CN102743790A - Extracellular matrix support material and preparation method thereof - Google Patents
Extracellular matrix support material and preparation method thereof Download PDFInfo
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Abstract
The invention belongs to the field of biological materials and discloses an extracellular matrix support material and a preparation method thereof. The extracellular matrix support material disclosed by the invention is prepared into a three-dimensional porous support material with a certain pore diameter and porosity through performing in-vitro monolayer high-density culture on immature rabbit bone marrow mesenchymal stem cells and collecting extracellular matrixes secreted by the immature rabbit bone marrow mesenchymal stem cells by applying the freeze-drying technology and a proper cross-linking agent. The material can be trimmed into proper sizes and shapes according to a cartilage defect zone or the requirement of an in-vitro experiment, and the material is sterilized by ethylene oxide to obtain an integral packaging bag. As the stem cells are used as material sources of an extracellular matrix support, the extracellular matrix support material disclosed by the invention is wide in tissue source and strong in multiplication capacity, therefore, the extracellular matrix support material can obtain enough cell quantity per se, and the problems of social ethics involved by embryonic stem cells are avoided.
Description
Technical field
The invention belongs to technical field of biological material, relate to a kind of extracellular matrix timbering material and preparation method thereof, be specifically related to a kind of extracellular matrix timbering material based on mesenchymal stem cells MSCs source and preparation method thereof.
Background technology
Articular cartilage is no blood vessel, lymph and innerv particular tissues.The self-repairing capability of articular cartilage own is very limited, in case the damaged diameter can't be repaired greater than 3mm voluntarily.The main method of treatment cartilage defect at present has: lavation art under the arthroscope, joint debridement art, subchondral bone Drilling (subchondralbone drilling), little fracture art (microfracture), from body or allograph bone cartilage grafting (auto or allo osteochondral transplantation), from body chondrocyte cell transplantation art (autologous chondrocyte implantation, ACI) and artificial joint replacement.Said method respectively has operative indication and excellent, shortcoming, is not most philanthropist's meaning.
In recent years, along with advancing by leaps and bounds of tissue engineering correlational study, brought hope for the regenerating bone or cartilage technology.The research of tissue engineering relates to the content of seed cell, Biodegradable material and three aspects of tissue construction.Wherein, successful key is the preparation of ideal degradable biomaterial.It is generally acknowledged to have following characteristic as the ideal stent of seed cell: excellent biological compatibility and biological degradability, three-dimensional porous stereochemical structure, plasticity and certain mechanical strength, good cell interface and antiseptic property.The high molecular polymer and the natural biologic material two big classes that biomaterial scaffolds can be divided at present, synthetic according to the source of material.High molecular polymer comprises polylactic acid (PLA), polyglycolic acid (PGA) and PGA/PLA copolymer (PLGA) etc.; Natural biologic material comprises collagen (Collagen), fibrin (Fibrin), gelatin (Gelatin), ammonia polyose of candy (GAG), Sargassum hydrochloric acid (Alginate) etc.Although these biomaterials have been observed good regenerating bone or cartilage ability in laboratory and zoopery, because of many deficiencies such as bio-toxicity, degradation speed and mechanical strength are failed to be applied to clinical.Sometimes even also the complex surface of need going is modified, so that to seed cell good artificial extracellular matrix's environment is provided.
Given this; Many scholars are devoted to the research of natural extracellular matrix (ECM) as carrier; Like submucous layer of small intestine (small intestinal submucosa; SIS), human amniotic membrane (human amniotic membrane, HAM), allosome takes off cell submucous layer of bladder, the excretory extracellular matrix of xenogenesis (pig) chondrocyte (ECM) etc.The biomaterial scaffolds of the natural or synthetic that extracellular matrix (ECM) support is more above-mentioned has the following advantages: 1, extracellular matrix (ECM) support main component is functional and structural albumen---collagen fiber (collagen), proteoglycan (proteoglycans), ammonia polyose of candy (GAGs), and they can organically be combined into ultrastructure and supply tissue construction; 2, the tripeptides RGD on extracellular matrix (ECM) surface, and be rich in the Dan Baijutang (like syndecan) and the CD44 of chondroitin sulfate, heparin sulfate, important effect is also arranged aspect cell adhesion; 3, it also contains multiple somatomedin, is playing important effect aspect chondrocyte differentiation that promotes stem cell and the promotion emiocytosis extracellular matrix (ECM) respectively like TGF-β, bFGF, VEGF etc., particularly TGF-β and bFGF; 4, has good natural biodegradability; 5, can be out of shape through the integral protein inducing cell, and then promote the growth and the differentiation of cell.At present, these extracellular matrixs (ECM) support has been widely used in skin wound reparation, ureter reconstruction, digestive tube and the cerebral dura mater reparation.The collagen support is also arranged in recent years and stimulate art to combine the report of regeneration of cartilage with little fracture bone marrow from body chondrocyte cell transplantation art and PGA support.
Above-mentioned allosome tissue source or heterogenous cell derived cell epimatrix (ECM) support have many good qualities, but still can not eliminate the risk of immunological rejection and pathophoresis fully.In order to address the above problem, the preparation of these extracellular matrixs (ECM) support all must be passed through the complicated cell processes that takes off.Take off employed protein denaturant and cosolvent SDS in the cell processes; Can make not only that the normal structure structure ruptures, amino protein sugar is lost and collagen destroys; And lose the raised growth factor (can up to 39～72%), and then reduced the biological function of extracellular matrix (ECM) support.What the present invention relates to is three-dimensional porous shape extracellular matrix (ECM) support in mesenchymal stem cells MSCs source; Can not only eliminate the risk of immunological rejection and pathophoresis; Can also provide that seed cell (chondrocyte or stem cell) sticks, propagation and the required microenvironment of chondrify differentiation; Be that orthopaedics is clinical and then explore a kind of simple and easy and new technique that effective cartilage defect is repaired in the future, for clinical practice from now on provides necessary theoretical foundation.
Summary of the invention
The objective of the invention is above-mentioned deficiency, a kind of extracellular matrix timbering material is provided to prior art.
Another object of the present invention provides the method for preparing of this extracellular matrix timbering material.
The object of the invention can be realized through following technical scheme:
A kind of extracellular matrix timbering material based on the mesenchymal stem cells MSCs source, this extracellular matrix support obtains through following method:
(1) former being commissioned to train of full bone marrow culture method supported young rabbit bone marrow mescenchymal stem cell: get 2-3 children's bifilar bone of rabbit in age in week and/or tibia, sterilization is rearmounted is equipped with in the culture dish of physiological saline solution, open femur and/or tibia two ends medullary cavity under aseptic condition; Use the normal saline flushing medullary cavity, collect bone marrow irrigation liquid, centrifugal 8 ~ 10 minutes of 800 ~ 1200 rev/mins of speed; Abandon supernatant; The mesenchymal stem cells MSCs deposition is with DMEM culture medium suspendible, and counting, with 1 * 10
8It is the 150mm culture dish that cell density is inoculated in diameter with mesenchymal stem cells MSCs; Every ware adds the DMEM culture fluid 20ml that contains 10% hyclone; Put and carry out the monolayer High Density Cultivation in the CO2 gas incubator; Carry out changing the first time liquid after 6 ~ 8 days, changed liquid once in per afterwards 3 days, whole cultivation cycle is 30～40 days;
(2) collecting cell epimatrix; And lyophilization: abandon the culture fluid in step (1) culture dish; Ware counterdie shape or gelatin extracellular matrix are collected in the brown glass container that the crosslinked fluid that contains 10mM EDC and 10mM NHS is housed, soak and use earlier 5mM Na after 20 ~ 25 hours
2HPO
4Wash 2 ~ 5 times; Each 20 ~ 30 minutes, reuse distilled water rinsing 2 ~ 5 times, each 20 ~ 30 minutes; 1200 ~ 1800 rev/mins of speed is centrifugal 8 ~ 15 minutes then, and the collecting precipitation thing obtains the extracellular matrix timbering material based on the mesenchymal stem cells MSCs source through lyophilization.
Wherein, the described extracellular matrix timbering material that obtains through lyophilizing is according to cartilage defect district or required suitable size and the shape of being trimmed to of experiment in vitro, and the ethane via epoxyethane sterilization obtains whole packing bag.
The described lyophilization of step (2) is described precipitate to be put into-80 ℃ profound hypothermia refrigerator or liquid nitrogen container freeze forming, after freezing at least 24 hours, can the extracellular matrix solids that obtain be put on the pallet of freezer dryer; Freezer dryer needs to start 1～1.5 hour pre-freeze in advance and reaches-65 ℃; Then with the extracellular matrix solids under-55～-75 ℃, vacuum 1～10KPa condition, handle to obtain the extracellular matrix timbering material that the silvery white or eburneous three-dimensional porous shape of white is originated based on mesenchymal stem cells MSCs in 24～48 hours through lyophilization.
The method for preparing of described extracellular matrix timbering material based on mesenchymal stem cells MSCs source comprises following steps:
(1) former being commissioned to train of full bone marrow culture method supported young rabbit bone marrow mescenchymal stem cell: get bifilar bone of young rabbit and/or tibia, sterilization is rearmounted is equipped with in the culture dish of physiological saline solution, open femur and/or tibia two ends medullary cavity under aseptic condition; Use the normal saline flushing medullary cavity, collect bone marrow irrigation liquid, centrifugal 8 ~ 10 minutes of 800 ~ 1200 rev/mins of speed; Abandon supernatant; The mesenchymal stem cells MSCs deposition is with DMEM culture medium suspendible, and counting, with 1 * 10
8Cell density is inoculated in the 150mm culture dish with mesenchymal stem cells MSCs; Every ware adds the DMEM culture fluid 20ml that contains 10% hyclone; Put and carry out the monolayer High Density Cultivation in the CO2 gas incubator; Carry out changing the first time liquid after 6 ~ 8 days, changed liquid once in per afterwards 2～3 days, whole cultivation cycle is 30～40 days;
(2) collecting cell epimatrix; And lyophilization: abandon the culture fluid in step (1) culture dish; Ware counterdie shape or gelatin extracellular matrix are collected in the brown glass container that the crosslinked fluid that contains 10mM EDC and 10mM NHS is housed, soak and use earlier 5mM Na after 20 ~ 25 hours
2HPO
4Wash 2 ~ 5 times; Each 20 ~ 30 minutes, reuse distilled water rinsing 2 ~ 5 times, each 20 ~ 30 minutes; 1200 ~ 1800 rev/mins of speed is centrifugal 8～15 minutes then, and the collecting precipitation thing obtains the extracellular matrix timbering material based on the mesenchymal stem cells MSCs source through lyophilization.
Wherein, The extracellular matrix timbering material that obtains through lyophilizing is according to cartilage defect district or required suitable size and the shape of being trimmed to of experiment in vitro; The ethane via epoxyethane sterilization obtains whole packing bag; Be aseptic condition before can keeping using and use, and can't harm its physicochemical property and space structure in order to experiment in vivo and vitro research.
The described lyophilization of described step (2) is described precipitate to be put into-80 ℃ profound hypothermia refrigerator or liquid nitrogen container freeze forming, after freezing at least 24 hours, can the extracellular matrix solids that obtain be put the pallet of freezer dryer; Freezer dryer needs to start 1～1.5 hour pre-freeze in advance and reaches-60 ℃; Then with the extracellular matrix solids under-55～-70 ℃, vacuum 1～10KPa condition, handle to obtain the extracellular matrix timbering material that the silvery white or eburneous three-dimensional porous shape of white is originated based on mesenchymal stem cells MSCs in 24～48 hours through lyophilization.
Bifilar bone of young rabbit of the present invention and tibia are taken from rabbit and are slaughtered the field, are preferably slaughtering bifilar bone and the tibia that separation obtains in back 1 hour.
The present invention is based on the physical and chemical indexes of the extracellular matrix timbering material in mesenchymal stem cells MSCs source: the extracellular matrix timbering material that the present invention is based on the mesenchymal stem cells MSCs source is three-dimensional porous shape timbering material, and pore size is 100-400 μ m, and porosity is not less than 90%; Mechanical strength is not less than 3.0KPa; High histocompatibility, the cell inoculation rate is not less than 55%, and the seed cell survival rate is more than 95%; Contain collagen, proteoglycan, and TGF-β 1 somatomedin such as grade.
Beneficial effect:
Extracellular matrix timbering material of the present invention is with the material source of stem cell as the extracellular matrix support; It is not only tissue-derived extensively, multiplication capacity is strong, can obtain competent cell concentration from body; Avoided the problem of the social ethics that embryonic stem cell involved; And overcome existing allosome tissue source or heterogenous cell derived cell epimatrix (ECM) drawback that support brought used both at home and abroad: the risk of immunological rejection and pathophoresis; Can also provide simultaneously that seed cell (chondrocyte or stem cell) sticks, propagation and the required microenvironment of chondrify differentiation, be a kind of biologic bracket material that clinical development prospect at a specified future date is arranged.
The preparation process of mesenchymal stem cells MSCs epimatrix support involved in the present invention; No matter be the highdensity In vitro culture of stem cell monolayer, or the vacuum lyophilization processing, or ethylene oxide sterilizing; All be the relatively more succinct row that is prone to, equipment is not had excessive demand.
Extracellular matrix timbering material ethane via epoxyethane of the present invention is sterilized and is obtained whole packing bag, is aseptic condition before can keeping using and uses in order to experiment in vivo and vitro research, and can't harm its physicochemical property and space structure.
Description of drawings
The schematic flow sheet of Fig. 1 mesenchymal stem cells MSCs epimatrix support preparation.
The figure of sight substantially of Fig. 2 mesenchymal stem cells MSCs epimatrix support.
Microgram under the scanning electron microscope of Fig. 3 mesenchymal stem cells MSCs epimatrix support.
General morphosis figure under Fig. 4 mesenchymal stem cells MSCs epimatrix support pathological section HE dyeing inverted microscope.
The sight substantially of Fig. 5 chondrocyte inoculation stem cell epimatrix support different time points.
The histochemical stain of Fig. 6 chondrocyte inoculation stem cell epimatrix support different time points is observed.
The specific embodiment
Embodiment 1
(1) schematic flow sheet of mesenchymal stem cells MSCs epimatrix support preparation is seen Fig. 1, and concrete grammar is following:
(1) former being commissioned to train of full bone marrow culture method supported young rabbit bone marrow mescenchymal stem cell: get the young rabbit of just having slaughtered in the slaughterhouse in fresh 3 ages in week; Separate and obtain bifilar bone in the left and right sides and tibia; Place and transport laboratory in the ice bag back and carry out following operation:, put in the culture dish that physiological saline solution is housed femur and the capable povidone iodine soaking disinfection of tibia.In the super-clean bench, open femur, tibia two ends medullary cavity are used the normal saline flushing medullary cavity, till medullary cavity is turned white.Collect bone marrow irrigation liquid, centrifugal 10 minutes of 1000 rev/mins of speed is abandoned supernatant, and cell precipitation is with DMEM base training suspendible, and counts.With 1 * 10
8Cell density is the 150mm culture dish with cell inoculation in diameter, and every ware adds the DMEM culture fluid 20ml that contains 10% hyclone, puts in the CO2 gas incubator in 37 ℃, 5%CO
2, carry out the monolayer High Density Cultivation under the saturated humidity condition and cultivate.Carry out changing the first time liquid after about 7 days, change the liquid frequency afterwards and be and changed liquid once in 2 days.Whole cultivation cycle need be cultivated 30 days approximately.
(2) collecting cell epimatrix; And lyophilization: abandon culture fluid in the ware;, be collected in the brown glass container that the crosslinked fluid that contains 10mM EDC and 10mM NHS is housed ware counterdie shape or gelatin extracellular matrix with the cell spatula, soak and use 5mMNa earlier after 24 hours
2HPO
4Wash each 30 minutes 3 times; Back reuse distilled water rinsing 3 times, each 30 minutes.Centrifugal 10 minutes of 1500 rev/mins of speed, the collecting precipitation thing is put in the suitable container, and the profound hypothermia refrigerator freeze forming of putting into-80 ℃ immediately obtains the extracellular matrix solids.After freezing at least 24 hours, can the extracellular matrix solids be put on the pallet of freezer dryer; Freezer dryer needs to start 1 hour pre-freeze in advance and reaches-65 ~-60 ℃; Then with the extracellular matrix solids under temperature-65～-75 ℃, vacuum 1-5KPa condition; Handled 36 hours through lyophilization, finally obtain the silvery white or eburneous three-dimensional porous shape timbering material of white.
(3) ethylene oxide sterilization processes and obtaining meets the whole packing bag of the relevant quality standard of country: this material can be required according to cartilage defect district or experiment in vitro; Be trimmed to suitable size and shape arbitrarily; But the ethane via epoxyethane sterilization obtains whole packing bag; Be aseptic condition before can keeping using and use, and can't harm its physicochemical property and space structure in order to experiment in vivo and vitro research.
(2) outside drawing of prepared stem cell epimatrix timbering material is as shown in Figure 2, and microgram is as shown in Figure 3 under its scanning electron microscope, and (40,100 and 400 times) HE coloration result is as shown in Figure 4 under the microscope different amplification.Its physical and chemical indexes: the pore size of three-dimensional porous shape timbering material is 100～400 μ m; Porosity is 91.5% ± 2.84%, and mechanical strength is 3.8 ± 0.6KPa, high histocompatibility; The cell inoculation rate is about 56%; The seed cell survival rate is 95.4%, contains collagen, proteoglycan, and TGF-β 1 somatomedin such as grade.Fig. 5 is seen in the sight substantially of chondrocyte inoculation stem cell epimatrix support different time points, and the histochemical stain of chondrocyte inoculation stem cell epimatrix support different time points is observed and seen Fig. 6.
Below be the detection method of each physical and chemical indexes of stem cell epimatrix timbering material:
(1) get the mesenchymal stem cells MSCs epimatrix support for preparing among the embodiment 1, as follows pathological section HE dyeed:
1) draw materials with fixing: backbone block cuts to 0.6 * 0.6 * 0.6cm size and is advisable, and drops in the 10% formaldehyde fixed liquid fixing 24 hours fast; Flowing water flushing afterwards 10 minutes is with the flush away fixative;
2) dehydration: 70% ethanol 0.5 hour; 80% ethanol 0.5 hour; 90% ethanol 0.5 hour; 100% ethanol (I) 0.5 hour; 100% ethanol (II) 0.5 hour;
3) transparent: xylene (I) 0.5 hour; Xylene (II) 0.5 hour;
4) waxdip: backbone block is put into melting wax, and 75 ℃ of incubators spend the night;
5) embedding: on BMJ-B type embedding machine with backbone block with FFPE (backbone block is embedded in the embedded box in principle) in embedded box; The working condition of embedding machine: operating temperature is 60 ℃, and storage tweezer deblocking temperature is 64 ℃, and the hawfinch temperature is 70 ℃; The embedded block that embedding finishes places cooling (25～-15 ℃) on the pathological tissue embedding freezing stage immediately;
6) section: on the Leica paraffin slicing machine, cut into slices immediately after the embedded block cooling, slice thickness is 2-3 μ m; Cut the support section of getting off and put in the 48 degree water that pathological tissue floats the baking appearance, gently it is affixed on the clean adhesion microscope slide, put afterwards on the constant temperature exhibition sheet platform and dry with tweezers; Dye preposition 65 ℃ 20 minutes with warm sheet;
7) conventional hematoxylin-eosin (HE) dyeing:
Slough paraffin: xylene (I) 5 minutes; Xylene (II) 5 minutes; Xylene (III) 5 minutes;
Aquation (making cell expansion): 100% ethanol (I) 3 minutes; 100% ethanol (II) 3 minutes; 90% ethanol 3 minutes; 80% ethanol 3 minutes; 70% ethanol 3 minutes; Flowing water flushing 10 minutes;
Transfect cell nuclear: brazilwood extract dyeing 5 minutes; Flowing water flushing 15 minutes;
Differentiation: 1% hydrochloride alcohol 3～5 seconds;
Return indigo plant: flowing water flushing 10～15 minutes;
Transfect cell matter: Yihong 5 minutes;
Dehydration: 70% ethanol 3 minutes; 80% ethanol 3 minutes; 95% ethanol 3 minutes; 100% ethanol (I) 3 minutes; 100% ethanol (II) 3 minutes;
Transparent: xylene (I) 5 minutes; Xylene (II) 5 minutes; Xylene (III) 5 minutes;
Mounting: cut into slices with neutral gum sealing support.
8) the painted experimental result of (40,100 and 400 times) observed and recorded support section HE under the microscope different amplification is seen Fig. 4.
(2) get the stem cell epimatrix timbering material for preparing in the present embodiment and detect porosity, mechanical strength according to following method:
1) porosity: the mercury injection method that adopts Nettles DL document to be reported is measured the brace aperture rate.Measure evacuation behind the support volume, hydrargyrum is charged to around the porous support, from the outside hydrargyrum is pressurizeed, get into the volume of hydrargyrum in the hole in record pressure and the pressure process, porosity is: the volume of the volume/porous material of hydrargyrum in the entering hole.
The experimental result that calculates the porosity of present embodiment stem cell epimatrix timbering material is: 91.5% ± 2.84%;
2) detection method of mechanical strength and result:
Backbone block is prepared into diameter and is 6mm, highly is to use the Instron electronics sound attitude universal testing machine test compressive property of model as model5943 by the cylinder of 6mm size, and compression speed is 0.1mm/min.The experimental result of stem cell epimatrix timbering material mechanical strength is: 3.8 ± 0.6Kpa.
(3) get the stem cell epimatrix timbering material for preparing in the present embodiment and detect the cell inoculation rate as follows:
1) backbone block to be prepared into diameter be 6mm, highly for the cylinder of 3mm size, soak more than 1 hour with the DMEM culture fluid that contains 10% hyclone in advance, note during immersion the gas in the support being extruded as much as possible with tweezers;
2) reached the chondrocyte of P2 or P3 with 0.25% trypsinization, counted and prepare needed cell suspension amount W1; The cell concentration of inoculation is not less than 5 * 10 for every milliliter
6Individual.
3) take the dynamic cellular inocalation method: add 1ml culture fluid and 1 support in each 1.5ml centrifuge tube, put on the table shake and hatched 1.5 hours with the frequency of 50 times/min;
4) the end back is taken out to prop up and is mounted in the 12 hole wares, and every hole adds the DMEM culture fluid that 2ml contains 10% hyclone, and it is interior in 37 ℃, 5%CO to put CO2 gas incubator
2, cultivate under the saturated humidity condition; Count simultaneously that remaining chondrocyte number is W2 in the 1.5ml centrifuge tube, support cell inoculation rate is=(W1-W2)/W1 * 100%.The cell inoculation rate of stem cell epimatrix timbering material is about 56%.
5) after 24 hours support is taken out, replace and continue in the 6 new hole wares to cultivate under the same condition, changed liquid once in 2 days, take out the detection of the seed cell survival rate of inoculating on the row support behind the experiment required time.
(4) the stem cell matrix scaffold material of getting present embodiment preparation detects the seed cell survival rate of inoculation as follows:
1) gets the support of seed cell inoculated and cultured after 3 days, remove culture fluid,, note culture fluid is rinsed well, in order to avoid influence experimental result with HBSS liquid flushing 3 times.
2) get the good SYTO of prior outfit 10 green fluorescent nucleic acid stain and DEAD Red nucleic acid stain, each support BIAO and BEN adds 500 μ l dye liquors, hatches 15 minutes under room temperature, the lucifuge condition.
3) dyeing liquor is removed in suction, with HBSS liquid flushing 3 times.The back is fixed 30～60 minutes with 4% glutaraldehyde, HBSS liquid flushing 3 times.
4) erythrophil under the field of view (R) and blue transfect cell number (B) under fluorescence microscope, erythrophil is dead cell, and blue transfect cell is survivaling cell.Seed cell survival rate=the B/ (R+B) * 100% in each visual field arbitrarily gets 5 different visuals field, calculates the mean of seed cell survival rate.
Experimental result: the survival rate of the seed cell of stem cell epimatrix timbering material inoculation is 95.4%.
(5) the stem cell epimatrix timbering material of getting the present embodiment preparation is measured collagen content as follows:
1) take by weighing the 20mg support, with the fine rearmounted 1.5ml Eppendorf pipe that shreds of shears, add 1～1.5ml papain solution again, in 65 ℃ of water-baths 24 hours, 12000g was centrifugal, and it is to be checked to get supernatant.
2) hydroxyproline standard substance and support BIAO and BEN mix according to table 1 and table 2 with 4NNaOH and deionized water respectively through the postdigestive supernatant of step 1 mutually, and final volume is 50 μ l.
Table 1
Table 2
| Stem cell ECM support | |
| Sample 20mg/ml (μ l) | ?5 |
| 4NNaOH（μl） | ?25 |
| Deionized water (μ l) | ?20 |
3) ready sample and standard QC were put in the autoclave sterilizer (120 ℃) hydrolysis 20 minutes.
4) add 450 μ l chloramine-T reagent, mixing at room temperature reacted 25 minutes gently.
5) add 500 μ l Ou Lixishi liquid, mixing gently, 65 ℃ of water-baths 20 minutes.
6) colorimetric reading under the ultraviolet spectrophotometer 550nm wavelength.
7), draw light absorption value and concentration curve according to the light absorption value of different hydroxyproline standard substance.
8) finally according to this curve, draw the concentration A1 of the hydroxyproline of support BIAO and BEN.
9), calculate stem cell epimatrix timbering material collagen content A2 again according to formula A1=A2 * 12.5%.
Experimental result: stem cell epimatrix timbering material collagen content accounts for dry weight ratio and is: 5.489%.
(6) the stem cell epimatrix timbering material of getting the present embodiment preparation is measured GAG content as follows:
1) take by weighing the 20mg support, with the fine rearmounted 1.5ml Eppendorf pipe that shreds of shears, add 1～1.5ml papain solution again, in 65 ℃ of water-baths 24 hours, 12000g was centrifugal, and it is to be checked to get supernatant.
2) take by weighing 50ul sample supernatant, add ddH
2O to 100 μ l, add 1ml Dye Reagent mixing after, place on the horizontal shaking table incubated at room 30min.
3) the centrifugal 10min of 12000rpm, the careful suction removed supernatant.
4) add 0.5ml Dissociation Reagent and heavily dissolve deposition 10min.
5) draw 200 μ l samples and add ELISA Plate, detect reading in the 656nm, 2h.
Experimental result: it is 1.259% that stem cell epimatrix timbering material sGAG (sulfate mucopolysaccharide) accounts for sample dry weight ratio.
(7) the stem cell epimatrix timbering material of getting the present embodiment preparation is measured somatomedin TGF-β 1 content as follows:
1) take by weighing the 20mg support, with the fine rearmounted 1.5ml Eppendorf pipe that shreds of shears, add 1～1.5ml papain solution again, in 65 ℃ of water-baths 24 hours, 12000g was centrifugal, and it is to be checked to get supernatant.
2) carry out application of sample according to the grow operating procedure of sub-factor beta1ELISA Kit of the conversion of Uscn Life Science Inc with this, use the optical density of ELIASA at last in each hole of 450nm wavelength measurement.
3), draw the concentration of the TGF-β 1 that surveys according to standard curve.
Experimental result: the content of stem cell epimatrix timbering material TGF-β 1 is 322.6 ± 16.9pg/10mg.
(8) the histochemical stain method of the external chondrocyte inoculation stem cell matrix scaffold material different time points of present embodiment preparation:
1) backbone block to be prepared into diameter be 6mm, highly for the cylinder of 3mm size, soak more than 1 hour with the DMEM culture fluid that contains 10% hyclone in advance, note during immersion the gas in the support being extruded as much as possible with tweezers;
2) reached the chondrocyte of P2 or P3 with 0.25% trypsinization, counted and prepare needed cell suspension amount W1; The cell concentration of inoculation is not less than 5 * 10 for every milliliter
6Individual.
3) take the dynamic cellular inocalation method: add 1ml culture fluid and 1 support in each 1.5ml centrifuge tube, put on the table shake and hatched 1.5 hours with the frequency of 50 times/min;
4) the end back is taken out to prop up and is mounted in the 12 hole wares, and every hole adds the DMEM culture fluid that 2ml contains 10% hyclone, and it is interior in 37 ℃, 5%CO to put CO2 gas incubator
2, cultivate under the saturated humidity condition; Changed liquid once in per 2～3 days.
5) in cultivating back 2 days, 1 week, 2 week and 4 weeks, sample is taken out, with the abundant soaking flushing of PBS liquid to the redness of not having culture fluid till, take with digital camera respectively and see substantially and carry out following histochemical stain observation;
6) draw materials with fixing: backbone block cuts to 0.6 * 0.6 * 0.6cm size and is advisable, and drops in the 10% formaldehyde fixed liquid fixing 24～48 hours fast.Flowing water flushing afterwards 10～20 minutes is with the flush away fixative.
7) dehydration: 70% ethanol 0.5 hour; 80% ethanol 0.5 hour; 90% ethanol 0.5 hour; 100% ethanol (I) 0.5 hour; 100% ethanol (II) 0.5 hour;
8) transparent: xylene (I) 0.5 hour; Xylene (II) 0.5 hour;
9) waxdip: backbone block is put into melting wax, and 75 ℃ of incubators spend the night;
10) embedding: on BMJ-B type embedding machine with backbone block with FFPE (backbone block is embedded in the embedded box in principle) in embedded box; The working condition of embedding machine: operating temperature is 60 ℃, and storage tweezer deblocking temperature is 64 ℃, and the hawfinch temperature is 70 ℃; The embedded block that embedding finishes places cooling (25～-15 ℃) on the pathological tissue embedding freezing stage immediately;
11) section: on the Leica paraffin slicing machine, cut into slices immediately after the embedded block cooling, slice thickness is 2～3 μ m; Cut the support section of getting off and put in the 48 degree water that pathological tissue floats the baking appearance, gently it is affixed on the clean adhesion microscope slide, put afterwards on the constant temperature exhibition sheet platform and dry with tweezers; Dye preposition 65 ℃ 20 minutes with warm sheet;
12) Safranin-O dyeing:
Slough paraffin: xylene (I) 5 minutes; Xylene (II) 5 minutes; Xylene (III) 5 minutes;
Aquation (making cell expansion): 100% ethanol (I) 3 minutes; 100% ethanol (II) 3 minutes; 90% ethanol 2 minutes; 80% ethanol 1 minute; 70% ethanol 1 minute; Flowing water flushing 5 minutes;
Transfect cell nuclear: brazilwood extract dyeing 5 minutes; Flowing water flushing 5 minutes;
Differentiation: 1% hydrochloride alcohol 2～3 seconds; Flowing water flushing 2 minutes;
0.02%Fast green dyed 5 minutes, fast rinsing in 1% acetic acid: be no more than 10～15 seconds; 0.1%safrain O dyed 5 minutes;
Dehydration: 80% ethanol 2 minutes; 95% ethanol 2 minutes; 100% ethanol (I) 2 minutes; 100% ethanol (II) 2 minutes;
Transparent: xylene (I) 2 minutes; Xylene (II) 2 minutes;
Mounting: cut into slices with neutral gum sealing support.
The painted experimental result of (40,100 and 400 times) observed and recorded support section HE is seen Fig. 6 under the microscope different amplification.
13) II Collagen Type VI immunohistochemical staining:
Slough paraffin: xylene (I) 5 minutes; Xylene (II) 5 minutes; Xylene (III) 5 minutes;
Aquation (making cell expansion): 100% ethanol (I) 3 minutes; 100% ethanol (II) 3 minutes; 90% ethanol 2 minutes; 80% ethanol 1 minute; 70% ethanol 1 minute; PBS flushing 2 minutes/inferior * 3 time;
The E.C. 3.4.21.64 method is repaired: every slice, thin piece adds 5ul, hatches 30 minutes for 37 ℃;
Paraffin section is put into the 3%H2O2-formalin and was soaked 10 minutes, eliminating the endogenous catalase, and PBS flushing 2 minutes/inferior * 3 time;
Add a reagent A (confining liquid is 10% lowlenthal serum) on tissue slice 37 ℃ hatched 10 minutes; Outwell or blot liquid; Every section adds an amount of one anti-(need cover tissue to be checked fully), hatches 30-60 minute PBS flushing 2 minutes/inferior * 3 time in the wet box for 37 ℃; Every section adds a reagent B (anti-mice IgG biotinylation two anti-), hatches 30～60 minutes PBS flushing 2 minutes/inferior * 3 time in the wet box for 37 ℃; Every section adds a reagent C (the new and plain labelling HRP of chain), hatches 30～60 minutes PBS flushing 2 minutes/inferior * 3 time in the wet box for 37 ℃; Preparation DAB colour developing liquid, every section adds an above-mentioned DAB colour developing liquid, and color development at room temperature 2～5 minutes, flowing water fully washed 5 minutes;
Transfect cell nuclear: brazilwood extract dyeing 5 minutes; Flowing water flushing 8～10 minutes;
Differentiation: 1% hydrochloride alcohol 20 seconds;
Return indigo plant: flowing water flushing 5 minutes;
Dehydration: 70% ethanol 5 minutes; 80% ethanol 5 minutes; 95% ethanol 5 minutes; 100% ethanol (I) 5 minutes; 100% ethanol (II) 5 minutes;
Transparent: xylene (I) 2 minutes; Xylene (II) 2 minutes;
Mounting: cut into slices with neutral gum sealing support.
The experimental result of (40,100 and 400 times) observed and recorded support section II Collagen Type VI SABC is seen Fig. 6 under the microscope different amplification.
The characteristic parameter of table 1 stem cell epimatrix support
Claims (6)
1. extracellular matrix timbering material based on mesenchymal stem cells MSCs source is characterized in that this extracellular matrix support obtains through following method:
(1) former being commissioned to train of full bone marrow culture method supported young rabbit bone marrow mescenchymal stem cell: get 2-3 children's rabbit femoral in age in week and/or tibia, sterilization is rearmounted is equipped with in the culture dish of physiological saline solution, open femur and/or tibia two ends medullary cavity under aseptic condition; Use the normal saline flushing medullary cavity, collect bone marrow irrigation liquid, centrifugal 8～10 minutes of 800～1200 rev/mins of speed; Abandon supernatant; The mesenchymal stem cells MSCs deposition is with DMEM base training suspendible, and counting, with 1 * 10
8It is the 150mm culture dish that cell density is inoculated in diameter with mesenchymal stem cells MSCs; Every ware adds the DMEM culture fluid 20ml that contains 10% hyclone; Put and carry out the monolayer High Density Cultivation in the CO2 gas incubator; Carry out changing the first time liquid after 6～8 days, changed liquid once in per afterwards 3 days, whole cultivation cycle is 30～40 days;
(2) collecting cell epimatrix; And lyophilization: abandon the culture fluid in step (1) culture dish; Ware counterdie shape or gelatin extracellular matrix are collected in the brown glass container that the crosslinked fluid that contains 10mM EDC and 10mM NHS is housed, soak and use 5mMNa earlier after 20 ~ 25 hours
2HPO
4Wash 2 ~ 5 times; Each 20 ~ 30 minutes, reuse distilled water rinsing 2 ~ 5 times, each 20 ~ 30 minutes; 1200 ~ 1800 rev/mins of speed is centrifugal 8～15 minutes then, and the collecting precipitation thing obtains the extracellular matrix timbering material based on the mesenchymal stem cells MSCs source through lyophilization.
2. extracellular matrix timbering material according to claim 1; The extracellular matrix timbering material that it is characterized in that obtaining through lyophilizing is according to cartilage defect district or required suitable size and the shape of being trimmed to of experiment in vitro, and the ethane via epoxyethane sterilization obtains whole packing bag.
3. extracellular matrix timbering material according to claim 1; It is characterized in that the described lyophilization of step (2) is described precipitate to be put into-80 ℃ profound hypothermia refrigerator or liquid nitrogen container freeze forming, puts the extracellular matrix solids that obtains on the pallet of freezer dryer; Freezer dryer needs to start 1～1.5 hour pre-freeze in advance and reaches-65 ~ 60 ℃; Then with the extracellular matrix solids under temperature-55～-75 ℃, vacuum 1～10KPa condition, handle to obtain the extracellular matrix timbering material that the silvery white or eburneous three-dimensional porous shape of white is originated based on mesenchymal stem cells MSCs in 24～48 hours through lyophilization.
4. the method for preparing of the described extracellular matrix timbering material based on mesenchymal stem cells MSCs source of claim 1 is characterized in that comprising following steps:
(1) former being commissioned to train of full bone marrow culture method supported young rabbit bone marrow mescenchymal stem cell: get 2-3 children's rabbit femoral in age in week and/or tibia, sterilization is rearmounted is equipped with in the culture dish of physiological saline solution, open femur and/or tibia two ends medullary cavity under aseptic condition; Use the normal saline flushing medullary cavity, collect bone marrow irrigation liquid, centrifugal 8～10 minutes of 800～1200 rev/mins of speed; Abandon supernatant; The mesenchymal stem cells MSCs deposition is with DMEM base training suspendible, and counting, with 1 * 10
8It is the 150mm culture dish that cell density is inoculated in diameter with mesenchymal stem cells MSCs; Every ware adds the DMEM culture fluid 20ml that contains 10% hyclone; Put and carry out the monolayer High Density Cultivation in the CO2 gas incubator; Carry out changing the first time liquid after 6～8 days, changed liquid once in per afterwards 3 days, whole cultivation cycle is 30～40 days;
(2) collecting cell epimatrix; And lyophilization: abandon the culture fluid in step (1) culture dish; Ware counterdie shape or gelatin extracellular matrix are collected in the brown glass container that the crosslinked fluid that contains 10mM EDC and 10mM NHS is housed, soak and use earlier 5mM Na after 20～25 hours
2HPO
4Wash 2 ~ 5 times; Each 20 ~ 30 minutes, reuse distilled water rinsing 2 ~ 5 times, each 20 ~ 30 minutes; 1200 ~ 1800 rev/mins of speed is centrifugal 8～15 minutes then, and the collecting precipitation thing obtains the extracellular matrix timbering material based on the mesenchymal stem cells MSCs source through lyophilization.
5. method for preparing according to claim 4, the extracellular matrix timbering material that it is characterized in that obtaining through lyophilizing are according to cartilage defect district or required suitable size and the shape of being trimmed to of experiment in vitro, and the ethane via epoxyethane sterilization obtains whole packing bag.
6. method for preparing according to claim 4; It is characterized in that the described lyophilization of step (2) is described precipitate to be put into-80 ℃ profound hypothermia refrigerator or liquid nitrogen container freeze forming, puts the extracellular matrix solids that obtains on the pallet of freezer dryer; Freezer dryer needs to start 1～1.5 hour pre-freeze in advance and reaches-60 ℃; Then with the extracellular matrix solids under-55～-70 ℃, vacuum 1～10KPa condition; Handled 24～48 hours through lyophilization, obtain the extracellular matrix timbering material of the silvery white or eburneous three-dimensional porous shape of white based on the mesenchymal stem cells MSCs source.
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103396982A (en) * | 2013-05-28 | 2013-11-20 | 中山大学附属第三医院 | Method for autocrine secretion of extracellular matrix by stem cells and induction of stem cells to become hepatocytes |
| CN104307046A (en) * | 2014-10-27 | 2015-01-28 | 王黎明 | Injectable mesenchymal stem cell extracellular matrix/agarose composite hydrogel as well as preparation method and application thereof |
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101085374A (en) * | 2007-07-09 | 2007-12-12 | 山东大学 | Tissue engineering bone complex and application |
| CN101134118A (en) * | 2007-07-13 | 2008-03-05 | 中国人民解放军第二军医大学 | Method of producing organization engineered cartilage with inner support |
| CN101653366A (en) * | 2009-06-11 | 2010-02-24 | 广州中大中山医科技开发有限公司 | Construction of gelatin sponge cylinder bracket used for repairing nerve injury |
| CN102188752A (en) * | 2011-04-12 | 2011-09-21 | 浙江大学 | Method and device for preparing bone marrow mesenchymal stem cells-tube scaffold compound |
-
2012
- 2012-07-11 CN CN201210240648.7A patent/CN102743790B/en not_active Expired - Fee Related
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101085374A (en) * | 2007-07-09 | 2007-12-12 | 山东大学 | Tissue engineering bone complex and application |
| CN101134118A (en) * | 2007-07-13 | 2008-03-05 | 中国人民解放军第二军医大学 | Method of producing organization engineered cartilage with inner support |
| CN101653366A (en) * | 2009-06-11 | 2010-02-24 | 广州中大中山医科技开发有限公司 | Construction of gelatin sponge cylinder bracket used for repairing nerve injury |
| CN102188752A (en) * | 2011-04-12 | 2011-09-21 | 浙江大学 | Method and device for preparing bone marrow mesenchymal stem cells-tube scaffold compound |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103396982A (en) * | 2013-05-28 | 2013-11-20 | 中山大学附属第三医院 | Method for autocrine secretion of extracellular matrix by stem cells and induction of stem cells to become hepatocytes |
| CN104726398A (en) * | 2013-12-20 | 2015-06-24 | 江阴司特易生物技术有限公司 | Preparation method of immobilized all-anthropogenic ECM coating matrix |
| CN104307046A (en) * | 2014-10-27 | 2015-01-28 | 王黎明 | Injectable mesenchymal stem cell extracellular matrix/agarose composite hydrogel as well as preparation method and application thereof |
| CN104307046B (en) * | 2014-10-27 | 2016-06-08 | 南京医科大学附属南京医院 | A kind of injectable bone bone marrow-drived mesenchymal stem epimatrix/agarose composite aquogel and its preparation method and application |
| CN108977405A (en) * | 2018-08-09 | 2018-12-11 | 西南大学 | A kind of instant 3D cell growth bracket and preparation method thereof |
| CN109777774A (en) * | 2019-01-30 | 2019-05-21 | 中国人民解放军军事科学院军事医学研究院 | The preparation method of macrophage derived extracellular matrix |
| CN112656991A (en) * | 2020-12-16 | 2021-04-16 | 中国人民解放军南部战区总医院 | Novel extracellular matrix biological scaffold material and preparation method thereof |
| CN116059451A (en) * | 2023-02-20 | 2023-05-05 | 青岛大学 | Aerogel artificial skin with three-dimensional porous structure and preparation method thereof |
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