CN105769381B - A kind of biological sticking patch for tissue damage reparation - Google Patents
A kind of biological sticking patch for tissue damage reparation Download PDFInfo
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- CN105769381B CN105769381B CN201510273678.1A CN201510273678A CN105769381B CN 105769381 B CN105769381 B CN 105769381B CN 201510273678 A CN201510273678 A CN 201510273678A CN 105769381 B CN105769381 B CN 105769381B
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Abstract
The invention discloses a kind of biological sticking patch for tissue damage reparation, including biomaterial basalis and cellular matrix layer, hypothallus adheres on the base layer, and basalis is equipped with several parallel grooves with the contact surface of cellular matrix layer.Biological sticking patch basalis of the present invention has porous and groove texture structure at the same time, not only can be grown and is distributed with the orientation of regulating cell, mechanical property can enable cellular matrix layer preferably adhere on the base layer possessed by groove texture structure, avoid coming off.Product of the present invention has stable structure, and suitable aperture, porosity and permeability, can simulate normal organization, using the teaching of the invention it is possible to provide the pipeline and cellular matrix of tissue growth, and can carry beneficial to tissue repair and regenerated cell.
Description
Technical field
The present invention relates to medical instruments field, more particularly to a kind of biological sticking patch for tissue damage reparation.
Background technology
Traffic accident, industrial accident, the movement event such as accident and clinical operation can cause tissue damage in society,
It is clinically to use the material that tissue is substituted and repaired as biological sticking patch more.Biological sticking patch refers to be derived from one kind of the same race
To tissue, completely retain the three-dimensional frame structure of extracellular matrix through taking off the various cells contained in cell processing removal tissue
And the material for repairing human body soft tissue can be used for.According to it is tissue-derived be divided into allogenic material such as corium acellular matrix,
Amnion, endocranium etc., heterogenous allosome material such as intestinal mucosa lower floor, ox, the pericardium of horse, ox peritonaeum etc..It is although this natural
Biological sticking patch commercialization, but prepare cumbersome, price is high.Find it is cheap, easy be easy to get, good biocompatibility
Artificial synthesized biological sticking patch substitutes the hot spot that natural biological sticking patch is still research.
The content of the invention：
It is an object of the invention to provide a kind of good biocompatibility, it is cheap, easy be easy to get be used for tissue damage
The biological sticking patch of reparation.
The present invention technical solution be：
A kind of biological sticking patch for tissue damage reparation, including biomaterial basalis and cellular matrix layer, hypothallus
On the base layer, basalis is equipped with several parallel grooves with the contact surface of cellular matrix layer for attachment.Groove can be prominent
For the convex groove of substrate surface, or the groove shape being recessed inwardly from substrate surface.Groove for laterally or
Longitudinal arrangement.
It is preferred that the size of groove is：Width is 10-50 μm, and depth is 10-30 μm.
Above-mentioned biological sticking patch, the porosity on the biomaterial basalis are 50%-90%, and aperture for 10 μm-
100μm。
Above-mentioned artificial synthesized biological sticking patch, the biomaterial base layer thickness are 0.1-3mm.
Above-mentioned artificial synthesized biological sticking patch, the biomaterial basalis is by fibroin albumen, chitosan, collagen, poly- breast
One or more in acid or polyglycolic acid are made.
Above-mentioned biological sticking patch, the cellular matrix layer is autologous or allogeneic derived cell is secreted and taken off carefully after being formed
Born of the same parents and obtain, the cell be schwann cells, the fibroblast of skin-derived, skin progenitor cell, mesenchymal stem cell,
One or more in navel blood stem cell or induced pluripotent stem cells.
Above-mentioned biological sticking patch does not contain foaming agent and/or crosslinking agent.
The invention also discloses the preparation method of above-mentioned biological sticking patch,
(1) PDMS elastomeric stamp lines is designed, biomaterial solution is added on seal, uses what is freezed after shaping and demoulding
Method, which prepares surface, has the biomaterial basalis of ditch slot texture；
(2) biomaterial basalis is cultivated with cell, obtains attaching the biomaterial basalis for growing and having cell；
(3) the biomaterial basalis that growth has cell is subjected to de- cell processing, it is further freezed, is obtained
Biological sticking patch.
The preferred solution of the present invention is as follows：
The principle influenced according to the size of very low power on the shape and spreading behavior of cell, design can guide nerve
The micrographics structure with channel form of the distribution of orientations of cell.
Elastomeric stamp used in the present invention is dimethyl silicone polymer (polydimethylsiloxane, PDMS), its
Preparation method is that prepared PDMS liquid and crosslinking agent will press 10 at room temperature:1 ratio after mixing, is cast to and adopts in advance
On the silicon chip base caster that the channel form pattern having laterally or longitudinally obtained with mask exposure is distributed, then put
Enter to vacuumize exclusion bubble in vacuum drying chamber, and carry out dry solidification, peeled off after finally being cured and obtain plane PDMS
Elastomeric stamp.
The solution of a certain concentration (1%, 2% and 5%) is prepared with acetic acid chitosan, then the solution is added dropwise
On PDMS, -60 DEG C are placed in -- 90 DEG C, when freezing 2-4 is small, chitosan ice body after freezing is placed in alkaline solution and fixed, is used
Water cleans；The chitosan-based bottom of sizing is freeze-dried, obtains being distributed with channel form with loose structure, surface
Porous chitosan basalis.
Porous chitosan basalis progress Three-dimensional cell culture is obtained surface attaching growth the chitosan-based bottom of cell
Layer takes out.The chitosan-based bottom that growth has cell is subjected to de- cell processing, it is further freezed, obtains the present invention
The biological sticking patch.
The aperture that biological sticking patch hole of the present invention is detected using scanning electron microscope is 10 μm -100 μm.Biology of the invention is mended
Piece basalis has porous and groove texture structure at the same time, can not only be grown and be distributed, ditch groove with the orientation of regulating cell
Mechanical property possessed by line structure can enable cellular matrix layer preferably adhere on the base layer, avoid coming off.This hair
The bright product has stable structure, and suitable aperture, porosity and permeability, can simulate normal organization, can
The pipeline and cellular matrix of tissue growth are provided, and can be carried beneficial to tissue repair and regenerated cell, production of the invention
Product material therefor is natural degradable material, has good biocompatibility with human body, obtained product is not contained by making
The foaming agent and crosslinking agent for the exogenous toxicity, side effect that standby technique is brought into.The present invention is with good tensile strength and with thin
Cytoplasmic matrix layer, is more advantageous to the nutriment needed for conveying cell growth process, good growing environment is provided to cell.Use
Effect is good.The method of the present invention is simple and easy to do.
Brief description of the drawings
Fig. 1 is biological sticking patch substrate surface ditch slot texture schematic diagram of the present invention.
For cell, the orientation on the basalis of biological sticking patch surface graphics of the present invention grows schematic diagram to Fig. 2.
For biological sticking patch structure diagram of the present invention, (A is the biological sticking patch schematic diagram of convex groove to Fig. 3, and B is spill
The biological sticking patch schematic diagram of groove, 1 is basalis, and 2 be groove, and 3 be cellular matrix layer).
Embodiment
With reference to embodiment, the invention will be further described.
Used term in the present invention, unless otherwise indicated, generally there are those of ordinary skill in the art usually to manage
The implication of solution.
The present invention is described in further detail with reference to specific embodiment and with reference to data.It is to be understood that these embodiments are only
It is in order to demonstrate the invention, rather than to limit the scope of the invention in any way.
In the examples below, the various processes and method not being described in detail are conventional methods as known in the art.
Embodiment 1
(1) culture and purifying of schwann cells
Newborn one day SD rat is taken, alcohol disinfecting after execution, takes bilateral sciatic nerve, is placed in ice bath operation console and removes nerve
Outer membrane and tissue is adhered, supernatant is abandoned in centrifugation afterwards completely for clostridiopetidase A/pancreatin mixture slaking, and cell, inoculation is resuspended in complete medium
Cultivated in the culture dish being coated with advance to PDL.24 are replaced with the complete medium culture containing cytarabine (10 μM) when small
48 it is small when, be changed to the complete medium containing HRG (50ng/ml) and Forsklin (2 μM) and continue to cultivate, change liquid within every 3 days, directly
To cell fusion.After cell fusion, centrifuged after pancreatin digestion and obtain cell precipitation, with complete mediums of the 1ml containing Thy1.1
(1:1000) cell is resuspended, when incubation 2 is small on ice；Supernatant is abandoned in centrifugation, with DMEM and the mixture (3 of complement:1) cell is resuspended,
When 37 DEG C of incubations 1 are small, complete medium cleans 2 times after centrifugation, is re-seeded into culture dish, liquid is changed every other day, after cell covers with
It can use.
(2) fibroblastic culture of skin-derived
SD rats 1 day newborn are taken, alcohol disinfecting after execution, takes out skin of back and be placed in the dissection liquid of precooling carefully
Reject subcutaneous tissue (fat and subcutaneous fascia layer, blood vessel etc.)；After PBS cleaning 3 times, be cut into small pieces with knife blade (<1mm×
1mm), supernatant is abandoned with centrifugation after type i collagen enzyme (1mg/ml) completely digestion, complete medium is resuspended cell, is inoculated into culture
Ware culture, secondary culture after cell fusion 90%.Fibroblast has epithelial cell pollution during original cuiture, mostly
Number heteroproteose cell (epithelium and endothelial cell) can be gradually dead after passing on several times.Therefore, cell used in the present invention is biography 3 more than generation
Cell.
(3) culture of skin progenitor cell and orientation are broken up along schwann cells
The culture of skin progenitor cell and differentiation reference literature《Isolation of skin-derived precursors
(SKPs)and differentiation and enrichment of their Schwann cell progeny》
(Jeffrey A Biernaskie, Ian A McKenzie, Jean G Toma＆Freda D Miller, NATURE
PROTOCOLS,2006(1):2803-2812), it is briefly described as follows：SD rats 1 day newborn are taken, alcohol disinfecting after execution, takes
Go out skin of back and be placed in the dissection liquid of precooling carefully to reject subcutaneous tissue (fat and subcutaneous fascia layer, blood vessel etc.)；PBS is clear
After washing 3 times, be cut into small pieces with knife blade (<1mm × 1mm), disappeared with 37 DEG C of 0.1% pancreatin or XI Collagenase Types (1mg/ml)
Change 45-60min, complete training terminates digestion centrifugation and abandons supernatant, with proliferated culture medium (DMEM/F12 (3:It is 1) dual anti-comprising 0.1%,
40 μ g/ml fungizone, 40ng/ml FGF2,20ng/ml EGF, 2%B27supplement) carry out suspension culture.Suspend
The cell ball of culture can carry out secondary culture and obtain sufficient amount of skin progenitor cell.
The skin progenitor cell of acquisition is subjected to differentiation culture to obtain schwann cells, is comprised the following steps that：Skin is done carefully
Born of the same parents are with differential medium I (DMEM/F12 (3:1) 0.1% dual anti-, 40 μ g/ml fungizone, 40ng/ml FGF2 are included,
20ng/ml EGF, 2%B27supplement, 10%FBS) culture 3 days after then at differential medium II (DMEM/F12 (3:1)
It is dual anti-comprising 0.1%, 5 μm of forskolin, 50ng/ml heregulin-1 β, 50 μ g/ml, 2%N2supplement, 1%
FBS culture can obtain schwann cells colony after 2-3 weeks in).A large amount of skin progenitor cells that obtain that picking collection falls behind amplification break up
Schwann cells (SKP-SC), Showed by immune group result is positive in S100.
(4) culture of mesenchymal stem cell
Adult SD rats are put to death in dislocation, and 75% alcohol soaks 5min, femur, shin bone are taken under aseptic condition, exposes ossis,
IMDM basic culture solutions rinse ossis, collect marrow.Syringe aspirates the marrow of harvest repeatedly, and single cell suspension is made.200
Mesh sieve net filtration, puts in horizontal centrifuge and centrifuges, and 1000rpm × 5min, abandons supernatant.With 4 × 105/cm2Density be inoculated in
IMDM complete culture solutions (contain hyclone 10%), are placed in 37 DEG C of cultures.Full dose changes liquid after 24h, removes non-attached cell, with
Per 3d, half amount changes liquid afterwards.Day by day cellular morphology and growing state are observed under inverted microscope.Melt when cell is paved with ware bottom up to 90%
Close, secondary culture.
The preparation of 2 biological sticking patch of embodiment
(1) preparation of elastomeric stamp
Elastomeric stamp used in the present invention is dimethyl silicone polymer (polydimethylsiloxane, PDMS), its
Preparation method is that prepared PDMS liquid and crosslinking agent will press 10 at room temperature:1 ratio after mixing, is cast to and adopts in advance
On the silicon chip base caster that the channel form pattern having laterally or longitudinally obtained with mask exposure is distributed, as shown in Figure 1.
Then put it into vacuum drying chamber and vacuumize exclusion bubble, and carry out dry solidification, peeled off after finally being cured to obtain the final product
To plane PDMS elastomeric stamps.
(2) preparation of the chitosan-based bottom of pattern
The solution of a certain concentration (1%, 2% and 5%) is prepared with acetic acid chitosan first, then the solution is added dropwise
On PDMS, -60 DEG C are placed in -- 90 DEG C, when freezing 2-4 is small, chitosan ice body after freezing is placed in alkaline solution and fixed,
Wash with water；The chitosan-based bottom of sizing is freeze-dried, obtains being distributed with channel form with loose structure, surface
Porous chitosan basalis；
(3) structure of cellular matrix layer
Complete medium is slowly injected into culture vessel, adds 2.5 × 107What a cell and aseptic process were crossed gathers shell
Glycosyl bottom, then complete medium is filled into whole container, it is 1 × 10 to control final cell density5/ ml, drains empty in system
After gas, start to rotate microgravity circumfusion culture；Rotary bioreactor is put into 37 DEG C of CO2In incubator, it is preceding 24 it is small when
Rotating speed is 10 revs/min, cell is come into full contact with chitosan-based bottom, is attached；24 it is small when after adjust rotary biological respinse
Device rotary speed, enables chitosan-based bottom to be suspended in nutrient solution；Differential medium culture is changed to after continuing culture 2 days,
To promote Extracellular Matrix Secretion；Culture is terminated after cultivating 2 weeks and attaches surface and grows the chitosan-based bottom taking-up for having cell.
Microscopy cell orientation on the chitosan-based bottom of surface graphics is grown, as shown in Figure 2.Growth there is into cell
Chitosan-based bottom carries out de- cell and handles, 37 DEG C of hypotonic 10min of deionization sterile water after PBS cleaning；Add extraction liquid of cell
In 37 DEG C of cell lysis 10-15min；37 DEG C of digestion 30min of Dnase I (4mg/ml) are added after PBS cleaning 3 times and remove DNA, will
It is further freezed, and obtains biological sticking patch of the present invention, as shown in figure 3, the biological sticking patch that A is convex groove is illustrated
Figure, B are the biological sticking patch schematic diagram of concave channels, and 1 is basalis, and 2 be groove, and 3 be cellular matrix layer.
The extracellular matrix of sertoli cell secretion has been uniformly distributed in the chitosan-based bottom surface of scanning electron microscope the results show
Component, the chitosan-based bottom containing n cell hypothallus, which has been made, to be saved backup at -80 DEG C, also can further freeze dry
Dry preservation.Then the freeze-dried chitosan biological sticking patch obtained containing cellular matrix, chitosan-based bottom surface and inside
Abundant hole is distributed with, cellular matrix layer is attached to basalis and pore surface.Porosity 89% after measured, average pore size
For 87-98 μm.
Claims (9)
1. a kind of biological sticking patch for tissue damage reparation, including biomaterial basalis and cellular matrix layer, hypothallus it is attached
On the base layer, basalis is equipped with several parallel grooves with the contact surface of cellular matrix layer, it is characterised in that described
Biological sticking patch is prepared using following steps：
（1）PDMS elastomeric stamp lines is designed, biomaterial solution is added on seal, using lyophilized side after shaping and demoulding
Method, which prepares surface, has the biomaterial basalis of ditch slot texture；
（2）Biomaterial basalis is cultivated with cell, obtains attaching the biomaterial basalis for growing and having cell；
（3) the biomaterial basalis that growth has cell is subjected to de- cell processing, it is further freezed, obtains biology
Sticking patch.
2. biological sticking patch as claimed in claim 1, it is characterised in that the groove is laterally or longitudinally to be distributed.
3. biological sticking patch as claimed in claim 1, it is characterised in that the size of the groove is：It is 10-50 μm wide, deep 10-
30µm。
4. biological sticking patch as claimed in claim 1, it is characterised in that the biomaterial basalis connects with cellular matrix layer
Abundant hole is distributed with contacting surface upper surface and inside.
5. biological sticking patch as claimed in claim 4, it is characterised in that the porosity on the biomaterial basalis is 50%-
90%, and aperture is 10 μm -100 μm.
6. biological sticking patch as claimed in claim 1, it is characterised in that the biomaterial base layer thickness is 0.1-3mm.
7. biological sticking patch as claimed in claim 1, it is characterised in that the biomaterial basalis is gathered by fibroin albumen, shell
One or more in sugar, collagen, polylactic acid or polyglycolic acid are made.
8. the biological sticking patch as described in one of claim 1-7 item, it is characterised in that the cellular matrix layer is autologous or same
Kind of xenogenic origin cell is secreted to be formed after take off cell and obtain.
9. biological sticking patch as claimed in claim 8, it is characterised in that the cell be schwann cells, skin-derived into fiber
One or more in cell, skin progenitor cell, mesenchymal stem cell, navel blood stem cell or induced pluripotent stem cells.
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Families Citing this family (5)
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| CN108815582A (en) * | 2018-07-11 | 2018-11-16 | 中国科学院上海高等研究院 | A kind of humidity response artificial skin and its preparation method and application |
| CN109793927A (en) * | 2019-01-24 | 2019-05-24 | 中国人民解放军军事科学院军事医学研究院 | The preparation method of silk fibroin porous scaffold based on extracellular derivative matrix modification |
| CN111359018B (en) * | 2020-05-06 | 2023-11-14 | 北京潞康科技有限公司 | Double-layer anti-seepage dura mater repairing sheet and preparation method thereof |
| 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 |
| CN116159185B (en) * | 2022-12-26 | 2024-04-09 | 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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