CN107349475B - The artificial organ engineering skin and preparation method thereof that nano fibrous membrane is layering with stem cell - Google Patents
The artificial organ engineering skin and preparation method thereof that nano fibrous membrane is layering with stem cell Download PDFInfo
- Publication number
- CN107349475B CN107349475B CN201710577134.3A CN201710577134A CN107349475B CN 107349475 B CN107349475 B CN 107349475B CN 201710577134 A CN201710577134 A CN 201710577134A CN 107349475 B CN107349475 B CN 107349475B
- Authority
- CN
- China
- Prior art keywords
- stem cell
- fibrous membrane
- nano fibrous
- cell
- artificial organ
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Classifications
-
- 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
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
- A61L27/60—Materials for use in artificial skin
-
- 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
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials 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
- A61L27/38—Materials 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
- A61L27/3804—Materials 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
- A61L27/3834—Cells able to produce different cell types, e.g. hematopoietic stem cells, mesenchymal stem cells, marrow stromal cells, embryonic stem cells
-
- 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
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
- A61L27/56—Porous materials, e.g. foams or sponges
-
- 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
- A61L2430/00—Materials or treatment for tissue regeneration
- A61L2430/40—Preparation and treatment of biological tissue for implantation, e.g. decellularisation, cross-linking
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Transplantation (AREA)
- Epidemiology (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- General Health & Medical Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Oral & Maxillofacial Surgery (AREA)
- Dermatology (AREA)
- Medicinal Chemistry (AREA)
- Cell Biology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Hematology (AREA)
- Botany (AREA)
- Developmental Biology & Embryology (AREA)
- Zoology (AREA)
- Urology & Nephrology (AREA)
- Dispersion Chemistry (AREA)
- Materials For Medical Uses (AREA)
Abstract
The purpose of the present invention is to provide the artificial organ engineering skins and preparation method thereof that a kind of nano fibrous membrane and stem cell are layering, and belong to high molecular material and biomedical material technology.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, is prepared by the method that stem cell and nano fibrous membrane are carried out LBL self-assembly co-cultivation.The artificial organization engineering skin can be used for the regeneration and reparation of various tissues, especially wound healing, the formation of reduction scar, skin regeneration etc..
Description
Technical field
The invention belongs to high molecular material and biomedical material technology, it is related to a kind of nano fibrous membrane and stem cell
Artificial organ engineering skin being layering and preparation method thereof.
Background technology
Currently, mankind's dermal tissue insult such as extensive burn is controlled by the method for self or heterogenous skin transplanting
It treats, this wound defect for not only causing skin donor site new, but also is often subject to the limitation for skin source.To solve this technology hardly possible
Topic is built using tissue engineering technique with the rise of polymeric biomaterial and imitates the dermal scaffold of extracellular matrix to control
Treating skin injury becomes good selection.But it is applied so far without really ideal Graftskin.
Ideal artificial skin needs to have the characteristics of soft, comfortable, air-moisture-permeable, can there is good patch with the surface of a wound
It closes, while needing certain ductility, toughness and mechanical strength again.In addition, artificial skin needs to provide a good material
Material-Cellular interfaces, in favor of itself epithelial cell the regeneration for growing into, promoting neoplastic skin appendicle, resist bacterial invasion, with
Play the regenerated purpose of wound healing, skin function.The prior art mainly adopts hydrogels and polymer compound film conduct
Artificial skin.But there are manufactured artificial skin is strong for the hydrogels such as chitosan, Sodium Hyaluronate, chondroitin sulfate, dermatan sulfate
The problem of degree is inadequate, poor air permeability, and as dosage increases, play the role of inhibiting 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, retention time is more long or even non-degradable, and foreign matter is caused to remain,
Cause aseptic inflammation, influences field planting and growth of the own cells in the surface of a wound, and since these materials cannot participate in being given birth to
Reason metabolism, can only often be used as outer layer dressing.So the interaction design using holder and cell is a kind of simple for process, it is easy to
Volume production is easy to preserve, and good biocompatibility, application is convenient, cheap artificial organ engineering skin, for quick, efficient
Wound repairing is of great significance.
Stem cell is a kind of initial cell with self-renewal capacity and polyphyly differentiation potential, be cell transplantation tissue again
Ideal 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 excessively high expression stem cell factor induction is also referred to as iPS cells, although dry thin with many embryos
The characteristic of born of the same parents, but because of preparation method complexity, also relate to using safety issues such as viral vectors, clinical application at present there is also
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
Extra fat on abundance, such as the body that can have drawn from, method of dissociating is easy, has self-renewing and pluripotent differentiation
Ability is a kind of ideal regeneration and repair materials.
Existing xenogenesis acellular matrix material is for clinic at present.Because being handled by taking off cell, there is no different in material
Kind cell, will avoid that the immune responses that human body generates dissimilar material.But because there is no cell component, acellular matrix
Therefore material is a lack of the material of bioactivity, limited to the repair of wound, tissue defect etc..Mescenchymal stem cell is big
Amount is present in tissue, such as from the fat stem cell of adipose tissue.But stem cell is used for histoorgan reparation at present
Cell therapy there are problems, although there is clinical application report, clinical effectiveness is uncertain.Especially repaiied for appearance wound
Multiple or injury tissue or organ replacement, if the not support of cell epimatrix material, stem cell are 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 that nano fibrous membrane is prepared by pure natural high polymer and combines layer-by-layer application
In the document of organization engineering skin, application No. is " CN201410423055.3 " and the patent of " 201610353226.9 " to report respectively
A kind of Properties of Chitosan Fibroin Blend albumen composite nano fiber multi-functional benefit for promoting regenerating heart tissue and stem cell to monitor in road
The preparation method of the preparation method of piece and a kind of antibacterial wound dressing of the high chitosan content of Static Spinning, but there is no carry out the surface of a wound
The correlative study of healing effect.Application No. is " 201610499353.X " and the patent of " 201611008057.1 " to 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 within the organization.
Invention content
The purpose of the present invention is to provide the artificial organ engineering skins that a kind of nano fibrous membrane and stem cell are layering
And preparation method thereof, by nano fibrous membrane and stem cell two parts, alternately assembling is combined, and can be used 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 is layering with stem cell, 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, described alternately laminated, refer to that layer is layering with nano fibrous membrane, the two passes 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 in nanofiber film surface.
Preferably, the stem cell is fat stem cell.
Preferably, the nano fibrous membrane is prepared by polycaprolactone and beta-cyclodextrin.
Preferably, envelope has brufen in beta-cyclodextrin.
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 the 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) step 21) and 22) is repeated, 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；Syringe needle is 5 at a distance from culture dish during electrostatic spinning
~20cm；Syringe is driven by syringe pump with the speed of 0.5~2.0mL/h 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
×104A cell/cm2。
Preferably, further include 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 comprising multilayer is received
Rice tunica fibrosa, nano fibrous membrane is porous and has three-dimensional structure, for cell stick and growth provides suitable configuration of surface, it is sharp
In the adherency and growth of cell；Stem cell can secrete various cell factors, promote the proliferation of cell, contribute to the regeneration of tissue
And reparation；Stem cell is divided into multilayer, alternately laminated with nano fibrous membrane, this makes stem cell in entire artificial organ engineering skin
In more uniformly spread.Zoopery proves that the artificial organization engineering skin can significantly improve cell and be distributed within the organization,
Wound healing.In this way, the artificial organization engineering skin can be used for the regeneration and reparation of various tissues, especially wound healing
Close, reduce scar formation, skin regeneration etc..
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 single layer nano fibrous membrane-fat stem cell compound is (red：Nanofiber；Blue：
DAPI), which shows, cell is only distributed in single layer nanofiber film surface (side).
Fig. 4-2 is that the coloration result of artificial organ engineering skin prepared by multilayer LBL is (red：Nanofiber；Blue：
DAPI), which shows, after nano fibrous membrane layer by layer-fat stem cell superposition, cell can be uniformly distributed in nanofiber
With interlayer total, cell density can obviously improve film surface compared with the inoculation of single layer fibre film.
The wound healing test result of Fig. 5-1, Fig. 5-2, the artificial organ engineering skin that Fig. 5-3 is the present invention.As a result it shows
Show, Different treatments obtain effect for wound healing, and there are notable differences, wherein Fig. 5-1 is control group (conventional gauze covering
Group), Fig. 5-2 is single 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 the artificial organ engineering skin flap coverage to 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 the artificial organ engineering skin flap coverage to 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.
Specific implementation mode
Technical scheme of the present invention 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 of the prior art and provide it is a kind of using fat stem cell as seed cell with
The artificial organ engineering skin and preparation method thereof that nano fiber scaffold is layering.Electrostatic spinning technique, macromolecule are complexed
Technology is combined with fiber-cell layer-by-layer, and preparing has good plasticity and suitable mechanical characteristic, height
The three-dimensional structure of porosity and the organization engineering skin of biocompatibility, the controllable biodegradation rate of the nano-bracket,
Suitable for skin regeneration organizational project.
Existing acellular matrix material is handled using xenogenesis skin through taking off cell, makes not and has antigenic biological material
Material.The covering that the tissue surface of a wound is provided by remaining cell epimatrix material (being mainly made of collagen and elastin laminin), provides group
It knits the holder of reparation and has certain value in clinical application.But since this host material lacks due and tissue repair
Related cell factor, purposes and validity are greatly affected.Numerous studies show that mescenchymal stem cell is such as free from fat
The fat stem cell of fat has more differentiation potentials, can be used as the ideal seed cell of tissue repair.Source for mesenchymal stem cells is wide
General, convenient material drawing, immunogenicity is low, and does not generate teratoma, still retains Multidirectional Differentiation after continuous passage culture and freezen protective
Potential can be used as the ideal seed cell of tissue repair.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 medicine 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, in artificial organ engineering skin field of stents, the material prepared for dermal scaffold is roughly divided into two classes：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 has the polyester such as polyglycolide, polycaprolactone, polyhydroxyalkanoates, polycarbonate-based
Class material；Most of dermal scaffolds are manufactured by electrostatic spinning technique with biomaterial.Although being obtained by electrostatic spinning
To nanofiber for cell stick and growth provides suitable configuration of surface, be conducive to adherency and life of the cell on holder
It is long, however the dermal scaffold prepared by electrostatic spinning is unfavorable for migration, the increasing of cell in the depth direction since aperture is too small
It grows, and is difficult to realize the Effective Regulation that cell is distributed on fibrous framework, which has limited artificial skin holders to cure in skin injury
Learn the application in therapy field.Meanwhile the holder as stem cell growth and epimatrix (ECM), it is good hydrophily, suitable
Surface charge, pore-size appropriate are more suitable for the apposition growth of cell.
According to the 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 suitble to cell growth differentiation of grade, and can be coated with as needed various
Cell factor or drug also carry out fiber-cell using LBL self-assembly (LBL) technology and are layering, promote from many aspects
The attachment and growth of stem cell, improve the efficiency of stem-cell therapy.Based on the above reasons, the present invention combines nanofiber-based material
Material and mescenchymal stem cell technology provide a kind of biomaterial having biological activity, which can be used for various tissues
Regeneration and reparation, especially wound healing, the formation of reduction scar, 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, to form artificial organ engineering skin.
Fat stem cell is separately cultured, purifies, identification 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 apparent connective group are cut off with eye scissors
It knits, is cleaned with PBS and shredded afterwards twice；The adipose tissue shredded is transferred in 50mL centrifuge tubes, 0.2% Type I collagen enzyme is added, sets
In 37 DEG C of 5%CO2It is primary per 30min mixings 2 hours in incubator；The tissue of digestion to paste is sieved through 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
Centrifugation after secondary is added the DMEM culture solutions containing 10%FBS and is gently blown and beaten with suction pipe, is allowed to be resuspended again；It is inoculated in and is pre-loaded with training
In the culture bottle for supporting base, it is placed in 37 DEG C of 5%CO2It is cultivated in incubator；Standing discard culture medium afterwards for 24 hours, with PBS clean twice with
The non-attached cell of removal, connective tissue and fragment, replace the fresh DMEM culture solutions containing 10%FBS, liquid were changed every 2 days, observe.
Embodiment 2, fat stem cell identification
The 3rd generation cell of culture is taken, after culture medium is sucked out, is first cleaned twice with PBS, 0.25% pancreatin that preheating is added disappears
Change, the DMEM containing 10%FBS is added later and terminates digestion, after gently blowing and beating into single cell suspension 1000rpm pelleted by centrifugation 5min,
It discards supernatant, PBS is cleaned twice；10 loading pipes are taken, often 1mL single cell suspensions are added in pipe, are 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；It is protected from light at room temperature and is incubated 20min；PBS cleanings are twice to remove not
Binding antibody, 500 μ L PBS use flow cytomery after being resuspended.
Embodiment 3, cell amplification
It is passed on after cell growth is converged to 80-90%, culture medium is sucked out, is first cleaned twice with PBS, is then added
0.25% pancreatin is placed in 37 DEG C of constant incubator 2min, and the DMEM culture solutions containing 10%FBS are then added and stop digestion, suction pipe
Gently piping and druming makes cell detachment, collects cell, moves into 15mL centrifuge tubes.1000rpm 5min abandon supernatant, and PBS is cleaned twice,
The DMEM containing 10%FBS is added, cell precipitation is resuspended, by 1:2 density are inoculated in culture bottle, and the DMEM containing 10%FBS is added and sets
In 37 DEG C of 5%CO2Continue to cultivate in incubator, microscopically observation cellular morphology obtains the fat stem cell of amplification.
Embodiment 4, a kind of preparation method of polycaprolactone-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 n,N-Dimethylformamide of (1~3)-dichloromethane mixed solution, it is 8% to be configured to mass concentration
Polycaprolactone solution；β-CD are added into polycaprolactone solution so that the mass fraction of β-CD is 0.1%, it is heated to 50~
It 70 DEG C, stirs evenly, mixing time is 6~12 hours, obtains polycaprolactone-β-CD solution；
Wherein, there can be brufen by envelope in advance in β-CD：It is 1 to weigh molal weight ratio:10~1:1 β-CD and cloth Lip river
In dry mortar 13% deionized water is added, 1~3h of grinding at room temperature will make in fragrant mixture in sealed environment into mortar
The packet and object got well 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 the speed of 1.0mL/h, and environment temperature is 25 DEG C, relative humidity 40%, and the electrostatic spinning time is
3min, obtains nano fibrous membrane, which is received in culture dish.
Embodiment 5, a kind of preparation method of polycaprolactone-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-
The mass concentration of CD is 0.02%；Syringe needle is 20cm at a distance from culture dish, and the DC voltage of application is 10 kilovolts；Syringe by
Syringe pump is driven with the speed of 2.0mL/h, and environment temperature is 35 DEG C, relative humidity 20%, and the electrostatic spinning time is 5min.
Embodiment 6, a kind of preparation method of polycaprolactone-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%；
The mass concentration of β-CD is 0.2%；Syringe needle is 5cm at a distance from culture dish, and the DC voltage of application is 35 kilovolts；Syringe by
Syringe pump is driven with the speed of 0.5m L/h, and environment temperature is 5 DEG C, relative humidity 80%, and the electrostatic spinning time is 1min.
Using layer-by-layer (LBL technologies) fat is alternately inoculated in polycaprolactone-β-CD nanofiber film surfaces
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 obtains 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 × 105A cell；Then again by the culture dish
Receiver as nano fibrous membrane carries out electrostatic spinning, fat stem cell inoculation is carried out again after electrostatic spinning, in this way, often
Layer inoculation 1 × 105A cell is inoculated with 10 layers in total.Because whole process is happened at media surface, cell is in packing engineering
It is always maintained at hydration status, a total of 10 confluent monolayer cells/nanofiber is alternately layering into three-dimensional structure.Culture dish is placed
In 37 DEG C of CO2Then DMEM/F12 supplementing culture mediums are added in incubator culture 30 minutes；Continue culture 1 week to form a kind of layer
The artificial organ engineering skin that layer is self-assembly of.Wherein DMEM/F12 culture solutions include 10% fetal calf serum and 1% penicillin/
Streptomysin.
Wherein, it if being only inoculated with a fat stem cell in nanofiber film surface, is received by layer-by-layer stacking
Rice tunica fibrosa and fat stem cell, by the way that single 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 × 106A 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 × 106A cell is inoculated with 20 layers in total.
The artificial organ engineering skin that LBL self-assembly prepared by 10 embodiment 7~9 of embodiment is formed, passes through epoxy second
It is packed after alkane sterilization, there is cryoprotector in packaging bag, freezen protective after packaging；Engineering skin after packaging can be cold
Freeze in -78~82 DEG C of refrigerator, can also be frozen in liquid nitrogen.
Embodiment 11
By 1 × 1cm2Polycaprolactone-beta-cyclodextrin fiber membrane sample carry out vacuum spray carbon processing after, swept using Flied emission
It retouches and observes fiber morphology on electron microscope, accelerating potential 15kV.Experimental results are 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.The results show that is prepared gathers oneself
For lactone-β-CD nano fibrous membranes in the three-dimensional 3D structures intersected, nanofiber is the uniform filament shape of thickness, diameter and hole point
Cloth is uniform, is in unordered arrangement, and avarage fiber diameter is 350 ± 72nm.
Embodiment 12
Fat stem cell is inoculated in after being co-cultured 3 days on polycaprolactone-beta-cyclodextrin nano fibrous membrane, using glutaraldehyde
After solution is fixed overnight at 4 DEG C, is cleaned with PBS, vacuum freeze drying is carried out after a series of Gradient elution using ethanols, it is dry
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 are respectively to be inoculated in polycaprolactone-for fat stem cell
1,3,5,7,9,11 days scanning electron microscope (SEM) photographs after β-CD single layer nano fibrous membranes.Scanning result shows, fat stem cell and nanometer
Tunica fibrosa sticks closely, can obviously observe that the filiform of cell is stretched with lamellipodia, Microfilaments In Cells wind mutually or mutually interconnect
It connecing, is in reticular 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
Born of the same parents' overlapping is in three dimensional growth, and iuntercellular forms pseudopodium identification, and forms cladding (Fig. 3-6), shows the nano fibrous membrane prepared tool
There is good biocompatibility, adherency and the proliferation of fat stem cell can be remarkably promoted.
Embodiment 13
Fat stem cell is inoculated in single layer polycaprolactone-beta-cyclodextrin nano fibrous membrane, co-cultures 3 days, is cleaned with PBS
3 times；30min, dry 5min are fixed using 4% formalin (being dissolved in PBS)；Then cleaned 3 times using PBS；It is used in combination 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：1:1000) dyeing 5min then, is carried out to nanofiber using rhodamine.It is cleaned with PBS unbonded to remove
Dyeing liquor, mounting carry out being copolymerized burnt observation.Experimental results show is in Fig. 4-1, wherein RED sector is nanofiber, blue
Part is DAPI；The figure shows that cell is only distributed in single layer nanofiber film surface (i.e. only in nano fibrous membrane side).
Fat stem cell using layer-by-layer replace with polycaprolactone-beta-cyclodextrin nano fibrous membrane and is folded
After adding, co-cultures 3 days, cleaned 3 times with PBS；30min, dry 5min are fixed using 4% formalin (being dissolved in PBS)；Then adopt
It is cleaned 3 times with PBS；It is used in combination 0.5%TRITON X-100 to permeate 20min, is then cleaned with PBS；Using DAPI to cell
Core carries out dyeing 10min (dilutions at room temperature：1:1000) dyeing 5min then, is carried out to nanofiber using rhodamine.
It is cleaned with PBS to remove unbonded dyeing liquor, mounting carries out being copolymerized burnt observation.Experimental results show is 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 nanofiber film surface with interlayer total, and 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, half male and half female, weight 200-250g, SPF grade, by the 4th medical university of army
Animal center is learned to provide.It is randomly divided into model group and control group by random digits table, every group 4, totally three groups of (control groups
(conventional gauze covering group), single 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 mold at back, periphery solid marks are cut off within the scope of solid marks
Full thickness skin to deep fascia layer, form full thickness dermal wounds.
Control group, single layer oil yarn flap coverage；
Single layer nanofiber-fat stem cell compound covering group：Single 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 grouping, 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
Subject to healing time.Wound healing situation is monitored in real time in different cycles after the surface of a wound is formed, it is soft using image analysis
Part analyzes photo before and after Wound treating, and healing rate is more than 90% and is 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 single layer nanofiber-fat stem cell is compound
Object covering group, Fig. 5-3 are artificial organization engineering skin covering group.The results show that Different treatments must imitate wound healing
Re-epithelialization degree is more there are notable difference, after the wound healing of the artificial organ engineering skin processing prepared through the present invention for fruit
Height illustrates that the material is best 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 paraffin embedding,
Slice is dyed using conventional hematoxylin-Yihong (HE) and is detected；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-corium layer separation),
Artificial organ engineering skin group does not observe apparent table-corium layer separation, 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, and baking piece is carried out after paraffin embedding, slice, is taken off
Wax, gradient enter water, after rinsing, R1, R2, R3, R4 liquid are used to be dyed (green skies staining kit) successively, 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 are aobvious
Show, artificial organ engineering skin prepared by the present invention has excellent degeneration energy, and has new life after material implantation
Angiogenesis.Experimental result confirms：Artificial organ engineering skin prepared by the present invention has better within the same implantation period
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 for the first time, made to penetrate
Stream solidification obtains the nanofiber of hyperfine structure, and the nanofiber prepared by this method has superfine fibre diameter, larger ratio
The unique advantages such as surface area and three-dimensional structure, this makes it be increasingly becoming the research hotspot that skin wound repairs field.This hair
It is bright that electrostatic spinning technique, reason is used to be:First, the tissue engineering bracket constructed by electrostatic spinning technique has in structure
There is the effect of simulation extracellular matrix (extracellularmatrix, ECM), fibroblast can be promoted to be formed with cutin
Adherency, proliferation 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.Third, β-CD (beta-cyclodextrin) have outer rim it is hydrophilic and
The hydrophobic special construction in inner cavity can improve the stability and medicament slow release energy of packet and object 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 adds
To realize that cell assembling is constituted three-dimensional structure, this three-dimensional structure can well control cell distribution, allow 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
A kind of emerging technology for adding realization cell assembling by nanofiber layer stackup is set up recently, and successfully by this skill
Art applies to build three-dimensional tissue (US 20080112998A1).It is layering in cell assembling this, 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
It needs 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, epidermal growth factor (EGF), blood are small
Plate derivative growth factor (PDGF) etc., can promote histocyte to regenerate；
4) because fat stem cell is derived from itself, therefore do not have antigenicity；
5) electrostatic spinning polycaprolactone tissue engineering bracket has simulation extracellular matrix in structure
The effect of (extracellular matrix, ECM), can promote adherency, proliferation and the migration of fat stem cell；And it can be notable
Accelerate wound healing；
6) β-CD have the special construction that outer rim is hydrophilic and inner cavity is hydrophobic, can be by forming Subjective and Objective inclusion complex to improve
The stability of packet and object and medicament slow release ability；
7) it carries out nanofiber layer stackup using the new technology of nanofiber-cell LBL self-assembly and is realized groups of cells
Dress constitutes three-dimensional structure tissue.This three-dimensional structure can well control cell distribution, allow co-culture various kinds of cell, and
Physically separating various cells does not influence its mass exchange but.
Claims (9)
1. the artificial organ engineering skin that a kind of nano fibrous membrane is layering with stem cell, which is characterized in that nano fibrous membrane
It is prepared by polycaprolactone and beta-cyclodextrin, including multilayer layer and the multi-layer porous Nanowire with three-dimensional structure
Film is tieed up, layer is alternately laminated with nano fibrous membrane.
2. the artificial organ engineering skin that nano fibrous membrane as described in claim 1 is layering with stem cell, 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 fiber film surface.
3. the artificial organ engineering skin that nano fibrous membrane as described in claim 1 is layering with stem cell, feature exist
In the stem cell is fat stem cell.
4. the artificial organ engineering skin that nano fibrous membrane as described in claim 1 is layering with stem cell, feature exist
In envelope has brufen in beta-cyclodextrin.
5. the artificial organ engineering skin that nano fibrous membrane as described in claim 1 is layering with stem cell, 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.
6. the artificial organ engineering skin that Claims 1 to 5 any one of them nano fibrous membrane is layering with stem cell
Preparation method, which is characterized 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) step 21) and 22) is repeated, until completing the inoculation number of plies of setting；
24) in culture dish, nano fibrous membrane and stem cell are co-cultured.
7. the preparation side for the artificial organ engineering skin that nano fibrous membrane as claimed in claim 6 is layering with stem cell
Method, which is characterized 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
Galvanic electricity pressure is 10~35kV；Syringe needle is 5~20cm at a distance from culture dish during electrostatic spinning；It is injected during electrostatic spinning
Device is driven by syringe pump with the speed of 0.5~2.0mL/h；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.
8. the preparation side for the artificial organ engineering skin that nano fibrous membrane as claimed in claim 6 is layering with stem cell
Method, which is characterized in that
In step 22), relative to the area of nano fibrous membrane, the quantity of stem cell inoculation is 1 × 104~2 × 104A cell/
cm2。
9. the preparation side for the artificial organ engineering skin that nano fibrous membrane as claimed in claim 6 is layering with stem cell
Method, which is characterized in that further include step:
3) artificial organ engineering skin prepared by step 2) is packed after sterilization, freezing.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710577134.3A CN107349475B (en) | 2017-07-14 | 2017-07-14 | The artificial organ engineering skin and preparation method thereof that nano fibrous membrane is layering with stem cell |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710577134.3A CN107349475B (en) | 2017-07-14 | 2017-07-14 | The artificial organ engineering skin and preparation method thereof that nano fibrous membrane is layering with stem cell |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN107349475A CN107349475A (en) | 2017-11-17 |
| CN107349475B true CN107349475B (en) | 2018-10-02 |
Family
ID=60292576
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710577134.3A Active CN107349475B (en) | 2017-07-14 | 2017-07-14 | The artificial organ engineering skin and preparation method thereof that nano fibrous membrane is layering with stem cell |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107349475B (en) |
Families Citing this family (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109529123B (en) * | 2018-11-08 | 2021-02-19 | 中国人民解放军第四军医大学 | Vascularized full-layer tissue engineering skin formed by assembling hydrogel, nanofiber scaffold and skin cells layer by layer and preparation method thereof |
| CN111484973B (en) * | 2020-06-04 | 2021-08-27 | 铜仁市泛特尔生物技术有限公司 | Purification method of adipose-derived stem cells |
| CN113893388A (en) * | 2021-09-23 | 2022-01-07 | 上海市第六人民医院 | Modular tissue engineering bone-ligament-bone graft and preparation method thereof |
| CN115558126A (en) * | 2021-11-29 | 2023-01-03 | 海诺生物科技有限公司 | Dynamically crosslinked hyaluronic acid hydrogels |
| CN114949364B (en) * | 2022-05-30 | 2022-12-27 | 四川大学 | Multilayer tissue engineering bionic periosteum scaffold and preparation method and application thereof |
| CN115804869A (en) * | 2022-10-26 | 2023-03-17 | 王丽 | BADSCs membrane and conductive nanofiber composite heart patch and preparation method thereof |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101361990A (en) * | 2008-09-03 | 2009-02-11 | 陕西瑞盛生物科技有限公司 | Double layer artificial skin and preparation method thereof |
| CN101829361A (en) * | 2009-03-10 | 2010-09-15 | 广州迈普再生医学科技有限公司 | Nano-bionic material for tissue repair and preparation method thereof |
| CN104984407A (en) * | 2015-07-01 | 2015-10-21 | 世科志扬（北京）医疗科技有限公司 | Tissue engineering artificial skin and preparation method thereof |
| CN106110401A (en) * | 2016-06-30 | 2016-11-16 | 上海大学 | Micro-nano composite double layer dermal scaffold and preparation method thereof |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20080112998A1 (en) * | 2006-11-14 | 2008-05-15 | Hongjun Wang | Innovative bottom-up cell assembly approach to three-dimensional tissue formation using nano-or micro-fibers |
-
2017
- 2017-07-14 CN CN201710577134.3A patent/CN107349475B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101361990A (en) * | 2008-09-03 | 2009-02-11 | 陕西瑞盛生物科技有限公司 | Double layer artificial skin and preparation method thereof |
| CN101829361A (en) * | 2009-03-10 | 2010-09-15 | 广州迈普再生医学科技有限公司 | Nano-bionic material for tissue repair and preparation method thereof |
| CN104984407A (en) * | 2015-07-01 | 2015-10-21 | 世科志扬（北京）医疗科技有限公司 | Tissue engineering artificial skin and preparation method thereof |
| CN106110401A (en) * | 2016-06-30 | 2016-11-16 | 上海大学 | Micro-nano composite double layer dermal scaffold and preparation method thereof |
Non-Patent Citations (1)
| Title |
|---|
| Polycaprolactone/Amino--Cyclodextrin Inclusion Complex Prepared by an Electrospinning Technique;Edgar Moyers-Montoya等;《Polymers》;20161118;第8卷(第395期);第2页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN107349475A (en) | 2017-11-17 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN107349475B (en) | The artificial organ engineering skin and preparation method thereof that nano fibrous membrane is layering with stem cell | |
| Fu et al. | Skin tissue repair materials from bacterial cellulose by a multilayer fermentation method | |
| US20190216984A1 (en) | Vascularized full thickness tissue-engineered skin assembled by hydrogel, nanofibrous scaffolds and skin cell layers and preparation method thereof | |
| Mohamed et al. | Nanomaterials and nanotechnology for skin tissue engineering | |
| Han et al. | Application of collagen-chitosan/fibrin glue asymmetric scaffolds in skin tissue engineering | |
| CN109529123B (en) | Vascularized full-layer tissue engineering skin formed by assembling hydrogel, nanofiber scaffold and skin cells layer by layer and preparation method thereof | |
| CN111714706B (en) | Vascular stent capable of promoting vascular cell proliferation and secreting extracellular matrix, preparation method of vascular stent and active artificial blood vessel | |
| Maghdouri-White et al. | Bioengineered silk scaffolds in 3D tissue modeling with focus on mammary tissues | |
| CN109999227B (en) | Preparation method and application of silk fibroin and chitin-based blended nanofiber embedded hydrogel cartilage bionic scaffold | |
| CN101302486B (en) | Acetobacter xylinum and method for preparing nano-cellulose skin tissue repair material by using the same | |
| CN102505184B (en) | Tissue engineering fiber bundle structure body and preparation method thereof | |
| CN110772669A (en) | Biological ink for 3D printing of artificial skin | |
| CN105854077B (en) | A kind of preparation method of new type nerve repair tissue engineering rack | |
| WO2014114043A1 (en) | Cell matrix modified tissue engineering nerve graft for repairing peripheral nerve injury and preparation method thereof | |
| Li et al. | Recent progress in tissue engineering and regenerative medicine | |
| JPS58501772A (en) | Method of seeding cells into fiber lattices by centrifugation | |
| CN110193098B (en) | Multilayer gradient biological membrane and preparation method thereof | |
| CN109154111A (en) | Cell culturing bracket yarn, the cell culturing bracket fabric comprising it | |
| CN106860915A (en) | A kind of mineralized collagen bionic bone repair material of hyaluronic acid oligosaccharide modification and preparation method thereof | |
| CN104399125A (en) | Method for differentiating epidermal stem cells to sweat gland-like epithelial cells | |
| CN107137763A (en) | A kind of study of vascularized tissue engineering bone and preparation method thereof | |
| CN102764451B (en) | Preparation method and application of porous biological cellulose gel material | |
| CN107823704A (en) | A kind of paradenlal tissue regeneration repair membrane and preparation method thereof | |
| JP2016524967A (en) | Functionalized three-dimensional scaffold using microstructure for tissue regeneration | |
| CN110201236A (en) | A kind of novel artificial blood vessel |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |