CN101491703B - Vitro tissue culturing method and multi-pore carrier - Google Patents
Vitro tissue culturing method and multi-pore carrier Download PDFInfo
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Abstract
The invention provides a method for culturing a tissue in vitro and a porous carrier used by the method. The method mainly comprises that: the tissue blocks are partially dissolved by enzyme and placed in a cavity of the porous carrier with hollow cavity, so that the tissue can grow in the pores of the porous carrier from the inside to the outside in the three-dimensional space mode, and the new tissue is directly grown in the state of the tissue. The method has the efficacy of culturing the necessary tissue in vitro by using the tissue quantity in the least volume and within the shortest time.
Description
The present invention is that application number is 01144738.9, the applying date be calendar year 2001 December 24 days, denomination of invention divide an application for " cultural method of vitro tissue and multi-pore carrier thereof ".
Technical field
The invention relates to a kind of cultural method and multi-pore carrier thereof of vitro tissue, it is the piece of tissue of acquisition to be inserted the hollow concrete dynamic modulus decompose in the carrier, and the supply nutrient makes tissue with the towards periphery hole growth of concrete dynamic modulus base material of three-dimensional space mode, to obtain newborn tissue.
Background technology
As everyone knows, when tissue or organ can't be repaired because the factors such as unexpected injury or disease are aging wreck, the life that tends to cause the disabled of sufferer limbs or threaten sufferer further caused great burden and the loss to family and society.Therefore seek a kind of suitable repairing and substitute tissue or organ, be the present continuous target of making great efforts of researcher institute.
In recent years, along with the progress of biotechnology, biological, medical material and histiocytic culture technique mutually combine, develop gradually this new research field of organizational project.Wish future, impaired tissue or organ can be via the reconstruction techniques of organizational project, in external repairing or rebuild a new tissue or organ, substitute impaired position, to recover the life of the healthy of sufferer or continuity sufferer.
Content according to present tissue engineering technique, the tissue that needs acquisition sufferer or donor's sub-fraction health, behind external a large amount of cultured tissue cells, implant the concrete dynamic modulus base material of a decomposability, utilize its three-dimensional space framework, allow histiocyte attach and growth thereon, when histiocyte gradually grow up become tridimensional piece of tissue after, replant go back to the position of the required repairing of sufferer.From histological viewpoint, one block organization is except containing its specific extracellular that forms, and cell and iuntercellular are coated by tridimensional extracellular matrix (extracellularmatrix, ECM), matter also can show the specific function of this tissue except keeping its cell framework around here.
Take the cultivation of cartilaginous tissue as example, cultivate at present external cartilage three-dimensional space, confirmed the three-dimensional space cultivation of chondrocyte in agar (agarosegel), can keep its original cellular morphology and function in tissue, and cultivate the chondrocyte that variation transforms through space twice, in the time of also can cultivating in the agar three-dimensional space, reply original chondrocyte form.Aspect the framework of analog cell outer room matter, developed at present multiple different materials such as collagen protein (collagen) or PLGA (poly (glycolic co-Lactic) acid (PLGA)) and different structure, such as fibrous web-like or mushy artificial base material.Facing at present maximum problem is:
1, histiocyte is in bidimensional culture dish during a large amount of cultured cell, because arranging, the three-dimensional space in the tissue transfers twice spatial growth to, cell will dedifferente the phenomenon of (dedifferentiation), thereby form and the function of lost cell in original tissue.
2, in addition, the implantation of cell (seeding) also is a problem that is difficult for overcoming, because provide cell enough to develop into the space of tissue, the aperture of concrete dynamic modulus base material needs the chi footpath greater than cell, when cell and culture medium mixed liquor implantation concrete dynamic modulus base material, cell tends to flow out from carrier, is difficult for remaining in the base material, for overcoming this problem, present way has two kinds:
A kind of is static implantation, first institute's cultured cells concentration is adjusted to 10
6The high concentration that cells/ml is above, then utilize the property of water-bearing of concrete dynamic modulus base material itself, the cell of implanting is contained in the base material, after cell is attached at base material, adding a large amount of culture medium cultivates again, although the method can know in the cell implantation base material of how many amounts, and the cell of implantable high concentration enters in the material, yet the factor that the distribution of cell still can be subject to gravity affects, and causes the cell distribution of levels uneven, in addition, for preventing that cell from overflowing, can with the volume limited of the culture medium of mixing with cells, the effect and the survival rate that therefore attach also need be considered.
Another kind is dynamic cell implantation, generally adopt at present stirring reactor (spinnerflask), bring cell into base material inside from culture medium in the mode that current stir, the method can obtain better cell attaching rate and compare the more uniform cell distribution of static implantation, yet, the cell number of using is higher, the actual cell number that is attached to material internal also is difficult for knowing, and cell is to implant from outside to inside, peripheral cell number can inner cell density be high still, when tissue culture, the higher one deck that will form of peripheral cell growth rate hinders, stop the nutrient exchange of inner cell, thereby cause the inside and outside Growth of Cells of material significantly uneven.
Summary of the invention
For shortcoming and the drawback of above-mentioned prior art, the inventor creates technical scheme of the present invention through studying chronically and testing.
Main purpose of the present invention provides a kind of cultural method of vitro tissue, comprises at least the following step: a multi-pore carrier with cavity is provided; Piece of tissue is partly dissolved through enzyme; To and dissolve the cavity that the histiocyte that places aforementioned multi-pore carrier through the partly soluble piece of tissue of enzyme; And include piece of tissue and histiocytic multi-pore carrier places culture medium to cultivate with aforementioned.Overcome to be organized in when external twice space plane is cultivated dedifferen tiation occurs, or the inside and outside histiocyte of the base material unequal shortcoming of growing, reach the purpose of effectively cultivating vitro tissue.
Another object of the present invention provides a kind of employed multi-pore carrier of cultural method of vitro tissue, develop a kind of multi-pore carrier, it mainly comprises main body, at least one is positioned at body interior and has the hollow cavity of an opening and the loam cake of covering hollow cavity opening, loam cake is to be made by the mushy material identical with main body, the particle diameter of piece of tissue is greater than the aperture of the peripheral base material of carrier cavity, reaches the purpose of avoiding the piece of tissue overflow to go out.
The object of the present invention is achieved like this: a kind of cultural method of vitro tissue is characterized in that: it comprises the following step at least:
(1) provides a multi-pore carrier with cavity;
(2) piece of tissue is partly dissolved through enzyme;
(3) will and dissolve the cavity that the histiocyte that places this multi-pore carrier through the partly soluble piece of tissue of enzyme;
(4) this is included piece of tissue and histiocytic multi-pore carrier places culture medium to cultivate.
Has the hollow cavity that at least one places piece of tissue in this multi-pore carrier.The particle diameter of this piece of tissue is the aperture greater than the peripheral base material of this carrier cavity.The particle diameter of this piece of tissue is 500 to 1000 μ m.Being shaped as of this multi-pore carrier is cylindrical, cubic type or required arbitrary shape.The pore diameter range of this multi-pore carrier is 50 to 500 μ m.This multi-pore carrier is the macromolecular material of Bioabsorbable, and this macromolecular material is selected from following at least a: polylactic acid, the acid of polylactic acid lactide, PLGA, polyanhydride, polycaprolactone, poly-two ketone esters or polyorthoesters.This multi-pore carrier is PLGA.This piece of tissue is animal tissue.This animal tissue is cartilaginous tissue.This enzyme is soluble protein enzyme or with piece of tissue cytolysis enzyme out.This culture fluid is the culture fluid that contains hyclone.
The present invention also provides a kind of cultural method of vitro tissue employed multi-pore carrier, it is characterized in that: it comprises a main body, at least more than one the loam cake that is positioned at this body interior and has the hollow cavity of an opening and cover this hollow cavity opening.
This main body is cylindrical, cubic type or required arbitrary shape.The pore diameter range of this multi-pore carrier is to be 50 to 500 μ m.This loam cake is by the mushy material identical with main body.
Major advantage of the present invention is: tissue mass that can minimal volumes, within the shortest time in the required tissue of In vitro culture.
Further specify below in conjunction with preferred embodiment and accompanying drawing.
Description of drawings
Fig. 1 is the flow process block schematic diagram of the cultural method of vitro tissue of the present invention.
Fig. 2 is the structural representation with multi-pore carrier of hollow cavity of the present invention.
Fig. 3 is the growth view that is organized in the multi-pore carrier with hollow cavity of the present invention.
Fig. 4 is the schematic flow sheet of cultural method of the vitro tissue of experimental group of the present invention.
Fig. 5 is the schematic flow sheet of cultural method of the vitro tissue of matched group of the present invention.
When Fig. 6 is the cartilaginous tissue cultivation fortnight of experimental group of the present invention, the tissue around the hollow cavity carrier filling cartilaginous tissue and the photo of Growth of Cells situation.
When Fig. 7 is the cartilaginous tissue cultivation fortnight of experimental group of the present invention, the tissue of the peripheral concrete dynamic modulus base material of carrier cavity and the photo of Growth of Cells situation.
Fig. 8 is that the cartilaginous tissue of experimental group of the present invention is cultivated four stars during the phase, reaches the tissue of the peripheral concrete dynamic modulus base material of carrier cavity and the photo (amplifying 40 times) of Growth of Cells situation around the hollow cavity carrier filling cartilaginous tissue.
Fig. 9 is that the cartilaginous tissue of experimental group of the present invention is cultivated four stars during the phase, reaches the tissue of the peripheral concrete dynamic modulus base material of carrier cavity and the photo (amplifying 200 times) of Growth of Cells situation around the hollow cavity carrier filling cartilaginous tissue.
Figure 10 is that the cartilaginous tissue of the matched group of the embodiment of the invention is cultivated four stars during the phase, the tissue of the peripheral concrete dynamic modulus base material of carrier cavity and the photo (amplifying 40 times) of Growth of Cells situation.
Figure 11 is that the cartilaginous tissue of the matched group of the embodiment of the invention is cultivated four stars during the phase, the tissue of the peripheral concrete dynamic modulus base material of carrier cavity and the photo (amplifying 200 times) of Growth of Cells situation.
Figure 12 is grow into cross-sectional area quantized result sketch map in the PLGA multi-pore carrier of the cartilaginous tissue of experimental group more of the present invention and matched group.
The specific embodiment
Consult shown in Figure 1ly, the cultural method of vitro tissue of the present invention mainly comprises the following step: a multi-pore carrier with cavity is provided; Piece of tissue is partly dissolved through enzyme; To and dissolve the cavity that the histiocyte that places aforementioned multi-pore carrier through the partly soluble piece of tissue of enzyme; And include piece of tissue and histiocytic multi-pore carrier places culture medium to cultivate with aforementioned.
Consulting shown in Figure 2ly, is to show multi-pore carrier 10 of the present invention, and it mainly comprises: a main body 1; At least more than one hollow cavity 2, it is to be positioned at aforementioned body 1 inside and to have an opening 5; And a loam cake 6, be as the usefulness that covers above-mentioned hollow cavity 2 openings 5.
Method of the present invention is that directly acquisition is organized, and shred and enzyme dissolving superficial cell after, not in addition in external in advance amplification culture, but piece of tissue is inserted in the hollow cavity of a multi-pore carrier 10 2, the periphery then is mushy base material, afterwards, the perforate 5 that more aforementioned loam cake 6 is covered hollow cavity 2 avoids piece of tissue to flow out.The aforementioned fragment of tissue that shreds can be controlled its size via the mode of sieving, and makes it greater than the aperture of the hollow cavity 2 peripheral base materials of multi-pore carrier 10.When piece of tissue is received in the hollow cavity 2 of multi-pore carrier 10, because of the aperture of peripheral base material less than piece of tissue, therefore, it is inner and can overflow not go out that piece of tissue will be restricted to carrier 10.
Consult shown in Figure 3, high concentration cell mass and the piece of tissue 4 through the enzyme dissolving will be contained in multi-pore carrier 10 inside (being hollow cavity 2), because the cell concentration of multi-pore carrier 10 inside is quite high, in incubation, histiocyte will be by interior to the lower peripheral concrete dynamic modulus base material climbing diffusion growth of density, from inside to outside along its piece of tissue structure originally, with the three-dimensional space mode to the periphery the hole 3 of concrete dynamic modulus base material grow newly-increased tissue 4 '.This cultural method is as external tissue culture, and be directly to cultivate new tissue with tissue morphology, the method of tissue culture outside conventional bulk can solve and cultivate the dedifferen tiation that causes in space twice, tissue mass that can also be minimum within the short time in the external tissue culture that carries out.
Aforementioned piece of tissue 4 can be any animal tissue, for example: cartilaginous tissue or os osseum tissue.
The particle diameter of aforementioned piece of tissue 4 is to be 500 to 1000 μ m.
The shape of aforesaid multi-pore carrier 10 can change arbitrarily according to actual needs, for example: cylindrical, cubic type or arbitrary shape.
Aforesaid multi-pore carrier 10 is the macromolecular materials for absorbability, and this material is to be selected from following group: polylactic acid (polyglycolic acid; PGA), polylactic acid lactide acid (polylacticacid; PLA), PLGA (poly (glycolic-co-Lactic) acid; PLGA), polyanhydride (polyanhydrides), polycaprolactone (polycapralactone), poly-two ketone esters (polydioxanone) and polyorthoesters (polyorthoester) wherein better multi-pore carrier material be PLGA (PLGA).
Aforesaid loam cake 6 is to be made by the mushy material identical with main body.
The pore diameter range of aforesaid multi-pore carrier 10 is to be 50 to 500 μ m.
Embodiment below by the cultivation of chondrocytes in vitro tissue is described in more detail method of the present invention.
Embodiment
A kind of cultural method of chondrocytes in vitro tissue:
(1) preparation has the Bioabsorbable multi-pore carrier 10 of hollow cavity 2:
Selected decomposability macromolecular material is the PLGA macromolecule (molecular weight is about 200,000) for preparing in the ring-opening polymerisation mode in the present embodiment.The PLGA macromolecular material of bulk is pulverized in pulverizer, and the granule of pulverizing can obtain particle diameter at the polymeric particles of 177-250 μ m after sieving by 60-80 hole purpose screen cloth; The water-soluble material that is added into creation pore space structure in the base material of selecting is the sodium chloride particle about particle diameter 250 μ m; The organic solvent of the dissolving polymeric particles of selecting is to be acetone.
PLGA polymeric particles and sodium chloride particle are complied with 10/90 weight ratio, evenly mix in the mode that stirs; Mixed PLGA polymeric particles and sodium chloride particle are poured termination into, and air extractor, diameter are arranged is in the filter of 7mm circle and compacting, pour into organic solvent-acetone in the hybrid particles and infiltrate this moment, open extraction valve and produce a negative pressure and take out unnecessary solvent downwards, and the polymeric particles that has dissolved on the surface is bondd mutually thereafter.After polymeric particles bonds mutually, pour a large amount of deionized waters in filter top, also open extraction valve and bleed simultaneously, a large amount of water by in the material, is separated out and solidified polymeric particles, the sodium chloride particle that it is inner is washed out simultaneously.Macromolecule after the curing takes out from filter, place the large beaker that deionized water is housed, under room temperature, changed water once in per 6 hours, with the mode soaking and water washing that stirs 24 hours, solvent and salt grain that inside is residual wash out, and place 50 ℃ vacuum drying oven to heat the universe dry 24 hours again, can obtain a pore-size distribution between 150 to 350 μ m, the about 90vol% of porosity, hole are the concrete dynamic modulus base material that is interconnected.
The multi-pore carrier with hollow cavity 2 10 that present embodiment is prepared, to cut diameter as 7mm take scalpel, it highly is the cylinder of 9mm, digging a diameter in base material inside is that the 3mm degree of depth is the hollow cylindrical cavity of 6mm, cutting a diameter is 3mm again, highly be the cylinder stopper of 3mm (being loam cake 6 shown in Figure 2), can be when piece of tissue be inserted, to seal inner hollow cavity 2.After multi-pore carrier 10 preparation of hollow is finished, the alcohol-pickled sterilization with 75% 6 hours, and include the ethanol of sterilization with a large amount of aseptic phosphate-buffered salt (phosphate buffered saline, PBS) solution displacements.
(2) processing of experimental group
Flow chart as shown in Figure 4, at first, cartilaginous tissue is the Thigh bone 12 that takes out about 2 to the 4 days rat 11 of new life with microinstrument, remove muscle and the periosteum on surface, be dipped among the DMEM that does not add hyclone (fetal calf serum, FCS), in the operating board (laminar flow) of cell culture, Thigh bone 12 is taken out, place the centrifuge tube 13 of 15ml, the phosphate buffered saline(PBS) that adds 10ml is cleaned, and repeats twice.After Thigh bone 12 cleaned, fall in the culture dish of 10cm, articular cartilage is separated and shred with the tissue shear of the bacterium of going out, piece of tissue 4 chip size are controlled between the 400-800 μ m with 20-40 hole purpose screen cloth.The garbage collection that shreds is poured the phosphate buffered saline(PBS) flushing of 10ml into, triplicate again in the centrifuge tube of 15ml.Cartilaginous tissue piece 4 fragments after the flushing, phosphate buffered saline(PBS) is removed, added again soluble protein enzyme (collagenase) 5ml (1mg/ml phosphate buffered saline(PBS)), place 37 ℃ incubator, left standstill 2 hours, the chondrocyte on surface is dissociated out.Afterwards, with soluble protein enzyme separate dissolved cartilage bone piece later, place centrifuge, with rotating speed 1500r.p.m. centrifugal 5 minutes, the soluble protein enzyme is separated with the cartilage piece.After centrifugal, draw the soluble protein enzyme supernatant of clarification, remaining fragment and cell tissue is with phosphate-buffered salt flushing twice, and is centrifugal twice, to remove residual soluble protein enzyme.Cartilage piece and histiocyte after cleaning are inserted it in hollow cavity 2 of multi-pore carrier 10 of volume 0.02ml, and cover the loam cake 6 of multi-pore carrier, run off to prevent the cartilage piece.
(3) processing of matched group
Flow chart as shown in Figure 5, at first, matched group is to adopt identical training method, take from equally the cartilage of above-mentioned Thigh bone 12, and with same method cartilage is peeled off and shredded, cartilage is placed on culture dish 8 through shredding, and is soaked in soluble protein enzyme 5ml (1mg/ml phosphate buffered saline(PBS)) and the hyaluronidase (Hyaluronidase), in 37 ℃ incubators (figure does not show), left standstill 24 hours, all chondrocytes are dissociated out.Usually the cartilage of 1ml volume has 1.5 * 10 approximately
7Individual chondrocyte, with respect to multi-pore carrier 10 hollow cavities 2 volume 0.02ml, the chondrocyte amount that matched group will add is 3 * 10
5Individual chondrocyte.The cell that adds is after centrifuge is centrifugal, counted with the cell counting dish, cell behind the counting is added in multi-pore carrier 10 hollow cavities 2 (the cell solution volume that adds is 200 μ l) by the upper end, for preventing that cell from flowing out, cover again loam cake 6 and place culture dish 8, in 37 ℃ of incubators that contain saturated aqueous vapor, leave standstill the cell that made adding in 6 hours and attach.
(4) co-treatment of experimental group and matched group
After the histiocyte of experimental group and matched group is added into multi-pore carrier 10, will contains histiocytic carrier 10 and put into the rotary incubator (spinner flask) 7 that contains cell culture fluid, such as Fig. 4 and last step shown in Figure 5.Cell culture fluid is the DMEM culture fluid that contains the 10%wt hyclone, and the nutrient of each carrier is 3ml/ days, and according to the fresh culture fluid of regular displacement of the time of cultivating, culture environment is the incubator that contains 37 ℃ of 5% carbon dioxide.In different incubation times multi-pore carrier 10 is taken out from reactor after cultivating, washed with phosphate buffered saline(PBS) first, then be dipped in the phosphate buffered saline(PBS) that contains 4% formalin test piece is fixed, mode with the paraffin organization embedding is cut into slices, method with haematoxylin-Yihong dyes again, and observes the result that its cartilaginous tissue is grown up.
(5) experimental result
Cultivate result such as Fig. 6, shown in Figure 7 of fortnight with the method for experimental group, can find out that hole has newborn cartilaginous tissue 9 around the carrier hollow cavity, the directly cartilage piece on every side to the periphery hole growth of kenel from implanting to organize, as shown in Figure 6, the hole at other positions of carrier also can be observed by the center and overgrows with cell to external diffusion, as shown in Figure 7.
Fig. 8, Fig. 9 is for cultivating the experimental result of four stars after date, can find in the carrier except the hole of hollow cavity periphery, the hole at other positions also grows many neocartilage tissues 9, as shown in Figure 8, can be observed with higher multiplying power has many chondrocyte lacunas (lacunae) in the cartilaginous tissue, as shown in Figure 9, chondrocyte in its lacuna has begun to split into microscler one-tenth isogenous group, show that formed cartilage is a quite active neocartilage tissue, and around the cartilaginous tissue also around many flat chondroblasts (chondroblast), this cartilaginous tissue is grown up sustainable expansion.
Figure 10 and Figure 11 show the result who is cultivated with the matched group method, in cultivating four stars during the phase, overgrowed with cell in the concrete dynamic modulus hole of PLGA hollow cavity carrier, as shown in figure 10, observe and to find with higher multiplying power, newborn cell kenel or fusoid fibrocartilage cells do not form cartilaginous tissue as shown in figure 11.
Figure 12 is with in microscopic structure image software analytical calculation experimental group and the matched group, the shared area of formed cartilaginous tissue in the peripheral hole of multi-pore carrier.Result by Figure 12 can learn:
The cartilaginous tissue of being cultivated with method of the present invention can be filled about 26% hole area in four stars during the phase, compared to the result of matched group obvious difference is arranged, and showing can be at short notice in the external neocartilage that grows volume with cultural method of the present invention.
Effect of the present invention:
The cultural method of vitro tissue of the present invention, mainly be piece of tissue to be inserted one have in the cavity of multi-pore carrier of hollow cavity, make the hole growth of the multi-pore carrier that tissue encloses from inside to outside in the three-dimensional space mode, the tissue that directly makes new advances with the form hypertrophy of tissue.The method can minimal volumes tissue mass, within the shortest time, cultivate required tissue in external connecing.The breakthrough of this technology will make the vitro tissue cultivation utilize cultured cell to come formative tissue by the past, turn to enter directly again and will come cultured tissue with tissue, to providing a quicker and effective culture technique with the organizational project industry that forms future.
Claims (3)
1. the employed multi-pore carrier of the cultural method of an external osseous tissue, it is characterized in that: it comprises a main body, at least more than one the loam cake that is positioned at this body interior and has the hollow cavity of an opening and cover this hollow cavity opening;
Wherein, described osseous tissue is the cartilaginous tissue piece, and the particle diameter of this cartilaginous tissue piece is the aperture greater than the peripheral base material of carrier cavity, and the particle diameter of this piece of tissue is 400 to 800 μ m; The pore diameter range of this multi-pore carrier is 150 to 350 μ m; The material of this multi-pore carrier is PLGA, and its porosity is 90vol%, and hole is for being interconnected.
2. the employed multi-pore carrier of the cultural method of external osseous tissue according to claim 1 is characterized in that: this main body is cylindrical, cubic type or required arbitrary shape.
3. the employed multi-pore carrier of the cultural method of external osseous tissue according to claim 1, it is characterized in that: this loam cake is the mushy material identical with main body.
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| CNB011447389A CN100532543C (en) | 2001-12-24 | 2001-12-24 | Culture method of external tissue and its porous carrier |
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| CNB011447389A Division CN100532543C (en) | 2001-12-24 | 2001-12-24 | Culture method of external tissue and its porous carrier |
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| CN101491703B true CN101491703B (en) | 2013-02-20 |
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| CNB011447389A Expired - Lifetime CN100532543C (en) | 2001-12-24 | 2001-12-24 | Culture method of external tissue and its porous carrier |
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| CN102586106B (en) * | 2006-01-23 | 2014-02-05 | 杨炜 | Three-dimensional space cell culture system preparation method |
| JP5851520B2 (en) * | 2011-12-01 | 2016-02-03 | 富士ソフト株式会社 | Long-term preservation method of porous body with chondrocytes attached |
| CN105265353B (en) * | 2015-06-07 | 2018-07-10 | 肖义军 | There are pearl nucleus of the hydrogel layer containing external carbon dosage and preparation method thereof on a kind of surface |
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| CN1297042A (en) * | 1999-11-16 | 2001-05-30 | 韩国科学技术院 | Method for preparing biocompatible supporter, and supporter prepared thereby |
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| CN1297042A (en) * | 1999-11-16 | 2001-05-30 | 韩国科学技术院 | Method for preparing biocompatible supporter, and supporter prepared thereby |
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| CN100532543C (en) | 2009-08-26 |
| CN1428418A (en) | 2003-07-09 |
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