CN204971702U - Be used for prosthetic biological patch of tissue lesion - Google Patents
Be used for prosthetic biological patch of tissue lesion Download PDFInfo
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- CN204971702U CN204971702U CN201520347479.6U CN201520347479U CN204971702U CN 204971702 U CN204971702 U CN 204971702U CN 201520347479 U CN201520347479 U CN 201520347479U CN 204971702 U CN204971702 U CN 204971702U
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- cell
- sticking patch
- biological sticking
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Abstract
The utility model discloses a be used for prosthetic biological patch of tissue lesion, including biomaterial stratum basale and cell matrix layer, the matrix layer is on attached to the stratum basale, is equipped with the parallel slot of a plurality of on the contact surface on stratum basale and cell matrix layer. The utility model discloses biological patch stratum basale has porous and ditch corrugation way structure simultaneously, not only can regulate and control the orientation of cell and grow and distribute, and mechanical properties that ditch corrugation way structure has can make adhesion that the cell matrix layer can be better on the stratum basale, avoids coming off. The product has the stable structure, and normal organizational structure can be simulated to suitable aperture, porosity and permeability, can provide the pipeline and the cell matrix of tissue growth to can bear and do benefit to repair of tissue and palingenetic cell.
Description
Technical field
This utility model relates to medical instruments field, particularly 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, are used as biological sticking patch to carry out substituting and repairing the material of tissue clinically more.Biological sticking patch refers to takes from one of the same race to tissue, removes the three-dimensional frame structure of the various cell that contains in tissue and complete reservation extracellular matrix and can be used for repairing the material of human body soft tissue through de-cell process.According to the tissue-derived allogenic material that is divided into as corium acellular matrix, amniotic membrane, cerebral dura mater etc., heterogenous allosome material is as the pericardium, cattle peritoneum etc. of intestinal mucosa lower floor, cattle, horse.Although the commercialization of this natural biological sticking patch, prepare loaded down with trivial details, price is high.Find cheap, to be easyly easy to get, the synthetic biological sticking patch of good biocompatibility carrys out the focus that alternative natural biological sticking patch remains research.
Summary of the invention:
The purpose of this utility model is to provide a kind of good biocompatibility, cheap, the easy biological sticking patch repaired for tissue injury be easy to get.
Technical solution of the present utility model is:
For the biological sticking patch that tissue injury repairs, comprise biomaterial basal layer and cellular matrix layer, on the base layer, basal layer and the contact surface of cellular matrix layer are provided with several parallel grooves in hypothallus attachment.Groove can, for protruding from the convex groove of substrate surface, also can be the groove shape caved inward from substrate surface.Groove is laterally or longitudinally arrange.
Preferred groove is of a size of: wide is 10-50 μm, is 10-30 μm deeply.
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 be autologous or the secretion of allogeneic derived cell formed after de-cell and obtaining, 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 is not containing foaming agent and/or cross-linking agent.
The invention also discloses the preparation method of above-mentioned biological sticking patch,
(1) design PDMS elastomeric stamp lines, biomaterial solution is added on seal, adopt the method for lyophilizing to prepare after shaping and demoulding biomaterial basal layer that surface has ditch slot texture;
(2) biomaterial basal layer and cell are cultivated, obtain attaching the biomaterial basal layer that growth has cell;
(3) there is the biomaterial basal layer of cell to carry out de-cell process growth, it is carried out lyophilizing further, obtains biological sticking patch.
A preferred version of the present utility model is as follows:
According to the principle that the size of very low power affects the shape of cell and spreading behavior, design can guide the micrographics structure with channel form of the distribution of orientations of neurocyte.
The elastomeric stamp that this utility model uses is polydimethylsiloxane (polydimethylsiloxane, PDMS), its preparation method is in after the ratio mix homogeneously of 10:1 by the PDMS liquid for preparing under room temperature and cross-linking agent, be cast to and adopt on the silicon wafer-based caster with horizontal or longitudinal channel form pattern distribution of mask exposure acquisition in advance, then put it into evacuation in vacuum drying oven and get rid of bubble, and carry out dry solidification, be finally cured rear stripping and namely obtain plane P DMS elastomeric stamp.
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, freezing rear chitosan ice body be placed in alkaline solution and fix, cleaning 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 and obtain the chitosan-based bottom taking-up that surface attaching growth has cell.There is the chitosan-based bottom of cell to carry out de-cell process growth, it is carried out lyophilizing further, obtains biological sticking patch described in the utility model.
The aperture using scanning electron microscope to detect biological sticking patch hole described in the utility model is 10 μm-100 μm.
This utility model biological sticking patch basal layer 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, avoids coming off.Product described in the utility model 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, product material therefor of the present utility model is natural degradable material, has good biocompatibility with human body, and obtained product does not contain foaming agent and the cross-linking agent of the exogenous toxicity, side effect brought into by preparation technology.This utility model has good tensile strength and has cellular matrix layer, is more conducive to carrying the nutrient substance needed for cell growth process, provides good growing environment to cell.Result of use is good.Method is simple for this utility model.
Accompanying drawing explanation
Fig. 1 is biological sticking patch substrate surface described in the utility model vertical ditch slot texture schematic diagram.
Fig. 2 is biological sticking patch substrate surface lateral trench lines schematic diagram described in the utility model.
Fig. 3 is cell orientation growth schematic diagram on the basal layer of biological sticking patch surface graphics described in the utility model.
Fig. 4 is the biological sticking patch structural representation (1 is basal layer, and 2 is groove, and 3 is cellular matrix layer) of convex groove described in the utility model.
Fig. 5 is the biological sticking patch structural representation (1 is basal layer, and 2 is groove, and 3 is cellular matrix layer) of concave channels described in the utility model.
Detailed description of the invention
Below in conjunction with embodiment, the utility model is described in further detail.
The term used in this utility model, except as otherwise noted, generally has the implication that those of ordinary skill in the art understand usually.
Below in conjunction with specific embodiment and comparable data describes in further detail this utility model.Should be understood that these embodiments just illustratively this utility model, but not limit scope of the present utility model by any way.
In the examples below, the various process do not described in detail and method are conventional methods as known in the art.
Embodiment 1
(1) cultivation of Scs and purification
Get newborn one day SD rat, alcohol disinfecting after putting to death, gets bilateral sciatic nerve, be placed in ice bath operating board remove epineurium and be adhered tissue, centrifugally after collagenase/pancreatin mixture slaking completely abandons supernatant, complete medium re-suspended cell, be inoculated into PDL and wrap in advance and cultivated by good culture dish.The complete medium be replaced with for 24 hours containing cytosine arabinoside (10 μMs) cultivates 48 hours, the complete medium be replaced by containing HRG (50ng/ml) and Forsklin (2 μMs) continues to cultivate, within every 3 days, change liquid, until cell fusion.After cell fusion, centrifugally after trypsinization obtain cell precipitation, with complete medium (1:1000) re-suspended cell of 1ml containing Thy1.1, hatch 2 hours on ice; Centrifugally abandon supernatant, with mixture (3:1) re-suspended cell of DMEM and complement, hatch 1 hour for 37 DEG C, centrifugal rear complete medium cleans 2 times, is re-seeded in culture dish, changes liquid every other day, namely can use after cell covers with.
(2) fibroblastic cultivation of skin-derived
Get the SD rat of newborn 1 day, alcohol disinfecting after putting to death, the dissection liquid that taking-up skin of back is placed in pre-cooling rejects subcutaneous tissue (fat and subcutaneous fascia layer, blood vessel etc.) carefully; After PBS cleans 3 times, be cut into small pieces (<1mm × 1mm) with knife blade, abandon supernatant, complete medium re-suspended cell with centrifugal after type i collagen enzyme (1mg/ml) catapepsis, be inoculated into culture dish to cultivate, Secondary Culture after cell fusion 90%.Fibroblast has epithelial cell and pollutes in original cuiture process, and most of heteroproteose cell (epithelium and endotheliocyte) can be dead gradually after going down to posterity several times.Therefore, this utility model cell used is biography 3 generation above cell.
(3) cultivation of skin progenitor cell and orientation are broken up along Scs
The cultivation of skin progenitor cell and differentiation reference literature " Isolationofskin-derivedprecursors (SKPs) anddifferentiationandenrichmentoftheirSchwanncellprogeny " (JeffreyABiernaskie, IanAMcKenzie, JeanGToma & FredaDMiller, NATUREPROTOCOLS, 2006 (1): 2803-2812), be briefly described as follows: the SD rat of getting newborn 1 day, alcohol disinfecting after putting to death, the dissection liquid that taking-up skin of back is placed in pre-cooling rejects subcutaneous tissue (fat and subcutaneous fascia layer carefully, blood vessel etc.), after PBS cleans 3 times, (<1mm × 1mm) is cut into small pieces with knife blade, pancreatin with 0.1% or XI Collagenase Type (1mg/ml) 37 DEG C digestion 45 – 60min, complete training termination digestion is centrifugal 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 the skin progenitor cell that Secondary Culture has obtained sufficient amount.
The skin progenitor cell of acquisition is carried out differentiation culture to obtain Scs, concrete steps are as follows: by skin progenitor cell, with division culture medium I, (DMEM/F12 (3:1) comprises 0.1% dual anti-, 40 μ g/mlfungizone, 40ng/mlFGF2, 20ng/mlEGF, 2%B27supplement, after 10%FBS) cultivating 3 days, in division culture medium II, (DMEM/F12 (3:1) comprises 0.1% dual anti-again, 5 μm of forskolin, 50ng/mlheregulin-1 β, 50 μ g/ml, 2%N2supplement, Scs colony can be obtained after cultivating 2-3 week 1%FBS).The Scs (SKP-SC) obtaining the differentiation of a large amount of skin progenitor cell increased after picking colony, Showed by immune group result is that S100 is positive.
(4) cultivation of mesenchymal stem cells MSCs
Adult SD rats is put to death in dislocation, and 75% alcohol-pickled 5min, gets femur, tibia under aseptic condition, exposes medullary cavity, and IMDM basic culture solution rinses medullary cavity, collects 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, 1000rpm × 5min, abandon supernatant.With 4 × 10
5/ cm
2density 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 is reached, Secondary Culture when cell is paved with at the bottom of ware.
The preparation of embodiment 2 biological sticking patch
(1) preparation of elastomeric stamp
The elastomeric stamp that this utility model uses is polydimethylsiloxane (polydimethylsiloxane, PDMS), its preparation method is in after the ratio mix homogeneously of 10:1 by the PDMS liquid for preparing under room temperature and cross-linking agent, be cast to and adopt on the silicon wafer-based caster with horizontal or longitudinal channel form pattern distribution of mask exposure acquisition in advance, as depicted in figs. 1 and 2.Then put it into evacuation in vacuum drying oven and get rid of bubble, and carry out dry solidification, be finally cured rear stripping and namely obtain plane P DMS 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, freezing rear chitosan ice body be placed in alkaline solution and fix, cleaning 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 culture vessel, then adds 2.5 × 10
7individual cell and aseptic process cross by chitosan-based bottom, then complete medium is filled whole container, controlling final cell density is 1 × 10
5/ ml, drains in system after air, starts to rotate microgravity circumfusion and cultivates; Rotary bioreactor puts into 37 DEG C of CO
2in incubator, within first 24 hours, rotating speed is 10 revs/min, and cell is fully contacted with chitosan-based bottom, attaches; Adjust rotary bioreactor rotary speed after 24 hours, chitosan-based bottom can be suspended in culture fluid; Continue cultivation and be replaced by division culture medium cultivation after 2 days, to promote Extracellular Matrix Secretion; Cultivating termination cultivation after 2 weeks has the chitosan-based bottom of cell to take out surface attaching growth.Microscopy cell is orientation growth on the chitosan-based bottom of surface graphics, as shown in Figure 3.The chitosan-based bottom of cell there is is to carry out de-cell process growth, deionization sterilized water 37 DEG C of hypotonic 10min after PBS cleaning; Add extraction liquid of cell in 37 DEG C of cell lysis 10-15min; PBS adds DnaseI (4mg/ml) 37 DEG C and digests 30min removal DNA after cleaning 3 times, it is carried out lyophilizing further, obtains biological sticking patch described in the utility model, as shown in Fig. 4 or Fig. 5,1 is basal layer, and 2 is groove, and 3 is cellular matrix layer.
Scanning electron microscope result shows the extracellular matrix components chitosan-based bottom surface being uniformly distributed sustenticular cell secretion, and the chitosan-based bottom made containing n cell hypothallus can save backup at-80 DEG C, also can lyophilization preserve further.Then obtain containing cytostromatic chitosan biological sticking patch through lyophilization, chitosan-based bottom surface and inside are distributed with abundant hole, and cellular matrix layer is attached to basal layer and pore surface.Porosity 89% after measured, average pore size is 87-98 μm.
Claims (8)
1. for the biological sticking patch that tissue injury repairs, it is characterized in that comprising biomaterial basal layer and cellular matrix layer, on the base layer, basal layer and the contact surface of cellular matrix layer are provided with several parallel grooves in hypothallus attachment.
2. biological sticking patch as claimed in claim 1, is characterized in that described groove is transverse direction or genesis analysis.
3. biological sticking patch as claimed in claim 1, is characterized in that described groove is of a size of: wide 10-50 μm, dark 10-30 μm.
4. biological sticking patch as claimed in claim 1, is characterized in that the contact surface upper surface of described biomaterial basal layer and cellular matrix layer and inside are distributed with abundant hole.
5. biological sticking patch as claimed in claim 4, it is characterized in that the porosity on described biomaterial basal layer is 50%-90%, and aperture is 10 μm-100 μm.
6. synthetic biological sticking patch as claimed in claim 1, is characterized in that described biomaterial base layer thickness is 0.1-3mm.
7. the biological sticking patch as described in one of claim 1-6 item, is characterized in that described cellular matrix layer is autologous or the secretion of allogeneic derived cell forms rear de-cell and obtains.
8. biological sticking patch as claimed in claim 7, is characterized 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.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201520347479.6U CN204971702U (en) | 2015-05-26 | 2015-05-26 | Be used for prosthetic biological patch of tissue lesion |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201520347479.6U CN204971702U (en) | 2015-05-26 | 2015-05-26 | Be used for prosthetic biological patch of tissue lesion |
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| Publication Number | Publication Date |
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| CN204971702U true CN204971702U (en) | 2016-01-20 |
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| CN201520347479.6U Expired - Fee Related CN204971702U (en) | 2015-05-26 | 2015-05-26 | Be used for prosthetic biological patch of tissue lesion |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105726160A (en) * | 2016-04-29 | 2016-07-06 | 陕西瑞盛生物科技有限公司 | Rehydration method of biological patch and rehydrated biological patch |
-
2015
- 2015-05-26 CN CN201520347479.6U patent/CN204971702U/en not_active Expired - Fee Related
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105726160A (en) * | 2016-04-29 | 2016-07-06 | 陕西瑞盛生物科技有限公司 | Rehydration method of biological patch and rehydrated biological patch |
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Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20160120 Termination date: 20210526 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |