CN107349475A - Artificial organ engineering skin that nano fibrous membrane is layering with stem cell and preparation method thereof - Google Patents

Artificial organ engineering skin that nano fibrous membrane is layering with stem cell and preparation method thereof Download PDF

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Publication number
CN107349475A
CN107349475A CN201710577134.3A CN201710577134A CN107349475A CN 107349475 A CN107349475 A CN 107349475A CN 201710577134 A CN201710577134 A CN 201710577134A CN 107349475 A CN107349475 A CN 107349475A
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stem cell
fibrous membrane
nano fibrous
artificial organ
cell
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CN107349475B (en
Inventor
李学拥
王红军
李跃军
黄容
李金清
李靖
边永钎
赵聪颖
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Fourth Military Medical University FMMU
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Fourth Military Medical University FMMU
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    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61LMETHODS 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
    • A61L27/00Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/60Materials for use in artificial skin
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61LMETHODS 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
    • A61L27/00Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
    • A61L27/3804Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
    • A61L27/3834Cells able to produce different cell types, e.g. hematopoietic stem cells, mesenchymal stem cells, marrow stromal cells, embryonic stem cells
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61LMETHODS 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
    • A61L27/00Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/56Porous materials, e.g. foams or sponges
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61LMETHODS 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
    • A61L2430/00Materials or treatment for tissue regeneration
    • A61L2430/40Preparation and treatment of biological tissue for implantation, e.g. decellularisation, cross-linking

Abstract

Artificial organ engineering skin being layering it is an object of the invention to provide a kind of nano fibrous membrane and stem cell and preparation method thereof, belongs to high polymer material and biology medical material technical field.Artificial organ engineering skin includes multilayer layer and the multi-layer porous nano fibrous membrane with three-dimensional structure, and layer is alternately laminated with nano fibrous membrane, and it is prepared by the way that stem cell and nano fibrous membrane to be carried out to the method for LBL self-assembly co-cultivation.The artificial organ engineering skin can be used for the regeneration and reparation of various tissues, particularly wound healing, reduce scar formation, skin regeneration etc..

Description

The artificial organ engineering skin and its preparation that nano fibrous membrane is layering with stem cell Method
Technical field
The invention belongs to high polymer material and biology medical material technical field, is related to a kind of nano fibrous membrane and stem cell Artificial organ engineering skin being layering and preparation method thereof.
Background technology
At present, mankind's dermal tissue insult such as extensive burn is controlled by the method for autologous or heterogenous skin transplanting Treat, this not only causes the new wound defect in skin donor site, and is often subject to the limitation for skin source.To solve this technology hardly possible Topic, with the rise of polymeric biomaterial, controlled using the dermal scaffold of tissue engineering technique structure imitation extracellular matrix Treating skin injury becomes good selection.But so far without really preferably Graftskin is applied.
Preferable artificial skin needs to have the characteristics of soft, comfortable, air-moisture-permeable, can there is good patch with the surface of a wound Close, while need certain ductility, toughness and mechanical strength again.In addition, artificial skin needs to provide a good material Material-Cellular interfaces, in favor of the regeneration for growing into, promoting neoplastic skin appendicle of itself epithelial cell, bacterial invasion is resisted, with Play wound healing, the purpose of skin function regeneration.It is in the prior art to use hydrogel and polymer compound film conduct more Artificial skin.But artificial skin made of the presence of the hydrogels such as chitosan, Sodium Hyaluronate, chondroitin sulfate, dermatan sulfate is strong Degree is inadequate, the problem of poor air permeability, and as dosage increases, plays the role of to suppress fibroblastic growth.In addition, poly- ammonia Although the medical macromolecular materials such as ester, silicon rubber, polyethylene glycol or ethylene glycol terephthalate can improve the strong of artificial skin Degree problem, but hydrophily is bad, and degradation speed is slow in vivo, RT is more long or even non-degradable, causes foreign matter to remain, Cause aseptic inflammation, influence field planting and growth of the own cells in the surface of a wound, and because these materials can not participate in being given birth to Reason metabolism, can only often be used as outer layer dressing.So it is simple using a kind of technique of interaction design of support and cell, it is easy to Volume production, easily preserve, good biocompatibility, application is convenient, cheap artificial organ engineering skin, for quick, efficient Wound repairing is significant.
Stem cell is a kind of initial cell with self-renewal capacity and polyphyly differentiation potential, be cell transplantation tissue again Preferable cell that is raw and repairing.Although embryonic stem cell is most original and most has the stem cell of differentiation potential, current source It is extremely limited.And the stem cell through too high expression stem cell factor induction is also referred to as iPS cells, although it has many embryos dry thin The characteristic of born of the same parents, but because preparation method is complicated, also relates to and also existed using the safety issues such as viral vector, at present clinical practice Many bottlenecks.Mescenchymal stem cell (Mesenchymal stem cells, MSC) is present in tissue with polyphyly point Change a kind of Subaerial blue green algae group of potential, such as stem cell, fat stem cell, cord blood stem cell.Fat stem cell Unnecessary fat on abundance, such as the body that can have drawn from, method of dissociating is easy, has self-renewing and pluripotent differentiation Ability, it is a kind of preferably regeneration and repair materials.
Have xenogenesis acellular matrix material at present and be used for clinic.Because being handled by de- cell, it is not present in material different Kind cell, will avoid that human body to immune response caused by foreign material.But because there is no cell component, acellular matrix Therefore material is a lack of the material of bioactivity, its repair to wound, tissue defect etc. is limited.Mescenchymal stem cell is big Amount is present in tissue, such as derives from the fat stem cell of adipose tissue.But stem cell is used for histoorgan reparation at present Cell therapy problems be present, although there is clinical practice report, clinical effectiveness is not known.Particularly repaiied for appearance wound Multiple or injury tissue or the replacement of organ, if without the support of cell epimatrix material, stem cell is difficult to play a role.
Although there is Many researchers to develop a variety of artificial organ engineering skins at present, most matrix are collagen gel Or sponge, it is not any at present to prepare nano fibrous membrane by pure natural high polymer and combine layer-by-layer application In the document of organization engineering skin, Application No. " CN201410423055.3 " and the patent of " 201610353226.9 " are reported respectively Road is a kind of to be used to promoting regenerating heart tissue and Properties of Chitosan Fibroin Blend albumen composite nano fiber multi-functional benefit that stem cell monitors The preparation method of piece and a kind of preparation method of the antibacterial wound dressing of the high chitosan content of Static Spinning, but the surface of a wound is not carried out The correlative study of healing effect.Application No. " 201610499353.X " and the patent of " 201611008057.1 " report respectively A kind of micro-nano composite double layer dermal scaffold and preparation method thereof and a kind of flexible artificial skin and preparation method thereof, but have no aobvious Writing improves cell is distributed and the correlative study of wound healing facilitation effect in tissue.
The content of the invention
It is an object of the invention to provide the artificial organ engineering skin that a kind of nano fibrous membrane and stem cell are layering And preparation method thereof, it is combined by nano fibrous membrane and the alternating assembling of stem cell two parts, available for various tissues again Raw and reparation.The present invention is to be achieved through the following technical solutions:
The artificial organ engineering skin that a kind of nano fibrous membrane and stem cell are layering, including multilayer layer and more The porous nano fibrous membrane with three-dimensional structure of layer, layer are alternately laminated with nano fibrous membrane.
Wherein, it is described alternately laminated, refer to that layer is layering with nano fibrous membrane, both pass through spaced Laminated structure so that the both sides of layer are nano fibrous membrane, and the both sides of nano fibrous membrane are layer.
Preferably, multi-layer nano tunica fibrosa is laminated to form nano fiber scaffold;Stem cell in layer is as seed Cell distribution is on nano fibrous membrane surface.
Preferably, the stem cell is fat stem cell.
Preferably, the nano fibrous membrane is prepared by polycaprolactone and beta-schardinger dextrin.
Preferably, envelope has brufen in beta-schardinger dextrin.
Preferably, the oxygen transmission rate of nano fibrous membrane is 50~60%, a diameter of 200 of the nanofiber in nano fibrous membrane ~600nm.
The preparation method for the artificial organ engineering skin that described nano fibrous membrane is layering with stem cell, including step Suddenly:
1) stem cell is prepared;
2) stem cell and nano fibrous membrane are subjected to LBL self-assembly co-cultivation;
Wherein, step 2) includes step:
21) using culture dish as receiver, nano fibrous membrane is prepared by electrostatic spinning technique;
22) stem cell is seeded in culture dish;
23) repeat step 21) and 22), until completing the inoculation number of plies of setting;
24) in culture dish, nano fibrous membrane and stem cell are co-cultured.
Preferably, in step 21), in superclean bench, nano fibrous membrane is prepared by electrostatic spinning technique;Its In, the DC voltage applied during electrostatic spinning is 10~35kV;The distance of syringe needle and culture dish is 5 during electrostatic spinning ~20cm;Syringe is driven by syringe pump with 0.5~2.0mL/h speed during electrostatic spinning;Electrostatic spinning process middle ring Border temperature is 5~35 DEG C;Envionmental humidity is 20~80% during electrostatic spinning;The time of electrostatic spinning is 1~5min.
Preferably, in step 22), relative to the area of nano fibrous membrane, the quantity of stem cell inoculation is 1 × 104~2 ×104Individual cell/cm2
Preferably, in addition to step:3) artificial organ engineering skin prepared by step 2) is packed after sterilization, Freezing.
Compared with prior art, the present invention has technique effect beneficial below:
The artificial organ engineering skin that nano fibrous membrane provided by the invention is layering with stem cell, it includes multilayer and received Rice tunica fibrosa, nano fibrous membrane is porous and has three-dimensional structure, sticking and growing and provide suitable configuration of surface for cell, sharp In the adhesion and growth of cell;Stem cell can secrete various cell factors, promote the propagation of cell, contribute to the regeneration of tissue And reparation;Stem cell is divided into multilayer, alternately laminated with nano fibrous membrane, and this causes stem cell in whole artificial organ engineering skin In more uniformly spread.Zoopery proves that the artificial organ engineering skin can significantly improve cell and is distributed in tissue, Wound healing.In this way, the artificial organ engineering skin can be used for the regeneration and reparation of various tissues, particularly wound healing Close, reduce scar formation, skin regeneration etc..
Brief description of the drawings
Fig. 1 is that the present invention utilizes fat stem cell and polycaprolactone-β-CD nanofiber film preparation artificial organ engineerings The process schematic of skin.
Fig. 2-1, Fig. 2-2 and Fig. 2-3 are microstructure (the Flied emission scanning electricity of nano fibrous membrane prepared by the present invention Mirror), wherein Fig. 2-1 amplifies 2000 times, and Fig. 2-2 amplifies 7000 times, and Fig. 2-3 amplifies 15000 times.
Fig. 3-1, Fig. 3-2, Fig. 3-3, Fig. 3-4, Fig. 3-5, Fig. 3-6 are that fat stem cell is inoculated in polycaprolactone-β-CD lists Scanning electron microscope (SEM) photograph after layer nano fibrous membrane, 1,3,5,7,9,11 days after being respectively inoculated with.
Fig. 4-1 is that the coloration result of individual layer nano fibrous membrane-fat stem cell compound is (red:Nanofiber;Blueness: DAPI), the figure is shown, cell is only distributed in individual layer nano fibrous membrane surface (side).
Fig. 4-2 is that the coloration result of artificial organ engineering skin prepared by multilayer LBL is (red:Nanofiber;Blueness: DAPI), the figure is shown, after nano fibrous membrane layer by layer-fat stem cell superposition, cell can be uniformly distributed in nanofiber In film surface and interlayer total, cell density can obviously improve compared with the inoculation of single layer fibre film.
Fig. 5-1, Fig. 5-2, the wound healing result of the test for the artificial organ engineering skin that Fig. 5-3 is the present invention.As a result show Showing, Different treatments obtain effect for wound healing and notable difference be present, wherein, Fig. 5-1 is control group (conventional gauze covering Group), Fig. 5-2 is individual layer nano fibrous membrane-fat stem cell compound covering group, and Fig. 5-3 covers for artificial organization engineering skin Group.
Fig. 6-1, Fig. 6-2 are that the HE after artificial organ engineering skin flap coverage to the complete healing of the present invention dyes knot Fruit, wherein Fig. 6-1 control groups (untreated fish group);Fig. 6-2 artificial organ engineering skin groups.
Fig. 6-3, Fig. 6-4 are the Masson dyeing after artificial organ engineering skin flap coverage to the complete healing of the present invention As a result, wherein, Fig. 6-3 is single layer fibre film-fat stem cell compound group;Fig. 6-3 is artificial organization engineering skin group.
Embodiment
Technical scheme is described further below by specific embodiment, its object is to help preferably Understand present disclosure, but these specific embodiments are not in any way limit the scope of the present invention.
The purpose of the present invention is overcome the deficiencies in the prior art, there is provided one kind using fat stem cell as seed cell and Artificial organ engineering skin that nano fiber scaffold is layering and preparation method thereof.Electrostatic spinning technique, macromolecule are complexed Technology is combined with fiber-cell layer-by-layer, and preparation has good plasticity and suitable mechanical characteristic, height The 3-D solid structure of porosity and the organization engineering skin of biocompatibility, the controllable biodegradation rate of the nano-bracket, It is suitable for skin regeneration organizational project.
Existing acellular matrix material is handled using xenogenesis skin through de- cell, makes not and has antigenic biological material Material.The covering of the tissue surface of a wound is provided by remaining cell epimatrix material (being mainly made up of collagen and elastin laminin), there is provided group Knit the support of reparation and have certain value in clinical practice.But because this host material lacks due and tissue repair Relevant cell factor, its purposes and validity are greatly affected.Numerous studies show that mescenchymal stem cell is as free from fat The fat stem cell of fat has more differentiation potentials, can be used as the preferable seed cell of tissue repair.Source for mesenchymal stem cells is wide General, convenient material drawing, immunogenicity is low, and does not produce teratoma, still retains Multidirectional Differentiation after continuous passage culture and freezen protective Potential, the preferable seed cell of tissue repair can be used as.Wherein, fat stem cell (AD-MSC) is largely present in adipose tissue, It is easy to expand, paracrine action and differentiation capability are good, as the waste material of medical science shaping, also can be recovered utilization, have good Potential applicability in clinical practice.So the present invention uses seed cell of the fat stem cell as organization engineering skin.
Currently, two classes are roughly divided into artificial organ engineering skin field of stents, the material prepared for dermal scaffold:One Class is natural biological derived material, such as collagen, chitosan, hyaluronic acid, carboxymethyl chitosan and fibroin albumen.It is another kind of to be Artificial synthesized bioabsorbable polymer material, mainly there is the polyester such as PGA, polycaprolactone, PHA, polycarbonate-based Class material;Most of dermal scaffolds are all to be manufactured with biomaterial by electrostatic spinning technique.Although obtained by electrostatic spinning To nanofiber sticking and growing suitable configuration of surface is provided for cell, beneficial to adhesion and life of the cell on support It is long, but by dermal scaffold prepared by electrostatic spinning because aperture is too small, it is unfavorable for migration, the increasing of cell in the depth direction Grow, and be difficult to the Effective Regulation that cell is distributed on fibrous framework, cured which has limited artificial skin support in skin injury Learn the application in therapy field.Meanwhile support and epimatrix (ECM) as stem cell growth, it is good hydrophily, suitable Surface charge, appropriate pore-size are more suitable for the apposition growth of cell.
According to above feature, the present invention makes polycaprolactone-β-CD nano fiber scaffolds in nanometer by electrostatic spinning technique The horizontal porous three-dimensional microstructure to interpenetrate for being processed as being adapted to cell growth differentiation of level, and can be coated with as needed various Cell factor or medicine, also carry out fiber-cell using LBL self-assembly (LBL) technology and be layering, promote from many aspects The attachment and growth of stem cell, improve the efficiency of stem-cell therapy.For the foregoing reasons, the present invention combines nanofiber-based material Material and mescenchymal stem cell technology provide a kind of biomaterial for having biological activity, and the biomaterial can be used for various tissues Regeneration and reparation, particularly wound healing, reduce scar formation, skin regeneration etc..
As shown in figure 1, fat stem cell is inoculated in polycaprolactone-β-CD nano fibrous membranes, by LBL self-assembly skill Art carries out nano fibrous membrane-fat stem cell and is layering, so as to form artificial organ engineering skin.
Fat stem cell is separately cultured, purified, authentication method
Embodiment 1, cell isolate and purify
Adipose tissue is transferred in the sterile petri dish for being pre-loaded with PBS, and peplos and obvious connective group are cut off with eye scissors Knit, shredded afterwards twice with PBS;The adipose tissue shredded is transferred in 50mL centrifuge tubes, 0.2% NTx enzyme is added, puts In 37 DEG C of 5%CO22 hours in incubator, mixed once per 30min;It is sieved through digesting to the tissue of pasty state by 250 mesh metals Filter, filtrate are transferred in 15mL centrifuge tubes, after 1500rpm 10min centrifugations, inhale the fat lump and supernatant for abandoning floating, PBS purgings 3 Centrifuged after secondary, add the DMEM nutrient solutions containing 10%FBS and gently blown and beaten with suction pipe, be allowed to be resuspended again;It is inoculated in and is pre-loaded with training In the blake bottle for supporting base, 37 DEG C of 5%CO are placed in2Cultivated in incubator;Stand 24h after discard culture medium, with PBS twice with Non- attached cell, connective tissue and fragment are removed, the fresh DMEM nutrient solutions containing 10%FBS is changed, liquid was changed every 2 days, is observed.
Embodiment 2, fat stem cell identification
The 3rd generation cell of culture is taken, after suctioning out culture medium, with PBS twice first, 0.25% pancreatin for adding preheating disappears Change, add the DMEM containing 10%FBS afterwards and terminate digestion, after gently blowing and beating into single cell suspension 1000rpm pelleted by centrifugation 5min, Supernatant discarding, PBS is twice;10 loading pipes are taken, often pipe adds 1mL single cell suspensions, is sequentially added into 10 μ L IgG- FITC, IgG-PE, mouse anti-human CD29-FITC, CD44-FITC, CD49d-FITC, CD73-FITC, CD90-FITC, CD105- FITC, CD34-PE, CD45-PE monoclonal antibody working solution;Lucifuge is incubated 20min at room temperature;PBS twice with remove not Binding antibody, 500 μ L PBS use flow cytomery after being resuspended.
Embodiment 3, cell amplification
Passed on after cell growth is converged to 80-90%, suction out culture medium, with PBS twice first, then add 0.25% pancreatin, 37 DEG C of constant incubator 2min are placed in, then add the DMEM nutrient solutions containing 10%FBS and stop digestion, suction pipe Gently piping and druming makes cell detachment, collects cell, moves into 15mL centrifuge tubes.1000rpm 5min, abandon supernatant, PBS twice, Add the DMEM containing 10%FBS and cell precipitation is resuspended, by 1:2 density are inoculated in blake bottle, are added the DMEM containing 10%FBS and are put In 37 DEG C of 5%CO2Continue to cultivate in incubator, micro- Microscopic observation cellular morphology, obtain the fat stem cell of amplification.
Embodiment 4, a kind of preparation method of PC-β-CD nano fibrous membranes
In superclean bench, polycaprolactone (PCL, Mn=70-90KDa, Sigma AldrichCo., USA) is dissolved It is 1 to mass ratio:In the DMF of (1~3)-dichloromethane mixed solution, mass concentration is configured to as 8% Polycaprolactone solution;β-CD are added into polycaprolactone solution so that β-CD mass fraction is 0.1%, it is heated to 50~ 70 DEG C, stir, mixing time is 6~12 hours, obtains polycaprolactone-β-CD solution;
Wherein, there can be brufen by envelope in advance in β-CD:Molal weight ratio is weighed as 1:10~1:1 β-CD and cloth Lip river Fragrant mixture adds 13% deionized water into mortar in sealed environment, 1~3h of grinding at room temperature, will made in drying in mortar Good bag and thing dry 6-12h in vacuum drying chamber at 35-60 DEG C, obtain β-CD/ brufen inclusion compounds.
In superclean bench, the polycaprolactone-β-CD solution of acquisition is sucked to the band metal needle of electrospinning device Plastic injector.For syringe needle with the culture dish as collector at a distance of 10 ㎝, the DC voltage of application is 20 kilovolts.Note Emitter is driven by syringe pump with 1.0mL/h speed, and environment temperature is 25 DEG C, relative humidity 40%, and the electrostatic spinning time is 3min, obtains nano fibrous membrane, and the nano fibrous membrane is received in culture dish.
Embodiment 5, a kind of preparation method of PC-β-CD nano fibrous membranes
With reference to the method for embodiment 4, wherein, in polycaprolactone-β-CD solution, the mass concentration of polycaprolactone is 4%;β- CD mass concentration is 0.02%;The distance of syringe needle and culture dish is 20cm, and the DC voltage of application is 10 kilovolts;Syringe by Syringe pump is driven with 2.0mL/h speed, and environment temperature is 35 DEG C, and relative humidity 20%, the electrostatic spinning time is 5min.
Embodiment 6, a kind of preparation method of PC-β-CD nano fibrous membranes
With reference to the method for embodiment 4, wherein, in polycaprolactone-β-CD solution, the mass concentration of polycaprolactone is 12%; β-CD mass concentration is 0.2%;The distance of syringe needle and culture dish is 5cm, and the DC voltage of application is 35 kilovolts;Syringe by Syringe pump is driven with 0.5m L/h speed, and environment temperature is 5 DEG C, and relative humidity 80%, the electrostatic spinning time is 1min.
Using layer-by-layer (LBL technologies) fat is alternately inoculated with PC-β-CD nano fibrous membranes surface Stem cell carries out three-dimensional layer by layer co-culture
Embodiment 7
In superclean bench, with reference to the method for embodiment 4~6, the nano fibrous membrane being received in culture dish is prepared, Wherein, a diameter of 30mm of culture dish;The fat stem cell for the amplification that embodiment 3 is obtained is seeded in nano fibrous membrane In culture dish, the volume of the cell suspending liquid of inoculation is 1mL, and cell quantity is 1 × 105Individual cell;Then again by the culture dish Receiver as nano fibrous membrane carries out electrostatic spinning, and electrostatic spinning carries out fat stem cell inoculation again after terminating, in this way, often Layer inoculation 1 × 105Individual cell, 10 layers are inoculated with altogether.Because whole process occurs in media surface, cell is in packing engineering Hydration status are always maintained at, a total of 10 confluent monolayer cells/nanofiber is alternately layering into three-dimensional structure.Culture dish is placed In 37 DEG C of CO2Incubator culture 30 minutes, then add DMEM/F12 supplementing culture mediums;Continue culture 1 week to form a kind of layer The artificial organ engineering skin that layer is self-assembly of.Wherein DMEM/F12 nutrient solutions include 10% hyclone and 1% penicillin/ Streptomysin.
Wherein, if being only inoculated with a fat stem cell on nano fibrous membrane surface, received by layer-by-layer stacking Rice tunica fibrosa and fat stem cell, by the way that individual layer nano fibrous membrane-fat stem cell compound can be formed after culture.
Embodiment 8
With reference to the preparation method of embodiment 6, wherein, a diameter of 100mm of culture dish, the fat stem cell of every layer of inoculation Quantity is 1 × 106Individual cell.
Embodiment 9
With reference to the preparation method of embodiment 6, wherein, a diameter of 100mm of culture dish, the fat stem cell of every layer of inoculation Quantity is 1 × 106Individual cell, 20 layers are inoculated with altogether.
The artificial organ engineering skin that LBL self-assembly prepared by the embodiment 7~9 of embodiment 10 is formed, passes through epoxy second Packed after alkane sterilization, there is cryoprotector in packaging bag, freezen protective after packaging;Engineering skin after packaging can be with cold Freeze in -78~82 DEG C of refrigerator, can also be frozen in liquid nitrogen.
Embodiment 11
By 1 × 1cm2Polycaprolactone-beta-schardinger dextrin fiber membrane sample carry out vacuum spray carbon processing after, swept using Flied emission Retouch and fiber morphology is observed on electron microscope, accelerating potential 15kV.Experimental results be illustrated in Fig. 2-1, Fig. 2-2 and Fig. 2- In 3, wherein, Fig. 2-1 amplifies 2000 times, and Fig. 2-2 amplifies 7000 times, and Fig. 2-3 amplifies 15000 times.As a result show, preparation gathers oneself For lactone-β-CD nano fibrous membranes in the three-dimensional 3D structures intersected, nanofiber is the homogeneous filament shape of thickness, diameter and hole point Cloth is uniform, and in unordered arrangement, avarage fiber diameter is 350 ± 72nm.
Embodiment 12
Fat stem cell is inoculated in after being co-cultured 3 days on polycaprolactone-beta-schardinger dextrin nano fibrous membrane, using glutaraldehyde After solution is fixed overnight at 4 DEG C, with PBS, vacuum freeze drying is carried out after a series of Gradient elution using ethanols, is dried After 6h carry out vacuum spray carbon processing, using field emission microscopy observation cell fiber surface growing state.
Fig. 3-1, Fig. 3-2, Fig. 3-3, Fig. 3-4, Fig. 3-5, Fig. 3-6 be respectively for fat stem cell be inoculated in polycaprolactone- The scanning electron microscope (SEM) photograph of 1,3,5,7,9,11 days after β-CD individual layer nano fibrous membranes.Scanning result shows, fat stem cell and nanometer Tunica fibrosa sticks closely, can substantially observe the thread and lamellipodia stretching, extension of cell, and Microfilaments In Cells wind mutually or mutually interconnected Connect, in network structure, cellular portions or whole migrate to material pore interior, cell surface and surrounding visible particulate structure, It may be the cell factor or protein of cell secretion, illustrate that cell growth state is good.As cultivated days increase, part is thin Overlapping born of the same parents are in three dimensional growth, and iuntercellular forms pseudopodium identification, and forms cladding (Fig. 3-6), show the nano fibrous membrane tool prepared There is good biocompatibility, adhesion and the propagation of fat stem cell can be remarkably promoted.
Embodiment 13
Fat stem cell is inoculated in individual layer polycaprolactone-beta-schardinger dextrin nano fibrous membrane, co-cultures 3 days, uses PBS 3 times;30min is fixed using 4% formalin (being dissolved in PBS), dries 5min;Then use PBS 3 times;And with 0.5% TRITON X-100 permeate 20min, are then cleaned with PBS;Dyeing 10min is carried out using DAPI at room temperature to nucleus (dilution factor:1:1000) dyeing 5min then, is carried out to nanofiber using rhodamine.Removed with PBS uncombined Dyeing liquor, mounting carry out being copolymerized burnt observation.Experimental results show in Fig. 4-1, wherein, RED sector is nanofiber, blueness Part is DAPI;The figure shows that cell is only distributed in individual layer nano fibrous membrane surface (i.e. only in nano fibrous membrane side).
Fat stem cell using layer-by-layer replace with polycaprolactone-beta-schardinger dextrin nano fibrous membrane and folded After adding, co-culture 3 days, with PBS 3 times;30min is fixed using 4% formalin (being dissolved in PBS), dries 5min;Then adopt With PBS 3 times;And 20min is permeated with 0.5%TRITON X-100, then cleaned with PBS;Using DAPI to cell Core carries out dyeing 10min (dilution factors at room temperature:1:1000) dyeing 5min then, is carried out to nanofiber using rhodamine. Uncombined dyeing liquor is removed with PBS, mounting carries out being copolymerized burnt observation.Experimental results show in Fig. 4-2, wherein, RED sector is nanofiber, blue portion DAPI;The figure is shown, it is superimposed by nanofiber-fat stem cell layer by layer Afterwards, cell can be uniformly distributed in nano fibrous membrane surface and in interlayer total, cell density can be bright compared with single layer fibre inoculation It is aobvious to improve.
Embodiment 14
Selection health, adult, cleaning grade SD rats, male and female half and half, weight 200-250g, SPF level, by the 4th medical university of army Animal center is learned to provide.Model group and control group are randomly divided into by random digits table, every group 4, totally three groups of (control groups (conventional gauze covering group), individual layer nano fibrous membrane-fat stem cell compound covering group, the covering of artificial organ engineering skin Group), the preoperative 12h fasting of every group of rat.Rat carries out intraperitoneal injection of anesthesia, shaves net mouse back wool, iodophor disinfection skin of back, paving Aseptic towel list.The skin ranges of 2cm diameters are measured with mould at back, periphery solid marks, are cut off in the range of solid marks Full thickness skin to deep fascia layer, form full thickness dermal wounds.
Control group, individual layer oil yarn flap coverage;
Individual layer nanofiber-fat stem cell compound covering group:Individual layer nano fibrous membrane-fat stem cell compound covers The lid surface of a wound;
Artificial organ engineering skin group:Artificial organ engineering skin covering group
According to packet, covering material is cut out according to surface of a wound size, flap coverage, the materials time is with the surface of a wound of each group animal Healing time is defined.Wound healing situation is monitored in real time in different cycles after the surface of a wound is formed, it is soft using graphical analysis Photo before and after part analysis Wound treating, healing rate are more than 90% and are judged to healing.Experimental results show Fig. 5-1, Fig. 5-2, In Fig. 5-3;Wherein, Fig. 5-1 is control group (conventional gauze covering group), and Fig. 5-2 is that individual layer nanofiber-fat stem cell is compound Thing covering group, Fig. 5-3 are artificial organization engineering skin covering group.As a result show, Different treatments must imitate for wound healing Notable difference be present in fruit, through the present invention prepare artificial organ engineering skin processing wound healing after re-epithelialization degree more Height, illustrate that the material is optimal for the facilitation effect of wound healing.
Embodiment 15
On the basis of embodiment 14, cambium (band edge of wound part) is taken after the surface of a wound heals completely, through FFPE, Section, dyed and detected using conventional hematoxylin-Yihong (HE);Testing result is illustrated in Fig. 6-1, Fig. 6-2, wherein, Fig. 6-1 pairs According to group (untreated fish group), Fig. 6-2 artificial organ engineering skin groups.Compared to control group (no basilar memebrane, table-skin corium separation), Artificial organ engineering skin group does not observe the separation of obvious table-skin corium, and skin corium can be observed has part new vessels.
Cambium (band edge of wound part) is taken after the surface of a wound heals completely, baking piece is carried out after FFPE, section, is taken off Wax, gradient enter water, and after rinsing, successively using R1, R2, R3, R4 liquid is dyed (green skies staining kit), neutral gum envelope Piece, using collagenous fibres, blood vessel and other skin textures in fluorescence microscope tissue.Experimental results show is in Fig. 6-3, figure In 6-4, wherein, Fig. 6-3 is control group (untreated fish group);Fig. 6-4 is artificial organization engineering skin group.Masson coloration results show Show, artificial organ engineering skin prepared by the present invention has degradation property inside excellent, and has new life after material implantation Angiogenesis.Experimental result confirms:Artificial organ engineering skin prepared by the present invention has more preferable within the same implantation cycle Degeneration energy, and new vessels generation can be observed in fibrous inside.
Formhals developed a kind of electrified jet by high-pressure electrostatic field excitation polymer in 1934 first, made to penetrate Stream solidification obtains the nanofiber of hyperfine structure, and the nanofiber prepared by this method possesses superfine fibre diameter, larger ratio The unique advantage such as surface area and 3-D solid structure, this makes it be increasingly becoming the study hotspot that skin wound repairs field.This hair Bright to use electrostatic spinning technique, its reason is:First, the tissue engineering bracket constructed by electrostatic spinning technique has in structure There is analog cell epimatrix (extracellularmatrix, ECM) effect, fibroblast can be promoted to be formed with cutin Adhesion, propagation and the migration of cell;Secondly, with gathering in oneself with good biocompatibility in electrostatic spinning solution of the invention Ester is raw material, and this nano fiber scaffold can substantially speed up wound healing.3rd, β-CD (beta-schardinger dextrin) have outer rim it is hydrophilic and The hydrophobic special construction in inner chamber, the stability of bag and thing and medicament slow release energy can be improved by forming Subjective and Objective inclusion complex Power.The present invention carries out nanofiber layer stackup on this basis, using the new technology of nano fibrous membrane-cell LBL self-assembly and added To realize that cell assembling is formed three-dimensional structure, this three-dimensional structure can well control cell distribution, it is allowed to co-culture a variety of thin Born of the same parents, and physically separate various cells and do not influence its mass exchange but.Laboratory and king Red Army of seminar where applicant Set up recently it is a kind of it is emerging add the technology for realizing cell assembling by nanofiber layer stackup, and successfully by this skill Art applies to build three-dimensional tissue (US 20080112998A1).It is layering this in cell assembling, cell is added to Between nano fibrous membrane.The composition and thickness of cell category and density per confluent monolayer cells layers and every layer of tunica fibrosa can bases Need to adjust.
Compared with prior art, the invention has the advantages that:
1) fat stem cell abundance, acquisition methods are simple;
2) fat stem cell has multiple differentiation potential, can be divided into the cell of various germinal layers, as epithelial cell, nerve are thin Born of the same parents, muscle cell, fibroblast etc.;
3) fat stem cell can secrete cytokine profiles, as angiogenesis factor, EGF (EGF), blood are small Plate derivative growth factor (PDGF) etc., can be with promotion organization cytothesis;
4) because fat stem cell is derived from itself, therefore do not have antigenicity;
5) electrostatic spinning polycaprolactone tissue engineering bracket has analog cell epimatrix in structure The effect of (extracellular matrix, ECM), can promote adhesion, propagation and the migration of fat stem cell;And can be notable Accelerate wound healing;
6) β-CD have the special construction that outer rim is hydrophilic and inner chamber is hydrophobic, can be improved by forming Subjective and Objective inclusion complex The stability of bag and thing and medicament slow release ability;
7) carry out nanofiber layer stackup using the new technology of nanofiber-cell LBL self-assembly and realized groups of cells Dress forms three-dimensional structure tissue.This three-dimensional structure can well control cell distribution, it is allowed to co-culture various kinds of cell, and Physically separating various cells does not influence its mass exchange but.

Claims (10)

1. the artificial organ engineering skin that a kind of nano fibrous membrane is layering with stem cell, it is characterised in that done including multilayer Cellular layer and the multi-layer porous nano fibrous membrane with three-dimensional structure, layer are alternately laminated with nano fibrous membrane.
2. the artificial organ engineering skin that nano fibrous membrane as claimed in claim 1 is layering with stem cell, its feature exist In multi-layer nano tunica fibrosa is laminated to form nano fiber scaffold;Stem cell in layer is distributed in as seed cell to be received Rice tunica fibrosa surface.
3. the artificial organ engineering skin that nano fibrous membrane as claimed in claim 1 is layering with stem cell, its feature exist In the stem cell is fat stem cell.
4. the artificial organ engineering skin that nano fibrous membrane as claimed in claim 1 is layering with stem cell, its feature exist In the nano fibrous membrane is prepared by polycaprolactone and beta-schardinger dextrin.
5. the artificial organ engineering skin that nano fibrous membrane as claimed in claim 4 is layering with stem cell, its feature exist In envelope has brufen in beta-schardinger dextrin.
6. the artificial organ engineering skin that nano fibrous membrane as claimed in claim 1 is layering with stem cell, its feature exist In the oxygen transmission rate of nano fibrous membrane is 50~60%, a diameter of 200~600nm of the nanofiber in nano fibrous membrane.
7. the artificial organ engineering skin that the nano fibrous membrane described in any one of claim 1~6 is layering with stem cell Preparation method, it is characterised in that including step:
1) stem cell is prepared;
2) stem cell and nano fibrous membrane are subjected to LBL self-assembly co-cultivation;
Wherein, step 2) includes step:
21) using culture dish as receiver, nano fibrous membrane is prepared by electrostatic spinning technique;
22) stem cell is seeded in culture dish;
23) repeat step 21) and 22), until completing the inoculation number of plies of setting;
24) in culture dish, nano fibrous membrane and stem cell are co-cultured.
8. the preparation side for the artificial organ engineering skin that nano fibrous membrane as claimed in claim 7 is layering with stem cell Method, it is characterised in that in step 21),
In superclean bench, nano fibrous membrane is prepared by electrostatic spinning technique;Wherein, what is applied during electrostatic spinning is straight Stream voltage is 10~35kV;The distance of syringe needle and culture dish is 5~20cm during electrostatic spinning;Injected during electrostatic spinning Device is driven by syringe pump with 0.5~2.0mL/h speed;Environment temperature is 5~35 DEG C during electrostatic spinning;Electrostatic spinning mistake Envionmental humidity is 20~80% in journey;The time of electrostatic spinning is 1~5min.
9. the preparation side for the artificial organ engineering skin that nano fibrous membrane as claimed in claim 7 is layering with stem cell Method, it is characterised in that
In step 22), relative to the area of nano fibrous membrane, the quantity of stem cell inoculation is 1 × 104~2 × 104Individual cell/ cm2
10. the preparation side for the artificial organ engineering skin that nano fibrous membrane as claimed in claim 7 is layering with stem cell Method, it is characterised in that also including step:
3) artificial organ engineering skin prepared by step 2) is packed after sterilization, freezing.
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