CN105597145A - Retina cell scaffold biological surgical binder and preparing method thereof - Google Patents
Retina cell scaffold biological surgical binder and preparing method thereof Download PDFInfo
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- CN105597145A CN105597145A CN201610097502.XA CN201610097502A CN105597145A CN 105597145 A CN105597145 A CN 105597145A CN 201610097502 A CN201610097502 A CN 201610097502A CN 105597145 A CN105597145 A CN 105597145A
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L24/00—Surgical adhesives or cements; Adhesives for colostomy devices
- A61L24/04—Surgical adhesives or cements; Adhesives for colostomy devices containing macromolecular materials
- A61L24/10—Polypeptides; Proteins
- A61L24/102—Collagen
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L24/00—Surgical adhesives or cements; Adhesives for colostomy devices
- A61L24/001—Use of materials characterised by their function or physical properties
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L24/00—Surgical adhesives or cements; Adhesives for colostomy devices
- A61L24/001—Use of materials characterised by their function or physical properties
- A61L24/0015—Medicaments; Biocides
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L24/00—Surgical adhesives or cements; Adhesives for colostomy devices
- A61L24/04—Surgical adhesives or cements; Adhesives for colostomy devices containing macromolecular materials
- A61L24/10—Polypeptides; Proteins
- A61L24/108—Specific proteins or polypeptides not covered by groups A61L24/102 - A61L24/106
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
- A61L2300/412—Tissue-regenerating or healing or proliferative agents
- A61L2300/414—Growth factors
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Abstract
The invention discloses a retina cell scaffold biological surgical binder and a preparing method thereof. The retina cell scaffold biological surgical binder comprises laminin, type-IV collagen, nidogen, human heparin sulphate protoglycans, fibroblast growth factors, matrix metalloproteinase and phosphate buffer salt solution. By the adoption of the retina cell scaffold biological surgical binder, degradable human iPSCs-derived retina nerve scaffold (iRMP) can be bound with a host retina nerve layer, and effective integration of a cell biological scaffold and a host retina is guaranteed. The tissue engineering technology and clinical practice are combined, and the retina cell scaffold biological surgical binder has certain theoretical and practical application value for regenerative medicine fundamental research and ophthalmology clinical treatment, promotes optic nerve system damage repair and nervous tissue engineering research, and lays a theoretical and experimental foundation for tissue engineering transplantation therapy of visual damage.
Description
Technical field:
The invention belongs to field of tissue engineering technology, be specifically related to biological surgical adhesive of a kind of retina cell's support and preparation method thereof.
Background technology:
The injuring and repairing of retinal neurons is that common problem in science occurs, develops and lapses to nerve impaired vision. In recent yearsAlong with developing rapidly of stem-cell research, induced multi-potent stem cells (inducedpluripotentstemcells, iPSCs) is optic nerveThe reparation of damage has brought new hope with treatment. Due to iPSCs self-replacation and many differentiation potentials, as repairing nervous systemThe donor of damage not only can meet cell concentration required while transplanting, and iPSCs can be induced to differentiate into and comprise god under certain conditionThrough unit at interior different neuronal cell line. Therefore, utilize humanized iPSCs transplantation treatment optic nerve damage disease to have heavilyThe theory significance of wanting and good clinical treatment prospect. At present, iPSCs replacement therapy is mainly by following two approach: 1. dry thinBorn of the same parents are implanted into subretinal space/vitreous chamber; 2. stem cell in vitro is divided into retinal neurons and is implanted into subretinal space/vitreumChamber. Research finds iPSCs cell to be grafted directly to after impaired retinal tissue, and more than 90% cell is all divided into starSpongiocyte, does not almost have neuronic; And the neuron of application iPSCs induction differentiation and host's retina conformability arePoor. Therefore, how by people iPSCs source property retina cell in vitro culture, and it is transplanted preferably and is integrated into impaired nethike embrane,That current stem-cell therapy, medical tissue engineering are learned and the common focus of paying close attention in ophthalmology field.
Summary of the invention:
The object of this invention is to provide a kind of can be by degradable people iPSCs source property retina neural support (iRMP) success of transplantingAdhere to host's cerebral stratum of retina, ensure the biological surgical adhesive of retina cell's support that cytoskeleton intraocular is effectively transplanted andIts preparation method.
The biological surgical adhesive of retina cell's support of the present invention, is characterized in that, comprise laminin, type Ⅳ collagen,Nestin, people's heparin sulfate glycoprotein, fibroblast growth factor, matrix metalloproteinase and PBS.
Preferably, the biological surgical adhesive of described retina cell's support, every milliliter contains laminin 30ng, type Ⅳ collagen30ng, nestin 10ng, people's heparin sulfate glycoprotein 10ng, desmocyte growth factor-21 0ng and matrix metalloproteinase10ng, the PBS that surplus is pH=7.
The preparation method of the biological surgical adhesive of retina cell's support of the present invention is: under 4 DEG C of environment, according to each componentContent, by laminin, type Ⅳ collagen, nestin, people's heparin sulfate glycoprotein, fibroblast growth factor and matrixMetalloproteinases joins in the PBS of pH=7, obtains the biological operation of retina cell's support sticky after mixingMixture.
The present invention has prepared the biological surgical adhesive of retina cell's support, utilizes it can be by degradable people iPSCs source property retinaNerve Scaffold (iRMP) and host's cerebral stratum of retina are bonding, provide guarantor for cell biological support and host's retina effectively integrateCard. The present invention combines tissue engineering technique with clinical practice, the basic research to regenerative medicine and ophthalmology clinical treatment are equalHave certain theory and actual application value, the research that the wound repair to optic nerve system and neural tissue engineering are learned also producesPositive impetus, for organizational project transplantation treatment impaired vision has been established theory and experiment basis.
Brief description of the drawings:
Fig. 1 is that Electronic Speculum is shown PLGA support aperture 50 μ m, thickness 60 μ m;
Fig. 2 is eye transplanting/co-culture of cells support in Matrigel-PLGA, diameter 4mm, and surface area is 12.56mm2；
Fig. 3 is that seed cell eye transplanting/co-culture of cells support in Matrigel-PLGA is cultivated visible cell appearance around after 7 days altogetherLonger projection, similar neuronal cell in form;
Fig. 4 is that in Matrigel-PLGA, eye transplanting/co-culture of cells support epineural axon growth is significantly better than control group, wherein PLGARepresent of the present invention seed cell to be seeded in Matrigel-PLGA on eye transplantings/co-culture of cells support in induction differentiation cultivationIn base, cultivate, Coverslip represents seed cell to be seeded on slide and to cultivate in inductive differentiation medium, Transwell tableShow seed cell is seeded on polycarbonate membrane and is cultivated in inductive differentiation medium;
Fig. 5 is the cell antibody Brn3b (A) of iRMP growth after 14 days, HuD (B) and Tju1 (C) immunofluorescence dyeing;
Fig. 6 is the action potential result that application patch clamp technique detects iRMP;
Fig. 7 is that applying biological surgical adhesive is by sticky to degradable people iPSCs source property retina neural support (iRMP) and host's retinaThe design drawing closing. IRMP is implanted to host eye, use biological surgical adhesive iRMP and retina top layer can be adhered to shapeBecome iRP-host's neural retina composite bed;
Fig. 8 is that experiment in vitro proves that this iRP-glue can effective bonding degradable people iPSCs source property retina neural support (iRMP)(A), 24h artifact surgical adhesive degraded (B), is used CCK-8 experiment, and spectrophotometric value shows biological surgical adhesive(iRP-glue) catabolite is to cell nontoxicity (C);
Fig. 9 uses iRP-glue that degradable people iPSCs source property retina neural support (iRMP) is adhered to rhesus macaque retina godThrough layer, form iRMP-neural retina composite bed;
Figure 10 is that eye-ground photography (A) shows that iRMP and host's retina successfully stick;
Figure 11 is that OCT (B) shows that iRMP and host's retina successfully stick;
Figure 12 is that eyeball anatomical slice (C) shows that iRMP and host's retina successfully stick.
Detailed description of the invention:
Following examples are to further illustrate of the present invention, instead of limitation of the present invention.
Embodiment 1: the preparation of biological surgical adhesive (iRP-glue):
The biological surgical adhesive (iRP-glue) of the present embodiment, every milliliter contains laminin 30ng (30%), type Ⅳ collagen30ng (30%), nestin 10ng (10%), people's heparin sulfate glycoprotein 10ng (10%), fibroblast growth factor10ng (10%) and matrix metalloproteinase (gelatinase) 10ng (10%), surplus is PBS (PBS, PH=7).The preparation method of the biological surgical adhesive of its 1ml is: under 4 DEG C of environment, according to the content of each component, by laminin 30ng,Type Ⅳ collagen 30ng, nestin 10ng, people's heparin sulfate glycoprotein 10ng, desmocyte growth factor-21 0ng and gelatinase10ng, joins the PBS of the pH=7 of 1ml, obtains biological surgical adhesive (iRP-glue), and it is effectively stickyClosing concentration is 10mg/ml, and this biology surgical adhesive, at 10 DEG C, glue freezing action occurs. This iRP-glue can protect at-20 DEG CDeposit, structure and physical characteristic that under normal body temperature condition, this biology surgical adhesive can analogue body inner cell basilar memebrane, form toolThere is the three dimensional matrix of BA.
Embodiment 2: the effect experiment of biological surgical adhesive (iRP-glue):
One, the preparation of degradable people iPSCs source property retina neural support (iRMP)
1, obtain degradable poly lactide copolymerization glycolide (PLGA) support:
PLGA (mol ratio 50:50, molecular weight is 80kg/mol) is dissolved in carrene, then joins 1,1,1,3,3,3-In hexafluoroisopropanol, in 25 DEG C of environment, stir PLGA polymer dissolution is mixed with in HFIPConcentration is spinning (polymer solution, the electrostatic spinning parameter: polymer solution flow velocity is 0.8-1.0ml/h, spinning electricity of 50wt%Press as 20kv, spinning is apart from 100mm); Polymer solution is packed in the syringe of 10ml into entry needle internal diameter 0.4mm;Syringe is connected with syringe pump, and receiving end device is for being coated with tetrafluoroethylene fiber film, and the rotating cylinder of rotating speed 4000-5500rpm is (straightFootpath 100mm, length 300mm); Support vacuum drying 48h at 25 DEG C thoroughly removes solvent, obtains aperture 50 μ m, thickSpend the PLGA support (Fig. 1) of 60 μ m.
2, make and meet the Matrigel-PLGA co-culture of cells support that interior eye transplanting requires:
Transplant requirement based on interior eye, PLGA support is prepared as to aperture 50 μ m, thickness 60 μ m, diameter 4mm, surface area is 12.56mm2Circular support (Fig. 2), be eye stent graft in PLGA. After 60Coradiation sterilizing, obtain asepticEye stent graft in PLGA; It is 7.2 PBS (PBS) that matrigel Matrigel (BD) is dissolved in to PHIn, obtaining concentration is the Matrigel solvent of mass fraction 10%; 10% Matrigel solvent is coated in to eye in PLGA to be movedPlant on support, under 4 DEG C of conditions, leave standstill 24h, form eye transplanting/co-culture of cells support in Matrigel-PLGA.
3, make degradable people iPSCs source property retina neural support (iRMP):
The way that adopts machinery to separate, by (the concrete preparation method's reference of the 3D retina of the cultivation people iPSc source property of 50-55 daysDocument: XiufengZhong, etal.Generationofthree-dimensionalretinaltissuewithfunct ionalPhotoreceptorsfromhumaniPSCs, NatCommun.2014Jun10; Nerve fibre layer 5:4047) is put intoIn D-Hanks liquid, just putting under microscope and using 0.45mm syringe needle chorista in 50 times, abandoning pigment in retinal tissue and sinkPart (uvea), retain flavous tissue part (nerve fibre layer); The nerve fibre layer tissue of separation is used and moves liquidPipe is transferred to 3.5cm culture dish, uses PBS to rinse 10 minutes; After absorbing PBS, use the digestion of accutase cell dissociation buffer thinBorn of the same parents, are placed in 37 DEG C of incubator 30min; The process later stage can suck cell centrifuge tube piping and druming, is just putting under microscope and is seeing at 10 timesCell mass is digested after unicellular and adds 2ml inductive differentiation medium to stop digestion. By after centrifugal suspension (1000 turn/5min)Sucking-off supernatant, obtains seed cell (precipitation), then uses inductive differentiation medium resuspended, then with 106Individual/mm2Density is inoculated inOn eye transplanting/co-culture of cells support, add inductive differentiation medium in Matrigel-PLGA, made to inoculate seed cellEye transplanting/co-culture of cells support is in inductive differentiation medium in Matrigel-PLGA, 37 DEG C, 5%CO2Under saturated humidity, trainSupport and form degradable people iPSCs source property retina neural support (iRMP). Seed cell 3-5 is as a child adherent, in vitro cultureAfter 24 hours, nerve cell aixs cylinder generates.
Described inductive differentiation medium is: every ml contains bFGF10ng, BDNF20ng, CNTF20ng, N2supplement1ng, nonessential amino acid (essentialmedia-nonessentialaminoacidsNEAAs) 1ng, heparin 2 μ g, surplusFor DMEM/F12 culture medium, described DMEM/F12 culture medium is according to volume by DMEM culture medium and F12 culture mediumMix than 1:1. Its compound method is: DMEM culture medium and F12 culture medium are mixed to shape according to volume ratio 1:1Become DMEM/F12 culture medium, then according to the content of above-mentioned composition, in DMEM/F12 culture medium, add bFGF, BDNF,CNTF, N2supplement, nonessential amino acid and heparin, mix and inductive differentiation medium.
4, the qualification of degradable people iPSCs source property retina neural support (iRMP):
(1) Morphological Identification: the iRMP that cultivates 14d is fixed to 15 minutes with 4% paraformaldehyde, and 0.01% PBS washes 3 timesAfter, 0.1%TritonX-100 is hatched 15 minutes, after 3% donkey serum sealing 30min, adds respectively after certain proportion dilutionGoat-anti rat Brn3b (santacruz), the mouse HuD of the rabbit Chinese People's Anti-Japanese Military and Political College (SantaCruz), it is 24 little that rabbit Chinese People's Anti-Japanese Military and Political College mouse Tju1 (Biolegend) hatchesTime, and add the anti-two marks of immunofluorescence that carry out of corresponding fluorescence two to dye.
(2) Function Identification: (Sigma contains 140mM sodium chloride solution, 5mM potassium chloride by the solution for iRMP of cultivating 14dLiquid, 1.5mM liquid calcium chloride, 1mM magnesium sulfate solution, 10mM Glucose Liquid and 10mM glutamic acid liquid, pH7.0) clean. LogicalCross electrode and draw instrument (PULL-100, the U.S.) drawing electrode, selective membrane smooth surface is RGC cell clearly, utilizes three-dimensionalExecutor traveling electrode is pressed close to target cell, and is gently pressed in cell surface, and negative pressure can form height more than 1G Ω level a littleImpedance sealing-in, then inhale broken cell film by larger negative pressure, form common full cell record form. In electrode, in liquid, be filled with both sexesMycin B (0.2-0.3g/L-1, sigma), in a few minutes, visible system impedance reduces gradually, forms and stablizes perforation to 100M Ω. ?Just putting under microscope (BX50WI, olympus) for 60 times and observing. Experimentation is by stimulating acquisition software Multiclamp700BAmplifier controls, and passes through analog-to-digital conversion image data through patch clamp amplifier (EPC10, Germany). Use pCLAMP10.2Version software is to data analysis.
5, result
Under Electronic Speculum, visible PLGA support aperture 50 μ m, thickness 60 μ m (Fig. 1), coated through Matrigel by this PLGA supportRear formation diameter is 4mm, and surface area is 12.56mm2Circular stent graft (eye transplanting/cell is common in Matrigel-PLGACultivate support) (Fig. 2); Seed cell is planted eye transplanting/co-culture of cells support in Matrigel-PLGA and is trained through induction differentiationSupport base and occur longer projection, similar neuronal cell (Fig. 3) in form around compared with the seed cell of endoplasmic reticulum as seen afterwards in 7 days; 14After it, noble cells quantity increases, and projection rises appreciably, and its growth axon length is significantly better than control group (seeing Fig. 4); Through applicationAfter Brn3b, HuD and the dyeing of Tju1 Double immunostaining, find, iRMP can express the specific stain of nerve cell, and iRMP is describedNoble cells be nerve cell (the results are shown in Figure 5); After stimulating by patch-clamp, can be observed iRMP action potential simultaneouslyObviously increase (50mV--20mv), show that this iRMP has basic bioelectrical activity (the results are shown in Figure 6).
Applying biological surgical adhesive of the present invention is by degradable retina neural support (iRMP) and bonding the establishing of host's retinaMeter figure as shown in Figure 7.
Two, the experiment in vitro of biological surgical adhesive (iRP-glue):
Under 4 DEG C of conditions, this iRP-glue is mixed with degradable people iPSCs source property retina neural support (iRMP), thisTime iRP-glue be liquid condition. The degradable people iPSCs source property retina neural support (iRMP) that adheres to iRP-glue is putUnder 37 DEG C of environment, cultivate, iRP-glue heat heats up and solidifies, and makes the iRMP can effective bonding culture dish. After 24h, adhesive fallsSeparate, use CCK-8 experiment, spectrophotometric value shows that biological surgical adhesive (iRP-glue) catabolite is to cell nontoxicity(Fig. 8)
Three, application in the body of biological surgical adhesive (iRP-glue):
Under gaseous environment, inject biological surgical adhesive in host's neural retina fibrage exposed region (approximately 2 × 2mm)(iRP-glue) (concentration 10mg/ml) 0.3ml; Meanwhile, transplant requirement based on interior eye, degradable people iPSCs source property is lookedNethike embrane Nerve Scaffold (iRMP) implantation glass body cavity, uses retina tweezer to adjust iRMP position, utilizes iRP-glue heat solidifyingGu adhesive effect, by iRMP targeted adhesion in host's neural retina fibrage exposed region; There is glue at 10 DEG C in iRP-glueFreezing action, iRMP can be bonding through iRP-glue and host's retina, forms iRMP-host's neural retina composite bed (Fig. 9);Check iRMP bonding firmly after, use gas filling host vitreous chamber;
Four, the observation of iRMP-host's neural retina composite bed:
(1) eye-ground photography is observed: after transplanting, art eye carries out eye-ground photography, to the position, eyeground of iRMP-host's neural retina composite bedThe situation of putting is observed;
(2) OCT observes: after transplanting, art eye carries out OCT scanning, to the nethike embrane interlayer of iRMP-host's neural retina composite bedIntegration checks;
(3) eyeball anatomical slice qualification: the art eye eyeball of transplanting latter 1 week is carried out to anatomical slice, iRMP-host's nerve is lookedThe integration of nethike embrane composite bed checks.
Five, result
The visible iRMP of eye-ground photography attaches to host's retina, has no iRMP displacement (Figure 10); OCT shows that iRMP is stickyInvest host's neural retina, form good iRMP-host's neural retina composite bed (Figure 11); Eyeball anatomical slice is aobviousShow transplant after 1 week iRMP still tight adhesion in host's retina (Figure 12), biological surgical adhesive of the present invention is described(iRP-glue) iRMP is had to good integration function.
Claims (3)
1. the biological surgical adhesive of retina cell's support, is characterized in that, comprises laminin, type Ⅳ collagen, nest eggIn vain, people's heparin sulfate glycoprotein, fibroblast growth factor, matrix metalloproteinase and PBS.
2. the biological surgical adhesive of retina cell's support according to claim 1, is characterized in that, described retina is thinThe biological surgical adhesive of born of the same parents' support, every milliliter contains laminin 30ng, type Ⅳ collagen 30ng, nestin 10ng, peopleHeparin sulfate glycoprotein 10ng, desmocyte growth factor-21 0ng and matrix metallopeptidase 1 0ng, surplus is pH=7PBS.
3. a preparation method for the biological surgical adhesive of retina cell's support claimed in claim 1, is characterized in that, according toThe content of each component, by laminin, type Ⅳ collagen, nestin, people's heparin sulfate glycoprotein, fibroblast is rawThe long factor and matrix metalloproteinase join in PBS, obtain retina cell's support raw after mixingThing surgical adhesive.
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Citations (4)
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| CN102016009A (en) * | 2008-04-14 | 2011-04-13 | Ucl商业股份有限公司 | Membrane |
| CN103989553A (en) * | 2014-03-25 | 2014-08-20 | 周辉 | Method for manufacturing and storing corneal injury scar-free repairing device |
| CN105013015A (en) * | 2014-04-20 | 2015-11-04 | 上海市第一人民医院 | Method for repairing nerve defects in tissue engineering |
| CN105013010A (en) * | 2015-07-07 | 2015-11-04 | 中山大学中山眼科中心 | Laminin membrane for assisting iPS-RPE transplantation |
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2016
- 2016-02-22 CN CN201610097502.XA patent/CN105597145A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102016009A (en) * | 2008-04-14 | 2011-04-13 | Ucl商业股份有限公司 | Membrane |
| CN103989553A (en) * | 2014-03-25 | 2014-08-20 | 周辉 | Method for manufacturing and storing corneal injury scar-free repairing device |
| CN105013015A (en) * | 2014-04-20 | 2015-11-04 | 上海市第一人民医院 | Method for repairing nerve defects in tissue engineering |
| CN105013010A (en) * | 2015-07-07 | 2015-11-04 | 中山大学中山眼科中心 | Laminin membrane for assisting iPS-RPE transplantation |
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