CN107050515A - A kind of corneal stroma, preparation method and application - Google Patents
A kind of corneal stroma, preparation method and application Download PDFInfo
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- A61L27/3687—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment characterised by the use of chemical agents in the treatment, e.g. specific enzymes, detergents, capping agents, crosslinkers, anticalcification agents
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- A61L27/3691—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment characterised by physical conditions of the treatment, e.g. applying a compressive force to the composition, pressure cycles, ultrasonic/sonication or microwave treatment, lyophilisation
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Abstract
The present invention provides a kind of corneal stroma, preparation method and application, the cornea of animal is used for raw material, corneal epithelium, the cell component in corneal stroma and DNA compositions are removed, retains bowman's lamina and segment thickness corneal stroma, retains complete extracellular matrix components；The corneal stroma is applied to the covering of pathological corneas, the invalid not yet perforation ulcer of the cornea of the medication that is particularly suitable for use in, needs the infected keratitis patient of flaggy transplantation treatment, and perforation of cornea provisional covering.
Description
Technical field
The present invention relates to medical biomaterial technical field, and in particular to a kind of corneal stroma, preparation method and application.
Background technology
Cornea is located at eyeball foremost, is directly contacted with external environment, subject to damage and infection, if do not defended with eye
It is raw, it is easy to cause corneal inflammation even muddy, cause hypopsia or even blindness.According to WHO Report, keratonosus
It is the second Etiological for causing visual loss, is only second to cataract.At present, unique effective means for the treatment of corneal blindness is exactly
Corneal transplantation, the cornea used in corneal transplantation mainly has three sources：Homologous allogeneic cornea donation, artificial cornea and organizational project
Cornea.The cornea limited source of donation, corneal donor critical shortage, it is impossible to meet the need of the blind patient's treatment of existing numerous keratonosuses
Will, it significantly limit corneal blindness patient and cast off illiteracy, this is also countries in the world urgent problem to be solved.The material of artificial cornea has firmly
Property artificial cornea material, silica gel material, PHEMA, PMMA material etc., although these materials have certain effect to alleviating keratonosus
Really, but all can not and patient tissue form good combination, some materials result even in eye's necrosis, produce
Complication.
Based on being understood in depth to artificial cornea synthetic material and cell-cell interaction, scientists start thin to live body
Extracellular matrix builds people's cornea equivalent and studied, it is desirable to which acquisition is similar to natural cornea structure, possess similar physicochemical performance
Organizational project organ, available for clinical treatment corneal blindness disease.Research of the mankind to tissue engineering comea, has carried out various taste
Examination, cultivates ox horn theca cell such as on collagen, successfully repairs Ocular surface damage, profit using amnion culture autologous corneal limbus epithelial cell
Ocular is repaired with the sensitive autologous oral mucosa cell of culture medium in vitro culture of temperature, utilize corneal limbal stem cell autograft in vitro culture
Corneal epithelial cell diaphragm etc., these researchs have established theoretical foundation for the research of tissue engineering comea, but can not still meet
Clinical practice requirement, such as collagen poor toughness, it is impossible to resist intraocular pressure effect and the mechanical stretch effect of suture, people's corneal
Wound healing mechanism still needs to further investigation.By years of researches, it was found that a kind of preferable tissue engineering comea material, it is immunized
Original removes porcine cornea host material, and it has very much like structure with people's corneal stroma, and with immunogenicity is low, source
Abundant the characteristics of.Heteroplasm's main component that immunogene is removed is collagen, elastic fibers etc., and form is smaller with change in toughness.
Immunogene, which is removed, can reduce inflammatory reaction and the immunological rejection of heteroplasm, the good biocompatibility with human body, in this
The growth factor in matrix can induce people's corneal limbal cells to be divided into corresponding keratocyte simultaneously, and immunogene removes pig angle
Membrane matrix material has turned into the only selection for the treatment of corneal blindness disease.
For the research of tissue engineering comea, China is walked in the forward position in the world.Shenzhen Ainier Cornea Engineering Co., Ltd.
The bioengineering cornea of global first listing is proposed on April 22nd, 2015, the cornea product has drawn from pig cornea, warp
Inactivation of virus and immunogene the technique such as remove and are prepared from, and are the extracellular matrix of porcine cornea, the main component of extracellular matrix
For collagen.This product has the collagen fiber structure of natural cornea, remains the bowman's lamina and part base of natural cornea
Matter layer.The patent of holding Guangzhou You get Qing bio tech ltd of Guangdong Guan Hao Biological Science ＆ Technology Co., Ltd, patent
Number CN201310527550.4, discloses a kind of preparation method of corneal material, using animal corneal as raw material, prepares the transparency
The heterogenic cornea material of high and low immunogenicity, bioactivity and good biocompatibility, and keep collagen three-dimensional structure and fresh angle
Film is close, and further reduces by collagen cross-linking the anti-degradability and immunogenicity of collagen.
The content of the invention
The present invention provides a kind of cornea using animal as raw material, is removed using multigelation method and enzyme solutions immunogene
Method, prepares that tensile strength is big, the corneal stroma of non-immunogenicity, good biocompatibility.
In order to solve the above-mentioned technical problem, the technical solution adopted in the present invention is：A kind of corneal stroma, using animal
Cornea is raw material, removes corneal epithelium, the cell component in corneal stroma and DNA compositions, retains bowman's lamina and part
Thickness corneal stroma, retains complete extracellular matrix components and (detects its mechanical property, its chemical property, biological property and drop
Solve performance).
The animal is mammal, preferably pig or ox.
The segment thickness corneal stroma is to be connected with bowman's lamina with certain thickness corneal stroma.
The extracellular matrix components are collagen, glycosaminoglycan.
The collagen is I-type collagen and IV collagen types, and I-type collagen and IV collagen types contain
Measure as 85-90%.
The glycosaminoglycan is chondroitin sulfate and keratan sulfate.
The above-mentioned corneal stroma of the present invention, it is diameter 7-10cm, thickness 0.1-0.2mm disk.
The removal corneal epithelium, the cell component in corneal stroma and DNA compositions are residual with cell residue amount, DNA
Allowance, galactosidase clearance rate evaluate, i.e., biological property be cell residue amount, DNA residual quantities, the sweet enzyme of galactolipin (α-
Gal) clearance rate.
The above-mentioned corneal stroma of the present invention, the cell residue amount is 0, and the DNA residual quantities are less than 10ng/mg, institute
It is more than 99% to state the sweet enzyme of galactolipin (α-Gal) clearance rate.
The present invention also provides a kind of preparation method of above-mentioned corneal stroma, it is characterised in that：It is swelled and is immunized by freeze thawing
The method that original is removed prepares the corneal stroma.
Specifically, the preparation method of the above-mentioned corneal stroma of the present invention, step includes：
(1) Feedstock treating：The eyeball of animal is taken, cornea (for example being drilled through using trepan) is taken, purified water is soaked in after cleaning
In；
(2) freeze thawing is swelled：Cornea is soaked in water, and then freezes；Take out, rinsed after after complete thaw with purified water, then
Immersion；Repeat the freeze-thaw process repeatedly, obtain the cornea of water swelling；
(3) inactivation of virus：Inactivation of virus is carried out using Peracetic acid-alcohol solution dipping cornea；
(4) cleaning process：Cornea is handled in Vltrasonic device with PBS solution and purified water respectively；
(5) immunogene is removed：It is the PBS solution containing trypsase and EDTA that immunogene, which removes liquid, and immunogene was removed
Journey is carried out in multiple frequency ultrasonic device, and wherein multiple frequency ultrasonic device can provide at least two supersonic frequencies；
(6) cleaning process：Cornea is handled in Vltrasonic device with PBS solution and purified water respectively；
(7) cornea repeatedly, is then immersed in purified water by repeat step (5) and (6)；
(8) cutting materials：Cornea lamellar blade is used under anatomical lens, lamina elastica corneae anterior and fraction matrix is cut, then
It is soaked in purified water；
(9) drying is dried：Cornea is open and flat in support surface, and placement is dried in an oven.
In Peracetic acid-ethanol solution described in step (3) concentration (percent by volume) of Peracetic acid be 0.1-5%,
The concentration (percent by volume) of ethanol is 5-40%, and the ratio (volume ratio) of Peracetic acid-ethanol solution and cornea is (30-
60) ︰ 1, inactivation time 2-4 hours, temperature range is 10-40 DEG C.
Cleaning fluid is pH6-8 PBS solution in the step (4), and PBS solution temperature is 10-40 DEG C, PBS solution and angle
The ratio (volume ratio) of film is (30-60) ︰ 1 are cleaned 2-4 times, each 10-30min, then using 10-40 DEG C of water for injection
I.e. purified water is cleaned, and purified water and cornea ratio (volume ratio) are (30-60) ︰ 1 extremely detect electrical conductivity for below 10 μ S/cm eventually
Only；Cleaning process is carried out in supersonic wave cleaning machine.
Immunogen in the step (5), which removes liquid, to be included：It is dissolved with PBS of trypsase and the EDTA pH value for 6-8
Solution；It is 0.01-0.2%, EDTA concentration 0.1- that the immunogen, which removes trypsase mass percent concentration in liquid,
1mmol/L；Further：The mass percent concentration that immunogen removes trypsase in liquid is 0.02-0.05%, EDTA's
Concentration is 0.4-0.8mmol/L, and it is 7.0-8.0, preferably 7.2-7.5 that immunogen, which removes liquid pH value,；The immunogen removes liquid
It is (30-60) ︰ 1 with cornea volume ratio；Multiple frequency ultrasonic at least includes two supersonic frequencies, and Frequency scope is 20-40KHz,
Higher frequency is 60-90KHz, wherein low frequency processing 5-40min, and high-frequency therapeutic treatment 5-40min, temperature range is 20-35 DEG C.
Cleaning fluid is pH6-8 PBS solution in the step (6), and PBS solution temperature is 10-40 DEG C, PBS solution and angle
The ratio (volume ratio) of film is (30-60) ︰ 1 are cleaned 2-4 times, each 10-30min, then using 10-40 DEG C of water for injection
Cleaning, water for injection and cornea ratio (volume ratio) are (30-60) ︰ 1, and detection cleaning water for injection is not with cleaning injection water power
The difference of conductance is terminated less than 1 μ S/cm.
Step (9) drying is dried to be carried out in heat-circulation oven：First baking oven is preheated to after 25-40 DEG C, then will
Material obtained by step (8) is put in baking oven and dried, and is air-dried moisture by thermal cycle wind, time 12-24 hour.
Above-mentioned preparation method also includes step (10) packaging and step (11) sterilizes and parsed, and packaging step is specially：Drying
Material is defended strong wrapping paper using spy and encapsulated with vacuum formed box；Sterilizing analyzing step be specially：First 20-40 DEG C of soaking time 2-4 of temperature
Hour, humidity 30-70% then passes to concentration 300-1000mg/L oxirane, sterilizes 4-8 hours；Then parse, parse
Journey is carried out in the Resolution Room of ventilation, and temperature control is between 10-30 DEG C, time 14-28d.
The present invention further provides application of the above-mentioned corneal stroma in ophthalmic implant, the ophthalmic implant is applied to disease
Become cornea covering, the medication that is particularly suitable for use in it is invalid not yet perforation ulcer of the cornea, need the infectious angle of flaggy transplantation treatment
Film inflammation patient, and perforation of cornea provisional covering.First by corneal trephine and cornea lamellar blade by patient's pathological corneas
Position excision is clean, and cutting depth meets injured depth, retains patient part's corneal stroma and descemet's membrane, thoroughly removes the surface of a wound
Foreign matter, it is to avoid burn hemostasis, normal saline flushing is clean.Then the outer packing of the present invention is opened, packing inside bag is taken out and cuts off,
Inner packaging box is taken out under aseptic condition, sterile saline is injected into inner packaging box, makes product rehydration 10-15min of the present invention,
Again by the convex surface (bowman's lamina face) of product of the present invention upwards, smooth be placed on the surface of a wound sutures fixation.Operation method, medication and art
Nursing is identical with conventional flaggy corneal graft afterwards, postoperative usable corneal epithelium protection medicine.
Compared with prior art, the present invention has following remarkable advantage and beneficial effect：
(1) by cryogenic freezing, the cell in cornea can be cracked in the presence of ice crystal to be crushed, simultaneously, due to water
Ice is changed into, volumetric expansion can increase corneal stroma flaggy spacing, during defrosting cornea, ice changes into water, volume reduces, can be from
Outside absorbs moisture, so that realize that cornea is swelled, thickness increase；
(2 are swelled increase corneal stroma interlamellar spacing by freeze thawing, are more beneficial for immunogene removal, are conducive to inhereditary material
Cleaning；
(3) trypsase and EDTA are used, the connection between cell and extracellular matrix is destroyed；Using low frequency ultrasound
Cell is crushed, while acting on broken cell and extracellular matrix using high frequency ultrasound, further makes cell detachment
Extracellular matrix, reaches de- cell purpose.Using aforesaid way, each step during whole cell detachment matrix is carried out
Reinforcing, makes cell be completely disengaged from from matrix.Reach optimal immunogene removal effect；Enzyme process cell elution processes：Using
Trypsase and EDTA composite solutions, the process for removing cell are gentle, reduce the destruction to matrix structure, retain the work in matrix
Property growth factor；
(4) high concentration immunogene removes solution and replaced with pure water, using the diffusion of solution high-concentration and low-concentration, in cornea base
Scutum interlayer formation scouring effect, improves inhereditary material cleaning efficiency；By air-dried cornea, corneal stroma flaggy can be reduced
Spacing, can eliminate the influence of cornea microstructure of corneal flaggy in frozen-thaw process, reduce corneal thickness, make matrix group
Knit more closely knit, improve the tensile strength of cornea；
(5) cornea after air-drying is planar transparent sheet film, it is to avoid cornea air-dries produced under free condition
Fold and contraction, cornea made from such a method, with certain internal stress, can quickly absorb water during rehydration
Point, it is rapid to recover elasticity；
(6) corneal stroma of the present invention can be used for lamellar keratoplasty.
Brief description of the drawings
Shown in Fig. 1 is the front SEM photograph of corneal stroma bowman's lamina of the embodiment of the present invention；
Shown in Fig. 2 is the front SEM photograph that corneal stroma of the embodiment of the present invention removes bowman's lamina；
Shown in Fig. 3 is the side SEM photograph of corneal stroma of the embodiment of the present invention；
Shown in Fig. 4 is the bowman's lamina and side SEM photograph of corneal stroma of the embodiment of the present invention；
Shown in Fig. 5 is the structural representation of corneal stroma of embodiment of the present invention cross section.
Embodiment
The present invention is further described in detail with reference to embodiments, but is not limited to this.
Pig cornea or buphthalmos cornea of the present invention are applied to embodiments of the invention.
Embodiment 1：
A kind of preparation method of corneal stroma of the present embodiment, including following operating procedure：
(1) Feedstock treating：
The eyeball of pig or ox within dead 4 hours is taken, is rinsed well watery blood with physiological saline, diameter 8.5mm rings are used
Brill drills through cornea, and purified water rinses rear purified water immersion well.
(2) freeze thawing is swelled：
Cornea is soaked with a small amount of water, is placed in -40 DEG C of refrigerator-freezers and is freezed 1 hour, is taken out and is used purified water after complete thaw
15min is rinsed, then sufficient purified water immersion 30min, repeats the freeze-thaw process 3 times, obtains the cornea of water swelling.
(3) inactivation of virus：
Inactivation of virus is carried out using Peracetic acid-alcohol solution dipping cornea, the process can be carried out in stainless steel cask.Cross
The concentration (percent by volume) of Peracetic acid is that the 1%, concentration (percent by volume) of ethanol is in fluoroacetic acid-ethanol solution
24%, the ratio (volume ratio) of Peracetic acid-ethanol solution and cornea is 40 ︰ 1, and inactivation time 2 hours, temperature is 20 DEG C.
(4) cleaning process：
Cornea is cleaned using cleaning fluid, cleaning fluid is pH7 PBS solution, and PBS solution temperature is 20 DEG C, PBS solution and angle
The ratio (volume ratio) of film is 40 ︰ 1, is cleaned 3 times, each 20min, is then cleaned using purified water, purified water and cornea ratio
(volume ratio) is 40 ︰ 1, is terminated to detection electrical conductivity for below 10 μ S/cm；Cleaning process is carried out in supersonic wave cleaning machine.
(5) immunogene is removed：
It is to be 2% trypsase comprising mass percent concentration and mass percent concentration is 0.3% that immunogene, which removes liquid,
EDTA pH value be 7 PBS solution, it is 40 ︰ 1 that immunogene, which removes liquid and cornea mixed proportion (volume ratio), and immunogene is removed
Process is carried out in supersonic wave cleaning machine, and multiple frequency ultrasonic includes at least two supersonic frequencies, and Frequency is 30KHz, high again and again
Rate is 70KHz, wherein low frequency processing 8min, and high-frequency therapeutic treatment 12min, temperature is 20 DEG C.
(6) cleaning process：
Cleaned using cleaning fluid, cleaning process is carried out in supersonic wave cleaning machine, the power of ultrasonic wave is 3000W
Below.Cleaning fluid is pH=7 PBS solution, and PBS solution temperature is 20 DEG C, and the ratio (volume ratio) of PBS solution and cornea is
40 ︰ 1, are cleaned 3 times, each 20min, are then cleaned using 20 DEG C of water for injection, and water for injection is 40 ︰ 1 with cornea volume ratio,
Detection cleans water for injection and does not clean the difference of water for injection electrical conductivity less than 1 μ s/cm terminations.
(7) repeat step (5), (6) 6 times, the outer solution concentration of cornea is changed repeatedly, solution infiltration direction change is formed
Scouring effect, the inhereditary material cleaned repeatedly in cornea, until inhereditary material on cornea is removed totally, then soaks cornea
In injection water.
(8) cutting materials：
Cornea lamellar blade is used under anatomical lens, lamina elastica corneae anterior and fraction matrix is cut, thickness is 2mm, then will
It is soaked in injection water.
(9) drying is dried：
Carried out in hundred grades of baking ovens of thermal cycle, baking oven is preheated to after 37 DEG C, step (8) resulting materials are put in into baking oven does
It is dry, moisture is air-dried by thermal cycle wind, air-dry time is 24 hours., need to be open and flat in stainless steel support by cornea in air drying process
Panel surface, makes it to keep flattened state after air-drying, irregular status otherwise can be shrunk in air drying process, or even pleat occur
Wrinkle deformation, influences product quality.
The preparation method of present embodiment can also include：
(10) pack：
Drying material is defended strong wrapping paper using spy and encapsulated with vacuum formed box.
(11) sterilizing parsing：
Sterilized using oxirane, sterilising conditions are：First 40 DEG C of temperature is incubated 4 hours, and humidity 70% is then passed to
Concentration 500mg/L oxirane, sterilizes 6 hours；Resolving is carried out in the Resolution Room of ventilation, temperature control 20 DEG C it
Between, 14 days time.
The sample cornea finally obtained, the structural representation of cross section as shown in Figure 5, includes hypothallus and the position of lower floor
Bowman's lamina in top；The cornea of the present invention can be by being positioned over the water treatment plants such as stainless steel mesh by the cornea after immersion
On, drainage is measured after more than 5 minutes with weighing utensil.
Embodiment 2：
Sample in embodiment 1 is observed by SEM, as shown in accompanying drawing 1 and 4, it is observed that through
Cross after immunogene removal processing, the bowman's lamina that immunogene removes cornea is intact, is not influenceed and destruction, has by enzyme solutions
Dividing a word with a hyphen at the end of a line, stick and breeding beneficial to corneal epithelial cell, the epithelialization for being conducive to cornea quick, complete, it is to avoid infection.Epithelium energy
Normal growth is covered in corneal stromal surface, it is possible to effectively completely cut off degraded of the various hydrolases to its collagen component, extension drop
The solution time, beneficial to the reconstruction of corneal stroma.
Remove after bowman's lamina as shown in Figures 2 and 3, it can be seen that three-dimensional net structure, three dimensional network are presented inside cornea
Network structure formation biological support, is conducive to growing into for patient's keratocyte, adds the attachment quantity of cell, be conducive to nutrients
The exchange of matter, the reconstruction of corneal stroma is removed beneficial to immunogene.
It can see from the side SEM electromicroscopic photographs of accompanying drawing 3 and 4, the laminar films structural integrity of bowman's lamina, corneal stroma
Inside is in layer structure, it is seen that collagen plate bedding void, and pore size is 20-150 μm.
Embodiment 3：
Physical property detection is carried out to sample obtained by preparation method of the present invention, it is specific as follows：
A. tensile strength is sutured：5 samples are prepared according to embodiment 1, with 3-0 non-absorbing suture on patch one end side
At 2 millimeters of edge, the other end of suture and patch is fixed on tensiometer, stretched with 20mm/min speed, directly
It is torn to stitch points, records maximal force, as a result show, maximum average value is up to 3.2N.
B. light transmittance：5 samples are prepared according to embodiment 1, air-dry sample, swollen sample, dehydrating glycerin sample are surveyed respectively
Light transmittance.It is prepared by air-dry sample：By inwall of the sample being swelled directly against cuvette printing opacity side, convection oven apoplexy is sent into
It is dry 24 hours；It is prepared by swollen sample：By inwall of the sample being swelled directly against cuvette printing opacity side；Dehydrating glycerin sample：Will
The sample being swelled is placed in glycerine and is dehydrated 12-48 hours, then takes out sample, cleans out the glycerine of sample surfaces, by sample
Directly against the inwall of cuvette printing opacity side.During test, by sample side be put in the right, respectively survey 800nm, 700nm, 600nm,
500nm, 400nm, 300nm light transmittance, take reading, and calculate average value.Air-dry sample light transmittance average value is 86.6%, molten
Swollen sample transmittance average value is 39.05%, and dehydrating glycerin sample transmittance average value is 80.0%；
C. expansion rate：5 samples are prepared according to embodiment 1, cornea are laid on flat board, upper lid slide is placed in 40
Air-dried 24-48 hour in DEG C temperature air dry oven, taking-up sample cuts into regular cuboid with scalpel, uses slide measure
The length, width and height of sample are measured, are taken reading, then sample is immersed in 24 hours water absorption and swellings in water, is measured and is swelled with slide measure
Length, width and height afterwards, take reading, and calculate be swelled front and rear volume V1, V2 respectively, calculate expansion rate and are：Result of calculation average value is 88.5%；
D. moisture content：Sample is prepared according to embodiment 1, cornea is laid on flat board, upper lid slide is placed in 40 DEG C of temperature
Spend in air dry oven and air-dry 24 hours, take out sample, claim dry weight W1.Sample is immersed in 24 hours water absorption and swellings in water again,
Claim weight in wet base W2, calculate moisture content：Result of calculation average value is 95.9%.
E. degradation property：Each a diameter of 8.5mm, the corneal stroma sample that thickness is 0.15mm, use 1ml mass percents
Soaked for 0.03% Proteinase K Solution, 56 DEG C of water-baths are weighed every 10min corneals, calculate cornea residual mass.
Test result is shown in table 1：
The embodiment sample degradation property of table 1
Embodiment 4：
Performance detection is carried out to sample obtained by preparation method of the present invention, it is specific as follows：
A. acid-base value：Sample is prepared according to embodiment 1, is entered according to method as defined in 5.4.1 in GB/T 14233.1-2008
OK, the difference of the pH value of sample survey liquid and blank control liquid should be no more than 1；
B. content of beary metal：Sample is prepared according to embodiment 1, lead, chromium are provided by 5.9.1 in GB/T 14233.1-2008
Atomic absorption spectrophotometer method experiment, mercury, the arsenic atomic fluorescence spectrophotometry as defined in 5.9.3 in GB/T 14233.1-2008
Method is tested, and testing result is shown, content of beary metal is less than 0.1 μ g/g.
C. residue on ignition：Sample is prepared according to embodiment 1, according to《Pharmacopoeia of People's Republic of China》(version four in 2015)
Method as defined in 0841 is determined, and residue on ignition is 1.0%.
D. bacterial endotoxin is determined：Sample is prepared according to embodiment 1, the method as defined in GB/T 14233.2-2005 is entered
Row operation, as a result shows that bacterial endotoxin is less than 2EU/g.
E. cell residue inspection：3 products are taken to carry out HE dyeing respectively, 3 visuals field are selected in each section, in 400 times of optics
Micro- Microscopic observation intact cell quantity divided by 3, as a result shows, average each visual field intact cell quantity is 0.
F.DNA residual quantities：Prepare sample according to embodiment 1, according to biological agent residual DNA detection method (《Middle traditional Chinese medicines
Allusion quotation》2010, annex IX-B Residual exogenous DNA amounts determination method), the sample that embodiment 1 is provided is detected using fluorescence colour
DNA residual quantities.As a result show, the DNA residual quantities in sample are less than 4.56 ± 0.01ng/mg.
G. galactosidase (α-Gal) clearance rate：Take animal derived biomaterial Gal positive reference product, Gal antigen negatives
Each 2mg of reference material, plus lysate 1mL, crack 30-90min, are configured to 20,10,5,2.5,1.25,0.625 μ g Gal standards
Curve sample, the test article before and after test immunogene is removed respectively takes 50mg, plus lysate 2mL, cracks 30-90min；Take cracking
Liquid and the supernatant after M86 antibody responses, are added 96 orifice plates, plus secondary antibody, plus developer, are detected and inhaled using ELISA method 450nm
Shading value, is calculated the Gal values of sample by standard curve, and the Gal values that immunogene removes before processing material are 25.69 ± 2.72 ×
1014The Gal values of sample are 0.12 ± 0.01 × 10 in/mg, embodiment 114/ mg, galactosidase (α-Gal) clearance rate exists
More than 99.52%.
H. collagen neuraminidase：I types and IV collagen types are detected using Immunohistochemical Staining, 3 μ m-thicks are continuous
Section, dimethylbenzene dewaxing, graded ethanol dehydration.Section is moved into electric cooker water-bath and (includes 0.01mol/L, pH6.0 Chinese holly
Rafter acid trisodium buffer solution), temperature is maintained at 95-100 DEG C, boils 20min, carries out antigen retrieval, naturally cold at room temperature after taking-up
But.Phosphate buffer (PBS) is washed, 5min × 3 time.Two step method SABC：I types and IV collagen types are added dropwise respectively
Monoclonal antibody primary antibody, concentration 1：100,4 DEG C of refrigerator overnights are incubated 60min at room temperature, and PBS is washed 3 times.Envision is added dropwise
Reaction solution, is incubated 30min at room temperature.PBS is washed 3 times.The 3 of 0.05%, the H2O2 colour developings of 3 one diaminobenzidines+0.03%
5-10min.Flowing water is washed, haematine lining dye.Incremental gradient ethanol dehydration, dimethylbenzene is transparent, usual resins sealing.As a result show,
The micro- all visible pale brown dyeing of four kinds of stained preparations of Microscopic observation, is positive, shows to can detect I and IV Collagen Type VI eggs in sample
In vain.
I. GAG content is detected：10 samples are taken, are sampled, extraction, with Biocolor chondroitin sulfates, keratosulfate
In plain detection kit test chondroitin sulfate and keratosulfate cellulose content, sample content of chondroitin sulfate average value be 573 ±
83μg/g；Sulfuric acid angle ossein content average value is 132 ± 13 μ g/g.
Embodiment 5：
Biological Detection is carried out to sample obtained by preparation method of the present invention, detection project includes：Pyrogen, cytotoxicity, late
Hair style hypersensitivity, intradermal reaction, Acute systemic toxicity, Salmonella reversion test, mouse lymphoma cell mutant test, chromosome are abnormal
Change, implantation, subchronic toxicity.
1) pyrogen
By 6cm2Sample adds 1ml to extract the ratio of medium, and 37 ± 1 DEG C, 72 ± 2hr prepares experimental liquid, extracts medium：Physiology
Salt solution.Method is carried out as defined in GB/T 14233.2-2005, the reaction of product apyrogeneity.
2) cytotoxicity
By 6cm2Sample adds 1ml to extract the ratio of medium, and 37 ± 1 DEG C, 24 ± 2hr prepares experimental liquid, extracts medium：Containing blood
Clear MEM culture mediums.The experimental liquid is taken to be tested according to test method specified in GB/T16886.5-2003, as a result product
Cell-cytotoxic reaction is not more than 1 grade.
3) delayed allergy
By 6cm2Sample adds 1ml to extract the ratio of medium, and 37 ± 1 DEG C, 72 ± 2hr prepares experimental liquid, extracts medium：Physiology
Salt solution and cottonseed oil.According to the parts of GB/T 16886.10-2005 the 10th：Stimulate and provided with delayed allergy test method
Tested, as a result product is without delayed allergy.
4) intradermal reaction
By 6cm2Sample adds 1ml to extract the ratio of medium, and 37 ± 1 DEG C, 72 ± 2hr prepares experimental liquid, extracts medium：Physiology
Salt solution and cottonseed oil.According to the parts of GB/T 16886.10-2005 the 10th：Stimulate and test test method with delayed allergy
Regulation is tested, as a result：The difference of test specimen and solvent control mean score is less than 1.0.
5) Acute systemic toxicity
By 6cm2Sample adds 1ml to extract the ratio of medium, and 37 ± 1 DEG C, 72 ± 2hr prepares experimental liquid, extracts medium：Physiology
Salt solution and cottonseed oil.Experimental liquid is taken to be tested according to test method as defined in GB/T16886.11-2011, as a result：Product without
Acute systemic toxicity reacts.
6) Salmonella reversion test
By 6cm2Sample adds 1ml to extract the ratio of medium, and 37 ± 1 DEG C, 72 ± 2hr prepares experimental liquid, extracts medium：Physiology
Salt solution and DMSO.Method is carried out as defined in GB/T16886.3-2008, as a result：The Salmonella reversion test of product is feminine gender.
7) mouse lymphoma cell mutant test
By 6cm2Sample adds 1ml to extract the ratio of medium, and 37 ± 1 DEG C, 72 ± 2hr prepares experimental liquid, extracts medium：Physiology
Salt solution and DMSO.Method is carried out as defined in GB/T16886.3-2008, as a result：The mouse lymphoma cell mutant test of product
For negative findings.
8) chromosomal aberration test
By 6cm2Sample adds 1ml to extract the ratio of medium, and 37 ± 1 DEG C, 72 ± 2hr prepares experimental liquid, extracts medium：Physiology
Salt solution and DMSO, method is carried out as defined in GB/T16886.3-2008, as a result：The chromosomal aberration test of product is feminine gender.
9) it is implanted into
Method is carried out as defined in GB/T16886.6-1997, as a result：Muscular grafting 1 week：Visible neutrophilia around sample
Granulocyte, lymphocyte and macrophages infiltration, should be formed without blister cavities；Muscular grafting 4 weeks：Visible a small amount of macrophage is thin around sample
Born of the same parents and lymphocyte, collagenous fibres and proliferation of fibroblast, have fiber blister cavities to be formed；Muscular grafting 12 weeks：Can around sample
See that a small amount of lymphocyte, collagenous fibres, fiber blister cavities are finer and close regular.
10) subchronic toxicity
Method is carried out as defined in GB/T 16886.11, as a result：Material subchronic toxicity is evaluated, without sub- chronic poison
Property reaction.
Embodiment 6：
Zoopery is carried out to sample obtained by preparation method of the present invention, it is specific as follows：
Sample in embodiment 1 is used 1：4000 gentamicin physiological saline clean cornea 15min rehydrations, with trepan diameter
7mm corneal grafts.Rabbit 12, body weight 1-2kg, the operation of left eye row, right eye blank control.1% amobarbital sodium is pressed
30mg/kg body weight is injected intraperitoneally, 0.5% cacaine surface anesthesia, 2% procaine hydrochloride 1mL retrobulbar injections, and massages eyeball, reduces
1% neophryn, 1% atropine and tropicamide eye drip is added dropwise in intraocular pressure, preoperative 30min, pupil is fully dissipated big.Routine disinfection is spread
Cloth, cuts off eyelashes near the eyes, makees four rectus index sutures, exposure and fixed eyeball, is placed in Central corneal with 6.5 trepans, downwards
Light pressure rotation, stops when there is aqueous humor outflow, the cornea not drilled is completed with corneal scissors, afterwards by the embodiment 1 after rehydration
Corneal transplantation is made 4 points of interruptions at defect, with 9/0 nylon wire and fixed, and remakes continuous suture and fixes.Postoperative daily check is added dropwise
0.4% gentamicin, 0.5% examine ground pine and 1% atropine liquid.12 animal implants are transparent in transparent 3,2 weeks in 1 week
5,1 death, 3 weeks transparent 3.There are 3 clearing times in 11 animals 43 days, 56 days, 58 days, remaining 8 clearing time
More than 90 days.Cornea keeps transparent, and cornea is without oedema, without infection, and cornea is without new vessels.Materials measurement corneal transplant is thick
Spend for 330 ± 20 μm, normal cornea thickness is 304 ± 11 μm, both no difference of science of statistics.Art eye intraocular pressure is 11 ± 1mmHg, just
Normal intraocular pressure is 10 ± 1mmHg, both no difference of science of statistics.
As fully visible, a kind of corneal stroma of the invention and preparation method, with following advantage：
(1) the three-dimensional space network structure of collagen fabric in corneal extracellular matrix is retained；
(2) active growth factor in corneal extracellular matrix is retained；
(3) by cryogenic freezing, the cell in cornea can be cracked in the presence of ice crystal to be crushed, simultaneously, due to water
Ice is changed into, volumetric expansion can increase corneal stroma flaggy spacing, during defrosting cornea, ice changes into water, volume reduces, can be from
Outside absorbs moisture, so that realize that cornea is swelled, thickness increase；
(4) increase corneal stroma interlamellar spacing, be more beneficial for immunogene removal, be conducive to the cleaning of inhereditary material；
(5) high concentration immunogene removes solution and replaced with pure water, using the diffusion of solution high-concentration and low-concentration, in cornea base
Scutum interlayer formation scouring effect, improves inhereditary material cleaning efficiency；
(6) DNA residuals can reach below 10ng/mg, and galactosidase clearance is higher, can reach more than 99%；
Moulding section：
(1) by air-dried cornea, corneal stroma flaggy spacing can be reduced, cornea can be eliminated right in frozen-thaw process
The influence of the microstructure of corneal lamellar, reduces corneal thickness, makes matrix organization more closely knit, improve the tensile strength of cornea；
(2) cornea after air-drying is planar transparent sheet film, it is to avoid cornea air-dries produced under free condition
Fold and contraction, the worth cornea of such a method, with certain internal stress, can quickly absorb water during rehydration
Point, it is rapid to recover elasticity；
Sterilization process：
By controlling sterilization process, effectively ethylene oxide gas can be made to penetrate into inside cornea, reach sterilization effect.
The above embodiment of the present invention is the description of the invention and cannot be used for the limitation present invention, the right with the present invention
Any change in claim suitable implication and scope, is all considered as being included within the scope of the claims.
Claims (10)
1. a kind of corneal stroma, it is characterised in that：The cornea of animal is used for raw material, corneal epithelium, corneal stroma is removed
In cell component and DNA compositions, retain bowman's lamina and segment thickness corneal stroma, retain complete extracellular matrix into
Point.
2. corneal stroma according to claim 1, it is characterised in that：The animal is mammal, preferably pig or ox.
3. corneal stroma according to claim 1, it is characterised in that：Described segment thickness corneal stroma be and preceding elastic force
What layer was connected has certain thickness corneal stroma, described extracellular matrix including collagen and glycosaminoglycan.
4. corneal stroma according to claim 3, it is characterised in that：
The collagen is I-type collagen and IV collagen types, and the I-type collagen and IV collagen types contain
Measure as 85-90%；
The glycosaminoglycan is chondroitin sulfate and keratan sulfate；
The corneal stroma is diameter 7-10cm, thickness 0.1-0.2mm disk；
The removal corneal epithelium, the cell component in corneal stroma and DNA compositions refer to the cell residue amount for 0,
It is more than 99% that DNA residual quantities, which are less than 10ng/mg, the sweet enzyme clearance rate of galactolipin,.
5. a kind of preparation method of corneal stroma, it is characterised in that：Prepare measured step includes suddenly：
(1) Feedstock treating：The eyeball of animal is taken, is soaked in after taking cornea, cleaning in purified water；
(2) freeze thawing is swelled：Cornea is first soaked in water, then freezes；Take out, rinsed after after complete thaw with purified water, so after freezing
Soak again afterwards；Repeat above-mentioned freezing, course of defrosting repeatedly, obtain the cornea of water swelling；
(3) inactivation of virus：Inactivation of virus is carried out using Peracetic acid-alcohol solution dipping cornea；
(4) cleaning process：Cornea is handled in Vltrasonic device with PBS solution and purified water respectively；
(5) immunogene is removed：Immunogene removes liquid for PBS solution, and trypsase and EDTA, cornea are included in the PBS solution
Immunogene removal process carried out in Vltrasonic device；
(6) cleaning process：Cornea is handled in Vltrasonic device with PBS solution and purified water respectively；
(7) cornea repeatedly, is then immersed in purified water by repeat step (5) and (6)；
(8) cutting materials：Under anatomical lens use cornea lamellar blade, cut lamina elastica corneae anterior and fraction matrix then by its
It is soaked in purified water；
(9) drying is dried：Cornea is open and flat in support surface, and acquisition corneal stroma is dried in placement in an oven.
6. the preparation method of corneal stroma according to claim 5, it is characterised in that：
The concentration of volume percent of Peracetic acid is 0.1-5%, the volume hundred of ethanol in step (3) Peracetic acid-ethanol solution
Divide specific concentration for 5-40%, the volume ratio of Peracetic acid-ethanol solution and cornea is (30-60) ︰ 1, inactivation time 2-4h, temperature
Scope is 10-40 DEG C；
Step (4) cleaning process, PBS solution pH6-8 therein, solution temperature are 10-40 DEG C, the volume of PBS solution and cornea
Than for (30-60) ︰ 1 are cleaned 2-4 times, each 10-30min；Then using 10-40 DEG C water for injection clean, water for injection with
Cornea volume ratio is (30-60) ︰ 1 are terminated to detection electrical conductivity for below 10 μ s/cm；Cleaning process is in supersonic wave cleaning machine
Carry out；
It is 0.01-0.2%, EDTA concentration that immunogen in step (5), which removes trypsase mass percent concentration in liquid,
0.1-1mmol/L, the pH value that immunogen removes liquid is 7-8；Multiple frequency ultrasonic at least includes two supersonic frequencies, Frequency scope
For 20-40KHz, higher frequency is 60-90KHz, wherein low frequency processing 5-40min, high-frequency therapeutic treatment 5-40min, and temperature range is
20-35℃；
The cleaning process of step (6), PBS solution pH6-8 therein, solution temperature are 10-40 DEG C, the body of PBS solution and cornea
Product is than for (30-60) ︰ 1, are cleaned 2-4 times, each 10-30min, are then cleaned using 10-40 DEG C of water for injection, water for injection
Volume ratio with cornea is (30-60) ︰ 1, detection cleans water for injection and do not clean the difference of water for injection electrical conductivity less than 1 μ s/
Cm is terminated；
Step (9) drying is dried to be carried out in heat-circulation oven, baking oven is preheated to after 25-40 DEG C, by obtained by step (8)
Material is put in oven drying, is air-dried moisture by thermal cycle wind, time 12-24 hour.
7. the preparation method of corneal stroma according to claim 6, it is characterised in that：Step (5) immunogen is removed in liquid
The mass percent concentration of trypsase is 0.02-0.05%, and EDTA concentration is 0.4-0.8mmol/L, and immunogen removes liquid
PH value is 7.2-7.5；It is (30-60) ︰ 1 that the immunogen, which removes liquid with cornea volume ratio,.
8. the preparation method of corneal stroma according to claim 5, it is characterised in that：Preparation process also includes step (10)
Packaging and step (11), which sterilize, to be parsed.
9. the preparation method of corneal stroma according to claim 8, it is characterised in that：Packaging step is specially：Dry material
Material is defended strong wrapping paper using spy and encapsulated with vacuum formed box；Sterilizing analyzing step be specially：First 20-40 DEG C of soaking time 2-4 of temperature is small
When, humidity 30-70% then passes to concentration 300-1000mg/L oxirane, sterilizes 4-8 hours；Then parse, resolving
Carried out in the Resolution Room of ventilation, temperature control is between 10-30 DEG C, time 14-28d.
10. a kind of application of corneal stroma in ophthalmic implant, it is characterised in that：It is invalid that the ophthalmic implant is used for medication
Still imperforated ulcer cornea, infected keratitis cornea and perforate cornea provisional covering.
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