CN109793927A - The preparation method of silk fibroin porous scaffold based on extracellular derivative matrix modification - Google Patents
The preparation method of silk fibroin porous scaffold based on extracellular derivative matrix modification Download PDFInfo
- Publication number
- CN109793927A CN109793927A CN201910066736.1A CN201910066736A CN109793927A CN 109793927 A CN109793927 A CN 109793927A CN 201910066736 A CN201910066736 A CN 201910066736A CN 109793927 A CN109793927 A CN 109793927A
- Authority
- CN
- China
- Prior art keywords
- silk
- fibroin
- bracket
- silk fibroin
- extracellular
- 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.)
- Pending
Links
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Materials For Medical Uses (AREA)
Abstract
The invention discloses a kind of preparation methods for the silk fibroin porous scaffold based on extracellular derivative matrix modification for belonging to technical field of biological materials.The present invention modifies silk fibroin porous scaffold using Cardiac Fibroblasts, obtained timbering material surface has extracellular matrix component, DNA residual is lower than 5% simultaneously, and the timbering material has good biocompatibility, provides good timbering material for the external structure of engineered myocardial tissue.
Description
Technical field
The invention belongs to technical field of biological materials, and in particular to a kind of fibroin egg based on extracellular derivative matrix modification
The preparation method of white porous support.
Background technique
Timbering material is the basis and cardiac muscle tissue engineering basis of seed cell adherency and growth.The spy of timbering material
Property and structure decide the form and function of engineering tissue.It is different from the tissue such as bone, cartilage, skin, the institutional framework of heart
It is complex, the physicochemical property of timbering material is required higher.In recent years, based on the outer component of imitative cardiac muscle cell, orientation texture,
Cardiac muscular tissue's progress of the more bionical characteristic of timbering material building of mechanics, electrophysiological characteristics is rapid, further utilizes day
So microenvironment design feature of cardiac muscle, research and development construct engineering based on imitative extracellular Matrix component and the timbering material of micro-structure
Change cardiac muscular tissue to receive significant attention, has been reported that utilize the natural extracellular matrix groups such as collagen, full organ acellular matrix at present
Part building engineered myocardial tissue is concerned.
The one kind of cell-derived extracellular matrix material as natural biologic material is to utilize chemical reagent, physics freeze thawing
The methods of acellular matrix is carried out to cell, retained to obtain no nucleus, but have natural extracellular matrix (ECM) special
The biologic bracket material of property.The advantages of this cell-derived cell epimatrix material, is, with tissue-derived ECM bracket phase
Than cell-derived ECM matrix is due to can effectively exclude pathogen contamination when its culture and amplification.It can keep good day
Right extracellular matrix components, and this cell-derived ECM matrix can retain the original trace of cell, have to cell is inoculated
Certain regulating and controlling effect.Meanwhile compared with tissue-derived ECM bracket, cell-derived extracellular ECM plasticity is strong, can be right
Other materials is effectively modified.But this cell-derived ECM material there is also to a certain degree the shortcomings that, such as de- thin
The physics and chemical method being related to during born of the same parents may be such that the factor in original cell is lost, cell-derived matrix and tissue
Derivative discrete phase is fewer etc..Cardiac Fibroblasts (CFs) account for about the 60%-70% of normal myocardium histocyte sum,
It is widely present in heart tissue, wraps cardiac muscle cell.The extracellular matrix of cardiac fibroblasts is as heart base
The important component of matter, it is closely related with heart development, structure, cell signaling system, electromechanical work energy etc..Therefore, the heart
Host material derived from myofibroblast is received significant attention as the important microenvironment factor for providing cell growth.Have and grinds
Study carefully report, Gu et al. rebuilds New-support by the ECM derived from schwann cell and chitosan material, is used for tissue engineering nerve
It repairs.And at present cell-derived extracellular matrix for cardiac muscular tissue building research there is not been reported.
Silk derives from Cocoon, is a kind of biomaterial of protein polymer for being widely used and studying.Silk
It is divided into sericin and fibroin albumen, wherein fibroin albumen is main component, accounts for 70-80%.Fibroin albumen is being formed not
With having significant mechanical performance when material, preferable biocompatibility is shown, there is controllable degradation rate, and can be with
By chemical modification to change surface nature or the fixed growth factor.Research can extract fibroin albumen from silk cocoon to develop at present
Hydrogel, sponge bracket, fibrous framework, microballoon and film etc..Fibroin material can be directly used as implants in vivo, can be used as
Bracket in organizational project and vitro disease research model, and for drug delivery etc..David L.Kaplan team is sharp
Adipose tissue, nephridial tissue, cartilaginous tissue and cerebral cortex are successfully constructed in vitro with silk-fibroin porous support.Shoei-
Shen Wang etc. also carries mesenchymal stem cell using silk fibroin porous scaffold and carries out rat heart infarction repairing research,
Achieve good effect, it was demonstrated that compound can effectively improve transplanted cells in the delay and survival in heart infarction area.However, fibroin
That there are material components is single for albumen bracket, does not have extracellular matrix component microenvironment component, the problem of biomimicking potential deficiency.
Summary of the invention
It is an object of the invention to propose a kind of system of silk fibroin porous scaffold based on extracellular derivative matrix modification
Preparation Method.
To realize the above-mentioned technical purpose, this invention takes the following technical solutions:
A kind of preparation method of the silk fibroin porous scaffold based on extracellular derivative matrix modification, by cardiac muscle at fiber finer
Born of the same parents are inoculated on silk fibroin bracket material and cultivate, the silk fibroin porous scaffold after being modified.
The preparation method of above-mentioned porous support carries out in accordance with the following steps:
S1. silk-fibroin pre-processes, the method is as follows: silk cocoon is shredded to and is put into the sodium carbonate boiled that mass fraction is 17%
It boils 30 minutes, take out cooling and squeezes the water out in aqueous solution, then rinsed silk-fibroin 20 minutes with water, it is dry to take out rear venting；
S2. silk protein solution is prepared, the method is as follows: dry silk-fibroin is dissolved in 20% 9.3M lithium-bromide solution,
It is dissolved 4 hours under the conditions of 60 DEG C, obtains 20% fibroin albumen, dialysed 2 days, be then centrifuged under the conditions of 4 DEG C of 9000rpm
It 20 minutes, takes out and is saved in 4 DEG C；
S3. silk-fibroin bracket is prepared, the method is as follows: silk fibroin protein solution is added in mold, and sprinkles salt particle gel
Change 1-2 days, the silk-fibroin bracket of gelation is desalted the sterilizing of laggard horizontal high voltage in distilled water immersion；
S4. Cardiac Fibroblasts are separated, the method is as follows: take newborn 1 day age SD rat, open chest, coring is dirty, is placed in pre-cooling
PBS liquid in, shred, and digest repeatedly using 0.05% pancreatin until tissue block digestion is complete, 1000rpm/7 minutes from
The heart obtains cell precipitation, is resuspended and is seeded on culture dish in DMEM in high glucose plus 15% fetal calf serum, discarded not after 1 hour
Adherent cardiac muscle cell obtains primary Cardiac Fibroblasts；And add 10% fetal calf serum culture medium culture using high sugar-DMEM
Cardiac Fibroblasts digest after merging to 85-90%；
S5. Cardiac Fibroblasts modify fibroin albumen, the method is as follows: are seeded in the Cardiac Fibroblasts digested
On timbering material, inoculum density is 1 × 107A/ml, each bracket are inoculated with 30 μ l, add 10% fetal calf serum using high sugar-DMEM
Culture medium culture 10 days, obtain the silk fibroin bracket of extracellularly derivative matrix modification.
Compared with prior art, the invention has the following beneficial effects: the present invention to utilize Cardiac Fibroblasts to fibroin
Albumen porous support is modified, and obtained timbering material surface has extracellular matrix component, while DNA residual is lower than 5%,
And the timbering material has good biocompatibility, provides good bracket for the external structure of engineered myocardial tissue
Material.
Detailed description of the invention
A is Activity determination figure in silk fibroin bracket in Fig. 1；B is Cardiac Fibroblasts in silk fibroin bracket
The expression figure of vimentin.
Fig. 2 is the silk fibroin bracket material figure that immunofluorescence dyeing detects cell-derived matrix modification.
Be in Fig. 3 A be DAPI dyeing detection nucleus residual figure；B is DNA residues detecton figure on timbering material.
It is the vital staining figure of brown fat stem cell on A timbering material in Fig. 4；B is to measure bracket material using DNA content
The vegetative map of brown fat stem cell in material.
Specific embodiment
The present invention is described in detail with reference to the accompanying drawings and examples, but implementation of the invention is not limited only to this.
Embodiment 1 prepares the silk fibroin bracket of Cardiac Fibroblasts modification
1, silk-fibroin pre-processes, the method is as follows: prepares that 2L ultrapure water is housed, 4.24g sodium carbonate is added, boils.Use scissors
Silk cocoon is cut into coin-size silk cocoon piece, and weighs 5g silk cocoon piece and is put into the aqueous sodium carbonate boiled, is boiled 30 minutes.Knot
Shu Houyong ultrapure water is cooling, and extra water is squeezed out from silk.It rinses silk-fibroin 20 minutes, and is stirred with stirring rod in water
It mixes.The silk-fibroin rinsed is placed in draught cupboard and is dried overnight.
2, silk protein solution is prepared, the method is as follows: dry silk-fibroin is dissolved in 20% 9.3M lithium-bromide solution, and
Silk-fibroin is set to dissolve 4 hours in 60 DEG C of baking oven to get 20% silk fibroin.Solution is subjected to dialysis 2 days, during which
A water was changed every 8 hours.By the solution after dialysis to carry out centrifugation 20 minutes under the conditions of 4 DEG C of 9000rpm, to remove impurity.
Carefully silk solution is sucked out, 4 DEG C of preservations.
3, silk-fibroin bracket is prepared, the method is as follows: take 2ml solution to be added in mold, and weigh 750 μm of 4g of salt
Grain, and salt particle is uniformly spread to progress gelation in 1-2 days in solution.The good silk-fibroin bracket of gelation is subjected to distilled water
It impregnates, desalts, replace 2-3 water daily, impregnate 2 days.It is subsequent to need size to cut by experiment on bracket, horizontal high voltage of going forward side by side
Sterilizing.
4, Cardiac Fibroblasts are separated, the method is as follows: take newborn 1 day age SD rat first, open chest, coring is dirty, in pre-
It in cold PBS liquid, shreds, and is digested repeatedly using 0.05% pancreatin until tissue block digests completely.1000rpm/7 minutes from
The heart obtains cell precipitation, is resuspended and is seeded on culture dish, 1 hour in DMEM in high glucose plus 15% fetal calf serum (gibco)
Not adherent cardiac muscle cell is discarded afterwards, obtains primary Cardiac Fibroblasts.And 10% fetal calf serum is added to train using high sugar-DMEM
It is digested after supporting base culture Cardiac Fibroblasts to 85-90% fusion.
5, Cardiac Fibroblasts modify fibroin albumen, the method is as follows: are seeded in the Cardiac Fibroblasts digested
On timbering material, inoculum density is 1 × 107A/ml, each bracket are inoculated with 30 μ l, add 10% tire ox blood using high sugar-DMEM
Clear culture medium culture 10 days, live/dead Activity determination, dead cell living were carried out using live/dead dyestuff (invitrogen)
Dyeing confirms that cell activity is good (Figure 1A).And Cardiac Fibroblasts vimentin further is detected using immunofluorescence dyeing
Expression, vimentin antibody buy from abcam, the Cardiac Fibroblasts on silk fibroin porous scaffold are significant as the result is shown
It expresses vimentin (Figure 1B).
The detection for the silk fibroin porous scaffold that embodiment 2 is modified
1. Immunofluorescence test: by the silk fibroin porous scaffold material after cultivating 10 days in 0.3% (volume/volume)
The NH of Triton X-100 and 20mM4OH PBS is handled 10 minutes under the conditions of 37 DEG C, is discarded digestive juice and is carried out immunofluorescence
Detection, observes whether cell-derived matrix can effectively modify on silk-fibroin bracket.As shown in Fig. 2, in silk fibroin bracket
There is the expression of a large amount of matrix, including Collagen I, Collagen III, α-tublin, fibronection and F-
actin.Antibody is purchased from abcam.
2. by detecting whether that nucleus can be effectively removed to the DNA content on de- cytoskeleton: by derivative matrix
It after the timbering material of modification fixes 15 minutes with 4% paraformaldehyde, is washed three times with PBS, and DAPI dyestuff (Zhong Shan Golden Bridge) is added,
DAPI coloration result shows that nucleus can be removed largely.And DNA further is detected using DNA detection kit (invitrogen)
Content, as shown in figure 3, DNA about remains 5%, the amount of the DNA fragmentation in external inflammation detection ECM bracket not will lead to significantly
Immune response.
3. the comparison of the silk fibroin bracket and simple silk fibroin bracket of derivative matrix modification
The separation for carrying out primary brown fat stem cell, by inoculum density grope and literature survey, it is final really
Inoculum density is determined with 1 × 106Cells/mL, the silk fibroin bracket and simple silk fibroin bracket of each derivative matrix modification
On be inoculated with 30 μ L.α-MEM of the addition containing 15% fetal calf serum is cultivated after being incubated for 15-20 minutes in 37 DEG C of incubators after inoculation
Base.The activity that brown fat stem cell is cultivated the 7th day on timbering material is detected by live/dead, as shown in figure 4, derivative
The activity of brown fat stem cell is preferable on timbering material after matrix modification.
Disclosed above is only a specific embodiment of the invention, and still, the present invention is not limited to this, any ability
What the technical staff in domain can think variation should all fall into protection scope of the present invention.
Claims (2)
1. a kind of preparation method of the silk fibroin porous scaffold based on extracellular derivative matrix modification, which is characterized in that by the heart
Myofibroblast is inoculated on silk fibroin bracket material and is cultivated, the silk fibroin porous scaffold after being modified.
2. the preparation method of the silk fibroin porous scaffold according to claim 1 based on extracellular derivative matrix modification,
It is characterized in that, carries out in accordance with the following steps:
S1. silk-fibroin pre-processes: silk cocoon being shredded and is put into the aqueous sodium carbonate boiled that mass fraction is 17% and boil 30
Minute, it takes out cooling and squeezes the water out, then rinsed silk-fibroin 20 minutes with water, it is dry to take out rear venting；
S2. it prepares silk protein solution: dry silk-fibroin being dissolved in 20% 9.3M lithium-bromide solution, dissolved under the conditions of 60 DEG C
4 hours, 20% fibroin albumen is obtained, is dialysed 2 days, then in being centrifuged 20 minutes under the conditions of 4 DEG C of 9000rpm, is taken out in 4 DEG C
It saves；
S3. it prepares silk-fibroin bracket: silk fibroin protein solution being added in mold, and sprinkles salt particle gelation 1-2 days, will be coagulated
The silk-fibroin bracket of gelatinization desalts the sterilizing of laggard horizontal high voltage in distilled water immersion；
S4. Cardiac Fibroblasts are separated: taking newborn 1 day age SD rat, opens chest, coring is dirty, it is placed in the PBS liquid of pre-cooling,
It shreds, and is digested repeatedly using 0.05% pancreatin until tissue block digests completely, it is heavy to obtain cell for centrifugation in 1000rpm/7 minutes
It forms sediment, is resuspended and is seeded on culture dish in DMEM in high glucose plus 15% fetal calf serum, not adherent cardiac muscle cell is discarded after 1 hour,
Obtain primary Cardiac Fibroblasts；And add 10% fetal calf serum culture medium culture Cardiac Fibroblasts extremely using high sugar-DMEM
It is digested after 85-90% fusion；
S5. Cardiac Fibroblasts modify fibroin albumen: the Cardiac Fibroblasts digested are seeded on timbering material,
Inoculum density is 1 × 107A/ml, each bracket are inoculated with 30 μ l, add 10% fetal calf serum culture medium culture 10 using high sugar-DMEM
It, obtains the silk fibroin bracket of extracellularly derivative matrix modification.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910066736.1A CN109793927A (en) | 2019-01-24 | 2019-01-24 | The preparation method of silk fibroin porous scaffold based on extracellular derivative matrix modification |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910066736.1A CN109793927A (en) | 2019-01-24 | 2019-01-24 | The preparation method of silk fibroin porous scaffold based on extracellular derivative matrix modification |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN109793927A true CN109793927A (en) | 2019-05-24 |
Family
ID=66560202
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910066736.1A Pending CN109793927A (en) | 2019-01-24 | 2019-01-24 | The preparation method of silk fibroin porous scaffold based on extracellular derivative matrix modification |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109793927A (en) |
Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20030100108A1 (en) * | 2001-11-16 | 2003-05-29 | Altman Gregory H. | Matrix for the production of tissue engineered ligaments, tendons and other tissue |
| CN101596327A (en) * | 2009-07-13 | 2009-12-09 | 北京理工大学 | A kind of preparation method of three-dimensional silk fibroin porous scaffold material |
| CN102892880A (en) * | 2010-01-14 | 2013-01-23 | 奥加诺吉尼西斯公司 | Bioengineered tissue constructs and methods for producing and using thereof |
| CN102933705A (en) * | 2009-12-17 | 2013-02-13 | 金斯顿女王大学 | Decellularized adipose tissue |
| CN104587528A (en) * | 2014-12-26 | 2015-05-06 | 上海交通大学医学院附属仁济医院 | Acellular matrix of human heart valve tissue and preparation and application of acellular matrix |
| CN105769381A (en) * | 2015-05-26 | 2016-07-20 | 南通大学 | Biological patch for tissue damage restoration |
| CN108714156A (en) * | 2018-05-03 | 2018-10-30 | 中国人民解放军军事科学院军事医学研究院 | The mescenchymal stem cell culture in people's umbilical cord source or the purposes of its culture supernatant |
-
2019
- 2019-01-24 CN CN201910066736.1A patent/CN109793927A/en active Pending
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20030100108A1 (en) * | 2001-11-16 | 2003-05-29 | Altman Gregory H. | Matrix for the production of tissue engineered ligaments, tendons and other tissue |
| CN101596327A (en) * | 2009-07-13 | 2009-12-09 | 北京理工大学 | A kind of preparation method of three-dimensional silk fibroin porous scaffold material |
| CN102933705A (en) * | 2009-12-17 | 2013-02-13 | 金斯顿女王大学 | Decellularized adipose tissue |
| CN102892880A (en) * | 2010-01-14 | 2013-01-23 | 奥加诺吉尼西斯公司 | Bioengineered tissue constructs and methods for producing and using thereof |
| CN104587528A (en) * | 2014-12-26 | 2015-05-06 | 上海交通大学医学院附属仁济医院 | Acellular matrix of human heart valve tissue and preparation and application of acellular matrix |
| CN105769381A (en) * | 2015-05-26 | 2016-07-20 | 南通大学 | Biological patch for tissue damage restoration |
| CN108714156A (en) * | 2018-05-03 | 2018-10-30 | 中国人民解放军军事科学院军事医学研究院 | The mescenchymal stem cell culture in people's umbilical cord source or the purposes of its culture supernatant |
Non-Patent Citations (1)
| Title |
|---|
| 王波: "《医学细胞生物学实验指导及习题集》", 31 August 2018, 中山大学出版社 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US6790454B1 (en) | Processes for the preparation of novel collagen-based supports for tissue engineering, and biomaterials obtained | |
| CN101757691B (en) | Preparation method of tissue engineering cornea | |
| CN102198292B (en) | Scaffolds of umbilical cord decellularized Wharton jelly for tissue engineering and preparation method thereof | |
| ES2677945T3 (en) | Production of artificial tissues by tissue engineering using fibrin and agarose biomaterials | |
| CN102946914B (en) | Methods and combination | |
| CN106075598A (en) | A kind of photo-crosslinking sericin hydrogel and its preparation method and application | |
| CN104399125B (en) | The method that epidermal stem cells breaks up to sweat gland sample epithelial cell | |
| CN109847102A (en) | A kind of preparation method of mescenchymal stem cell artificial langerhans ' islet | |
| CN106667899B (en) | Injectable hydrogels incorporating IGF-1C polypeptides | |
| WO2011094965A1 (en) | Tissue engineering cornea producing method | |
| CN109550080A (en) | A kind of artificial bilayer's skin and preparation method thereof | |
| CN108546674A (en) | Pre-stimulation stem cell and its preparation method and application | |
| CN110354311A (en) | Extracellular matrix composite transparent matter acid gel and preparation method thereof, application and biomaterial | |
| CN104225667A (en) | Temperature-sensitive hydrogel powder for promoting angiogenesis and temperature-sensitive hydrogel prepared from same | |
| WO2018120672A1 (en) | Bioactive stent for inducting regeneration of tendon tissue, preparation method therefor and use thereof | |
| CN109112101A (en) | A kind of fibroblast culture medium and its application | |
| CN106929474A (en) | A kind of M2 macrophages derivant | |
| CN112870445A (en) | Preparation method and application of soft tissue repair material | |
| JP2009538854A (en) | Isolated natural natural collagen | |
| CN114246988B (en) | Collagen nanocluster composite material and preparation method thereof | |
| CN104971382B (en) | The mounted artificial active mass of wound and its construction method that a kind of use serum-free and ox pituitary extract nutrient solution are built | |
| CN102105581B (en) | Artificial kidney precursor and process for production thereof | |
| CN109793927A (en) | The preparation method of silk fibroin porous scaffold based on extracellular derivative matrix modification | |
| CN110464877A (en) | A kind of preparation method and its effect evaluation method of acellular nerve allografts | |
| CN109666633A (en) | A method of improving brown fat stem cell myocardiac differentiation efficiency |
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 | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20190524 |