CN103966158A - Preparation method of specific extracellular matrix ECM of periodontium and application thereof - Google Patents
Preparation method of specific extracellular matrix ECM of periodontium and application thereof Download PDFInfo
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Abstract
The invention relates to a preparation method of a specific extracellular matrix ECM of periodontium. The preparation method comprises the following steps: 1) separating and culturing a human periodontal ligament cell; and 2) preparing the specific extracellular matrix ECM of periodontium; applying the specific extracellular matrix ECM of periodontium and applying the specific extracellular matrix ECM of periodontium as a human periodontal ligament stem cell in in-vitro amplification; and promoting directional differentiation of the ECM. The preparation method is simple and can remarkably promote in-vitro amplification and directional differentiation of the periodontal ligament stem cell.
Description
Technical field
The invention belongs to biological technical field, relate to the preparation method of the extracellular matrix in a kind of periodontal tissue source, with and in the application of the aspects such as periodontal ligament stem cell cultivation, amplification.
Background technology
Periodontitis is one of ancient, the most general disease of the mankind.It is the chronic infectious disease by the caused periodontal support tissue of the microorganism in plaque, can cause inflammation, periodontal pocket formation, carrying out property attachment loss and the frontal resorption of periodontal support tissue, finally causes odontoseisis and is pulled out.According to statistics, the ill rate of periodontitis and seriousness increase with the age and increase, and after 35 years old, morbidity obviously increases, and 50 years old to 60 years old time, peaks.At present, periodontitis has exceeded 80% in the morbidity of China, is the first reason that China grownup loses tooth.Meanwhile, for example important risk factor of diabetes, cardiovascular disorder of periodontitis or whole body systemic disease.
At present, comprise following four-stage for the treatment procedure of periodontitis: the first stage, Primary Care, uses the ordinary methods such as scaling, interlock adjustment, pharmacological agent to eliminate paathogenic factor, controls inflammation; Subordinate phase, periodontal surgery treatment, removes infected tissue by flap operation, bone grafting operation, guided tissue regeneration etc., promotes periodontal regenerative; Phase III, repair and/or orthodontic treatment, fixing agomphiasis in repairing agomphosis, sets up stable interlock; Finally, periodontal support treatment, so that curative effect is kept for a long time.But all there is limitation separately in existing methods for the treatment of, the periodontal regenerative that still can not realize ideal and recover periodontal tissue structure completely.
In recent years, the fast development of stem-cell research and regenerative medicine has brought dawn for treating mankind's difficult diseases.Seo etc. isolate a kind of adult stem cell first from people's periodontium tissue, called after periodontal ligament stem cell (periodontal ligament stem cells, PDLSCs) (Seo BM, Miura M, Gronthos S, et al. Investigation of multipotent postnatal stem cells from human periodontal ligament. Lancet, 2004,364:149-55.).Periodontal ligament stem cell is considered to possess the potential to cementoblast, inoblast and osteoblast differentiation, can form respectively dental cement, periodontal ligament and alveolar bone, recover complexity and meticulous " sandwich sample " periodontal tissue structure, for patients with periodontitis brings glad tidings.But adult stem cell comprises that periodontal ligament stem cell in vitro after repeatedly going down to posterity, can lose the characteristic of its stem cell gradually, Spontaneous Differentiation or aging occurs, and cannot meet the demand of tissue regeneration and reconstruction.Therefore, how to amplify in a large number in vitro high-quality periodontal ligament stem cell? how accurately to control the directed differentiation of periodontal ligament stem cell? these are all key issues urgently to be resolved hurrily during realization successfully transforms from laboratory to clinical application.
Extracellular matrix (extracellular matrix, ECM) refers to the network structure general name that is arranged in the organism inner cell external space, is made up of protein and the polysaccharide of emiocytosis.It or between cell and cell, form iuntercellular cement substance with the form filling of extracellular matrix, or be positioned at reticular tissue form its important component part, or be positioned at epithelium and reticular tissue junction region with basilar membrane form, adhering to, breeding and breaking up and can produce material impact for cell.Meanwhile, the physicals of matrix also can affect the state of cell.The researchs such as Engler find that the Young's modulus of matrix can instruct mescenchymal stem cell generation directed differentiation, the soft matrix that is for example similar to cerebral tissue can induced dry-cell differentiating into nerve cells, the matrix that is similar to muscle tissue can be broken up to myocyte by induced dry-cell, the hard matrix that is similar to osseous tissue induced dry-cell to osteoblast differentiation (Engler AJ, Sen S, Sweeney HL, et al. Matrix elasticity directs stem cell lineage specification. Cell, 2006,126:677-89).Chen etc. taken the lead in taking the lead in the world proposing " ECM in medullary cell (Bone marrow cells, BMCs) source can analogue body in microenvironment, keep the stem cell characteristic of mescenchymal stem cell " hypothesis, and carried out effective checking.They cultivate BMCs on common culture plate, make BMCs secrete a large amount of ECM, after effectively de-cell is processed, and the remaining membranaceous ECM with complete structure and protein ingredient.First they studied the ECM in mouse BMCs source, compared with cultivating with common Plastic, this ECM can promote mouse BMMSCs propagation, the bone that simultaneously keeps cell to, fat is to differentiation capability, in body, transplantation experiments result is presented at ECM bone amount and marrow amount that above the mouse BMMSCs of cultivation generates has increased by 5 times and 8 times of (Chen XD than Plastic respectively, Dusevich V, Feng JQ, et al. Extracellular matrix made by bone marrow cells facilitates expansion of marrow-derived mesenchymal progenitor cells and prevents their differentiation into osteoblasts. J Bone Miner Res, 2007, 22:1943-56).They have further studied the effect of people BMCs source ECM for hBMMSCs, find that ECM can promote cell proliferation, keep stem cell property, reduce the generation that produces oxyradical simultaneously, improve telomerase activation, strengthen the osteogenic response to BMP2; In body, experimental result shows, the hBMMSCs that amplification is gone down to posterity on ECM can keep good bone formation performance, (the Lai Y and the osteogenic ability of common cultured cells falls sharply conventionally after 6 ~ 7 generations, Sun Y, Skinner CM, et al. Reconstitution of marrow-derived extracellular matrix ex vivo:a robust culture system for expanding large-scale highly functional human mesenchymal stem cells. Stem Cells Dev, 2010,19:1095-107).Why ECM can bring into play its mechanism of such effect may 2 points: first, the Endogenous Growth Factors of temporarily " binding " emiocytosis of ECM is as BMP etc., play slow release effect, thereby make cell obtain sufficient generation time, and exogenous somatomedin keeps the susceptibility of height; The second, scanning electron microscope shows that the structure of ECM is as " track " of ordered arrangement, and cell thereon can be along " track " migration, and just few generation " collision ", has reduced contact inhibition, thereby promoted cell proliferation, has reduced the probability of differentiation.
According to above research, we infer " extracellular matrix in periodontium source can provide suitable growing environment for periodontal ligament stem cell, promotes its propagation and directed differentiation ".We utilize periodontal ligament cell successfully to construct periodontal tissue specificity ECM, and find this ECM periodontal ligament stem cell that can efficiently increase in vitro, and maintain its higher dryness.Up to now, do not see about the preparation of periodontal tissue specificity ECM and the report on periodontal ligament stem cell impact thereof.
Summary of the invention
In order to overcome above-mentioned the deficiencies in the prior art, the object of the present invention is to provide a kind of preparation method and its usage of periodontal tissue specific cell epimatrix, and utilize this tissue specificity ECM high quality amplification periodontal ligament stem cell promote its directed differentiation in vitro, have preparation method simple, can significantly promote the in-vitro multiplication of periodontal ligament stem cell and the feature of directed differentiation.
To achieve these goals, the technical solution used in the present invention is: a kind of preparation method of periodontal tissue specific cell epimatrix, comprises the following steps:
1) separation of people's periodontal ligament cell and cultivation, specific practice is:
Collect fresh permanent bicuspid and the third molar that within 12～29 years old, need to pull out because of correction clinically, periodontal health, bad without dental caries, without tip of a root inflammation, in Bechtop with 75% alcohol disinfecting corona, PBS liquid rinsing 3～5 times, 1/3 periodontium in scraping root, move into centrifuge tube, add type i collagen enzyme and the 2.4units/mlDispase light shaking of 625units/ml, digestion 40min in 37 DEG C of incubators, every 5min vibration 1 time, centrifugal rotational speed 1200r/min, centrifugal 5min, abandoning supernatant; Add the α-MEM nutrient solution of 0.5～1mL containing 15% foetal calf serum, move into 25mL culturing bottle diapire, evenly inoculation, inserts 37 DEG C, 5%CO culturing bottle
2in the incubator of saturated humidity, hatch 24h, after cell attachment, add gently nutrient solution to 3mL; After 3d, change nutrient solution and discard not attached cell, change liquid 1 time later every 2～3d, the culture supernatant of the periodontal ligament cell of collection logarithmic phase is for subsequent use, and Growth of Cells reaches when 8O%～90% converges uses 0.25% tryptic digestion, goes down to posterity with 1:2 ratio;
2) preparation of periodontal tissue specificity ECM, specific practice is:
By P3 for people's periodontal ligament cell hPDLCs with 2x10
4the density of cells/ml is inoculated in 6 orifice plates, cultivates 15 days, and every 3-4 days changes liquid once, within last 8 days, changes film induced liquid into, PBS liquid rinses 2 times, adds de-cell treatment solution, reacts 5 minutes under room temperature, and PBS liquid rinses 3 times, obtain hPDLCs-ECM, be attached to 6 orifice plates, 4 ° of C cryopreservation.
Described de-cell treatment solution contains 0.5%TritonX-100 and 20mM NH
4the PBS liquid of OH.
Described PBS liquid is containing 100U/ml penicillin, 100 μ g/ml Streptomycin sulphates and amphotericin 0.25 μ g/ml.
Described α-MEM nutrient solution is containing 15% foetal calf serum, 2mmol/L mono-glutamine, 100U/ml penicillin and 100 μ g/ml Streptomycin sulphates.
Described film induced liquid contains xitix 50 μ M.
An application for periodontal tissue specific cell epimatrix, is characterized in that, periodontal tissue specificity ECM is as the application of amplification in vitro Periodontal ligament stem cell, and promotes its directed differentiation.
Compared with prior art, the present invention has following useful technique effect:
1) the prepared periodontal tissue specificity ECM of the present invention can altitude simulation periodontal tissue microenvironment, significantly promotes in-vitro multiplication and the directed differentiation of periodontal ligament stem cell, and without adding somatomedin or cell is carried out to genetic modification in nutrient solution.
2) the prepared periodontal tissue specificity ECM of the present invention has set up an external model simple, that effectively periodontal ligament stem cell is grown, contribute to study the impact of microenvironment on stem cell, be also conducive to the conversion to clinical application by periodontal ligament stem cell research from now on simultaneously.
Brief description of the drawings
Fig. 1 is periodontal tissue specificity ECM(hPDLCs-ECM of the present invention) result under the microscope of de-cell processing front and back; Wherein, Figure 1A is before de-cell is processed, 4 ×; Figure 1B is before de-cell is processed, 10 ×; Fig. 1 C is after de-cell is processed, 4 ×; Fig. 1 D is after de-cell is processed, 10 ×.
Fig. 2 is periodontal tissue specificity ECM(hPDLCs-ECM of the present invention) result under the scanning electron microscope of de-cell processing front and back; Wherein, Fig. 2 A is de-cell figure before treatment; Fig. 2 B is de-cell figure after treatment.
Fig. 3 is periodontal tissue specificity ECM(hPDLCs-ECM of the present invention) result under the transmission electron microscope of de-cell processing front and back; Wherein, Fig. 3 A is for taking off before cell processing; Fig. 3 B is for taking off after cell processing.
Fig. 4 is periodontal tissue specificity ECM(hPDLCs-ECM of the present invention) process the type i collagen immunofluorescence dyeing of front and back through de-cell; Wherein, Fig. 4 A is for taking off before cell processing; Fig. 4 B is for taking off after cell processing.
Fig. 5 is periodontal tissue specificity ECM(hPDLCs-ECM of the present invention) process the Biglycan immunofluorescence dyeing of front and back through de-cell; Wherein, Fig. 5 A is for taking off before cell processing; Fig. 5 B is for taking off after cell processing.
Fig. 6 is periodontal tissue specificity ECM(hPDLCs-ECM of the present invention) process the Decorin immunofluorescence dyeing of front and back through de-cell; Wherein, Fig. 6 A is for taking off before cell processing; Fig. 6 B is for taking off after cell processing.
Fig. 7 is periodontal tissue specificity ECM(hPDLCs-ECM) process the Perlecan immunofluorescence dyeing of front and back through de-cell; Wherein, Fig. 7 A is for taking off before cell processing; Fig. 7 B is for taking off after cell processing.
Fig. 8 is the Fibronectin immunofluorescence dyeing of periodontal tissue specificity ECMhPDLCs-ECM of the present invention before and after de-cell is processed; Wherein, Fig. 8 A is de-cell figure before treatment; Fig. 8 B is de-cell figure after treatment.
Fig. 9 is the microscopic examination figure of inventor's periodontal ligament stem cell hPDLSCs upgrowth situation on tissue specificity ECM.
Figure 10 is the growth curve chart after inventor's periodontal ligament stem cell hPDLSCs cultivates on tissue specificity ECM.
Figure 11 is that the clone of inventor's periodontal ligament stem cell hPDLSCs on tissue specificity ECM forms detection and skeletonization becomes fat differentiation, gross examination of skeletal muscle figure.
Figure 12 is that the clone of inventor's periodontal ligament stem cell hPDLSCs on tissue specificity ECM forms detection and skeletonization becomes fat differentiation, microscopic examination figure.
Embodiment
Below in conjunction with specific embodiments and the drawings, the present invention is described in further detail, the explanation of the invention is not limited.
The invention provides a kind of simple, periodontal tissue specificity ECM(hPDLCs-ECM efficiently) preparation method, utilize can increase in a large number in vitro Periodontal ligament stem cell hPDLSCs promote its directed differentiation of prepared ECM.
Taking the preparation method of people's periodontium tissue specificity ECM as example, concrete steps comprise the former culture of people's periodontal ligament cell, the preparation and determination methods of cultivation, ECM that goes down to posterity, and apply this ECM amplification in vitro Periodontal ligament stem cell hPDLSCs.
1) separation of people's periodontal ligament cell hPDLCs and cultivation, specific practice is:
Collect fresh permanent bicuspid and the third molar that within 12～29 years old, need to pull out because of correction clinically; periodontal health, bad without dental caries, without tip of a root inflammation; in Bechtop with PBS liquid rinsing after 75% alcohol disinfecting corona 3～5 times; 1/3 periodontium in scraping root; move into centrifuge tube; add type i collagen enzyme and the 2.4 units/ml Dispase light shaking of 625units/ml; digestion 40 min in 37 DEG C of incubators; every 5min vibration 1 time; centrifuge speed 1200r/min; centrifugal 5min, abandoning supernatant.Add the α-MEM nutrient solution of 0.5～1mL containing 15% foetal calf serum, move into 25mL culturing bottle diapire, evenly inoculation. culturing bottle is put to 37 DEG C of people, 5%CO
2in the incubator of saturated humidity, hatch 24 h, after cell attachment, add gently nutrient solution to 3mL.After 3d, change nutrient solution and discard not attached cell, change liquid 1 time later every 2～3d, the culture supernatant of the periodontal ligament cell of collection logarithmic phase is for subsequent use, and Growth of Cells reaches when 8O%～90% converges uses 0.25% tryptic digestion, goes down to posterity with 1:2 ratio.
2) preparation of periodontal tissue specificity ECM
By P3 for hPDLCs with 2x 10
4the density of cells/ml is inoculated in 6 orifice plates, cultivates 15 days, and every 3-4 days changes liquid, within last 8 days, changes film induced liquid into, PBS liquid rinses 2 times, adds de-cell treatment solution, reacts 5 minutes under room temperature, and PBS liquid rinses 3 times, obtain hPDLCs-ECM, it is attached to 6 orifice plates, 4 ° of C cryopreservation.
Described de-cell treatment solution contains 0.5%TritonX-100 and 20mM NH
4the PBS liquid of OH.
Described PBS liquid is containing 100U/ml penicillin, 100 μ g/ml Streptomycin sulphates and amphotericin 0.25 μ g/ml.
Described α-MEM nutrient solution is containing 15% foetal calf serum, 2mmol/L mono-glutamine, 100U/ml penicillin and 100 μ g/ml Streptomycin sulphates.
Described film induced liquid contains xitix 50 μ M.
the structure observation of periodontal tissue specificity ECM
Ordinary optical microscope is observed: before and after Cell extraction, under inverted microscope, directly observe hPDLCs-ECM configuration of surface, as shown in Figure 1.
Scanning electron microscope (SEM) morphological observation hPDLCs-ECM: by PBS washing for hPDLCs-ECM three times, fixes after 1 hour with the 0.1M sodium cacodylate buffer liquid (pH value 7.2) containing 2% glutaraldehyde, in the 0.1M cacodylate buffer of transferring to.Sample dewaters (from 70% to 100% ethanol) through gradient concentration, metal spraying processing, and sample uses field emission scanning electron microscope (S-4800, Hitachi, Japan) to observe hBMCs-ECM ultrastructure.As shown in Figure 2.
Transmission electron microscope ultrastructure observation of experimental: with the fixing hPDLCs-ECM of 2% glutaraldehyde PBS stationary liquid and hPDLCs 2 h, collecting precipitation thing, through rinsing, fix, replace, soak into, embedding, section, with lead citrate electron stain, JEM mono-1400 transmission electron microscopes (Philips, Holland) observation of cell ultrastructure.As shown in Figure 3.
The immunofluorescence dyeing of periodontal tissue specificity ECM
P3 is rinsed 2 times with PBS for hPDLCs and hPDLCs-ECM cell climbing sheet, 4% paraformaldehyde is fixed 20 minutes on ice, with PBS rinsing 3 times, hPDLCs cell climbing sheet adds 0.5%Triton-100,1% BSA37 DEG C of sealing 20min, 37 DEG C of sealing 20min of hPDLCs-ECM cell climbing sheet 1% BSA, add COL I, Biglycan, Decorin, Perlecan, Fibronectin primary antibodie (1:500), 4 DEG C are spent the night, and after next day rewarming, PBS washes 3 times.Under lucifuge condition, add respectively the goat antirabbit of FITC mark and goat anti-mouse two anti-(1:200), hatch 1 hour for 37 DEG C, the DAPI 5min that dye, PBS washes 3 times, mounting, fluorescence microscope, experimentation lucifuge operates.Negative control group replaces primary antibodie with PBS.As shown in Fig. 4 to Fig. 8.
The separation and Culture of Periodontal ligament stem cell hPDLSCs
limiting dilution assay clone culture purified periodontal ligament stem cell: the culture supernatant of the logarithmic phase periodontal ligament cell of above-mentioned collection is pressed to centrifugal 10 min of 1500 r/min, after the filter of 0.22 μ m diameter filters, mix as adaptability substratum using 1:1 ratio with substratum. the 1st generation cell in the vegetative period of taking the logarithm adaptability substratum doubling dilution, adjust cell density to 1O～15/mL, piping and druming mixes, be inoculated in 96 well culture plates, lO0 μ l/ hole, cultivate the adherent hole of mark individual cells after 12 h, and fluid infusion to 200 μ l/ hole, after 5 days, change liquid, light Microscopic observation clone formational situation.Cellar culture 7～14 d, to occurring cell clone (cell count >=5O is criterion), at the bottom of clone grows to hole, trysinization after 1/3～1/2, is transferred to 24 orifice plate enlarged culturing.
Periodontal tissue specificity ECM is for the effect of hPDLSCs propagation
By P3 for hPDLSCs with 2x 10
4the density of cells/ml is inoculated in respectively common culture plate, hPDLCs-ECM culture plate and hBMCs-ECM culture plate, at inoculation latter 4 hours, 3 days and 7 days difference observation of cell growing states.As shown in Figure 9, the adherent and growth vigor of hPDLSCs on hPDLCs-ECM culture plate is obvious.
Get the P3 of growth logarithmic phase for hPDLSCs, be inoculated in common culture plate, hPDLCs-ECM culture plate and hBMCs-ECM culture plate, trysinization respectively afterwards in seven days.After cell counting, adjusting cell density is 2 × 10
3/ hole is inoculated in 96 orifice plates, and every hole adds liquid 100 μ L, inoculates altogether 48 holes, overnight incubation.Change nutrient solution next day, treat that gaging hole adds 100 μ L CCK8 liquid, measure 12 holes at every turn, incubator is cultivated 4 hours.After 4 hours enzyme-linked immunosorbent assay instrument 450 nm go out measure 12 hole light absorption values remove mean value.Replicate measurement next day, continuously measured 7 days, repeats 3 times.As shown in figure 10, hPDLSCs cultivates and breeds later obviously on hPDLCs-ECM culture plate.
Periodontal tissue specificity ECM is for the effect of hPDLSCs directed differentiation
P3 is inoculated in respectively to three groups of common six orifice plates for hPDLSCs lower concentration (100 cells/well),, hPDLCs-ECM six orifice plates and hBMCs-ECM six orifice plates, conventional α-MEM nutrient solution is (containing 15% foetal calf serum, 2 mmol/L mono-glutamines, 100 U/ml penicillin, 100 μ g/ml Streptomycin sulphates) cultivate, within every two days, change liquid, cultivate and after 14 days, use one group of six orifice plate to detect CFU-F clone, with PBS rinse, methyl alcohol is fixed, 0.1% violet staining 10min; Other two group of six orifice plate continues to cultivate, and (osteogenic induction liquid is the α-MEM nutrient solution containing 15%FBS, 10mmol/L β Sodium Glycerophosphate, 0.05mmol/L vitamins C and 100mmol/L dexamethasone with becoming fat induction to carry out respectively osteogenic induction; Becoming fat induced liquid is the α-MEM nutrient solution containing 15%FBS, 10 μ g/mL Regular Insulin, 1mmoL/L dexamethasone, 0. 5 mmoL/L isobutyl methylxanthine (IBMX), 200 μ moL/L indomethacins), after 20 days, carry out respectively Von kossa dyeing and oil red dyeing, detect CFU-OB and CFU-AD.As shown in Figure 11,12, clonality, the Osteoblast Differentiation of hPDLSCs on hPDLCs-ECM and become fat differentiation obviously to strengthen, wherein become that fat differentiation is better than hBMCs-ECM group, Osteoblast Differentiation is weaker than this group, has shown the specific effect of periodontal tissue ECM.
The invention discloses the preparation method of a kind of periodontal tissue specificity ECM, and apply this ECM periodontal ligament stem cell is carried out to amplification in vitro and directed differentiation.The prepared periodontal tissue specificity ECM of the present invention has set up an external model simple, that effectively periodontal ligament stem cell is grown, contribute to study the impact of microenvironment on stem cell, be also conducive to from now on periodontal ligament stem cell research be transformed to clinical application simultaneously.
Claims (6)
1. a preparation method for periodontal tissue specific cell epimatrix, is characterized in that, comprises the following steps:
1) separation of people's periodontal ligament cell and cultivation, specific practice is:
Collect fresh permanent bicuspid and the third molar that within 12～29 years old, need to pull out because of correction clinically, periodontal health, bad without dental caries, without tip of a root inflammation, in Bechtop with 75% alcohol disinfecting corona, PBS liquid rinsing 3～5 times, 1/3 periodontium in scraping root, move into centrifuge tube, add type i collagen enzyme and the 2.4units/mlDispase light shaking of 625units/ml, digestion 40min in 37 DEG C of incubators, every 5min vibration 1 time, centrifugal rotational speed 1200r/min, centrifugal 5min, abandoning supernatant; Add the α-MEM nutrient solution of 0.5～1mL containing 15% foetal calf serum, move into 25mL culturing bottle diapire, evenly inoculation, inserts 37 DEG C, 5%CO culturing bottle
2in the incubator of saturated humidity, hatch 24h, after cell attachment, add gently nutrient solution to 3mL; After 3d, change nutrient solution and discard not attached cell, change liquid 1 time later every 2～3d, the culture supernatant of the periodontal ligament cell of collection logarithmic phase is for subsequent use, and Growth of Cells reaches when 8O%～90% converges uses 0.25% tryptic digestion, goes down to posterity with 1:2 ratio;
2) preparation of periodontal tissue specificity ECM, specific practice is:
By P3 for people's periodontal ligament cell hPDLCs with 2x10
4the density of cells/ml is inoculated in 6 orifice plates, cultivates 15 days, and every 3-4 days changes liquid once, within last 8 days, changes film induced liquid into, PBS liquid rinses 2 times, adds de-cell treatment solution, reacts 5 minutes under room temperature, and PBS liquid rinses 3 times, obtain hPDLCs-ECM, be attached to 6 orifice plates, 4 ° of C cryopreservation.
2. the preparation method of a kind of periodontal tissue specific cell epimatrix according to claim 1, is characterized in that, described de-cell treatment solution contains 0.5%TritonX-100 and 20mM NH
4the PBS liquid of OH.
3. the preparation method of a kind of periodontal tissue specific cell epimatrix according to claim 1, is characterized in that, described PBS liquid is containing 100U/ml penicillin, 100 μ g/ml Streptomycin sulphates and amphotericin 0.25 μ g/ml.
4. the preparation method of a kind of periodontal tissue specific cell epimatrix according to claim 1, is characterized in that, described α-MEM nutrient solution is containing 15% foetal calf serum, 2mmol/L mono-glutamine, 100U/ml penicillin and 100 μ g/ml Streptomycin sulphates.
5. the preparation method of a kind of periodontal tissue specific cell epimatrix according to claim 1, is characterized in that, described film induced liquid contains xitix 50 μ M.
6. an application for periodontal tissue specific cell epimatrix, is characterized in that, periodontal tissue specificity ECM is as the application of amplification in vitro Periodontal ligament stem cell, and promotes its directed differentiation.
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