The preparation method of silk fibroin porous scaffold based on extracellular derivative matrix modificationTechnical field
The invention belongs to technical field of biological materials, and in particular to a kind of fibroin egg based on extracellular derivative matrix modificationThe 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 materialProperty 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 heartIt 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 daySo microenvironment design feature of cardiac muscle, research and development construct engineering based on imitative extracellular Matrix component and the timbering material of micro-structureChange 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 presentPart 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 thawingThe methods of acellular matrix is carried out to cell, retained to obtain no nucleus, but have natural extracellular matrix (ECM) specialThe biologic bracket material of property.The advantages of this cell-derived cell epimatrix material, is, with tissue-derived ECM bracket phaseThan cell-derived ECM matrix is due to can effectively exclude pathogen contamination when its culture and amplification.It can keep good dayRight extracellular matrix components, and this cell-derived ECM matrix can retain the original trace of cell, have to cell is inoculatedCertain regulating and controlling effect.Meanwhile compared with tissue-derived ECM bracket, cell-derived extracellular ECM plasticity is strong, can be rightOther materials is effectively modified.But this cell-derived ECM material there is also to a certain degree the shortcomings that, such as de- thinThe 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 tissueDerivative 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 baseThe important component of matter, it is closely related with heart development, structure, cell signaling system, electromechanical work energy etc..Therefore, the heartHost material derived from myofibroblast is received significant attention as the important microenvironment factor for providing cell growth.Have and grindsStudy carefully report, Gu et al. rebuilds New-support by the ECM derived from schwann cell and chitosan material, is used for tissue engineering nerveIt 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.SilkIt is divided into sericin and fibroin albumen, wherein fibroin albumen is main component, accounts for 70-80%.Fibroin albumen is being formed notWith having significant mechanical performance when material, preferable biocompatibility is shown, there is controllable degradation rate, and can be withBy chemical modification to change surface nature or the fixed growth factor.Research can extract fibroin albumen from silk cocoon to develop at presentHydrogel, sponge bracket, fibrous framework, microballoon and film etc..Fibroin material can be directly used as implants in vivo, can be used asBracket in organizational project and vitro disease research model, and for drug delivery etc..David L.Kaplan team is sharpAdipose 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, fibroinThat 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 modificationPreparation 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 finerBorn 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 9000rpmIt 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 gelChange 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-coolingPBS liquid in, shred, and digest repeatedly using 0.05% pancreatin until tissue block digestion is complete, 1000rpm/7 minutes fromThe 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 hourAdherent cardiac muscle cell obtains primary Cardiac Fibroblasts；And add 10% fetal calf serum culture medium culture using high sugar-DMEMCardiac Fibroblasts digest after merging to 85-90%；
S5. Cardiac Fibroblasts modify fibroin albumen, the method is as follows: are seeded in the Cardiac Fibroblasts digestedOn timbering material, inoculum density is 1 × 107A/ml, each bracket are inoculated with 30 μ l, add 10% fetal calf serum using high sugar-DMEMCulture 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 fibroinAlbumen 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 tissueMaterial.
Detailed description of the invention
A is Activity determination figure in silk fibroin bracket in Fig. 1；B is Cardiac Fibroblasts in silk fibroin bracketThe 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 contentThe 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 scissorsSilk 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.KnotShu 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 waterIt 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, andSilk-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 whichA 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 saltGrain, 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 waterIt 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 sideSterilizing.
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 fromThe 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-DMEMIt 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 digestedOn timbering material, inoculum density is 1 × 107A/ml, each bracket are inoculated with 30 μ l, add 10% tire ox blood using high sugar-DMEMClear 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 dyeingExpression, vimentin antibody buy from abcam, the Cardiac Fibroblasts on silk fibroin porous scaffold are significant as the result is shownIt 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 immunofluorescenceDetection, observes whether cell-derived matrix can effectively modify on silk-fibroin bracket.As shown in Fig. 2, in silk fibroin bracketThere 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 matrixIt 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 significantlyImmune 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 reallyInoculum density is determined with 1 × 106Cells/mL, the silk fibroin bracket and simple silk fibroin bracket of each derivative matrix modificationOn 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 inoculationBase.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, derivativeThe 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 abilityWhat the technical staff in domain can think variation should all fall into protection scope of the present invention.