CN101433478A - Whole layer biological cornea as well as construction method and use thereof - Google Patents
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Abstract
The invention aims at providing a novel full-thickness biological cornea used for transplantation. The full-thickness biological cornea takes an animal cornea acellular matrix as a carrier, and the acellular matrix comprises animal cornea matrix cells, epithelial cells and endothelial cells which are cultured and augmented in vitro. The cornea is characterized in that the xenogenic corneal acellular matrix prepared by a biochemical method can be used as a good carrier for in vitro constructing the biological cornea in the aspects of shape, structure and biological compatibility, the matrix cells, epithelial cells and endothelial cells of the cornea are planted respectively, and dynamically cultured in the simulated in vivo environment of a biological reactor, so that the full-thickness biological cornea with nearly normal tissue structure and characteristics can be constructed in vitro, and the biological cornea can be used to simulate the physiological cornea for fundamental research on physiology, pathology and pharmacology; moreover, the biological cornea can also be directly used as the donator for corneal transplantation.
Description
Technical field
The present invention relates to the field of medical materials in medical science and the biological engineering, relating in particular to the heterogenic cornea acellular matrix is that carrier is cultivated the biological cornea of holostrome that structure forms in bioreactor, this biological cornea can be used for physiology, pharmacology and pathologic basis research, can be used as donor again and is applied to corneal transplantation and refractive surgery.
Background technology
Corneal injury, ulcer, cicatrix and edema muddiness due to the intractable diseases causing blindness that keratopathy is a kind of sickness rate height, treatment is difficult, a variety of causes are the one of the main reasons of world today's blinding.The main method of the treatment corneal opacity is flaggy or penetrating keratoplasty at present, but has been subjected to the restriction of present situations such as cornea donor source shortage, the aging of cornea donor, post-operative complication and postoperative immunological rejection.The clinical practice of materials such as macromolecule PMMA has promoted artificial cornea's development, make it become another kind of treatment approach after the donor source corneal transplantation, but the consistency problem between heterogeneous material and the biological tissue does not solve as yet fully, cause severe complications such as rejection, seepage infection thus, its design and implantation mode are ripe not enough.
Utilize emerging biological engineering and tissue engineering technique to transplant new thinking and and the method that provide to substitute donor's cornea for external structure goes out the biological cornea of holostrome.Yet the structure of biological cornea makes its research rest on the elementary step owing to lack suitable carriers and approximate physiological condition of culture, though the first tool normal structure 26S Proteasome Structure and Function of external structure artificial cornea model can't be used for transplanting.Plant corneal epithelium and endotheliocyte again after the nineties in 20th century, Minami etc. went out hypothallus with collagen mixing angle membrane matrix cell construction the earliest, obtain the blank of tool cornea three confluent monolayer cells structures just, Griffith etc. is with the collagen of the glutaraldehyde cross-linking support as the artificial cornea in recent years, the cornea fibroblast is blended in the collagen to be cultivated, epithelial cell is cultivated on the support top layer, endotheliocyte is cultivated the bottom at collagen scaffold, thereby constructs and the intimate cornea equivalent of people's cornea form, structure and tissue characteristics.And Chinese patent CN 1579342A also discloses " a kind of acellular hetero stroma of cornea and preparation method and purposes ", the acellular hetero stroma of cornea that it provides a kind of artificial cornea of can be used for to make up.
Existing research is used for complex that carrier material that biological cornea makes up comprises natural extracellular matrix, natural and synthesized polymer material, the organic material complex with inorganic material.Common have collagen, polylactic acid, chitin, chitosan, a nano material etc., but not bionical as yet preparing and the akin fibrolamellar structure of cornea.Cornea makes up these carriers commonly used also because of parameters such as the transparency, mechanical strength, degradation speed are not the best, and the cornea that constructs is all not ideal enough.
Construct the biological cornea of holostrome, its core is to set up the three dimensions complex of keratocyte and biomaterial, and carrier bracket is the basic framework and the metabolism place of cell attachment, and its form and function directly influence the tissue morphology and the function of formation.The cornea its specific structure is to keep the key factor of normal physiological functions such as corneal transparency, dioptric, permeability, take off cell tissue substrate as support by biochemical method with what cell component was removed the extracellular matrix composition keep normal structure and structure, have good biological characteristics, and wide material sources.Cell-eliminating coanea matrix can keep normal cornea flaggy arrangement architecture, and has a physiology microenvironment of good toughness and suitable cell growth, though the corneal stroma of pig is xenogeneic natural homogeneous material simultaneously, but has excellent biological compatibility, especially it is extremely low to take off cell processing back antigenicity, is the good carrier that makes up biological cornea.
Traditional cornea construction method all is a static culture in culture dish, be unfavorable for that tissue and cell carry out nutrition exchange and metabolism, and bioreactor plays an increasingly important role in cell in vitro cultivation and tissue reconstruction research field in recent years, by dynamic environment in the analogue body, for the cell growth provides constantly fresh nutrient substance and takes away metabolite, thereby improve the condition of culture of isolated cells; Simultaneously cell and be organized in dynamically in more help the secretion and the intercellular signal transduction of extracellular matrix; Cell has three dimensions freedom to a certain degree in bioreactor, help being in contact with one another by histological characteristic between cell-cell, the cell-matrix, and be difficult for forming downright bad center.The ideal device that the These characteristics of bioreactor makes it to become engineered organ and organizes reconstruction in vitro.
Summary of the invention
The purpose of this invention is to provide the biological cornea of a kind of new holostrome that can be used for transplanting, the biological cornea of this holostrome is a carrier with the animal corneal acellular matrix, also has animal corneal stromal cell, epithelial cell and the endotheliocyte of In vitro culture and amplification on described acellular matrix.
Another object of the present invention provides the construction method of the biological cornea of this holostrome, and this method is achieved through the following technical solutions:
(1) as the preparation of the acellular matrix of carrier: get the fresh animal cornea, Mechanical Method removes epithelial layer, remove descemet's membrane after, be dipped in and contain 1%TritonX-100, cell was taken off in 4 ℃ of vibrations in 72 hours, reuse PBS buffer shakes repeatedly and washes, flaggy and metaplax layer before harvesting of tissue's excision, / 3rd thick hypothalluses in the middle of keeping, after leaving standstill-80 ℃ of frozen spending the night, changed vacuum drying treatment over to 24 hours, rehydration in the DMEM of PBS buffer and serum-free culture medium respectively before described acellular matrix is used, 4 ℃ of preservations are standby;
(2) cultivation of substrate, epithelium and endotheliocyte and amplification: aseptic another animal corneal of getting, microscopically is peeled off described corneal epithelium, hypothallus, along the Descemet film endodermis of tearing, get limbal epithelium 1mm * 1mm piece of tissue, adherent method is incubated in the culture medium of the DMEM:F12=1:1 that contains 10% hyclone; Hypothallus 3%II Collagen Type VI enzyme, digestion method is made cell suspension and is planted in culture dish; Endothelial cell on the trypsinization descemet's membrane is in the planted medium; Each cell leaves standstill 37 ℃, 5%CO
2The saturated humidity incubator is cultivated, the next day change liquid, when the cell growth is merged greater than 80%, trypsinization, centrifugal, make cell suspension, distribute by 1:2 or 1:3 to be inoculated in cultivations of going down to posterity of new culture dish, treat that the cell growth is near merging back repetition above-mentioned steps go down to posterity successively digestion, the cultivation of going down to posterity;
(3) cultivate three-dimensional corneal stroma in the bioreactor: with keratocyte transfection green fluorescence protein gene, pair cell carries out fluorescent labeling, and making concentration then is 2 * 10
6The cell suspension of individual/ml is in centrifuge tube, the acellular matrix carrier of preparation is inserted, evacuation is dipped in the cell suspension carrier, take out the cell carrier complex after 2 hours and place 24 orifice plates, make the keratocyte suspension once more, inject the carrier central part with 1ml OT syringe, be the 0.1ml/ carrier, leave standstill cultivation and change bioreactor over to after 4 hours;
(4) corneal endothelium and epithelial cell are planted in substrate and bioreactor and cultivated: 24 orifice plates are put in the hypothallus taking-up of cultivating a week at first with bioreactor, endothelium is towards last, with endothelial cell system cell suspension, drip on interior surface, leave standstill cultivation and after 4 hours, change the reactor internal rotation over to, 20 rev/mins, subculture took out during one week, epithelial surface is upwards in culture dish, the corneal epithelial cell suspension is dripped the surface, leave standstill cultivation equally and change bioreactor culture over to after 4 hours, continue to cultivate week back termination.
A further object of the present invention is the application as medical material in corneal transplantation, cornea refractive surgery of the biological cornea of holostrome.
The heterogenic cornea acellular matrix that main feature of the present invention is to prepare by biochemical method is in form, structure and biocompatibility are the good carriers as the biological cornea of external structure, by planting keratocyte respectively, endotheliocyte and epithelial cell, simulated in vivo environment is dynamically cultivated in bioreactor, can go out to be similar to the biological cornea of holostrome of normal structure structure and characteristic in external structure, this biological cornea can be used for simulating the physiological cornea and carries out physiology, pathology and pharmacological basic research also can be used as donor simultaneously and are directly used in the therapeutic corneal transplanting.
Description of drawings
Fig. 1 has shown the porcine cornea acellular matrix carrier bracket structure of preparation;
Fig. 2 has shown that xenogenesis acellular matrix carrier implants host's cornea behind the rabbit corneal substrate flaggy;
Fig. 3 has shown that the acellular matrix carrier plants sheet in host's corneal stroma interlayer good knitting;
Fig. 4 has shown that the keratocyte of GFP labelling in the three dimensional matrix layer that makes up is embedded in matrix scaffold inside;
Fig. 5 has shown the biological cornea tissue general form of the holostrome that makes up;
Fig. 6 has shown holostrome cornea tool stratified squamous epithelium layer, substrate flaggy and monolayer endothelial cell;
Fig. 7 has shown that biological anterior corneal surface epithelial cell is layer-by-layer growth under the scanning electron microscope, arranges closely;
Fig. 8 has shown that the polygon cell that biological endothelial cell under the scanning electron microscope is monolayer inlays arrangement;
Fig. 9 has shown that keratocyte is attached on the carrier collagen fiber under the transmission electron microscope, the justacrine extracellular matrix;
It is positive that Figure 10 has shown that biological corneal epithelium cell-specific keratin CK3 expresses;
Figure 11 has shown that keratocyte Vimentin vimentin expresses positive in the biological cornea;
Figure 12 has shown that biological endothelial cell water is positive by road protein A QP1 expression.
The specific embodiment
Below will the invention will be further described by embodiment.
Embodiment 1The preparation of porcine cornea acellular matrix and biocompatibility thereof
Get the fresh pig cornea, Mechanical Method removes epithelial layer, remove descemet's membrane after, be dipped in and contain 1%TritonX-100, cell was taken off in 4 ℃ of vibrations in 72 hours, reuse PBS buffer shakes repeatedly and washes, flaggy and metaplax layer before harvesting of tissue's excision, 1/3rd thick hypothalluses in the middle of keeping, after leaving standstill-80 ℃ of frozen spending the night, changed vacuum drying treatment over to 24 hours.Acellular matrix rehydration in the DMEM of PBS buffer and serum-free culture medium respectively before application, 4 ℃ of preservations are standby.The porcine cornea acellular matrix of preparation is kept original grown form of cornea and toughness, is the translucent of edema, and substrate longitudinal section flaggy arrangement of collagen fibers is more neat, wavy under the scanning electron microscope, and is fine and close, the hole (see figure 1) that as seen differs in size between fiber.
The porcine cornea acellular matrix of preparation is planted sheet to be implanted in the new zealand white rabbit corneal stroma capsule bag: white rabbit with the speed new 2mg/kg intramuscular injection general anesthesia of sleeping, is equipped with the anesthesia of 2% lignocaine 1ml retrobulbar injection; The trepan that with the diameter is 7mm is marked at anterior corneal surface, cut the cornea degree of depth to 1 o ' clock position mark at 11 o'clock in cornea top and reach 1/3～1/2 corneal thickness approximately, make interlayer then separates on this flaggy plane, the border reaches mark between passivity isolation medium flaggy, form a capsule bag shape, use the normal saline flushing plant bed; Acellular matrix made 0.3mm is thick, diameter is the graft of 5mm, move into the rabbit corneal capsule bag from incision, iris repositor will be planted sheet and be flattened 10-0 nylon wire interrupted suture otch.Big mycin of 40,000 units and 2.5mg dexamethasone are celebrated in the postoperative subconjunctival injection, are coated with tetracycline cortisone spongaion.
Transplant the back in one week the animal corneal graft area edema is arranged, 4 week the back corneal transparences recover gradually, observe 6 months corneas of postoperative and keep transparent, tangible rejection does not take place, also do not have the new vessels (see figure 2) of growing into.Cornea tissue is learned observation by drawing materials, acellular matrix is implanted behind the receptor always in the matrix substrate interlayer, merge better with host's corneal stroma, implant the 8th when week visible host's keratocyte in the implant two ends (see figure 3) in the acellular matrix fiber of growing into up and down, implanting tissue and host's corneal lamellar merge gradually and are difficult to observe out the demarcation line, limit afterwards.
Embodiment 2Separating and cultivation of rabbit corneal epithelium, substrate and endotheliocyte
The aseptic rabbit corneal of getting, microscopically is peeled off rabbit corneal epithelium, hypothallus, along the Descemet film endodermis of tearing.The piece of tissue planting method is cultivated former generation corneal epithelial cell: after removing the shallow-layer epithelial cell limbus of corneae is cut into 1mm * 2mm piece of tissue, epithelial surface is affixed at the bottom of the culture dish down, add DMEM/F12 (1:1) the culture medium submergence piece of tissue just that contains 10% hyclone on a small quantity in the incubator after dry half an hour for well, back interpolation culture fluid spends the night.Stromal cell obtained to adopt and united digestion and tissue block method former generation: the hypothallus piece of tissue shreds, and puts in the centrifuge tube, adds the digestion of 3%II Collagen Type VI enzyme heat, in about 37 ℃ of jolting 40min.To observe piece of tissue loose when microscopically, form netted collagen silk, stop digestion therebetween during round adrift bright cell, add the serum-free medium dilution of 5 times of volumes, behind the mixing agitator in centrifuge centrifugal 5 minutes of 1500r/min, abandon supernatant, add the washing of PBS buffer again, recentrifuge 5 minutes, abandon supernatant, DMEM/F12 (1:1) culture medium that adds 10%FBS then, the piping and druming mixing is resuspended, and cell suspension and residual indigested piece of tissue all are inoculated in culture dish, add a little culture medium earlier and allow cell and tissue block adherent, add culture medium next day.Obtain the cultivation of former generation endothelial cell by the endotheliocyte of digestion on the descemet's membrane: the descemet's membrane of tearing is placed sterile petri dish, endothelium adds towards last flattening back and contains blood serum medium, put earlier to hatch in the incubator after 1 day and discard culture fluid, add the washing of PBS buffer once, add 0.25% pancreatin-0.02%EDTA mixture slaking liquid then, about 37 ℃ of digestion 15min.Microscopically is observed on the transparent descemet's membrane hexagonal cell and is inlayed arrangement, sharpness of border, cell shrinkage gradually, it is big that intercellular substance becomes, when rocking culture dish, most of cell adds and contains the hyclone culture medium and stop digestion when descemet's membrane comes off, piping and druming back is in centrifuge 1500r/min centrifugal 5 minutes repeatedly, abandon supernatant, add the washing of PBS buffer again, recentrifuge 5 minutes, abandon supernatant, add the culture medium that contains 15% hyclone and make 2 * 10
4～3 * 10
4Individual/ml cell suspension plantation diameter is in the 35mm culture dish.Each cell leaves standstill 37 ℃, 5%CO
2The saturated humidity incubator is cultivated, the next day change liquid.When cell growth was merged greater than 80%, cell suspension was made in trypsinization, centrifugal, distributed by 1:2 or 1:3 to be inoculated in cultivations of going down to posterity of new culture dish, treated that the cell growth goes down to posterity successively near merging back repetition above-mentioned steps.
Each keratocyte growth in vitro propagation of cultivating is good, and epithelial cell is polygon, and iuntercellular is connected mutually to be inlayed, and is the mosaic shape; Keratocyte is that the cell of fusiformis is arranged closely, and spectrum is swirl shape, radiation braiding shape pattern; Endotheliocyte is hexagon or polygon, and iuntercellular is inlayed closely, is the paving stone shape and is laid at the bottom of the culture dish.Growing multiplication is vigorous behind each passage, can reach more than 10 generations, and cellular morphology is still similar substantially to former generation with the rate of increase, and sufficient amount is planted carrier as seed cell, makes up the biological cornea of holostrome.
Embodiment 3Cultivate three-dimensional corneal stroma in the bioreactor
At first (green fluorescent protein, GFP), pair cell carries out fluorescent labeling with keratocyte transfection green fluorescence protein gene.When keratocyte adherent growth nearly 70%～80% merges, inhale and abandon culture fluid, fresh medium and GFP recombinant adenovirus venom are added culture dish with the ratio of 2:1, put 37 ℃, 5%CO
2Spend the night in the saturated humidity incubator, discard mixed liquor next day, with PBS buffer washed twice, remove residual virion, and then add the fresh blood serum medium that contains, continue to cultivate 24 hours, behind the observation of cell high expressed green fluorescence, can be used for planting cornea acellular matrix carrier under the fluorescence microscope.
Making concentration with the digestion of the keratocyte of GFP labelling, after centrifugal is 2 * 10
6The cell suspension of/ml injects the carrier central part with the 1mlOT syringe, and about 0.1ml/ carrier leaves standstill to cultivate after 4 hours and changes bioreactor over to, in the bioreactor cornea tissue under the swiveling wheel drive, slight fluctuation in culture fluid.After cultivating a week, take out the cell-scaffold complex tissue, collect the three dimensional structure of observing stromal cell and carrier under the focusing microscope altogether in laser, the green fluorescence that sends by GFP labelling keratocyte, as seen stromal cell is embedded in the carrier bracket, growth is distributed between different flaggies more uniformly, forms the hypothallus (see figure 4) of 3-D solid structure.
Embodiment 4Corneal endothelium and epithelial cell are planted in substrate and bioreactor and are cultivated
At first the hypothallus of cultivating a week in the bioreactor is taken out and put in the culture dish, endothelium with the endothelial cell system cell suspension of cultivating, drips on interior surface then towards last, leaves standstill cultivation and dynamically cultivates greater than changing the reactor inner suspension over to after 4 hours.Subculture one took out during week, and up in culture dish, drip its surface with the corneal epithelial cell suspension of cultivating with the complex epithelial surface this moment, left standstill equally to cultivate to change bioreactor after 4 hours over to and suspend and dynamically cultivate, continue to cultivate one week the back stop.
After stopping cultivating, take out cornea tissue, its general form transparency of perusal and the thickness situation of rebuilding.Portion of tissue is fixed 24 hours with neutral formalin, the dehydration of ethanol gradient, and paraffin embedding, section HE dyeing, optical microscope is observed down and is rebuild the cornea tissue structure.Scanning electron microscopic observation epithelium and interior surface cell growthform structure on the acellular matrix carrier, the transmission electron microscope observing stromal cell is in growth of carrier board interlayer and vigor situation.Carry out the SABC detection to rebuilding each confluent monolayer cells secretory protein of cornea tissue, epithelial layer detects corneal epithelium specificity keratin CK3; The hypothallus cell is carried out Vimentin vimentin to be detected; Endodermis is detected and the closely-related aquaporin AQP1 of endothelial cell pumping function.
The biological cornea of the holostrome that makes up in the bioreactor is thin and transparent, for discoid, certain curvature and camber is arranged, diameter 8-10mm, and thickness 400-600 μ m is with the basic identical (see figure 5) of normal mammalian cornea tissue form.By tissue slice HE dyeing its structure is observed, the cornea tissue that discovery goes out with cornea acellular matrix vector construction is the holostrome structure of tool cornea just, epithelial layer is multiple layer pinacocyte layer, hypothallus is for the collagen fiber flaggy and inlay the fusiformis stromal cell of growth therebetween, interior surface is the successive pinacocyte layer of a monolayer, more approximate normal cornea organizational structure (see figure 6) on the histology.As seen each confluent monolayer cells of biological cornea of cultivating in the bioreactor is carried out Ultrastructural observation, epithelial surface at the acellular matrix carrier, cell is the overlapping growth of multilamellar, marshalling, cell is a pancake, and the figure is full, closely connects mutually, and on form, make a variation to fusiformis, the cell surface that has has the microvillus (see figure 7); At the carrier endothelium long cell of looking unfamiliar is that the polygon cell of monolayer is inlayed arrangement, presents typical endotheliocyte and inlays the sample outward appearance, and cellular morphology is fuller, and iuntercellular interconnects tight (see figure 8); Keratocyte is attached on the carrier collagen fiber, organelles such as the interior many endoplasmic reticulum of cell, and as seen engulf granule, the other extracellular matrix components (see figure 9) that emiocytosis is arranged of cell.Biological each the confluent monolayer cells secretory protein of cornea of holostrome is detected corneal epithelium cellular expression corneal epithelium specificity keratin CK3 (see figure 10); The hypothallus cell has brown particle in the endochylema, promptly express Vimentin (seeing Figure 11); Endodermis is detected the aquaporin AQP1 relevant with pumping function, and SABC result is strong positive, endothelial cell high expressed aquaporin (seeing Figure 12).
Need not further to elaborate, believe read the above-mentioned disclosed content of the present invention after, those skilled in the art can use the present invention to greatest extent, can make various changes or modifications, therefore, the embodiment of front is interpreted as only illustrating, but not limits the scope of the invention by any way.
Claims (4)
1, the biological cornea of a kind of holostrome is a carrier with the animal corneal acellular matrix, it is characterized in that, also has animal corneal stromal cell, epithelial cell and the endotheliocyte of In vitro culture and amplification on described acellular matrix.
2, the preparation method of the biological cornea of holostrome as claimed in claim 1 is characterized in that being achieved through the following technical solutions:
(1) as the preparation of the acellular matrix of carrier: get the fresh animal cornea, Mechanical Method removes epithelial layer, remove descemet's membrane after, be dipped in and contain 1%TritonX-100, cell was taken off in 4 ℃ of vibrations in 72 hours, reuse PBS buffer shakes repeatedly and washes, flaggy and metaplax layer before harvesting of tissue's excision, / 3rd thick hypothalluses in the middle of keeping, after leaving standstill-80 ℃ of frozen spending the night, changed vacuum drying treatment over to 24 hours, rehydration in the DMEM of PBS buffer and serum-free culture medium respectively before described acellular matrix is used, 4 ℃ of preservations are standby;
(2) cultivation of substrate, epithelium and endotheliocyte and amplification: aseptic another animal corneal of getting, microscopically is peeled off described corneal epithelium, hypothallus, along the Descemet film endodermis of tearing, get limbal epithelium 1mm * 1mm piece of tissue, adherent method is incubated in the culture medium that contains 10% hyclone and DMEM:F12=1:1; Hypothallus 3%II Collagen Type VI enzyme, digestion method is made cell suspension and is planted in culture dish; Endothelial cell on the trypsinization descemet's membrane is in the planted medium; Each cell leaves standstill 37 ℃, 5%CO
2The saturated humidity incubator is cultivated, the next day change liquid, when the cell growth is merged greater than 80%, trypsinization, centrifugal, make cell suspension, distribute by 1: 2 or 1: 3 to be inoculated in cultivations of going down to posterity of new culture dish, treat that the cell growth is near merging back repetition above-mentioned steps go down to posterity successively digestion, the cultivation of going down to posterity;
(3) cultivate three-dimensional corneal stroma in the bioreactor: with keratocyte transfection green fluorescence protein gene, pair cell carries out fluorescent labeling, and making concentration then is 2 * 10
6The cell suspension of individual/ml is in centrifuge tube, the acellular matrix carrier of preparation is inserted, evacuation is dipped in the cell suspension carrier, take out the cell carrier complex after 2 hours and place 24 orifice plates, make the keratocyte suspension once more, inject the carrier central part with 1ml OT syringe, be the 0.1ml/ carrier, leave standstill cultivation and change bioreactor over to after 4 hours;
(4) corneal endothelium and epithelial cell are planted in substrate and bioreactor and cultivated: 24 orifice plates are put in the hypothallus taking-up of cultivating a week at first with bioreactor, endothelium is towards last, with endothelial cell system cell suspension, drip on interior surface, leave standstill cultivation and after 4 hours, change the reactor internal rotation over to, 20 rev/mins, subculture took out during one week, epithelial surface is upwards in culture dish, the corneal epithelial cell suspension is dripped the surface, leave standstill cultivation equally and change bioreactor culture over to after 4 hours, continue to cultivate week back termination.
3, the biological cornea of holostrome as claimed in claim 1, its in corneal transplantation, cornea refractive surgery as the application of medical material.
4, the biological cornea of holostrome of the biological cornea preparation method preparation of holostrome as claimed in claim 2, its in corneal transplantation, cornea refractive surgery as the application of medical material.
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