sulfate solution (1% wt in PBS) was added to each well. Shaking vigorously for 1 hour, eluting the surface-adhered proteins. The BCA kit was used to quantitatively analyze the concentration of bovine serum albumin adhered to the surface of the sample.
As shown in FIG. 4, the results of FIG. 4 show that after the surface of the inner surface of the decellularized biological cornea of the invention is modified by the zwitterionic polymer, the protein adhered to the surface is reduced from 71.58+/-10.68 mug/cm 2 to 11.93+/-2.09 mug/cm 2, and the surface has good protein adhesion resistance.
Example 7
Hydrophilicity test of decellularized biological cornea.
After drying the pDCSM prepared and pDCSM-PMPC samples prepared in example 3, they were glued on cover slips with double-sided adhesive. The water contact angle of the sample surface was measured with an optical contact angle meter.
The results are shown in FIG. 5, and the results in FIG. 5 show that the water contact angle is reduced and the hydrophilicity is significantly improved after the zwitterionic polymer is modified on the inner surface of the decellularized biological cornea of the invention.
Example 8
The light transmittance and mechanical property of the decellularized cornea of the invention are tested.
The prepared pDSM, pDSM-MA and pDCSM-PMPC samples prepared in example 3 were placed on millimeter paper for photographing. Light transmittance was then measured in the visible range (380-800 nm) using an ultraviolet visible-near infrared spectrophotometer. The samples pDSM, pDSM-MA and pDCSM-PMPC prepared in example 3 were prepared as long strips 4cm wide, and tensile test was performed using a tensile machine until the samples broke, at a tensile rate of 2mm/min.
The light transmittance test results are shown in fig. 6, the mechanical property test results are shown in fig. 7, and the left graph in fig. 7 shows the tensile strength at break and the right graph shows the stress-strain curve. The results of FIG. 6 show that the transmittance of the acellular biological cornea modified zwitterionic polymer of the invention is still kept above 90% and the acellular biological cornea modified zwitterionic polymer has good light transmittance. FIG. 7 illustrates that the present invention, having a zwitterionic polymer modified on the inner surface of the decellularized biological cornea, has less difference in tensile strength at break than the decellularized corneal stroma and is able to withstand surgical thread suturing. pDCSM-PMPC has a higher modulus of elasticity than the acellular cornea stroma, probably due to the lower water content of the acellular cornea, and a denser network.
The embodiments of the present invention have been described in detail above, but the present invention is not limited to the described embodiments. It will be apparent to those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, and yet fall within the scope of the invention.
Claims (10)
1. A decellularized biological cornea, wherein a surface of one side of the cornea is chemically modified with a zwitterionic polymer, and wherein the zwitterionic polymer forms a polymer molecular brush on the surface of the cornea; the polymer molecular brush is prepared by reacting a zwitterionic monomer or a polymer with a zwitterionic group on the surface of the decellularized biological cornea;
the zwitterionic monomer includes at least one of methacryloyloxyethyl sulfobetaine, methacryloyloxyethyl carboxylic betaine, 2-methacryloyloxyethyl phosphorylcholine, trimethylammonium, and a sulfonate mixture.
2. A method of preparing a decellularized biological cornea according to claim 1, comprising the steps of:
Immersing the decellularized cornea matrix into an acrylic anhydride compound solution for reaction, washing after the reaction, covering one side surface by using a mask, immersing in a reaction solution containing zwitterionic groups to initiate grafting reaction on the other side surface of the decellularized cornea matrix, washing after the reaction, and immersing in deionized water to obtain the decellularized biological cornea;
the acrylic anhydride compound solution comprises an aqueous solution of methacrylic anhydride;
The reaction solution containing the zwitterionic groups contains a zwitterionic monomer or a polymer with the zwitterionic groups, and the reaction solution containing the zwitterionic groups also contains an initiator.
3. The method of claim 2, wherein the decellularized biological cornea stroma is prepared by a method comprising: taking an animal eyeball to be washed by using a washing liquid; peeling the obtained cornea slice by using a keratome; and (3) performing decellularization treatment on the cornea slice to obtain a decellularized cornea stroma.
4. A method of preparing a decellularized biological cornea according to claim 3, wherein said animal eyeball is derived from a mammal, said mammal comprising one of a human, pig, sheep, cow, dog.
5. The method for producing a decellularized biological cornea according to claim 2, wherein the concentration of the acrylic anhydride compound solution is 0.5 to 10 (v/v)%.
6. The method for preparing a decellularized biological cornea according to claim 5, wherein the pH of the acrylic anhydride compound solution is in the range of 5 to 9.
7. The method for producing a decellularized biological cornea according to claim 2, wherein the reaction solution containing a zwitterionic group contains a zwitterionic monomer or a polymer having a zwitterionic group; the concentration of the zwitterionic monomer is 0.1-1mol/L.
8. The method of claim 2, wherein the initiator is one of a redox initiator, a thermal initiator, and a photoinitiator; the redox initiator comprises one of dibenzoyl peroxide and N-N-dimethylaniline mixture, dibenzoyl peroxide and N-N-dimethyl-p-toluidine mixture, ammonium persulfate and sodium bisulfite mixture; the thermal initiator concentration comprises potassium persulfate and/or azo diisobutyl amidine hydrochloride; the photoinitiator comprises 2-hydroxy-4' - (2-hydroxyethoxy) -2-methylpropionacetone.
9. The method for producing a decellularized biological cornea according to claim 8, wherein when the initiator is a redox initiator, the concentration of the redox initiator in the reaction solution containing the zwitterionic group is 0.01 to 0.5mol/L; when the initiator is a thermal initiator, the concentration of the thermal initiator in the reaction solution containing the zwitterionic groups is 0.05-0.1mol/L; when the initiator is a photoinitiator, the concentration of the photoinitiator in the reaction solution containing the zwitterionic groups is 0.05-0.1 (v/v)%.
10. The method for preparing a decellularized biological cornea according to claim 9, wherein when the initiator is a redox initiator, the grafting reaction temperature is 10-30 ℃ and the reaction time is 0.5-24h; when the initiator is a thermal initiator, the grafting reaction temperature is 30-40 ℃ and the reaction time is 0.2-2h; when the initiator is a photoinitiator, the illumination wavelength is 290-450 nm, the illumination intensity is 2-10mW/cm 2, and the illumination time is 40-120s.
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