CN109069696A - A kind of dry cornea of de- cell pig plate layer and its application method and purposes - Google Patents
A kind of dry cornea of de- cell pig plate layer and its application method and purposes Download PDFInfo
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Abstract
A kind of dry cornea of de- cell pig plate layer, is made of the bowman's lamina of porcine cornea and hypothallus；The hypothallus maintains the regularly arranged structure of collagenous fibres；In visible-range, the moisture content of the dry cornea is not more than 20%；The light transmittance of dry cornea is not less than 70%.On the basis of the present invention is used using porcine cornea as raw material, the optimal combination scheme of cornea product necessity characteristic of proposition, the advantageous normalization for realizing the dry cornea product quality of de- cell pig.
Description
Fields
The present invention relates to one kind to be used for corneal transplantation, the allosome xenogenesis artificial cornea and its application method and purposes of substitution people's cornea.It specifically refers to a kind of use porcine cornea for the dry cornea of the de- cell plate layer of material source.
Make great efforts to develop to take the lead in having carried out clinical test in worldwide by artificial cornea prepared by raw material of porcine cornea by more than ten years of Chinese Scientists, the product of Some Enterprises acquires the examination & approval of national correlation department, and the people's cornea for solving the problems, such as that domestic various big hospital is generally existing at present to a certain extent is in short supply.According to statistics, China is accumulative because a variety of causes causes the patient of canthus membrane damage to be up to ten thousand people more than 500, if the artificial cornea that porcine cornea is raw material can be produced in batches, can provide possibility to cure more patients.
Current patents and pertinent literature report, are mostly at present non-dry corneas by artificial cornea used in the artificial cornea of raw material and its clinical test of porcine cornea.Therefore, from the point of view of according to clinical test results disclosed in correlation, since the preparation method of this kind of artificial cornea is different, final product obtained, which exists, has any different, therefore clinical effectiveness obtained is also not quite similar.The present inventor proves by a large amount of test and clinical test, artificial cornea carries out main problem existing for large batch of popularization and use as a kind of product: quality is different, there are certain differences for product final performance, thus there is also have bigger difference for clinical use effect.(for example, time of Effectiveness and delay of recovering lost eyesight etc., differences were just quite big after graft according in 40 many cases clinical experiment report of Wuhan Concord Hospital.) wherein, since country does not work out specific standard for the biological nature of transplanting cornea at present, entering the product of the artificial cornea of clinical test (partially using) in the market now is including: that there are biggish differences in biological nature, biomechanics characteristic, product form and its application method, and these differences are due to caused by the difference of preparation method.In numerous patent and correlation are offered, there is also be relatively entirely different for the product as defined in preparation method.
It is proposed in the first Chinese CN101947144 patent that the present inventor proposed in 2010, is also using the pig lamellar cornea under undried state by the test data of the plate layer artificial cornea of raw material of porcine cornea.And the present inventor's research in recent years and clinical success show an important factor for transplanting with product quality of the cornea especially lamellar cornea in clinical use is effect after determining transplanting.The stability of product quality can determine the stability of transplantation effect, and in other words, cornea product with the same characteristics, transplantation effect is also substantially the same.Therefore for the angle of bulk article, it is necessary to which providing a kind of can ensure that transplanting safety and transplantation effect
Lamellar cornea product.The quality of the product should being consistent property, rather than it is different with the difference of the preparation method of cornea.Accordingly, in recent years, the present inventor has carried out a large amount of test and clinic for the lamellar cornea of different performance index, it is desirably to obtain the prerequisite product characteristic of institute that can be used as lamellar cornea product, overcome defect currently different as the lamellar cornea product quality of raw material using porcine cornea, the alternative humanized's cornea of product, to realize the target of the larger scale clinical application of lamellar cornea.
Cornea is located at the front of eyeball, is the transparent collagen tissue that a kind of structure height is regular, relatively cell-free.It only includes bowman's lamina and hypothallus that existing major part, which takes off cell plate layer corneal stroma,.By the heterogeneous cell ingredient in necessary de- cell processing removal hypothallus, the immunological rejection of porcine cornea transplanting is reduced.The germ-free condition for controlling corneal transplantation by sterilization treatment again, so that lamellar cornea be made to reach biological indicator required by corneal graft.
Corneal stroma is made of the collagenous fiber bundle thin plate of nearly 150 layers of queueing discipline, there is keratocyte therebetween.The stroma cell of cornea remains static, and about of the total volume 3~10%, thus opposite acellular tissue is called by some scientists.The design feature of corneal stroma is the type i collagen of triple helix orderly and the arrangement that is parallel to each other, and this arrangement mode constitutes the structure basis of the equal physics characteristics of corneal elasticity mechanical strength and its transparency.This important feature of cornea can after the implantation, and inducing receptor keratocyte orderly, is equably grown into, and cornea is made to keep transparent.
The analysis of the characteristics of based on to above-mentioned lamellar cornea matrix, the height rule of corneal stroma arrangement of collagen fibers structure are to realize lamellar cornea transparent an important factor for waiting biophysical properties.However, current method for removing cells all inevitably to varying degrees damages the ordered arrangement of hypothallus.
Existing method for removing cells is broadly divided into physical method, chemical method, enzyme process and electrophoresis.The method of physics is such as: freeze thawing, drying, and the efficiency for removing cell is low, and the residual of keratocyte ingredient is more, and immunological rejection is easily caused after implantation.The reagent that chemical method uses mainly has: ionic species detergent, nonionic class detergent, alkaline reagent, acid reagent；Although the efficiency that these reagents remove cell is higher, destroy the structure of cornea while removing cell, so that the transparency of corneal stroma is decreased obviously.
The reagent of enzyme process mainly has: lipidase, nuclease and protease；Different enzymes is directed to specific cell component, and the lipid components of lipidase degradation cell membrane, the cell membrane although protease can degrade, to corneal extracellular matrix, there is also apparent destructions, and the transparency of cornea is caused to decline.It is also affected to corneal transparence using the cell component in the method removal corneal stroma of electrophoresis.
In addition to physics method for removing cells, the method for other de- cell corneas all refers to use de- cell reagent.
There is influence for transparency, epithelial repair and the corneal stroma Reconstruction of The Function after taking off the implantation of cell cornea in the residual of de- cell reagent.Existing de- cell technology is while completely removing cell, or the transparency of cell-eliminating coanea matrix cannot be kept well, or since residual excessively causes cornea reconstruction process very long, easily cause corneal neovascularization, hence it is evident that reduce the transparency of cell-eliminating coanea matrix or transplantation is caused to fail.
It is well known that the reconstitution process in cornea is so that cornea softening, reaches the purpose that can be convenient transplanting.But cornea can make cornea water content substantially increase in reconstitution process, transparency can also be greatly reduced.In clinic, institute's water content is transparent artificial lamellar cornea after transplanting gradually by half dehydration mechanism possessed by people's cornea backstage in the cornea after transplanting.Therefore the clinical data of existing procucts is shown, post-transplantation cornea restores the transparent period up to 3 months or more, and the time of corneal transparency is also uncontrollable in each case.
In addition, cornea product should be under the requirement with product quality identity as a kind of cornea product with batch production characteristic, it is also desirable to have and facilitate preservation, transport and the product attribute used.Currently, de- cell porcine cornea substitution people's cornea technology enters a Rapid development stage, the quality identity and product attribute of cornea product are the emphasis for solving China's corneal transplantation donor and lacking.Therefore, it is necessary to the optimal combination scheme in the necessary characteristic such as the integrative biology of a kind of pair of corneal transplant product, physics and its product form is found, and the cornea product independent of cornea preparation method, to realize the marketization of cornea product.
Summary of the invention
The purpose of the present invention is to provide a kind of dry corneas of de- cell pig plate layer, from the most basic physiological function of cornea " transparency " this angle, on the basis of using using porcine cornea as raw material, comprehensively consider the necessary characteristic such as biology, physics and its product form of transplanting cornea, the preferred plan of de- cell cornea product characteristic is proposed, the dry cornea product quality identity of de- cell pig plate layer is realized.To overcome cornea product spotty defect due to preparation method difference in the prior art, reach the technical requirements of batch production, de- cell plate layer cornea is set to can be used as the substitute of humanized's cornea, the development for promoting China's ophthalmic medical level solves the problem of numerous sufferers cannot be cured effectively because of corneal donor shortage.
Another object of the present invention is to provide a kind of dry corneas of de- cell pig plate layer, make it have the product form for facilitating preservation, transport and using, and improve the product attribute that artificial cornea is suitable for market, meet artificial cornea product high-volume and promote the use of requirement.
A further object of the present invention is to provide a kind of dry corneas of de- cell pig plate layer, are easy to specification and simplification
The operation of diagonal film process, is allowed to facilitate clinical application in clinical transplantation art.
The object of the present invention is achieved like this, and a kind of dry cornea of de- cell pig plate layer provided by the invention is made of the bowman's lamina of porcine cornea and hypothallus；The hypothallus maintains the regularly arranged structure of collagenous fibres；In visible-range, the light transmittance of the dry cornea is not less than 70%；The moisture content of the dry cornea is not more than 20%.
De- cell pig plate layer of the invention dries cornea, and DNA residual is not more than 100ng/mg in hypothallus after de- cell.
In optional embodiment of the invention, it is at this time the Product Status of the dry cornea that the light transmittance, which is detected value after sterilizing,.
In optional embodiment of the invention, the dry cornea is externally provided with the airtight package that can be played a supporting role, and can effectively keep the Product Status of cornea after terminal sterilization, avoids transporting, cornea product is polluted and damaged in preservation.
In optional embodiment of the invention, the airtight package is arranged before or after dry cornea terminal sterilization.
The dry cornea of de- cell pig plate layer of the invention, hypothallus maintain the regularly arranged structure of collagenous fibres；Refer under transmission electron microscope observing, the average headway of type i collagen is 25nm ± 10nm in dry corneal extracellular matrix.The especially described corneal stroma keeps original regularly arranged structure of collagenous fibres to refer under transmission electron microscope observing, type i collagen sharpness of border in corneal extracellular matrix, and cross section is in the same size.This collagen fiber structure substantially maintains the original regularly arranged structure of porcine cornea hypothallus, to realize porcine cornea after taking off the complicated preparation process such as cell, it is still able to maintain the most basic physiological function of " transparent " this cornea, to reach preferable transplantation effect.
The dry cornea of de- cell pig plate layer of the invention, the corneal lamellar thickness 300um~700um drawn.
In the present invention, the dry cornea people light transmittance selection of the plate layer measures the absorbance value A of more than two different wave lengths, calculates gained according to formula " T=1/10A × 100% ".
In a preferred embodiment of the present invention, it is 380-780nm that the light transmittance, which selects measurement wave-length coverage, and measurement step-length is not more than 10nm.
In the present invention, the dry cornea at least bowman's lamina surface is smooth, the ridge shape protrusion or microscopic folds of no macroscopic make it be more advantageous to the growth of epithelial cell.
The application method of the dry cornea of de- cell pig plate layer of the present invention, opens airtight package in the preoperative；It takes out
Dry cornea, transfer operation can be carried out by being put into after impregnating 15~30 minutes in physiological saline.
In the dry cornea application method of the present invention, the cornea rehydration Best Times are 15~30 minutes.
The heretofore described dry cornea of de- cell pig plate layer is applied in corneal transplantation.
The heretofore described dry cornea of de- cell pig plate layer is performed the operation in refractive correction to be applied.
When the dry cornea of de- cell pig plate layer of the present invention is applied to refractive correction, in refractive correction operation consent, the drying cornea for adapting to diopter or preoperative foundation diopter demand can be selected to carry out Precision Machining to dry cornea according to dioptric demand, reach required power prescription.
The solution have the advantages that significant: first, the present inventor is according to during the last ten years to using porcine cornea as the statistic analysis result of great number tested data obtained in the research of the de- cell plate layer cornea of material, to the necessary characteristic of transplanting cornea and its to the influence of transplantation effect in complex clinical, from the angle of the clinically basic demand of cornea product, it is carried out in a large amount of experimental basis using the cornea of distinct methods preparation simultaneously, proposes a kind of dry cornea of de- cell pig plate layer.Reach an optimal combination scheme in the necessary characteristic that drying cornea of the invention can be required in corneal transplantations such as integrative biology, physics and its product form, realize that the quality of the dry cornea product of de- cell pig is uniform, to overcome the defect that quality is different or properties of product are different due to preparation method is different of cornea product in the prior art.And this defect causes to be difficult specification clinical manipulation, while can not obtain a foreseeable transplantation effect, so that hindering pig takes off cell cornea in clinical large-scale application.
Make a general survey of the research progress up to more than ten years, initially it is believed that substituting people's cornea as transplanting medical material using porcine cornea, first have to solve the problems, such as be to solve dissimilar material to the immunogenicity of human body, and propose many method for removing cells and its various combination with reference to the method for removing cells of other class xenogenesis alternative materials (such as skin, bone, lung organ).As described above, domestic existing de- cell technology can effectively remove the cell of corneal stroma, i.e. standard of the DNA residual quantity no more than 100ng/mg at present.But cornea has the different distinguishing features with all other tissues: transparent, this is also the most important basis that cornea executes its physiological function.And the transparent collagen arrangement for being derived from hypothallus height rule of cornea.However it is a large amount of facts proved that, existing method for removing cells is pursuing the de- cell effect of high level simultaneously, the ultra microstructure that the hypothallus height rule arrangement of cornea will be destroyed to varying degrees, to have ignored " transparency " of cornea this mostly important optical property for cornea.Accordingly, the applicant's this most basic and most important performance from the most important feature of cornea product " transparent " first proposes a kind of dry cornea of de- cell pig plate layer.Reach an optimal combination scheme in the necessary characteristic that drying cornea of the invention can be required in corneal transplantations such as integrative biology, physics and its product form, reaches cornea product quality consistency and trackability.So that artificial cornea
Product reaches " transparency " requirement necessary to transplanting clinic on the basis of guaranteeing that dissimilar material cornea requires human immunity originality.,.Realize that batch production meets wilderness demand clinically.The current clinical case of applicant proves that cornea product provided by the invention can be used as the substitute of humanized's cornea.
Secondly, it is applicant's understanding that dry cornea is undoubtedly the best Product Status of cornea product, is convenient to save, transport as can be with batch application in clinical cornea product.Compared with glycerol store method, the drying regime of cornea product can keep the performance of dry cornea product quality to stablize in the market links such as preservation, transport.Meanwhile dry cornea described herein has 5~20% moisture content, has certain toughness again while can keep its intrinsic shape, to reduce because of the brittle defect of the excessively high increase cornea product of aridity.Drying cornea of the invention can greatly extend the validity period of product relative to the cornea that glycerol saves.
And another important technology effect of drying cornea of the invention is, preoperative rehydration is easy to operate, and rehydration time is short.In reconstitution process, operation can be reached in the water content of cornea and required, and can properly control the water content of cornea after rehydration, this is extremely important to controlling or shortening cornea time of recovering lost eyesight.And the present invention completely can be by preoperative rehydration regularized operation, the problem of reaching water content after efficiently controlling cornea rehydration.And the current all clinical cases of the applicant can prove, drying cornea of the invention carries out in the transplantation carried out after rehydration, cornea, which restores the transparent time, after transplanting can control within 3 days, wherein cornea is gradually recovered transparent state, the postoperative transparent effect for just reaching at least postoperative March stated in existing literature of surgeon's knot to 50% case in the course of surgery (referring to Wuhan Union Medical College clinical report).In addition, the simple rehydration operation of specification can make the moisture content after the preoperative rehydration to cornea accurately be controlled, and therefore greatly reduce the difficulty of surgical procedure.
Third, test and clinic prove that the high product form characteristic of surface smoothness possessed by drying cornea of the invention is conducive to the attaching and proliferation of epithelium confluent monolayer cells.The flatness of especially lamina elastica corneae anterior, which is highly advantageous to, improves the speed of growth and growth quality of cornea implantation epithelium posterius cell.
4th, currently on the market into clinical using porcine cornea as the bio-artificial cornea of material, DNA residue criterion is not all provided.The applicant it has been investigated that, cornea is different from other organs, and stroma cell is seldom, about of the total volume 3~10%, thus be called opposite acellular tissue by some scientists.So in allograft, i.e., people's corneal transplantation when this index would generally be ignored.Therefore the DNA residue criterion for exceedingly pursuing cornea can take off cell strength disposal because increasing cornea during cornea preparation, and it is regularly arranged inevitably to destroy original collagen in corneal stroma.It is applicant's understanding that since cornea is there is no blood vessel structure and with the lower biological characteristics of immunogenicity, the appropriate selection right and wrong of this biological indicator value of cornea
It is often necessary.Accordingly, applicant carried out a large amount of animal experiments, based on result of study, special optical characteristic needed for comprehensively considering cornea product, formulating DNA residual quantity for dry cornea product is no more than 100ng/mg, both achieved the purpose that take off cell processing in cornea preparation, in the case where meeting Corneal Immune originality basic demand, the ultra microstructure for keeping corneal stroma collagen regularly arranged to the maximum extent, so that artificial cornea product has " transparency " that can guarantee transplantation effect, as shown in Figure 2.
In conclusion the present invention is on the basis of the optimal combination scheme in the necessary characteristic of integrative biology, physics and its product form etc. for corneal transplant product, proposition it is a kind of using porcine cornea as the de- cell plate layer cornea of material.De- cell porcine cornea of the invention can be used as the substitute of source of people cornea, and the present invention solves the problems, such as that the performance that cornea product quality arrives by preparation method is different, quality is inhomogenous, provides safeguard for the production of cornea product batch and clinical application.
The present invention and its specific embodiment and its effect are briefly described with reference to the accompanying drawing, attached drawing below is only to select some specific test examples explanations of the invention, and the whole of non-present invention.
The collagen of Figure 1A people's cornea arranges cross section arrangement architecture；
The porcine cornea collagen cross section arrangement architecture of the not de- cell processing of Figure 1B；
Collagen cross section arrangement architecture of Fig. 1 C present invention through de- cell treated porcine cornea；
Fig. 2 pig of the present invention takes off the dry lamellar cornea product photo of cell.
Fig. 3 pig of the present invention takes off the dry lamellar cornea HE stained photographs of cell.
Fig. 4 pig of the present invention takes off the dry lamellar cornea of cell to new zealand white rabbit plate layer post-transplantation photo.
The preoperative photo of Fig. 5 A clinical transplantation of the invention；
Fig. 5 B is the postoperative 3 days photos of Fig. 5 A clinical transplantation；
Fig. 5 C is the postoperative 2 months photos of Fig. 5 A clinical transplantation；
Fig. 5 D is the postoperative 6 months photos of Fig. 5 A clinical transplantation；
Fig. 5 E is the postoperative 1 year photo of Fig. 5 A clinical transplantation
Below in conjunction with the embodiment of the present invention, technical solution of the present invention is clearly and completely described, it is clear that described embodiments are only a part of the embodiments of the present invention, instead of all the embodiments.Based on the present invention
In embodiment, every other embodiment obtained by those of ordinary skill in the art without making creative efforts shall fall within the protection scope of the present invention.
Embodiment 1
In the present embodiment, the dry cornea of de- cell pig plate layer is only made of the bowman's lamina of porcine cornea and hypothallus using fresh porcine cornea as raw material, does not include epithelial layer, descemet's membrane and endodermis.The hypothallus maintains the regularly arranged structure of collagenous fibres；In visible-range, the light transmittance of the dry cornea is not less than 70%；The moisture content of the dry cornea is not more than 20%.
Specifically in the present embodiment 1, before terminal sterilization processing, light transmittance measured by the dry cornea of the plate layer of moisture content 15% ± 2% is 86 ± 5%.The transmission electron microscope observing of the dry cornea of the plate layer provided in the present embodiment, as described in Fig. 1 C, type i collagen aligned gaps are uniform in corneal extracellular matrix, and type i collagen sharpness of border in corneal extracellular matrix, cross section is in the same size, and average headway is about 25 ± 10nm.And the structure of this collagen arrangement and people's cornea in Figure 1A, the porcine cornea that is handled in Figure 1B without de- cell are extremely similar.A large amount of animal experiment proves that the structure of lamellar cornea provided by the invention can after the implantation, and inducing receptor keratocyte orderly, is equably grown into, and cornea is made to restore pellucidity quickly after the transfer.
It is well known that regularly arranged and " half dewatering state " of corneal stroma collagenous fibres maintains the transparency of cornea jointly: the regularly arranged of corneal stroma plays the role of diffraction grating, reduces scattering by destroying interference；And the light transmission of cornea is then related with the relative water content of cornea, water content increases the decline that will lead to corneal transparence in cornea.Therefore " transparency " and " water content " parameter area for properly selecting cornea product is the link for realizing that the standardization of cornea product quality is important.
Research of the present inventor in more than ten years, and pass through the statistic analysis result to great number tested data, from clinically thinking to the angle of cornea product basic demand, for it can be applied to the dry cornea product of clinical plate layer, the transparency of cornea and the moisture content of cornea are important and essential fundamental characteristics, and otherwise cornea product does not have the value of clinical application.And the angle that the present invention is based on physiological function essential to cornea is set out, and proposes most basic cornea physics characteristic, reaches an optimal combination scheme, realizes the quality conformance of the dry cornea product of de- cell pig.It is irrefragable the fact is that, currently used any one cornea preparation method, different methods all can generate different influences to certain characteristics of cornea, and this is exactly the main reason for anterior corneal product quality is inconsistent.But it is applicant's understanding that clinically cornea product can be widely used in as one kind, it is necessary to ensure that product quality is consistent or the identity of properties of product.Therefore when selecting cornea preparation method, it is contemplated that the most basic requirement of cornea product proposed by the present invention first.
In one embodiment of the present embodiment 1, in carrying out lamellar cornea preparation, removal epithelial layer, descemet's membrane and endodermis retain bowman's lamina, produce the hypothallus thickness of fresh cornea are as follows: 500 ± 50um constitutes lamellar cornea.
In 1 preferable embodiment of the present embodiment, the drying lamina elastica corneae anterior surface is smooth, the ridge shape protrusion or microscopic folds being visible by naked eyes.A large amount of animal experiment and clinical test prove that smooth cornea elastic force layer surface is more advantageous to the growth of epithelium confluent monolayer cells.As shown in Fig. 5 A to Fig. 5 E, drying cornea described in the present embodiment 1 has whole surface flatness.It is shown from post-transplantation effect, simultaneously, and is being conducive to the recovery of Postoperative visual acuity with splendid transparency with the dry cornea of whole surface flatness.
In another the preferable embodiment of the present embodiment 1, DNA residual quantity is not more than 100ng/mg in hypothallus after de- cell.Specifically in the present embodiment 1, DNA residual is 47ng/mg in hypothallus after de- cell.Using porcine cornea substitution people's cornea as transplanting medical device product, first have to solve the problems, such as to be the Immunogenicity for solving dissimilar material.And the research about xenogenesis substitution cornea up to more than ten years at home and abroad is always seen, the de- cell processing to heterogenic cornea is the most important thing link of all preparation methods.Based on the research conclusion of the dissimilar material organ transplant in other fields, it is conventionally believed that, DNA remains one of an important factor for index is decision heterogenic cornea transplantation effect in hypothallus.Therefore increase in a variety of different methods to the cell free strength disposal of cornea, make every effort to reach lower DNA residual level.However inventors believe that, while pursuing lower DNA residual level, it must be to pay close attention to the destruction that de- cell processing reduces the collagen arrangement architecture to rule original in corneal stroma as much as possible, the transparency for ensuring cornea product guarantees that the post-transplantation of cornea has the effect of preferably recovering lost eyesight.
In a large amount of animal experiment, the lamellar cornea that the present embodiment 1 proposes, country can be reached to the animal derived remaining requirement of biomaterial DNA, also it can satisfy the requirement of transplantation immunological rejection rate, and make to keep original regular arrangement of collagen fibers structure in corneal stroma, product post-transplantation obtains preferable " transparent " effect.
One optional embodiment of the present embodiment 1 is that the dry cornea is placed in the airtight package that is equipped with and can play a supporting role.Dry cornea provided by the invention can greatly extend the storage time of product relative to the cornea that glycerol saves, and have the advantage convenient for storage, transport.Since any sterilizing methods can all generate certain influence to the transparency of cornea product.Therefore, specifically in the present embodiment, measured cornea light transmittance is 78% ± 5% after using 25kgy to irradiate terminal sterilization.
The light transmittance selection of the dry cornea of plate layer of the invention measures the absorbance value A of more than two different wave lengths,
Gained is calculated according to formula " T=1/10A × 100% ".It is wherein described to select spectral scanning range for 380-780nm, step-length 10nm." light transmittance " of the dry cornea of the present invention is not limited only to method as described above and obtains, and also can choose other optical means and is measured, but corresponding with light transmittance proposed by the invention after its light transmittance values progress equivalent conversion obtained.
The application method of the dry cornea of de- cell pig plate layer of the present invention has the characteristics that easily operated, opens sealed package in the preoperative；Dry cornea is taken out, transfer operation can be carried out by being put into after impregnating 15~30 minutes in physiological saline.Specifically in the present embodiment, cornea rehydration time is 15~20 minutes.
The specific clinical case of one of the present embodiment proves that applicant can reach preferable transplantation effect in the effective proofs of clinical case all at present, the product index of the dry cornea of plate layer proposed by the present invention.Clinical case Fig. 5 A~Fig. 5 E of the invention shown by Fig. 5 A~Fig. 5 E is that the human cornea of the dry cornea of the de- cell plate layer of pig as described in the embodiment of the present invention 1 transplants clinic operation later group photo, the preoperative case photo of respectively Fig. 5 A, and Fig. 5 B is postoperative 3 days；Fig. 5 C is postoperative 2 months；Fig. 5 D is postoperative 6 months；Fig. 5 E is postoperative 1 year.For the existing clinical effectiveness being documented, 3 days after surgery of the dry cornea of the plate layer that the present embodiment 1 proposes are that transparent state, corneal epithelium have been repaired substantially, have no obvious rejection, month after operation cornea restores transparent completely, and no new vessels are grown into, and has no rejection.Postoperative 1 year fully transparent, substantially achieves essentially identical with people's corneal transplantation effect, and correct vision is up to 1.0.
Test proves, for transplanting cornea product, it is most important that " transparent " this most basic performance.The invention proposes the technical indicators of cornea product, make it on the basis of with preferable transparency, guarantee the mass production of product, meet clinical demand.
Embodiment 2
In the present embodiment, the dry cornea of de- cell pig plate layer is only made of the bowman's lamina of porcine cornea and hypothallus using fresh porcine cornea as raw material, does not include epithelial layer, descemet's membrane and endodermis.By all de- cell preparation process, the regularly arranged structure of collagenous fibres is still maintained in hypothallus；Specifically in the present embodiment 2, before terminal sterilization processing, light transmittance measured by the dry cornea of plate layer when moisture content is about 5% is 83 ± 5%.The transmission electron microscope observing of the dry cornea of the plate layer provided in the present embodiment 2, type i collagen aligned gaps are uniform in corneal extracellular matrix, type i collagen sharpness of border in corneal extracellular matrix, cross section size is almost the same, and average headway is about 25 ± 10nm and this collagen arrangement architecture and people's cornea, the collagen arrangement architecture of the porcine cornea handled in Figure 1B without de- cell in Figure 1A are substantially similar.Test result is aobvious
Show, in the state that moisture content is close to 5%, the brittleness of cornea slightly increases the dry cornea of the present embodiment 2.Measured cornea light transmittance is 73% ± 2% after using 25kgy to irradiate terminal sterilization.
The present embodiment 2 in, in carrying out lamellar cornea preparation, removal epithelial layer, descemet's membrane and endodermis retain bowman's lamina and produce 600 ± 50um of hypothallus thickness of fresh cornea and collectively form lamellar cornea structure.
In the present embodiment 2, the drying lamina elastica corneae anterior surface is smooth, the ridge shape protrusion or microscopic folds of no macroscopic.
DNA remains 86ng/mg in hypothallus after de- cell in the present embodiment 2.
One optional embodiment of the present embodiment 2 is that the dry cornea is externally provided with the sealed package that can be played a supporting role, so that the dry cornea of plate layer of the invention is maintained at solid product form, is convenient for storage and transport.
The detection method of light transmittance described in the present embodiment 2 is substantially the same manner as Example 1, and so it will not be repeated.The application method of the dry cornea of de- cell pig plate layer of the present invention, opens sealed package in the preoperative；Dry cornea is taken out, transfer operation can be carried out by being put into after impregnating 25 minutes in physiological saline.
Embodiment 3
In the present embodiment, the dry cornea of de- cell pig plate layer is only made of the bowman's lamina of porcine cornea and hypothallus using fresh porcine cornea as raw material, does not include epithelial layer, descemet's membrane and endodermis.The regularly arranged structure of collagenous fibres is still maintained in hypothallus；Specifically in the present embodiment 3, before terminal sterilization processing, light transmittance measured by the dry cornea of plate layer when moisture content is about 20% is 85% ± 5%.The transmission electron microscope observing of the dry cornea of the plate layer provided in the present embodiment 3, type i collagen aligned gaps are uniform in corneal extracellular matrix, type i collagen sharpness of border in corneal extracellular matrix, cross section size is almost the same, average headway be about the arrangement of its collagen of 25 ± 10nm structure and people's cornea in Figure 1A, the arrangement of collagen fibers structure of the porcine cornea handled without de- cell in Figure 1B it is substantially similar.Test result shows, for the dry cornea of the present embodiment 3 in the state that moisture content is close to 20%, lamellar cornea has preferable toughness.Measured cornea light transmittance is 78% ± 2% after using 25kgy to irradiate terminal sterilization.
In the present embodiment 3, in carrying out lamellar cornea preparation, removal epithelial layer, descemet's membrane and endodermis, reservation bowman's lamina and the hypothallus thickness 600um ± 50um for producing fresh cornea collectively form lamellar cornea structure.
In this implementation 3, the drying lamina elastica corneae anterior surface is smooth, the ridge shape protrusion or microscopic folds of no macroscopic.
DNA remains 56ng/mg in hypothallus after de- cell in the present embodiment 3.
One optional embodiment of the present embodiment 3 is that the dry cornea is placed in the sealed package that is equipped with and can play a supporting role, so that the dry cornea of plate layer of the invention keeps solid product form, is convenient for storage and transport.
The detection method of light transmittance described in the present embodiment 3 is substantially the same manner as Example 1, and so it will not be repeated.
The application method that the dry cornea of cell pig plate layer is taken off described in the present embodiment, opens airtight package in the preoperative；Dry cornea is taken out, transfer operation can be carried out by being put into after impregnating 20~30 minutes in physiological saline.
The dry cornea of plate layer described in the above embodiment of the present invention is obtained using different preparation methods.From the point of view of the effect of test result and clinical transplantation art, reach an optimal combination scheme in the necessary characteristic that drying cornea of the invention can be required in corneal transplantations clinics such as biology, physics and its product form, realizes the specifications of quality of the dry cornea product of de- cell pig.Ensuring that cornea product quality is identical, properties of product properly select or combine under the same conditions different preparation methods and are very important.
The dry cornea of de- cell pig plate layer of the present invention corneal transplantation suitable for applying.
The dry cornea of de- cell pig plate layer of the present invention is performed the operation in refractive correction to be applied.The dry cornea of plate layer of the present invention has preferable machinability, in refractive correction operation consent, can carry out Precision Machining to dry cornea according to the demand of diopter, reach required power prescription.
For explaining in detail for the respective embodiments described above, its purpose, which is only that, explains the present invention, in order to better understood when the present invention, but, these descriptions cannot be with any explanation at being limitation of the present invention, especially, the each feature described in various embodiments can also mutual any combination, to form other embodiments, in addition to there is clearly opposite description, these features should be understood to can be applied in any one embodiment, and be not limited merely to described embodiment.
[correcting 07.03.2017 according to detailed rules and regulations 26]
Claims (16)
- A kind of [correcting 07.03.2017 according to detailed rules and regulations 26] dry cornea of de- cell pig plate layer, is made of the bowman's lamina of porcine cornea and hypothallus；It is characterized in that, the hypothallus maintains the regularly arranged structure of collagenous fibres；In visible-range, the light transmittance of the dry cornea is not less than 70%；The moisture content of the dry cornea is not more than 20%.
- [correcting 07.03.2017 according to detailed rules and regulations 26] takes off the dry cornea of cell pig plate layer as claimed in claim 1, which is characterized in that the DNA residual of the dry cornea of pig plate layer is no more than 100ng/mg after de- cell.
- The dry cornea of de- cell pig plate layer, feature exist as described in claim 1, and the light transmittance is detected value after terminal sterilization.
- The dry cornea of de- cell pig plate layer as claimed in claim 1, which is characterized in that the dry cornea is externally provided with the airtight package that can be played a supporting role.
- The dry cornea of de- cell pig plate layer as claimed in claim 4, which is characterized in that the airtight package is arranged before or after dry cornea terminal sterilization.
- The dry cornea of de- cell pig plate layer as claimed in claim 1, which is characterized in that hypothallus maintains the regularly arranged structure of collagenous fibres；Refer under transmission electron microscope observing, the average headway of type i collagen is 25nm ± 10nm in the extracellular matrix of dry cornea.
- Such as the dry cornea of the de- cell pig plate layer of claim 1 or 6, which is characterized in that hypothallus keeps original regularly arranged structure of collagenous fibres to refer under transmission electron microscope observing, type i collagen sharpness of border in corneal extracellular matrix, and cross section is in the same size.
- The dry cornea of de- cell pig plate layer as claimed in claim 1, which is characterized in that the drying anterior angle diaphragm plate thickness degree 300um~700um produced.
- In the present invention, the dry cornea people light transmittance selection of the plate layer measures the absorbance value A of more than two different wave lengths, calculates gained according to formula " T=1/10A × 100% ".
- The dry cornea of de- cell pig plate layer as claimed in claim 9, which is characterized in that it is 380-780nm that the light transmittance, which selects measurement wave-length coverage, and measurement step-length is not more than 10nm.
- The dry cornea of de- cell pig plate layer as claimed in claim 1, which is characterized in that the dry cornea at least bowman's lamina surface is smooth, the ridge shape protrusion or microscopic folds of no macroscopic.
- A kind of application method taking off the dry cornea of cell pig plate layer as described in above-mentioned 1 to 10 any one claim, which is characterized in that preoperative opening airtight package；Dry cornea is taken out, transfer operation can be carried out by being put into physiological saline after 15~30 minutes rehydrations.
- Such as the drying cornea application method of claim 12, which is characterized in that the cornea rehydration Best Times are 15~20 minutes.
- A kind of dry cornea of cell pig plate layer that take off as described in above-mentioned 1 to 12 any one claim is applied in corneal graft.
- A kind of [correcting 07.03.2017 according to detailed rules and regulations 26] dry cornea of cell pig plate layer that take off as described in above-mentioned 1 to 12 any one claim is applied in refractive correction operation.
- [correcting 07.03.2017 according to detailed rules and regulations 26] takes off the dry cornea of cell pig plate layer as claimed in claim 15, in refractive correction operation consent, the drying cornea for adapting to diopter or preoperative foundation diopter demand can be selected to process dry cornea according to dioptric demand, reach required power prescription.
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| PCT/CN2016/110462 WO2018107485A1 (en) | 2016-12-16 | 2016-12-16 | Decellularized dried swine lamellar cornea, used method for same, and uses thereof |
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