CN109125808A - A kind of biodegradable collagen-based cornea substitute and preparation method thereof - Google Patents
A kind of biodegradable collagen-based cornea substitute and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a kind of biodegradable cornea substitutes and preparation method thereof, it is to handle collagen and high molecular polymer by composite crosslinking mode in water phase, obtain after mold cured molding；Or by collagen and high molecular polymer use electrostatic spinning for nano fibrous membrane after, further progress composite crosslinking, it is dry obtain three-dimensional corneal stent, used after rehydration swelling when clinical.Cornea substitute degradability of the invention is controllable, has good phototropism and mechanical strength, and the growth of corneal epithelial cell and corneal corpuscles has significant inducing action.
Description
Technical field
The invention belongs to tissue engineering comea graft material technical fields, in particular it relates to a kind of collagen-based
Cornea substitute and preparation method thereof.
Background technique
The blind more than 10,000,000 people is caused by keratonosus in the world, for the second largest reason for leading to blind, is only second to
Cataract.Penetrating Keratoplasty, which is that keratonosus is main, is in harmony treatment means, but for severe dry eye, Stevens-
The prognosis of Johnson syndrome, severe chemical burn etc. is poor, and faces graft rejection, the problems such as donor is insufficient.Artificial cornea
Application and the research of tissue engineering comea be that blind patient brings hopes, currently used cornea constructs material and mainly makes a living
Object timbering material, high molecular material etc..
Biomaterial scaffolds are grown frequently as machinery mount sertoli cell, mitigate cell effect, and over time,
Gradually change their chemistry and mechanical property.Biocompatibility is its most important characteristic, and biomaterial is closer to group
Microenvironment is knitted, biocompatibility is better.Ideal tissue engineering comea material should meet the following conditions: (1) good biocompatibility,
Three-dimensional space is maintained, promotes normal cell growth and differentiation, does not interfere other physiology courses；(2) without bad tissue reaction；(3)
Material production is simple, and can remold by different shapes and sizes；(4) after implanting, material can be degraded or absorb.
Application No. is the Chinese invention patent applications of 201510250565.X to disclose a kind of artificial cornea and its preparation side
Animal cell-eliminating coanea matrix is first soaked in photosensitizer by method, is then crosslinked under ultraviolet light uniform.Invention narration
It can effectively improve the biomechanics characteristics such as the mechanical strength of animal cell-eliminating coanea matrix and its stability.But this method
There are crosslinking degrees it is inhomogenous, xenogenesis collagen immunological rejection risk is high the deficiencies of, may cause product quality problem.
Application No. is 201510453581.9 Chinese invention patent applications to disclose a kind of preparation of tissue engineering comea
Method, specially by the way of the timbering material of people's corneal limbal cells composite moving material resource cornea preparation, by bowman's lamina, base
Matter layer and descemet's membrane separation, the cell suspension of coating people's corneal limbal cells preparation, after completed by the modes such as being crosslinked, bonding
The building of celliferous tissue engineering comea, the tissue engineering comea of the method preparation have in use histocompatbility it is high,
Cell is not easy the advantages that breaking up, can preparing with industrialization.But this method does not consider to carry the storage life problem of cellular biological material,
It is difficult to form market-oriented cornea substitute, is unfavorable for marketing, and exogenous cells and animal source material mixed type product
Immunological rejection risk is higher, product implantation after provide light phototropism effect also it is difficult to ensure that.
Application No. is 200910068343.0 Chinese invention patent applications to disclose a kind of biologically active collagen
Based composite cornea substitute and preparation method thereof, mainly with collagen, 3- (Methacrylamide) propyl-dimethyl (3- sulphur the third)
Amine (MPDSAH) is raw material, takes water as a solvent, is crosslinked with carbodiimide (EDC) to collagen；It is light with IRGACURE 2959
Initiator, polyethyleneglycol diacrylate (PEGDA) are that crosslinking agent is carried out causing polymerization and be crosslinked to MPDSAH.It is uniformly mixed
Afterwards, curing molding in mold is injected.The composite cornea substitute of this method preparation can induce and promote cornea regeneration, can be with cornea
Regeneration and it is biodegradable, the introducing of growth factor can promote the enrichment of autologous growth factor, reduce the appearance of postoperative complications.
But polymer MPDSAH and photoinitiator used in this method are stimulation class reagent, and biocompatibility is poor, and the later period saves and adopts
It is highly-safe less as terminal sterilizations modes such as preceding damp and hot or irradiation with chloroformic solution.
All there is certain deficiency in artificial cornea disclosed in above-mentioned patent, cannot be considered in terms of biocompatibility, bio-safety
Validity after property and corneal transplantation, while portioned product is difficult to the marketization, does not have clinical value.
Summary of the invention
It is an object of the invention to the deficiency for existing product and technology of preparing, provide that a kind of quality is stable, biology peace
The preparation method of Quan Xinggao, biodegradable collagen-based cornea substitute and the cornea substitute.
For above-mentioned purpose, biodegradable collagen-based cornea substitute of the invention is prepared by the following method to obtain:
1, in mass ratio it is that 0.1~10:1 is mixed by collagen and high molecular polymer, obtains collagen-based blend.
2, collagen-based blend is added in deionized water, and crosslinking agent is added, be subsequently placed in mold, carry out compound friendship
Connection；Or after nano fibrous membrane is made by electrostatic spinning in collagen-based blend, immerse the ethanol solution or crosslinking agent of crosslinking agent
Aqueous solution in, carry out composite crosslinking.
3, crosslinking agent is removed after cross-linking reaction is complete, dry, sterilizing obtains cornea substitute.
In above-mentioned steps 1, the collagen is the recombined collagen that microbial fermentation technology obtains, molecular weight
For 30~300kDa；The high molecular polymer is that polyvinyl alcohol, polylactic acid, polyglycolic acid, poly lactic-co-glycolic acid are total
One of polymers is a variety of, preferably polyvinyl alcohol.
In above-mentioned steps 1, the mass ratio of further preferred collagen and high molecular polymer is 2~8:1.
In above-mentioned steps 2, the crosslinking agent is glutaraldehyde, carbodiimides, glutamine transaminage, epoxychloropropane
One of or it is a variety of, preferably the additive amount of crosslinking agent be collagen-based be blended amount of substance 0.1%~3%.
In above-mentioned steps 2, by collagen-based blend nano fibrous membrane made of electrostatic spinning with a thickness of 0.1~
0.2mm, wherein the diameter of nanofiber is 50~500nm.
In above-mentioned steps 3, the method for crosslinking agent is removed as any one in dialysis, PBS rinsing, ultrasonic cleaning.
In above-mentioned steps 3, the sterilizing is low temperature irradiation sterilizing, and temperature is -20~10 DEG C, irradiation dose is 10~
25kGy。
Compared with the existing technology, collagen-based cornea substitute and preparation method thereof of the invention has the following beneficial effects:
1, the collagen that the present invention uses is the recombination human source collagen obtained in the method for genetic engineering, is had same
Source property is high, and sample purity is high, non-immunogenicity risk, cell adhesion excellent.
2, the high molecular polymer that the present invention is blended with collagen, is the medical raw material of high-biocompatibility, and can drop safely
Solution, has no toxic side effect, while clinical required mechanical performance can be provided for corneal stroma.
3, the present invention uses composite crosslinking mode, it is ensured that is further reduced while cross-linking effect (improving mechanical strength)
Crosslinking agent usage amount, while can control residual crosslinker, final products cytotoxicity detection knot subsequently through technique is effectively removed
Fruit is shown as 0~1 grade of cytotoxicity, highly-safe, is suitble to the tight requirement of implantation material.
4, the present invention is irradiated using low temperature environment, can both guarantee sterile horizontal (SAL≤10-6) of product, prevented also from
Collagen and the damage of high molecular polymer molecular structure.
5, the mechanical strength of collagen-based cornea substitute of the present invention is good, and phototropism is good, and biodegradable.
Specific embodiment
Below with reference to embodiment, the present invention is described in more detail, but protection scope of the present invention is not limited only to these realities
Apply example.
Collagen in the following example is according to application No. is a kind of 201610388271.8, entitled " recombined humans
The series recombination that method disclosed in the application for a patent for invention of source collagen and its encoding gene and preparation method " prepares
Human-like collagen, and obtain by adjusting the molecular cut off of Hollow Fiber Ultrafiltration system the recombination human source of target molecular weight
Collagen, confirmation molecular weight ranges are 35~95kDa, and purity is about up to 99% or more.The recombination human source collagen is without exempting from
Epidemic focus, can be degradable in vivo, has excellent cell adhesion effect.
Embodiment 1
1, by 18g molecular weight be 60kDa recombination human source collagen and 2g number-average molecular weight be 170,000 polyvinyl alcohol
124 are uniformly mixed, and obtain collagen-based blend.
2, the collagen-based blend that step 1 obtains is settled to 100mL with deionized water, stirs evenly, is then added
Then gained mixed liquor is placed in mold by 0.02g carbodiimides, the cross-linking reaction 2h at 25 DEG C.
3, the sample after step 2 cross-linking reaction is placed in the bag filter of 1kDa retention and carries out crosslinking agent removal processing, often
6h replaces external environment ultrapure water, replaces 4 times altogether；The sample for removing residual crosslinker is placed in the sterile guarantor of phosphate under clean environment
It in liquid storage and encapsulates, in -10 DEG C of processing 10h, 25kGy Co60Irradiation sterilization obtains biodegradable collagen-based cornea substitution
Object.
Embodiment 2
1, by 8g molecular weight be 90kDa recombination human source collagen and 8g number-average molecular weight be 120,000 polyvinyl alcohol
124 are uniformly mixed, and obtain collagen-based blend.
2, the collagen-based blend that step 1 obtains is settled to 100mL with deionized water, stirs evenly, is then added
Then gained mixed liquor is placed in mold by 0.032g glutamine transaminage, the cross-linking reaction 48h at 4 DEG C；Products therefrom is used
PBS cleans 30min, is then added in 100mL deionized water, stirs evenly, and 0.016g glutaraldehyde is added, and is crosslinked at 25 DEG C anti-
Answer 2h.
3, the sample ultrasonic cleaning after step 2 cross-linking reaction is removed into crosslinking agent, then hands over removal under clean environment
The connection remaining sample of agent is placed in phosphate Preservation in sterile condition liquid and encapsulates, in -10 DEG C of processing 10h, 25kGy Co60Irradiation sterilization,
Obtain biodegradable collagen-based cornea substitute.
Embodiment 3
1, the poly- cream for being 40,000 with 1g number-average molecular weight by the recombination human source collagen freeze-dried powder that 8g molecular weight is 38kDa
Acid, the polyglycolic acid that 1g number-average molecular weight is 20,000 are uniformly mixed, and obtain collagen-based blend.
2, above-mentioned collagen-based blend is subjected to rack forming using electrostatic spinning technique, obtains the netted branch of nanoscale three-dimensional
Timbering material is cut to required size by frame, and is immersed in 100mL and is contained in the ethanol solution of 0.3g carbodiimides, 25
Cross-linking reaction 9h at DEG C；Products therefrom rinses 30min with PBS, then is immersed in the water that 100mL contains 0.2g glutamine transaminage
In solution, the cross-linking reaction 16h at 4 DEG C.
3, the corneal stent by step 2 cross-linking reaction after complete cleans the remaining crosslinking agent of removing with PBS repeatedly, freeze-dried
It after processing, is packaged in medical aluminium foil bag, seals, in -10 DEG C of freezing processing 2h, 15kGy Co60Irradiation sterilization obtains collagen-based
Corneal stent.It is used after rehydration swelling when clinical.
In order to prove beneficial effects of the present invention, the collagen-based cornea substitute that inventor obtains embodiment 1 is carried out
Performance test, specific test are as follows:
1, degradability is tested
The collagen-based cornea substitute 0.05g for weighing the preparation of embodiment 1 respectively, is added 200 μ of collagenase type I of 200U/mL
L, acted on respectively in 37 DEG C of water-baths 1h, 2h, 4h, 8h, 16h, for 24 hours, 48h, calculate the degradation rate of different time, the results are shown in Table
1。
Table 1
Regeneration and Repair is the emphasis direction of Tissue Engineering Study, and material degradation simultaneously is organized itself gradually to substitute, repair to be
The purpose of tissue reconstruction, therefore degradability is one of tissue engineering material basic demand, according to the testing result in table 1, originally
Invention collagen-based cornea substitute external degradation is gentle, controllable, meets the technical requirements of In vivo study.
2, mechanical strength is tested
Literature Consult people's cornea mechanical strength related data, and collagen-based cornea substitute prepared by embodiment 1 is carried out
Tensile strength and elasticity modulus detection, comparing result are as shown in table 2.
Table 2
| Tensile strength MPa | Elasticity modulus MPa | |
| Collagen-based cornea substitute | 2.99±0.7 | 4.3±0.5 |
| Natural human cornea | 3.81±0.4 | 3-13 |
As seen from the results in Table 2, collagen-based cornea substitute prepared by the present invention has and natural human in terms of tensile strength
The identical order of magnitude of cornea, and be closer to, it can meet and operate requirement in clinical operation, while elasticity modulus is in natural cornea model
In enclosing, quality is stablized.
3, translucency
Light transmittance inspection is carried out to collagen-based cornea substitute prepared by embodiment 1 using the spectrophotometer with recorder
It surveys, and is compared with natural human cornea light transmittance reported in the literature, the results are shown in Table 3.
Table 3
| Collagen-based cornea substitute | Natural human cornea (document) | |
| Light transmittance % | 91.6 ± 2.6% | > 87% |
By the comparing result in above-mentioned table 3 it is found that collagen-based cornea substitute prepared by the present invention has good light transmission
Rate, it is possible to provide later period preferable vision restoration effect.
4, efficacy is tested
Collagen-based cornea substitute prepared by Example 1 carries out efficacy research, chooses conventional corneal injury mould
Type --- new zealand rabbit Corneal Alkali Burns model, wherein left eye is control eye, and right eye is test eye, carries out cornea substitution to right eye
The transplanting of object plate layer, is observed, and compare with control group January, March, June after suture, the results are shown in Table 4.
Table 4
| Control group | Experimental group | |
| January | Damage is not healed | Transplantation site gradually heals, and epithelial cell is climbed attached |
| March | Wound is further aggravated | Cornea is more transparent clear, and epithelial cell is complete |
| June | Wound becomes larger, and opaque scar is formed at the healing of part | Edge of cornea heals completely, and capilaryization is surround, intermediate transparent |
By upper table 4 it is found that after the progress collagen-based cornea substitute transplanting of new zealand rabbit right eye, without obvious inflammatory reaction, nothing
It is implanted into adverse reaction, and biocompatibility is good, material is gradually degraded, and synkaingenesis tissue gradually substitutes, the suction of endothelial cell
Water effect also makes cornea that good translucency be presented.
Claims (10)
1. a kind of preparation method of biodegradable collagen-based cornea substitute, it is characterised in that:
(1) in mass ratio it is that 0.1~10:1 is mixed by collagen and high molecular polymer, obtains collagen-based blend；
(2) collagen-based blend is added in deionized water, and crosslinking agent is added, be subsequently placed in mold, carry out composite crosslinking；
Or after nano fibrous membrane is made by electrostatic spinning in collagen-based blend, immerse the ethanol solution or crosslinking agent of crosslinking agent
Aqueous solution in, carry out composite crosslinking；
(3) crosslinking agent is removed after cross-linking reaction is complete, dry, sterilizing obtains cornea substitute；
Above-mentioned collagen is the recombined collagen that microbial fermentation technology obtains；Above-mentioned high molecular polymer is poly- second
One of enol, polylactic acid, polyglycolic acid, poly lactide-glycolide acid are a variety of.
2. the preparation method of cornea substitute according to claim 1, it is characterised in that: the collagen and macromolecule
The mass ratio of polymer is 2~8:1.
3. the preparation method of cornea substitute according to claim 1 or 2, it is characterised in that: point of the collagen
Son amount is 30~300kDa.
4. the preparation method of cornea substitute according to claim 1 or 2, it is characterised in that: the high molecular polymer
For polyvinyl alcohol.
5. the preparation method of cornea substitute according to claim 1, it is characterised in that: the crosslinking agent is penta 2
One of aldehyde, carbodiimides, glutamine transaminage, epoxychloropropane are a variety of.
6. the preparation method of cornea substitute according to claim 5, it is characterised in that: the additive amount of the crosslinking agent is
The 0.1%~3% of amount of substance is blended in collagen-based.
7. the preparation method of cornea substitute described according to claim 1 or 5 or 6, it is characterised in that: the nano fibrous membrane
Film with a thickness of 0.1~0.2mm, wherein the diameter of nanofiber is 50~500nm.
8. the preparation method of cornea substitute according to claim 1, it is characterised in that: the method for removing crosslinking agent
For any one in dialysis, PBS rinsing, ultrasonic cleaning.
9. the preparation method of cornea substitute according to claim 1, it is characterised in that: the sterilizing is low temperature irradiation
Sterilizing, temperature is -20~10 DEG C, and irradiation dose is 10~25kGy.
10. the biodegradable collagen-based cornea substitute that method of claim 1 is prepared.
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111234104A (en) * | 2020-03-26 | 2020-06-05 | 四川大学 | Water-soluble cross-linking agent, preparation method thereof and prepared electro-spinning collagen fiber |
| CN112295021A (en) * | 2020-10-19 | 2021-02-02 | 四川大学 | Skin graft with improved topological structure |
| CN112494722A (en) * | 2020-12-15 | 2021-03-16 | 中新国际联合研究院 | Collagen-based cornea regeneration repair material capable of realizing rapid epithelialization and preparation method thereof |
| CN113773380A (en) * | 2021-09-28 | 2021-12-10 | 山东汉肽医美生物科技有限公司 | Recombinant human collagen, encoding gene and application thereof in preparation of biodegradable collagen-based cornea substitute |
| CN113926001A (en) * | 2021-09-13 | 2022-01-14 | 熹微(苏州)生物医药科技有限公司 | Bionic cornea and preparation method thereof |
| EP4023266A1 (en) * | 2020-12-31 | 2022-07-06 | Industrial Technology Research Institute | Non-fibrous film and cell sheet |
| CN115785256A (en) * | 2022-11-09 | 2023-03-14 | 武汉轻工大学 | Method for regulating and controlling binding capacity of collagen and cell receptor |
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