CN105769381A - Biological patch for tissue damage restoration - Google Patents
Biological patch for tissue damage restoration Download PDFInfo
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- CN105769381A CN105769381A CN201510273678.1A CN201510273678A CN105769381A CN 105769381 A CN105769381 A CN 105769381A CN 201510273678 A CN201510273678 A CN 201510273678A CN 105769381 A CN105769381 A CN 105769381A
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Abstract
The invention discloses a biological patch for tissue damage restoration. The biological patch comprises a biological material substrate layer and a cell substrate layer, wherein the substrate layer is attached onto the substrate layer; and a plurality of parallel grooves are formed in the contact surface of the substrate layer and the cell substrate layer. The substrate layer of the biological patch is simultaneously provided with porous and groove vein structures; the orientation growth and distribution of cells can be regulated and controlled; the cell substrate layer can be well attached onto the substrate layer through the mechanical property of the groove vein structures; and the falling is avoided. A product of the biological patch has the advantages that the structure is stable; the pore diameter, the porosity and the permeability are proper; the normal tissue structure can be simulated; the tissue growth pipeline and cell substrates can be provided; and cells favorable for tissue restoration and regeneration can be carried.
Description
Technical field
The present invention relates to medical instruments field, particularly to a kind of biological sticking patch repaired for tissue injury.
Background technology
The events such as vehicle accident in society, industrial accident, motion accident and clinical operation all can cause tissue injury, and use carries out substituting and repairing the material of tissue as biological sticking patch more clinically.Biological sticking patch refers to takes from one of the same race to tissue, processes, through de-cell, the various cells contained in removal tissue and completely retains the three-dimensional frame structure of extracellular matrix and can be used for repairing the material of human body soft tissue.Being divided into allogenic material such as corium acellular matrix, amniotic membrane, cerebral dura mater etc. according to tissue-derived, heterogenous allosome material is intestinal mucosa lower floor, cattle, the pericardium of horse, cattle peritoneum etc. such as.Though this natural biological sticking patch commercialization, prepare loaded down with trivial details, and price is high.Find cheap, easy be easy to get, the synthetic biological sticking patch of good biocompatibility remains the focus of research to substitute natural biological sticking patch.
Summary of the invention:
It is an object of the invention to provide a kind of good biocompatibility, the biological sticking patch repaired for tissue injury being easy to get cheap, easy.
The technical solution of the present invention is:
A kind of biological sticking patch repaired for tissue injury, including biomaterial basal layer and cellular matrix layer, hypothallus adheres on the base layer, and basal layer is provided with several parallel grooves with the contact surface of cellular matrix layer.Groove can be the convex groove protruding from substrate surface, it is also possible to for the groove shape caved inward from substrate surface.Groove is for laterally or longitudinally to arrange.
Preferred groove is of a size of: wide for 10-50 μm, is deeply 10-30 μm.
Above-mentioned biological sticking patch, the porosity on described biomaterial basal layer is 50%-90%, and aperture is 10 μm-100 μm.
Above-mentioned synthetic biological sticking patch, described biomaterial base layer thickness is 0.1-3mm.
Above-mentioned synthetic biological sticking patch, described biomaterial basal layer is made up of one or more in fibroin albumen, chitosan, collagen, polylactic acid or polyglycolic acid.
Above-mentioned biological sticking patch, described cellular matrix layer is autologous or takes off cell after the secretion formation of allogeneic derived cell and obtain, and described cell is one or more in Scs, the fibroblast of skin-derived, skin progenitor cell, mesenchymal stem cells MSCs, navel blood stem cell or induced pluripotent stem cells.
Above-mentioned biological sticking patch does not contain foaming agent and/or cross-linking agent.
The preparation method that the invention also discloses above-mentioned biological sticking patch,
(1) design PDMS elastomeric stamp lines, is added on seal by biomaterial solution, adopts the method for lyophilizing to prepare surface and have the biomaterial basal layer of ditch slot texture after shaping and demoulding；
(2) biomaterial basal layer and cell being cultivated, obtaining attaching growth has the biomaterial basal layer of cell；
(3) growth has the biomaterial basal layer of cell carry out de-cell to process, it is carried out further lyophilizing, obtains biological sticking patch.
One preferred version of the present invention is as follows:
The principle that shape and the spreading behavior of cell are affected by the size according to very low power, design can guide the micrographics structure with channel form of the distribution of orientations of neurocyte.
Elastomeric stamp used in the present invention is polydimethylsiloxane (polydimethylsiloxane, PDMS), its preparation method is in after the ratio mix homogeneously of 10:1 by the PDMS liquid prepared under room temperature and cross-linking agent, what be cast to employing mask exposure acquisition in advance has on the silicon wafer-based caster that channel form pattern laterally or longitudinally is distributed, then put it into evacuation in vacuum drying oven and get rid of bubble, and carry out dry solidification, peel off after finally solidifying and namely obtain plane PDMS elastomeric stamp.
Prepare the solution of finite concentration (1%, 2% and 5%) with acetic acid chitosan, then this solution dripped on PDMS, be placed in-60 DEG C--90 DEG C, freezing 2-4 hour, chitosan ice body after freezing is placed in alkaline solution and fixing, cleans with water；To sizing chitosan-based bottom carry out lyophilization, obtain that there is loose structure, surface have channel form distribution porous chitosan basal layer.
Porous chitosan basal layer is carried out Three-dimensional cell culture obtain surface attach growth have the chitosan-based bottom of cell to take out.Growth has the chitosan-based bottom of cell carry out de-cell process, it is carried out further lyophilizing, obtains biological sticking patch of the present invention.
Using the aperture that scanning electron microscope detects biological sticking patch hole of the present invention is 10 μm-100 μm.Biological sticking patch basal layer of the present invention has porous and groove texture structure simultaneously, not only can regulating cell orientation growth and distribution, the mechanical property that groove texture structure has can enable cellular matrix layer better adhere on the base layer, it is to avoid comes off.Product of the present invention has stable structure, suitable aperture, porosity and permeability, normal organization can be simulated, pipeline and the cellular matrix of tissue growth can be provided, and the cell being beneficial to tissue repair and regeneration can be carried, the product material therefor of the present invention is natural degradable material, has good biocompatibility, prepared product not to contain foaming agent and the cross-linking agent of the exogenous toxicity, side effect brought into by preparation technology with human body.The present invention has good tensile strength and has cellular matrix layer, is more beneficial for the nutrient substance needed for conveying cell growth process, provides good growing environment to cell.Result of use is good.The inventive method is simple and easy to do.
Accompanying drawing explanation
Fig. 1Illustrate for biological sticking patch substrate surface ditch slot texture of the present inventionFigure。
Fig. 2For cell orientation growth signal on the basal layer of biological sticking patch surface graphics of the present inventionFigure。
Fig. 3For biological sticking patch structural representation of the present inventionFigure (A is the biological sticking patch signal of convex grooveFigure, B is the biological sticking patch signal of concave channelsFigure, 1 is basal layer, and 2 is groove, and 3 is cellular matrix layer).
Detailed description of the invention
Below in conjunction with embodiment, the invention will be further described.
The term used in the present invention, except as otherwise noted, generally has the implication that those of ordinary skill in the art are generally understood that.
Also with reference to data, the present invention is described in further detail below in conjunction with specific embodiment.Should be understood that these embodiments present invention solely for the purpose of illustration, but not the scope being intended to limit the present invention in any manner.
In the examples below, the various processes not being described in detail and method are conventional methods as known in the art.
Embodiment 1
(1) cultivation of Scs and purification
Take newborn one day SD rat, locate after death alcohol disinfecting, take bilateral sciatic nerve, it is placed in ice bath operating board remove epineurium and be adhered tissue, collagenase/pancreatin mixture slaking is centrifugal completely afterwards abandons supernatant, complete medium re-suspended cell, is inoculated in the PDL culture dish being coated in advance and cultivates.Within 24 hours, it is replaced with the complete medium containing cytosine arabinoside (10 μMs) to cultivate 48 hours, it is replaced by the complete medium containing HRG (50ng/ml) and Forsklin (2 μMs) to continue to cultivate, within every 3 days, change liquid, until cell fusion.After cell fusion, it is centrifuged after trypsinization and obtains cell precipitation, with 1ml complete medium (1:1000) re-suspended cell containing Thy1.1, hatch 2 hours on ice；Being centrifuged and abandon supernatant, with mixture (3:1) re-suspended cell of DMEM and complement, hatch 1 hour for 37 DEG C, after centrifugal, complete medium cleans 2 times, is re-seeded in culture dish, changes liquid every other day, and namely cell can use after covering with.
(2) fibroblastic cultivation of skin-derived
Take the SD rat of newborn 1 day, locate after death alcohol disinfecting, take out skin of back and be placed in the dissection liquid of pre-cooling and reject subcutaneous tissue (fat and subcutaneous fascia layer, blood vessel etc.) carefully；After PBS 3 times, it is cut into small pieces (< 1mm × 1mm) with knife blade, abandons supernatant, complete medium re-suspended cell with centrifugal after type i collagen enzyme (1mg/ml) catapepsis, it is inoculated into culture dish to cultivate, Secondary Culture after cell fusion 90%.Fibroblast has epithelial cell in original cuiture process and pollutes, and most of heteroproteose cells (epithelium and endotheliocyte) can be dead gradually after going down to posterity several times.Therefore, cell used by the present invention is for passing 3 generation above cells.
(3) cultivation of skin progenitor cell and orientation are broken up along Scs
nullThe cultivation of skin progenitor cell and differentiation reference literature " Isolationofskin-derivedprecursors (SKPs) anddifferentiationandenrichmentoftheirSchwanncellprogeny " (JeffreyABiernaskie,IanAMcKenzie,JeanGToma&FredaDMiller，NATUREPROTOCOLS,2006(1):2803-2812)，It is briefly described as follows: take the SD rat of newborn 1 day，Locate after death alcohol disinfecting，Take out skin of back to be placed in the dissection liquid of pre-cooling and reject subcutaneous tissue (fat and subcutaneous fascia layer carefully、Blood vessel etc.)；After PBS 3 times, it is cut into small pieces (< 1mm × 1mm) with knife blade, pancreatin or XI Collagenase Type (1mg/ml) 37 DEG C with 0.1% digest 45 60min, complete training terminates digesting being centrifuged abandons supernatant, with proliferated culture medium, (DMEM/F12 (3:1) comprises 0.1% dual anti-, 40 μ g/mlfungizone, 40ng/mlFGF2,20ng/mlEGF, 2%B27supplement) carry out suspension culture.The cell ball of suspension culture can carry out Secondary Culture and obtain sufficient amount of skin progenitor cell.
The skin progenitor cell of acquisition is carried out differentiation culture to obtain Scs, specifically comprise the following steps that (it is dual anti-that DMEM/F12 (3:1) comprises 0.1% with division culture medium I by skin progenitor cell, 40 μ g/mlfungizone, 40ng/mlFGF2,20ng/mlEGF, 2%B27supplement, after 10%FBS) cultivating 3 days, then at division culture medium II, (it is dual anti-that DMEM/F12 (3:1) comprises 0.1%, 5 μm of forskolin, 50ng/mlheregulin-1 β, 50 μ g/ml, 2%N2supplement, 1%FBS) Scs colony can be obtained after middle cultivation 2-3 week.The Scs (SKP-SC) obtaining the differentiation of a large amount of skin progenitor cell of amplification after picking colony, Showed by immune group result is that S100 is positive.
(4) cultivation of mesenchymal stem cells MSCs
Adult SD rats, 75% alcohol-pickled 5min are put to death in dislocation, take femur, tibia under aseptic condition, expose medullary cavity, and IMDM basic culture solution rinses medullary cavity, collect bone marrow.Syringe aspirates the bone marrow of results repeatedly, makes single cell suspension.200 eye mesh screens filter, and put in horizontal centrifuge centrifugal, and 1000rpm × 5min abandons supernatant.With 4 × 105/cm2Density be inoculated in IMDM complete culture solution (containing hyclone 10%), be placed in 37 DEG C of cultivations.After 24h, full dose changes liquid, removes non-attached cell, and every 3d half amount changes liquid later.Day by day observation of cell form and growing state under inverted microscope.90% fusion, Secondary Culture is reached when cell is paved with at the bottom of ware.
The preparation of embodiment 2 biological sticking patch
(1) preparation of elastomeric stamp
Elastomeric stamp used in the present invention is polydimethylsiloxane (polydimethylsiloxane, PDMS), its preparation method is in after the ratio mix homogeneously of 10:1 by the PDMS liquid prepared under room temperature and cross-linking agent, it is cast to having on the silicon wafer-based caster that channel form pattern laterally or longitudinally is distributed of employing mask exposure acquisition in advanceSuch as Fig. 1Shown in.Then put it into evacuation in vacuum drying oven and get rid of bubble, and carry out dry solidification, peel off after finally solidifying and namely obtain plane PDMS elastomeric stamp.
(2) preparation of the chitosan-based bottom of pattern
First with the solution of acetic acid chitosan preparation finite concentration (1%, 2% and 5%), then this solution is dripped on PDMS, is placed in-60 DEG C--90 DEG C, freezing 2-4 hour, chitosan ice body after freezing is placed in alkaline solution and fixing, cleans with water；To sizing chitosan-based bottom carry out lyophilization, obtain that there is loose structure, surface have channel form distribution porous chitosan basal layer；
(3) structure of cellular matrix layer
Complete medium is slowly injected into culture vessel, adds 2.5 × 107Individual cell and aseptic process cross by chitosan-based bottom, then complete medium is filled whole container, controlling final cell density is 1 × 105/ ml, drains in system after air, starts to rotate microgravity circumfusion and cultivates；Rotary bioreactor puts into 37 DEG C of CO2In incubator, within first 24 hours, rotating speed is 10 revs/min, makes cell and chitosan-based bottom be fully contacted, attaches；Adjust rotary bioreactor rotary speed after 24 hours, make chitosan-based bottom can be suspended in culture fluid；It is replaced by division culture medium after continuing cultivation 2 days to cultivate, to promote Extracellular Matrix Secretion；Terminating after cultivating 2 weeks cultivating attaching to grow by surface has the chitosan-based bottom of cell to take out.Microscopy cell is orientation growth on the chitosan-based bottom of surface graphics,Such as Fig. 2Shown in.Growth has the chitosan-based bottom of cell carry out de-cell process, 37 DEG C of hypotonic 10min of deionization sterilized water after PBS；Add extraction liquid of cell in 37 DEG C of cell lysis 10-15min；Add DnaseI (4mg/ml) 37 DEG C after PBS 3 times to digest 30min and remove DNA, it carried out further lyophilizing, obtains biological sticking patch of the present invention,Such as Fig. 3Shown in, A is the biological sticking patch signal of convex grooveFigure, B is the biological sticking patch signal of concave channelsFigure, 1 is basal layer, and 2 is groove, and 3 is cellular matrix layer.
Scanning electron microscope result shows the extracellular matrix components being uniformly distributed support emiocytosis in chitosan-based bottom surface, and having made the chitosan-based bottom containing n cell hypothallus can save backup at-80 DEG C, it is possible to lyophilization preserves further.Then freeze-dried obtaining has abundant hole containing cytostromatic chitosan biological sticking patch, chitosan-based bottom surface and internal distribution, and cellular matrix layer is attached to basal layer and pore surface.Porosity 89% after measured, average pore size is 87-98 μm.
Claims (10)
1. the biological sticking patch repaired for tissue injury, it is characterised in that include biomaterial basal layer and cellular matrix layer, hypothallus adheres on the base layer, and basal layer is provided with several parallel grooves with the contact surface of cellular matrix layer.
2. biological sticking patch as claimed in claim 1, it is characterised in that described groove is for be laterally or longitudinally distributed.
3. biological sticking patch as claimed in claim 1, it is characterised in that described groove is of a size of: wide 10-50 μm, deep 10-30 μm.
4. biological sticking patch as claimed in claim 1, it is characterised in that described biomaterial basal layer has abundant hole with the contact surface upper surface of cellular matrix layer and internal distribution.
5. biological sticking patch as claimed in claim 4, it is characterised in that the porosity on described biomaterial basal layer is 50%-90%, and aperture is 10 μm-100 μm.
6. biological sticking patch as claimed in claim 1, it is characterised in that described biomaterial base layer thickness is 0.1-3mm.
7. biological sticking patch as claimed in claim 1, it is characterised in that described biomaterial basal layer is made up of one or more in fibroin albumen, chitosan, collagen, polylactic acid or polyglycolic acid.
8. the biological sticking patch as described in one of claim 1-7 item, it is characterised in that described cellular matrix layer is autologous or takes off cell after the secretion formation of allogeneic derived cell and obtain.
9. biological sticking patch as claimed in claim 8, it is characterised in that described cell is one or more in Scs, the fibroblast of skin-derived, skin progenitor cell, mesenchymal stem cells MSCs, navel blood stem cell or induced pluripotent stem cells.
10. the preparation method of the biological sticking patch as described in one of claim 1-9 item, it is characterised in that comprise the steps:,
(1) design PDMS elastomeric stamp lines, is added on seal by biomaterial solution, adopts the method for lyophilizing to prepare surface and have the biomaterial basal layer of ditch slot texture after shaping and demoulding；
(2) biomaterial basal layer and cell being cultivated, obtaining attaching growth has the biomaterial basal layer of cell；
(3) growth has the biomaterial basal layer of cell carry out de-cell to process, it is carried out further lyophilizing, obtains biological sticking patch.
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| CN113150321A (en) * | 2021-04-09 | 2021-07-23 | 南通大学 | Preparation method of hydrogel with different elastic chitosan/acrylamide micro-nano topological structures |
| CN116159185A (en) * | 2022-12-26 | 2023-05-26 | 烟台德胜海洋生物科技有限公司 | Composite acellular matrix material and preparation method thereof |
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