CN101584883A - Full-implanted artificial corneal stroma and preparation method thereof - Google Patents
Full-implanted artificial corneal stroma and preparation method thereof Download PDFInfo
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- CN101584883A CN101584883A CNA2008100376230A CN200810037623A CN101584883A CN 101584883 A CN101584883 A CN 101584883A CN A2008100376230 A CNA2008100376230 A CN A2008100376230A CN 200810037623 A CN200810037623 A CN 200810037623A CN 101584883 A CN101584883 A CN 101584883A
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Abstract
The invention belongs to the field of biomedical engineering, in particular to a novel full-implanted artificial corneal stroma and a preparation method thereof. The artificial corneal stroma uses free isolated adipose-derived stem cells as seed cells which are inoculated in polylactic acid polyglycolic acid copolymer scaffold materials and can absorb nourishment and excrete waste in a three-dimensional space formed by biomaterial scaffolds, and the suitable scaffold materials not only can maintain the positions and the forms of the seed cells, but also can participate the adjustment and the control of cell differentiation states. Proved by animal experiments, the corneal stroma has favorable biocompatibility, avoids the toxic reaction and the induced exclusive reaction of a traditional corneal stroma to a human body, and can be fully degraded over time after being implanted so that the cornea can recover the transparency, thereby satisfying the requirement of the human body to the vision.
Description
Technical field
The invention belongs to the biomedical engineering field, relate to a kind of novel full-implanted artificial corneal stroma (totally implantable artificial corneal stroma) and preparation method thereof.
Background technology
See because of the patient of keratopathy blindings such as cornea burn, corneal ulcer, corneal leukoma is more clinically.The allogeneic corneal graft is the main and effective and efficient manner of treatment these corneal blindnesses patient.At present, China corneal blindness patient reaches 4,000,000 more than, and along with laser surgery progressively carry out continuous aggravation with aged tendency of population, allosome cornea source is deficient day by day, the ophthalmologist is urgent all the more to the demand of donor's cornea clinically.Practice shows, is that the high-risk corneal graft long-term prognosis of donor is not good with the allosome cornea.It is its main cause that the destruction of eye table local immunity microenvironment is caused the occurred frequently of postoperative transplant rejection.At present, tissue engineering technique successfully is applied to multiple tissue construction and repair deficiency.Therefore, make up have good biological learn active and can satisfy that certain physiological function requires tissue engineering comea have great exploration value and clinical practice application value.In recent years, the engineered artificial cornea that reaches its maturity of technology provides a new thinking for this area.It is to be support with the degradation material, at the external cell culture that carries out, allows cell carry out three dimensional growth, to be divided into multi-layer cellular at material surface and inside, obtains being similar to " the artificial donor's cornea " of donor's cornea at last.
Corneal stroma is the major part that cornea constitutes, and accounts for 90% of corneal thickness.It mainly is made up of keratocyte, collagen fiber and other extracellular matrixs.Under the normal condition, keratocyte remains static, and in addition, the collagen fiber stratification between keratocyte is arranged in parallel, the diameter unanimity, and spacing equates.The distinctive histological structure of corneal stroma is an essential condition of keeping corneal transparency, is that its hetero-organization is irreplaceable.Application organizes engineering structure corneal stroma can be corneal epithelium, endothelial cell growth on the one hand and organizes to provide in the forming process and contains the physiological environment that is necessary somatomedin, signal.On the other hand, it also can be used as the carrier of tissue engineering comea epithelium, endothelium, overcomes the soft directly sutured of epithelial tissue, endothelial tissue quality of In vitro culture, the defective that is difficult to shift.Therefore, the structure of corneal stroma tissue is main task, difficult point and the key point that tissue engineering technique makes up cornea.
Owing to use the auto corneal stromal cell bigger, the probability of using the allosome keratocyte to exist rejection to take place again to the strong side angle membrane damage of individuality.Open up new seed cell source and use it for and make up the difficult problem that corneal stroma is organized just becomes the urgent need solution.Reported successively in the recent period both at home and abroad the outer cell of multiple cornea tissue particularly some adult stem cell may participate in the formation of corneal stroma.Yamagami in 2006 etc. studies show that, have the derived from bone marrow cell in normal person's corneal stroma, although should study the meaning that not clear and definite derived from bone marrow cell exists, have illustrated that at least the derived from bone marrow cell has participated in the formation of corneal stroma.Along with the development of stem-cell research, being that seed cell makes up tissue engineering comea substrate and more and more shows its superiority especially from the body adult stem cell.
(Adult Stem Cells ASCs) is the undifferentiated cell that is present in in the periplasts such as human organism's bone marrow, fat, skin to adult stem cell, and is from the horse's mouth and have to the multiple directed differentiation potential of organizing, and is one of focus of present seed cell research.(Bone Marrow Mesenchymal Stem Cells is the ASCs that is present in the myeloid tissue BMSCs) to bone marrow stroma stem cell, has multidirectional differentiation potential, and the amplification in vitro ability is strong.But BMSCs content in myeloid tissue is few, contain 1 BMSCs in average 100,000 nucleated cell of adult's bone marrow, and along with passage number increase or donor's age increase, it is big that BMSCs becomes gradually, the cell of self renewal reduces, and cloning efficiency reduces, and cell loses multidirectional differentiation potential, therefore, also limited the application of BMSCs.
ADSCs (Adipose-Derived Stem Cells) is the ASCs that is present in the fatty tissue.Widely distributed at human body because of fatty tissue, the source is sufficient, and it is less to the body wound to be obtained from body adipose tissue, and patient is easy to accept, and is ideal seed cell source.Since two thousand one, reports such as Zuk contain a large amount of ADSCs through the aspirate that suction lipectomy obtains, and can be under given conditions to osteoblast, chondrocyte, adipose cell differentiation.And with age growth, cell number does not reduce, and still keeps the potential of multidirectional differentiation.ADSC not only has similar to the bone marrow stroma stem cell abilities to the many differentiation of cells such as skeletonization, cartilage, fat, muscle and nerve, and expression surface marker such as CD29, CD105, the CD106 identical with MSC, reaches CD166 etc.At present do not see with the fat stem cell to be the report that seed cell is used to make up the artificial cornea as yet both at home and abroad.
The core of organizational project is a simulation body normal structure, sets up cell and biological support complex external, and uses it for tissue or the organ that substitutes body disease.This shows that good cell epimatrix substitute is that timbering material also is the important component part that makes up engineered corneal stroma.Suitable timbering material can be kept position, the form of seed cell, also can participate in the regulation and control of cell differentiation state.Meanwhile, seed cell can absorb nourishment in the formed three dimensions of biological support, drains refuse.Generally speaking, the timbering material of ideal tissue engineering comea should possess following characteristics: excellent biological compatibility, and avoid the toxic reaction of body and bring out rejection; Have certain tenacity, can satisfy the needs of three-dimensional stereo forming, return the mechanical force when planting again in the ability receptor; The speed of the degradation rate of timbering material and organism normal cell or seed cell synthetic cell epimatrix is complementary, and relies on cambium to repair pathological tissues or organ as far as possible; After being arranged, certain light transmission, particularly absorbed should satisfy the requirement of body to vision as far as possible.
Polylactic acid (PLA), polyglycolic acid (PGA) copolymer p LGA belong to the synthetic degradable high polymer material, have excellent biological compatibility, do not find the reaction of serious type tissue reaction and toxicity in body, and no antigen.
Summary of the invention
The purpose of this invention is to provide a kind of novel full-implanted artificial corneal stroma (totally implantableartificial corneal stroma).
Full-implanted artificial corneal stroma of the present invention can be applied to the cornea tissue of clinical alternative body disease.
Another object of the present invention provides the preparation method of above-mentioned artificial corneal stroma.
The technical scheme of full-implanted artificial corneal stroma of the present invention is: by simulation body normal cornea matrix organization, set up cell and biological support complex external, and use it for the cornea tissue that substitutes body disease.Meanwhile,, when this artificial corneal stroma repair tissue is damaged, progressively improve corneal transparence, finally make corneal transparency, recover patient's vision by the natural degradation characteristic of degradable biomaterial.
Full-implanted artificial corneal stroma of the present invention mainly is made up of seed cell and suitable timbering material.
Described seed cell adopts fat stem cell, be inoculated in timbering material, can in the formed three dimensions of biological support, can absorb nourishment, drain refuse, described fat stem cell is that described cell inoculation density is 3～5 * 10 from the stripped fat stem cell of body
6/ cm
3Preferred 4 * 10
6/ cm
3Described suitable timbering material adopts the polylactic acid poly co-glycolic acid, wherein the mass ratio of polylactic acid and polyglycolic acid is 65～75: 35～25, preferred 70 to 30, this timbering material can be kept position, the form of seed cell, also can participate in the regulation and control of cell differentiation state.
Full-implanted artificial corneal stroma of the present invention adopts stripped autologous fat stem cell and the polylactic acid poly co-glycolic acid complex with good biocompatibility, has avoided traditional artificial corneal stroma to the toxic reaction of body and bring out rejection; This complex has certain tenacity, can satisfy the needs of three-dimensional stereo forming, returns the mechanical force when planting again in the ability receptor; In addition, the speed of the scaffold degradation speed of this complex and organism normal cell or seed cell synthetic cell epimatrix is complementary, and can rely on cambium to repair pathological tissues or organ; The result proves through zoopery, and after this complex implanted as time passes degraded fully, it is transparent that cornea can recover, thereby satisfy the requirement of body to vision.
Technical scheme of the present invention realizes by following step:
1. preparation timbering material
At first polylactic acid and polyglycolic acid are dissolved in 1 with certain proportioning, 4 dioxane are configured to 10% solution, the NaCl granule that in solution, adds certain proportion, selected particle diameter then, and stirring makes even suspension on magnetic stirrer, suspension is splashed into surface plate, and curtain coating becomes the PLGA film, behind the natural drying material film is taken out from surface plate, use distilled water immersion, take out material film at last, respectively soak with standby with PBS and culture medium respectively after the drying;
Timbering material of the present invention, wherein polylactic acid is 65～75 to 35～25 with the polyglycolic acid mass ratio, returns in animal body and plants experimental verification, this timbering material has better biocompatibility and suitable vivo degradation speed.
2. select the stripped autologous fat stem cell concentration of inoculation
ADSCs is inoculated in respectively by different cell concentrations makes the cell material complex on the timbering material, each 5; After in incubator, placing certain hour, the digestion collecting cell, the calculating instrument counting draws the cell concentration of loss; The cell material complex was cultivated after 24 hours, the cell material complex is rocked in culture fluid, and change to another culture dish cultivation, collect culture dish culture fluid and the adherent cell of digestion collection, cell in the original fluid is counted in the lump, drawn not adherent cell number;
Cell adhesion rate=(cell number of the cell number-loss of inoculation-not adherent cell number)/(cell number of the cell number-loss of inoculation).Experimental result shows 3～5 * 10
6/ cm
3Cell inoculation density in the scope has higher material adherence rate and cell adhesion amount.
3. preparation full-implanted artificial corneal stroma
DMEM with serum-free adjusts cell concentration to 1 * 10
6/ ml, every ml cells suspension adds the DiO reagent of 5 microlitres, mix homogeneously is put into incubator and is hatched, and removes supernatant, repeat to wash twice back the good cell of labelling by 4 * 10
6/ cm
3Be seeded on the PLGA material, standby after 1 week of cultivation in incubator.
4. full-implanted artificial corneal stroma uses experiment
With the rabbit autologous fat stem cell of external structure and PLGA material tissue engineering complex Hui Zhi to rabbit corneal substrate: get healthy purebred new zealand white rabbit, after intramuscular injection ketamine and the diazepam anesthesia, change the auxiliary corneal epithelium substrate lobe of making down at ring, about 1/2 corneal thickness, on the plant bed that exposes, excise the certain diameter corneal stroma then, again above-mentioned rabbit autologous fat stem cell and the engineered complex of PLGA corneal stroma are placed between corneal lamellar, sew up with section between the nylon wire of 10-0 at last, 3 weeks were used in the complete 0.3% tobramycin/0.1% dexamethasone eye drop part of giving of art.The result shows that can degrade fully as time passes after above-mentioned artificial corneal stroma implants, it is transparent that cornea also progressively recovers, and repairing corneal substrate is damaged better, satisfies the requirement of body to vision.
The advantage of the artificial corneal stroma of the present invention is:
1, traditional artificial corneal stroma is to use keratocyte as seed cell, because this cell is bigger to the strong side angle membrane damage of individuality if derive from the auto corneal stromal cell; If derive from the probability that the allosome keratocyte then exists rejection to take place; The artificial corneal stroma of the present invention adopts fat stem cell as seed cell: fatty tissue is widely distributed at human body, and the source is sufficient, and it is less to the body wound to be obtained from body adipose tissue, and the patient is easy to accept, and is ideal seed cell source; Studies confirm that fat stem cell can be under given conditions to osteoblast, chondrocyte, adipose cell differentiation; Fatty tissue is with age growth, and cell number does not reduce, and still keeps the potential of multidirectional differentiation.
2, traditional corneal stroma stent comprises collagen and the gelatin that extracts from animal, as timbering material, they are very easily degraded by the effect of collagenase in vivo on the one hand, bring out the cornea rejection in addition easily, and not satisfactory and poor stability is not suitable for transplant operation with the corneal epithelial cell compatibility.The polylactic acid of the extra fine quality ratio in the artificial corneal stroma of the present invention and the copolymer of polyglycolic acid can be processed into various planforms on request, and simultaneously they have excellent biological compatibility and catabolite is nontoxic.
3, the artificial corneal stroma of the inventive method structure only need excision pathological changes corneal stroma earlier during use, and then with this complex Hui Zhi, operation is easy to grasp, and the conventional antiinflammatory of postoperative is handled, and nursing is convenient.
Description of drawings
The former generation rADSC of Fig. 1 cultivates 72h, inverted microscope * 40.
Fig. 2 rADSC cultivates 96h, inverted microscope * 40.
Fig. 3 PLGA material is seen substantially.
Fig. 4 PLGA material electron-microscope scanning figure.
Fig. 5 fluorescence inverted microscope is observed the adhesion growing state of DiO labelling ADSCs in material,
Wherein, A, cell material complex In vitro culture 1 day, * 100;
B, cell material complex In vitro culture 1 day, * 200;
C, cell material complex In vitro culture 7 days, * 100;
D, cell material complex In vitro culture 7 days, * 200.
Fig. 6 scanning electric mirror observing cell material composite,
Wherein, rADSCs is at the material surface well-grown for the A diagram, and visible cell is evenly distributed, and * 50,
B diagram cellular morphology is fiber-like, sees through the visible below of intercellular substance PLGA fiber, * 300.Fig. 7 two-photon microscope observing cell material composite,
Wherein, A, cell material complex sketch map; B, two-photon microscope choose the imaging of 30um tomoscan, and visible cell is evenly distributed; C, the simple cell scanning imagery; D, simple scanning of materials imaging; E, the video picture altogether of cell material complex.
Fig. 8 slit lamp observation corneal stroma is repaired situation.
Fig. 9 HE dyeing viewing angle membrane matrix obturator overview.
The burnt corneal microscope of Figure 10 live body copolymerization dynamic observes cornea, and scale is 50um.
Figure 11 immunohistochemical detection Vimentin and smooth muscle actin express * 100.
Figure 12 transmission electron microscope observing corneal stroma fiber alignment direction and size,
Wherein, A is a normal cornea substrate, and B is the complex group.
The specific embodiment
The external preparation and the detection of embodiment 1 full-implanted artificial corneal stroma
1, experimental apparatus and reagent 6 month female new zealand white rabbits, body weight 2～2.5kg (the Shanghai City Chinese science grinds the zooscopy center).DMEM (Dulbeco ' smodified eagle ' smedium) powder is a U.S. GIBCO company product; The type i collagen enzyme is a U.S. Worthington company product; (Fetal Bovine Serum FBS) is U.S. Hyclone company product to hyclone; Dexamethasone and transferrins are U.S. Sigma company product; L-glutaminate, ascorbic acid and trypsin are the Shanghai Bioisystech Co., Ltd's product that grows directly from seeds; Penicillin and streptomycin are North China pharmaceutical Co. Ltd product; NaHCO
3Be last marine rainbow photoinitiator chemical factory product.LSM-510 two-photon microscope is available from German Zeiss company.The JSM-6701F scanning electron microscope is available from Japanese Jeo company.
2, rabbit fat fat stem cell former is commissioned to train and supports and go down to posterity by the stable intramuscular injection anesthesia of 1mg/kg ketamine associating 0.5mg/kg.Treating excess syndrome is tested the about 2～3g of animal rabbit subcutaneus adipose tissue, and fatty tissue is packed under aseptic condition in the 10ml DMEM culture fluid 50ml centrifuge tube that contains 5%FBS; With streptomycin solution and PBS; Wash rabbit fat fat tissue repeatedly; Fatty tissue is put in the culture dish, shred; The fatty tissue that shreds places the 50ml centrifuge tube, adds 0.075%I Collagen Type VI enzymatic solution 40ml, at 37 ℃ of constant temperature digestion 30min, the centrifugal 10min of 1500rpm, remove supernatant, obtain highdensity cell precipitation thing, add DMEM culture fluid+10%FBS culture fluid and make cell resuspended; By 10
6/ ml cell concentration is inoculated in culture dish, adds culture fluid and puts 37 ℃, 5%CO to 8ml
2, 100% saturated humidity condition under, cultivate and change liquid first after 48～72 hours; Inverted phase contrast microscope is observed down a large amount of cell attachments and buoyant hemocyte, washes repeatedly with PBS and removes buoyant cell, adds the DMEM+10%FBS culture fluid; Change liquid every other day, cell fusion to 80% goes down to posterity; Wash with PBS before going down to posterity, add 0.25% trypsin and 0.02%EDTA 2ml, observe most of cell cytoplasm retraction, form change circle, add the DMEM culture fluid termination digestion that 2ml contains serum, collecting cell suspension, counting are with 0.5 * 10
6/ cm
2Cell concentration is inoculated in the new culture dish; Cell reaches nearly fusion state after 3～4 days, goes down to posterity once more; Inverted microscope observation of cell form is taken pictures.
3, preparation PLGA timbering material, at first with PLGA material (LA/GA=70/30, Mw=104900) be dissolved in 1,4 dioxane are configured to the solution of 10% (mass volume ratio), the NaCl granule that in solution, adds certain proportion, selected particle diameter then, magnetic agitation makes even suspension, and suspension is splashed into surface plate, and curtain coating to become thickness be the PLGA film of 20um; Natural drying 48h behind the vacuum drying 24h, takes out the material film that makes from surface plate, distilled water immersion 48h changes distilled water when 8h; Take out material film, respectively soak 1d with PBS and culture medium respectively after the drying, standby.
4, fat stem cell is inoculated in the PLGA material, and the cell that step 1 is made is by 4 * 10
6/ cm
3Be inoculated on the PLGA material of 5mm * 5mm * 1mm; Inverted fluorescence microscope observation of cell form is taken pictures.
5, the result shows:
(1) it is adherent that ADSCs is inoculated in behind the culture dish 6h beginning, 24h～48h inner cell poor growth; Behind the 72h, have cell clone to form, cell stretches and is the fibroblast sample, and growth has directivity (Fig. 1); But P1 for cell after observation of cell still be fibroblast sample (Fig. 2).
(2) PLGA timbering material gross examination of skeletal muscle, the thick 1mm of material (Fig. 3); Scanning electron microscope inspection prompting PLGA material evenly spreads out and forms cavernous space (Fig. 4).
(3) ADSCs of DiO labelling and PLGA material composite are observed under laser confocal microscope, and the cell of In vitro culture visible green fluorescence after 1 day evenly adheres to (Fig. 5 A) on the PLGA material; Cellular morphology is fiber-like or ovum circle shape (Fig. 5 B); Increase when cell concentration was obviously than the 1st day when the cell material complex was cultured to the 7th day altogether, most of regional cell fusion is in blocks, visible obviously green fluorescence (Fig. 5 C); Cellular morphology is based on ovum circle shape (Fig. 5 D).
(4) after the cell material complex cultivated for 1 week, cell and material adhesion were good, and emiocytosis substrate is more vigorous, with PLGA fiber fuse (Fig. 6).
(5) the microscopical tomoscan of two-photon, a certain tomography of visible cell material composite, and carry out three-dimensional imaging; Visible cell evenly attaches to material internal after cultivating for 1 week, and cellular morphology is to become fiber-like (Fig. 7).
(animal) experiment in the body of embodiment 2 full-implanted artificial corneal stromas
1, experimental apparatus 6 month female new zealand white rabbits, body weight 2～2.5kg (the Shanghai City Chinese science grinds the zooscopy center).DMEM (Dulbeco ' s modified eagle ' s medium) powder is a U.S. GIBCO company product; The type i collagen enzyme is a U.S. Worthington company product; (Fetal Bovine Serum FBS) is U.S. Hyclone company product to hyclone; Dexamethasone and transferrins are U.S. Sigma company product; L-glutaminate, ascorbic acid and trypsin are the Shanghai Bioisystech Co., Ltd's product that grows directly from seeds; Penicillin and streptomycin are North China pharmaceutical Co. Ltd product; NaHCO
3Be last marine rainbow photoinitiator chemical factory product; The Vimentin monoclonal antibody is available from U.S. Lab Vision company; The smooth muscle protein monoclonal antibody is available from U.S. Lab Vision company; 0.3% tobramycin/0.1% dexamethasone eye drop is available from U.S. Alcon company; 0.4% times of promise happiness is available from the towering drugmaker of Japan.LSM-510 two-photon microscope is available from German Zeiss company.The JSM-6701F scanning electron microscope is available from Japanese Jeo company.Medical material commonly used and apparatus are commercial.
2, transplantation experiments in fat stem cell and the PLGA composite body
With the experimental rabbit autologous fat stem cell of external structure and PLGA material tissue engineering complex Hui Zhi to rabbit corneal substrate: get healthy purebred new zealand white rabbit, after the ketamine of intramuscular injection 15mg/kg and the anesthesia of the diazepam of 4mg/kg, changeing the auxiliary diameter of making down at ring is the corneal epithelium substrate lobe of 8mm, about 1/2 corneal thickness, the excision diameter is the corneal stroma of 6.5mm on the plant bed that exposes, the above-mentioned stripped rabbit autologous fat stem cell and the engineered complex of PLGA corneal stroma that with diameter are 7mm again are placed between corneal lamellar, sew up with section between the nylon wire of 10-0 at last; Art finishes gives local 3 weeks of use of 0.3% tobramycin/0.1% dexamethasone eye drop.Operation back in 1 week every day examination with slitlamp microscope, slit lamp is taken pictures when 3,6 and 9 weeks.
3, the result shows:
(1) after the transplant operation, cell and material composite group and simple material group occupy cornea central authorities, are opaque white color, the beginning of the 3rd week, material degradation quickens, central cornea is organized and is recovered transparent gradually, and during the 6th week, the visible material degraded obviously, but still have small quantity of material residual, so show as corneal nebula, during the 9th week, cornea central authorities substantially transparent (Fig. 8).
(2) 9 all cornea HE stained (Fig. 9) show after the transplant operation, and complex group cambium mesostroma cellular morphology is rule comparatively, and normal cornea substrate is bigger relatively on every side for cell; Newborn collagen is arranged comparatively rule in the graft area, with be connected between adjacent collagen closely, do not see tangible demarcation line (Figure 10 A) between the two, normal cornea substrate (Figure 10 B) is approaching, show as collagenous fiber bundle queueing discipline, neat, keratocyte is evenly distributed; Measure the paraffin section corneal thickness, the complex group is (231.0 ± 2.9) mm, and normal cornea matrigel fibril diameter is (29.2 ± 2.9) nm; Through rank test method statistical analysis, Z
Complexes=0.286, P=0.775, the two no difference of science of statistics.
(3) the burnt corneal microscope of live body copolymerization dynamic observes each experimental group cornea: normal top layer epithelial cell has bright, intermediate light and three kinds of dark performances, epidermis cell is arranged loose, be polygon, based on hexagon, bright nucleus is visible or invisible; The epithelial layer of experimental group does not have obviously unusual (Figure 10 A), visible keratocyte nuclear bright under dark relatively background in the hypothallus, separated by background material, can not observe endochylema, cell boundaries and collagen layer, repair near visible obviously nerve fiber growth (shown in Figure 11 B arrow) district; Each experimental group endodermis all shows as the hexagonal mosaics of rule, and cell boundaries is black, and cell space is bright, does not see nucleus (Figure 11 C).
(4) immunohistochemistry detects Vimentin and Smooth Muscle Actin protein expression: all express negative (Figure 11) as the Vimentin of fibroblast labelling and the Smooth muscle actin of fibroblast activation migration labelling in each group corneal stroma, the reparation of prompting corneal stroma is good.
(5) aspect arrangement of collagen fibers, each experimental group corneal collagen arrangement disorder has obviously different (Figure 12) with the normal cornea collagen bundle of marshalling, rule; At the collagen fiber diametrically, normal cornea matrigel fibril diameter is 29.2 ± 2.9nm (Figure 12 .A); The complex group is 28.5 ± 2.9nm (Figure 12 .B), and the statistical value of experimental group collagen fiber diameter and the comparison of normal cornea substrate is respectively Zt
Omplexes=-1.725, P=0.086＞0.05, no difference of science of statistics between experimental group collagen fiber diameter and the normal cornea substrate has confirmed the reason that the damaged back of complex repairing corneal substrate cornea is why transparent.
Claims (6)
1, full-implanted artificial corneal stroma is characterized in that being made up of seed cell and timbering material, and described seed cell is inoculated in described timbering material, and described seed cell is selected from from the stripped fat stem cell of body, and cell inoculation density is 3～5 * 10
6/ cm
3Described timbering material adopts the polylactic acid poly co-glycolic acid, and wherein the mass ratio of polylactic acid and polyglycolic acid is 65～75: 35～25.
2, by the described full-implanted artificial corneal stroma of claim 1, it is characterized in that described seed cell inoculum density is 4 * 10
6/ cm
3Described timbering material, wherein polylactic acid is 70 to 30 with the mass ratio of polyglycolic acid.
3, by the preparation method of the described full-implanted artificial corneal stroma of claim 1, it is characterized in that comprising the steps:
(1) preparation timbering material
Polylactic acid and polyglycolic acid are dissolved in 10% solution that 1,4 dioxane is made into by mass ratio; In solution, add the stirring of NaCl granule and make even suspension, suspension is splashed into surface plate, and curtain coating becomes the PLGA film; Dry back distilled water immersion is again after the drying, respectively with PBS and culture medium immersion, standby;
(2) select inoculating cell concentration
The stripped fat stem cell of variable concentrations is inoculated in respectively on the timbering material, places, digest collecting cell in the incubator, get the drain cell amount; Cultivate after 24 hours, the cell material complex is rocked and changes the culture dish cultivation in culture fluid, collect culture fluid and the adherent cell of digestion collection, cell in the original fluid is counted in the lump, get not adherent cell number and cell adhesion rate;
(3) preparation full-implanted artificial corneal stroma
DMEM with serum-free adjusts cell concentration to 1 * 10
6/ ml, cell suspension add the DiO reagent mix, put into incubator and hatch, and remove supernatant, behind the repeated washing, the cell of labelling are pressed 3～5 * 10
6/ cm
3Be seeded on the timbering material, standby after 1 week of cultivation.
4, by the method for claim 3, it is characterized in that the polylactic acid of described step 1 and polyglycolic acid mass ratio are 65～75 to 35～25.
5,, it is characterized in that cell adhesion rate=(cell number of the cell number-loss of inoculation-not adherent cell number)/(cell number of the cell number-loss of inoculation) of described step 2 by the method for claim 3.
6, by the method for claim 3, the cell inoculation density that it is characterized in that described step 2 is 3～5 * 10
6/ cm
3
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105641750A (en) * | 2016-02-22 | 2016-06-08 | 中山大学中山眼科中心 | Bioengineering retinal nerve scaffold with cell tracing function and preparation method of bioengineering retinal nerve scaffold |
| CN113425909A (en) * | 2021-06-30 | 2021-09-24 | 上海交通大学医学院附属第九人民医院 | Biological material for repairing corneal injury and preparation method and application thereof |
-
2008
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105641750A (en) * | 2016-02-22 | 2016-06-08 | 中山大学中山眼科中心 | Bioengineering retinal nerve scaffold with cell tracing function and preparation method of bioengineering retinal nerve scaffold |
| CN113425909A (en) * | 2021-06-30 | 2021-09-24 | 上海交通大学医学院附属第九人民医院 | Biological material for repairing corneal injury and preparation method and application thereof |
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