CN106890363A - A kind of preparation method for being engineered dental pulp - Google Patents
A kind of preparation method for being engineered dental pulp Download PDFInfo
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Abstract
The present invention relates to a kind of preparation method for being engineered dental pulp, rebuild for root of the tooth.The dental pulp is made up of the extracellular matrix that pulp cells and its secretion synthesize, i.e. first by pulp cells through amplification cultivation, after forming the pulp cells condensate of cladding, then multiple pulp cells condensates is carried out into stacking placement and moulding, formation engineering dental pulp.The dental pulp of the present invention, can form new Dentin Structure in vivo so that root of the tooth continues to develop.The dental pulp has the advantages that preparation is easy, plasticity is strong, cell density is suitable, Extracellular Matrix Content is abundant, short without timbering material, Time in Vitro.
Description
Technical field
The invention belongs to tissue engineering technique field, and in particular to a kind of engineering tooth rebuild for root of the tooth with regeneration
The preparation method of marrow.
Background technology
After tooth eruption, root of the tooth relies on the pulp cells living in root pipe and periapex, continues development and forms root of the tooth, about 3
Root of the tooth could complete development within~5 years.When root of the tooth in growth course because wound, dental caries or central cusp deformity etc. cause dental pulp sense
Dye, meronecrosis, can make tooth root stop development.The not full permanent teeth of root of the tooth, its root length and thickness are not enough,
Apical foramen of tooth is in open shape, and such tooth cannot bear normal masticatory force, and service life shortens, therefore, make permanent teeth after
Supervention is educated, apical closure, and periapical tissue is got well, with important clinical meaning.
The Apexification of current clinical practice is on the basis of infection control, to be protected using medicine or operation method
The dental pulp and periapex soft tissue of root tip remaining are deposited and induced, promotes root of the tooth to continue to develop or formed calcification barrier closing root
End.Its cell biology basis is using Dental Pulp Cells and the histiocytic propagation of tip of a root dental papilla and differentiation activity, hair
Educate to form root of the tooth periapical tissue.But clinically the curative effect of Apexification is depending in the permanent teeth of pulp necrosis
The living cells quantity of remaining, if periapex inflammation seriously causes these cells largely downright bad, periapical tissue can not rebuild,
Root development curtailment is in turn resulted in, root canal wall is thin, tip of a root end paramophia, therefore cause its clinical efficacy not good.
The research that application organizational project regenerates with the technology promotion organization of regenerative medicine at present causes more and more extensive weight
Depending on concern.In recent years, Gronthos etc. is successfully separated out dental pulp stem cell from people pulp tissue, applies soughing of the wind in the pines etc. from the breast that comes off
The deciduous teeth stem cell that comes off is isolated in tooth（Stem cell from human exfoliated deciduous teeth,
SHED）, the immature cynodontin end such as Sonoyama is separately cultured out tip of a root dental papilla stem cell（stem cells from
Apical papilla, SCAP）, using with high proliferation ability, height self-renewal capacity, Multidirectional Differentiation ability it is dry thin
Born of the same parents, it would be possible to construct the pulp tissue of engineering, so as to the continuation for realizing root of the tooth is developed.Therefore carried out using stem cell transplantation
The new strategy for turning into treatment permanent teeth periapical disease is rebuild in regeneration.Permanent teeth causes pulp necrosis disease due to wound,
Now children are in and replace tooth period, still there is deciduous teeth to be replaced in oral cavity, and deciduous teeth can provide richer for therapy-related disease
Rich source of human stem cell.
American scholar Huang GT et al. after Fibrin Glue and butt dental papilla stem cell combine to form cell mass,
Implantation root cap is interior and is implanted into immunodeficient mouse dorsal sc, and implant is taken out after 3~4 months, is clearly observed regeneration
Dentin layer, odontoblast layer and the pulp tissue containing blood vessel, but do not possess the morphosis of natural tooth root.At present
There is no and set up engineering dental pulp for root of the tooth reconstruction and the report of regeneration.
The content of the invention
In view of the shortcomings of the prior art, it is an object of the invention to：A kind of engineering rebuild for root of the tooth with regeneration is provided
Change dental pulp and preparation method thereof.
The preparation method of the engineering dental pulp of the present invention, is to use pulp cells, after amplification cultivation, forms the tooth of cladding
Myelocyte condensate；Multiple pulp cells condensate stackings, moulding rear formation are engineered dental pulp.
Engineering dental pulp prepared by the present invention has pulp cells and its extracellular matrix of secretion synthesis to constitute, with system
For simplicity, plasticity is strong, cell density is suitable, Extracellular Matrix Content is abundant, short etc. without timbering material, Time in Vitro
Advantage；New Dentin Structure can be formed in vivo so that root of the tooth continues to develop.Specific steps include：
Step one, pulp cells culture：The pulp tissue of deciduous teeth or permanent teeth is shredded, with the NTx enzyme of 0.1~0.5g/L and
The Dispase enzymic digestions of 0.1~0.4 g/L extremely organize loose for 0.5~2 hour, single cell suspension are obtained after sieving, after centrifugation
Abandoning supernatant, adds pulp cells nutrient solution re-suspended cell, is placed in 37 DEG C, 5%CO2Under the conditions of cultivate, the next day change liquid, carefully
Born of the same parents cultivate to 90 % and passed on by converging, and obtain second generation pulp cells.
The composition of the pulp cells nutrient solution is：Contain in commercial α-MEM nutrient solutions, 10%（v/v）Hyclone,
0.2~0.4mg/mL glutamine, 75~125ng/mL basic fibroblast growth factors.
Step 2, pulp cells screening：Taking the second generation pulp cells that step one is obtained, adjustment cell density is 50~
200/mL, in 37 DEG C, 5%CO2Under the conditions of cultivate 12 hours, the next day change liquid, be expanded to after 70~80 % converge i.e. after cell
Can pass on, the pulp cells after being screened.
It is prepared by step 3, pulp cells condensate：It is 1 × 10 by the pulp cells adjustment cell density after screening5~5 ×
106It is inoculated with after individual/mL, adds pulp cells condensate nutrient solution, changes within every 2~3 days liquid, 37 DEG C, 5%CO2Under the conditions of cultivate, even
Continuous culture 1~3 week, milky film sample material can occurs in culture dish bottom, form pulp cells condensate.
The composition of the pulp cells condensate nutrient solution is：Contain in commercial α-MEM nutrient solutions, 10%（v/v）Tire ox
Serum, 0.2~0.4mg/mL glutamine, 75~125ng/mL basic fibroblast growth factors, 1 × 10-5~5 × 10-4M Ostholes, 1 × 10-7~5 × 10-68- isopentene groups naringenin, 1 × 10 of M-6~5 × 10-5M anhydroicartins, 1
×10-5~8 × 10-4M licochalcone As, 50~100 μ g/mL vitamin Cs, 1 × 10-4~5 × 10-3M aspirin, 1 × 10-6~5 × 10-5M rapamycins and 1 × 10-7~5 × 10-5M resveratrols.
This step uses pulp cells condensate nutrient solution culture pulp cells, pulp cells is changed under normal condition
The contact inhibition of cell and the property of density contact.In pulp cells condensate nutrient solution, vitamin C is by anti-oxidant acceleration
Cell propagation, the synthesis secretion for promoting extracellular matrix, aspirin maintain Cell Telomerase Activity, rapamycin enhancing cell
Autophagy simultaneously maintains stem cell dryness, the suppression cell ageing of resveratrol active cell Sirt signals during cell fast breeding,
The traditional Chinese medicine monomer compounds such as Osthole, 8- isopentene groups naringenin, anhydroicartin, liquorice chalcone are by activation
Smad, p38, MAPK and Wnt signal path promote Proliferation of Dental Pulp Cell largely to synthesize, extracellular matrix secretion, promote collagen
The secretions such as albumen, laminin, fibronectin splicing variants and dense arrangement, form multiple layered structure, and are formed in vitro containing thin
The pulp cells condensate of extracellular matrix, it is close with natural pulp tissue from structure and composition.
The preparation of step 4, engineering dental pulp：It is streak by being rolled into after 1~5 pulp cells condensate overlap, reuse
Biogum is fixed, and is subsequently adding induction broth and is continued to cultivate 1~3 day, that is, obtain engineering dental pulp.
The composition of the induction broth is：Contain in commercial α-MEM nutrient solutions, 10%（v/v）Hyclone,
0.292mg/mL glutamine, 75~125ng/mL basic fibroblast growth factors, 5~10ng/mL conversion growth because
Son-β 1,1 × 10-8~1 × 10-58- isopentene groups naringenin, 1 × 10 of M-6~5 × 10-4M licochalcone As, 1 × 10-7~
1×10-5M icariin, 10~20mM β-phosphoglycerol sodium, 5~20nM dexamethasone and 50~100 μ g/mL vitamin Cs.
Multiple pulp cells condensates are carried out stacking placement by this step, and the shape by natural dental pulp is moulding, by induction
Culture, the ability for making it have into dentine after breaking up, can form dentine after implanting, continue to complete the development of root of the tooth.
And in the induction broth for using, transforminggrowthfactor-β1 is able to maintain that cell state and reconstruction in-vitro cell growth microenvironment dimension
Hold stem cell stabilization；8- isopentene groups naringenin, licochalcone A and icariin can activate respectively stem cell Smads,
BMPs, Wnt signal path, promote cell bone to differentiation；β-phosphoglycerol sodium, dexamethasone and vitamin C can promote cell
Bone promotes biomineralization to the deposition of differentiation and calcium microcosmic salt.
Engineering dental pulp prepared by the present invention, its advantage is embodied in：
（1）The dental pulp of the present invention, with the composition and structure similar with natural dental pulp, therefore is engineered energy after dental pulp implants
The normal function of dental pulp is enough played, odontoblast can be differentiated, promote root of the tooth to continue to develop, ultimately formed normal complete
Root form；
（2）The dental pulp preparation method of the present invention, uses the polymeric culture technique of pulp cells so that pulp cells is dashed forward in vitro
The limitation of broken cellar culture, forms multiple layered structure, and a large amount of extracellular matrix secretions, as autocrine natural scaffold group
Fill, build engineering three-dimensional tissue structures, it is ensured that the effect microenvironment of signal transduction and growth factor after implanting.So that system
Standby engineering dental pulp is close with natural pulp tissue from structure and composition, it is to avoid conventional stent material and local microenvironment
The not good shortcoming of compatibility.
（3）The dental pulp preparation method of the present invention, with preparing, easy, plasticity is strong, cell density is suitable, extracellular matrix
It is abundant, without synthetic material, it is to avoid the advantages of the loss cell caused during cell inoculation material.
Brief description of the drawings
Fig. 1 is to prepare the polymeric picture of pulp cells using the inventive method
Fig. 1-A are the microphotos of pulp cells in vitro culture, and Fig. 1-B are the polymeric substantially photos of pulp cells, and Fig. 1-C are
The polymeric HE stained photographs of pulp cells.
Fig. 1-A, Fig. 1-B, Fig. 1-C explanations：Pulp cells condensate prepared by the present invention is made up of pulp cells, with day
The structure and composition of right dental pulp.
Fig. 2 is the outward appearance photo of engineering pulp cells prepared by the present invention
It can be seen that：Engineering dental pulp can facilitate clinical practice.
Fig. 3 is the Histological results that engineering dental pulp prepared by the present invention is used for pig dental pulp transplantation treatment
Masson trichrome stains；RF is regenerated fiber tissue；RD is regeneration dentine tissue；Black arrow meaning is into dentine
Cell, showing the engineering dental pulp of implantation can in vivo promote root of the tooth to continue to develop, and form dentine.
Fig. 4 is the iconography testing result that engineering dental pulp prepared by the present invention is used for pig dental pulp transplantation treatment
Fig. 4-A are the iconography testing results before tooth treatment, and Fig. 4-B are to use trimestral image after engineering endodontic treatment
Learn testing result.It can be seen that using three months after engineering endodontic treatment, the root canal orifice at white arrow meaning started closure, root pipe
Length, illustrates that engineering dental pulp can effectively induce the tip of a root to shape.
Specific embodiment
In conjunction with the embodiment of experiment, the invention will be further described.
Following embodiment, during preparation, first by pulp cells through amplification cultivation, forms the pulp cells polymerization of cladding
Body, then multiple pulp cells condensates are carried out into stacking placement and moulding, formation engineering dental pulp.
One of embodiment 1, preparation method of engineering dental pulp
Its preparation process is as follows：
Step one, pulp cells culture：The pulp tissue of Deciduous Teeth of Minipigs is shredded, with the NTx enzyme and 0.1g/ of 0.2g/L
The Dispase enzymic digestions of L extremely organize loose for 1 hour, and being blown and beaten with suction pipe makes pulp tissue fully dispersed.Sieve to form list with screen cloth
Cell suspension, abandoning supernatant after centrifugation adds pulp cells nutrient solution and suspends cell again, is transferred to Tissue Culture Flask, 37
℃、5%CO2Cultivated in incubator, the next day change liquid, cell culture converges to 90 % can passage, acquisition second generation pulp cells.
The composition of the pulp cells nutrient solution A is：Contain 10% in commercial α-MEM nutrient solutions（v/v）Hyclone,
0.2mg/mL glutamine, 75ng/mL basic fibroblast growth factors.
Step 2, pulp cells screening：After by the pulp cells digestion of the second generation, cell is blown outstanding using nutrient solution, carefully
Born of the same parents' density is adjusted to 50~60/mL, in addition culture vessel, by 37 DEG C, 5%CO2After being cultivated 12 hours in incubator, every
Day changes liquid, is passed on by cell is expanded to after 70~75 % converge, the pulp cells after being screened.
It is prepared by step 3, pulp cells condensate：By the good pulp cells of the growth conditions after screening with 1 × 106Individual/
ML is inoculated in culture dish, and adds pulp cells condensate nutrient solution, changes within every 2 days liquid, 37 DEG C, 5%CO2Cultivated in incubator,
Continuous culture 3 weeks, milky film sample material can occurs in culture dish bottom, form pulp cells condensate.
The composition of the pulp cells condensate nutrient solution is：Contain in commercial α-MEM nutrient solutions, 10%（v/v）Tire ox
Serum, 0.3mg/mL glutamine, 75ng/mL basic fibroblast growth factors, 1 × 10-5M Ostholes, 5 × 10-7M
8- isopentene groups naringenin, 1 × 10-6M anhydroicartins, 5 × 10-4M licochalcone As, 50 μ g/mL vitamin Cs, 1 ×
10-3M aspirin, 1 × 10-5M rapamycins and 1 × 10-5M resveratrols.
The preparation of step 4, engineering dental pulp：Pulp cells condensate nutrient solution is discarded, by pulp cells condensate along training
Support ware edge separation, using apparatus will 3 pulp cells condensates overlap after be rolled into streak, reuse Fibrin Glue parcel
It is fixed, be subsequently adding induction broth and continue to cultivate 3 days, that is, obtain engineering dental pulp.
The composition of the induction broth is：Contain in commercial α-MEM nutrient solutions, 10%（v/v）Hyclone, 0.292
Mg/mL glutamine, 75ng/mL basic fibroblast growth factors, 10ng/mL transforming growth factor-beta 1s, 1 × 10-5M's
8- isopentene groups naringenin, 5 × 10-4M licochalcone As, 1 × 10-6M icariin, the β-phosphoglycerol sodium of 10mM, 10nM
Dexamethasone, 50 μ g/mL vitamin Cs.
Engineering dental pulp prepared by this example is schemed as shown in figure 1, Fig. 1-A are the microphotos of pulp cells in vitro culture
1-B is the substantially photo of pulp cells Cell-aggregates, and Fig. 1-C are the polymeric HE stained photographs of pulp cells, it is seen that dental pulp
Cell-aggregates are made up of pulp cells, and structure and composition with natural dental pulp, are implanted into miniature pig Permanent incisors tooth
Marrow, top closing calcium hydroxide and glass ion, temporarily envelope observation.It is preoperative that root length, width are detected by Imaging Method,
The tip of a root mouthful width.Measure same index after materials within 6 months after the implantation, as a result show apical closure, root of the tooth after treating 6 months
Extension, root tube chamber reduces.
The two of embodiment 2, the preparation method of engineering dental pulp
Step one, pulp cells culture：The pulp tissue of miniature pig permanent teeth is shredded, with the NTx enzyme and 0.3g/ of 0.5g/L
The Dispase enzymic digestions of L extremely organize loose for 0.5 hour, and being blown and beaten with suction pipe makes pulp tissue fully dispersed.With screen cloth sieving shape
Into single cell suspension, abandoning supernatant after centrifugation adds special culture solution and suspends cell again, is transferred to Tissue Culture Flask, 37
℃、5 %CO2Cultivated in incubator, the next day change liquid, cell culture converges to 90 % can passage, acquisition pulp cells.
The composition of the pulp cells nutrient solution is：Contain 10% in commercial α-MEM nutrient solutions（v/v）Hyclone,
0.3mg/mL glutamine, 100ng/mL basic fibroblast growth factors.
Step 2, pulp cells screening：After by the pulp cells digestion of the second generation, cell is blown outstanding using nutrient solution, carefully
Born of the same parents' density is adjusted to 100~150/mL, in addition culture vessel, by 37 DEG C, 5%CO2After being cultivated 12 hours in incubator,
Check cell attachment situation, mark unicellular adherent hole, the next day change liquid, after cell is expanded to can biography after 70~80 % converge
Generation, conventional pancreatin digestion, Amplification Culture.
It is prepared by step 3, pulp cells condensate：By the good pulp cells of the growth conditions after screening with 2 × 106Individual/
ML is inoculated in culture dish, and adds special culture solution, changes within every 2 days liquid, 37 DEG C, 5%CO2Cultivated in incubator, continuously cultivate 2
In week, milky film sample material can occur in culture dish bottom, form pulp cells condensate.
The composition of the pulp cells condensate nutrient solution is：Contain in commercial α-MEM nutrient solutions, 10% hyclone,
0.2mg/mL glutamine, 100ng/mL basic fibroblast growth factors, 5 × 10-4M Ostholes, 1 × 10-6The 8- of M
Isopentene group naringenin, 5 × 10-6 M anhydroicartins, 5 × 10-5M licochalcone As, 100 μ g/mL vitamin Cs, 5 × 10-4M aspirin, 5 × 10-6M rapamycins, 1 × 10-6M resveratrols.
The preparation of step 4, engineering dental pulp：Special culture solution is discarded, by pulp cells condensate along culture dish edge point
From, using apparatus will 5 pulp cells condensates overlap after be rolled into streak, reuse collagen glue and wrap, then
Add induction broth to continue to cultivate 3 days, that is, obtain engineering dental pulp.
The composition of the induction broth is：Contain in commercial α-MEM nutrient solutions, 10%（v/v）Hyclone,
0.292mg/mL glutamine, 125ng/mL basic fibroblast growth factors, 6ng/mL transforming growth factor-beta 1s, 1 ×
10-88- isopentene groups naringenin, 1 × 10 of M-5M licochalcone As, 1 × 10-7β-the phosphoglycerol of M icariin, 20 mM
Sodium, 15nM dexamethasone, 100 μ g/mL vitamin Cs.
Cell-aggregates prepared by this example have certain form and elasticity, and the engineering dental pulp of preparation was as shown in Fig. 2 should
Engineering pulp cells epimatrix is enriched, and plasticity is strong, and clinical practice is convenient and swift.
The three of embodiment 3, the preparation method of engineering dental pulp
Step one, pulp cells culture：The pulp tissue of Deciduous Teeth of Minipigs is shredded, with the NTx enzyme of 0.1 g/L and 0.4
The Dispase enzymic digestions of g/L extremely organize loose for 1.5 hours, and single cell suspension is obtained after sieving, abandoning supernatant after centrifugation, then
Pulp cells nutrient solution re-suspended cell is added, 37 DEG C, 5%CO is placed in2Under the conditions of cultivate, the next day change liquid, cell culture to 90 % converges
Closing can passage, acquisition second generation pulp cells.
The composition of the pulp cells nutrient solution is：Contain in commercial α-MEM nutrient solutions, 10%（v/v）Hyclone,
0.4mg/mL glutamine, 125ng/mL basic fibroblast growth factors.
Step 2, pulp cells screening：Taking the second generation pulp cells that step one is obtained, adjustment cell density is 150~
200/mL, in 37 DEG C, 5%CO2Under the conditions of cultivate 12 hours, the next day change liquid, be expanded to after 70~80 % converge i.e. after cell
Can pass on, the pulp cells after being screened.
It is prepared by step 3, pulp cells condensate：It is 1 × 10 by the pulp cells adjustment cell density after screening5Individual/mL
After be inoculated with, add pulp cells condensate nutrient solution, change within every 3 days liquid, 37 DEG C, 5%CO2Under the conditions of cultivate, continuous culture 3 weeks,
Milky film sample material can occur in culture dish bottom, form pulp cells condensate.
The composition of the pulp cells condensate nutrient solution is：Contain in commercial α-MEM nutrient solutions, 10%（v/v）Tire ox
Serum, 0.4mg/mL glutamine, 80ng/mL basic fibroblast growth factors, 5 × 10-5M Ostholes, 8 × 10-7M
8- isopentene groups naringenin, 1 × 10-5M anhydroicartins, 1 × 10-4M liquorice chalcones, 75 μ g/mL vitamin Cs, 5 ×
10-3M aspirin, 5 × 10-5M rapamycins and 5 × 10-5M resveratrols.
The preparation of step 4, engineering dental pulp：It is streak by being rolled into after 2 pulp cells condensates overlaps, reuse life
Thing glue is fixed, and is subsequently adding induction broth and is continued to cultivate 1 day, that is, obtain engineering dental pulp.
The composition of the induction broth is：Contain in commercial α-MEM nutrient solutions, 10%（v/v）Hyclone,
0.292mg/mL glutamine, 100ng/mL basic fibroblast growth factors, 5ng/mL transforming growth factor-beta 1s, 5 ×
10-88- isopentene groups naringenin, 5 × 10 of M-6M licochalcone As, 1 × 10-5β-the phosphoglycerol of M icariin, 15mM
Sodium, 5nM dexamethasone and 75 μ g/mL vitamin Cs.
Engineering dental pulp prepared by this example is implanted into pig Permanent incisors dental pulp, iconography testing result as shown in figure 4, planting
After entering three months, root canal orifice is gradually closed, and sono-explorer is elongated, is illustrated the engineering dental pulp of implantation and can be played the normal of dental pulp
Function, promotes root development in vivo.
The four of embodiment 4, the preparation method of engineering dental pulp
Step one, pulp cells culture：The pulp tissue of deciduous teeth or permanent teeth is shredded, with the NTx enzyme and 0.2g/ of 0.4g/L
The Dispase enzymic digestions of L extremely organize loose for 2 hours, and single cell suspension is obtained after sieving, and abandoning supernatant after centrifugation is added
Pulp cells nutrient solution re-suspended cell, is placed in 37 DEG C, 5 %CO2Under the conditions of cultivate, the next day change liquid, cell culture to 90% converges i.e.
Can pass on, obtain second generation pulp cells.
The composition of the pulp cells nutrient solution is：Contain in commercial α-MEM nutrient solutions, 10%（v/v）Hyclone,
0.25mg/mL glutamine, 90ng/mL basic fibroblast growth factors.
Step 2, pulp cells screening：Taking the second generation pulp cells that step one is obtained, adjustment cell density is 150~
180/mL, in 37 DEG C, 5%CO2Under the conditions of cultivate 12 hours, the next day change liquid, be expanded to after 70~80 % converge i.e. after cell
Can pass on, the pulp cells after being screened.
It is prepared by step 3, pulp cells condensate：It is 5 × 10 by the pulp cells adjustment cell density after screening5Individual/mL
After be inoculated with, add pulp cells condensate nutrient solution, change within every 2 days liquid, 37 DEG C, 5%CO2Under the conditions of cultivate, continuous culture 2 weeks,
Milky film sample material can occur in culture dish bottom, form pulp cells condensate.
The composition of the pulp cells condensate nutrient solution is：Contain in commercial α-MEM nutrient solutions, 10%（v/v）Tire ox
Serum, 0.25mg/mL glutamine, 90ng/mL basic fibroblast growth factors, 1 × 10-4M Ostholes, 5 × 10- 68- isopentene groups naringenin, 5 × 10 of M-5M anhydroicartins, 8 × 10-4M licochalcone As, 80 μ g/mL vitamin Cs, 9
×10-4M aspirin, 8 × 10-6M rapamycins and 1 × 10-7M resveratrols.
The preparation of step 4, engineering dental pulp：It is streak by being rolled into after 1 pulp cells condensate overlap, reuse life
Thing glue is fixed, and is subsequently adding induction broth and is continued to cultivate 3 days, that is, obtain engineering dental pulp.
The composition of the induction broth is：Contain in commercial α-MEM nutrient solutions, 10%（v/v）Hyclone, 0.292
Mg/mL glutamine, 95ng/mL basic fibroblast growth factors, 8ng/mL transforming growth factor-beta 1s, 2 × 10-7The 8- of M
Isopentene group naringenin, 1 × 10-6M licochalcone As, 5 × 10-6M icariin, the β-phosphoglycerol sodium of 12mM, 20nM ground
Sai meter Song and 90 μ g/mL vitamin Cs.
Engineering dental pulp prepared by this example is carried out into histological observation, it is seen that root inside pipe wall attached to one layer and new be formed
Dentin layer, a large amount of blood vessels in the odontoblast layer on its surface, and root pipe.Illustrate the engineering dental pulp for preparing from structure
And close to natural pulp tissue on composition.
The five of embodiment 5, the preparation method of engineering dental pulp
Step one, pulp cells culture：The pulp tissue of deciduous teeth or permanent teeth is shredded, with the NTx enzyme of 0.3g/L and
The Dispase enzymic digestions of 0.15g/L extremely organize loose for 1 hour, and single cell suspension is obtained after sieving, abandoning supernatant after centrifugation,
Pulp cells nutrient solution re-suspended cell is added, 37 DEG C, 5%CO is placed in2Under the conditions of cultivate, the next day change liquid, cell culture to 90%
Passed on by converging, obtain second generation pulp cells.
The composition of the pulp cells nutrient solution is：Contain in commercial α-MEM nutrient solutions, 10%（v/v）Hyclone,
0.35mg/mL glutamine, 80ng/mL basic fibroblast growth factors.
Step 2, pulp cells screening：Taking the second generation pulp cells that step one is obtained, adjustment cell density is 90~
100/mL, in 37 DEG C, 5%CO2Under the conditions of cultivate 12 hours, the next day change liquid, be expanded to after 70~80 % converge i.e. after cell
Can pass on, the pulp cells after being screened.
It is prepared by step 3, pulp cells condensate：It is 5 × 10 by the pulp cells adjustment cell density after screening6Individual/mL
After be inoculated with, add pulp cells condensate nutrient solution, change within every 2 days liquid, 37 DEG C, 5%CO2Under the conditions of cultivate, continuous culture 1 week,
Milky film sample material can occur in culture dish bottom, form pulp cells condensate.
The composition of the pulp cells condensate nutrient solution is：Contain in commercial α-MEM nutrient solutions, 10%（v/v）Tire ox
Serum, 0.35mg/mL glutamine, 115ng/mL basic fibroblast growth factors, 2 × 10-5M Ostholes, 1 × 10-78- isopentene groups naringenin, 2 × 10 of M-6M anhydroicartins, 1 × 10-5M licochalcone As, 60 μ g/mL vitamin Cs, 1
×10-4M aspirin, 1 × 10-6M rapamycins and 5 × 10-6M resveratrols.
The preparation of step 4, engineering dental pulp：It is streak by being rolled into after 4 pulp cells condensates overlaps, reuse life
Thing glue is fixed, and is subsequently adding induction broth and is continued to cultivate 2 days, that is, obtain engineering dental pulp.
The composition of the induction broth is：Contain in commercial α-MEM nutrient solutions, 10%（v/v）Hyclone,
0.292mg/mL glutamine, 110ng/mL basic fibroblast growth factors, 9ng/mL transforming growth factor-beta 1s, 5 ×
10-68- isopentene groups naringenin, 2 × 10 of M-4M licochalcone As, 2 × 10-6β-the phosphoglycerol of M icariin, 18mM
Sodium, 8nM dexamethasone and 65 μ g/mL vitamin Cs.
Engineering dental pulp prepared by this example is carried out into histological observation, it is seen that root inside pipe wall attached to one layer and new be formed
Dentin layer, a large amount of blood vessels in the odontoblast layer on its surface, and root pipe.Illustrate the engineering dental pulp for preparing from structure
And close to natural pulp tissue on composition.
Claims (4)
1. it is a kind of be engineered dental pulp preparation method, it is characterised in that：The extracellular matrix synthesized by pulp cells and its secretion
Constitute, i.e., first by pulp cells is through amplification cultivation and forms the pulp cells condensate of cladding, then multiple pulp cells are gathered
Zoarium carries out stacking placement and moulding, formation engineering dental pulp.
2. the preparation method of dental pulp is engineered according to claim 1, it is characterised in that the pulp cells amplification cultivation step
Rapid and method is：
1）The pulp tissue of deciduous teeth or permanent teeth is shredded, with the NTx enzyme of 0.1~0.5 g/L and 0.1~0.4 g/L
Dispase enzymic digestions obtain single cell suspension in 0.5~2 hour to organizing loosely after sieving, abandoning supernatant after centrifugation, then add
Enter pulp cells nutrient solution re-suspended cell, be placed in 37 DEG C, 5 %CO2Under the conditions of cultivate, the next day change liquid, cell culture to 90 % converges
Closing can passage, acquisition second generation pulp cells；
The composition of the pulp cells nutrient solution is：Contain 10% in commercial α-MEM nutrient solutions（v/v）Hyclone, 0.2~
0.4 mg/mL glutamine, 75~125ng/mL basic fibroblast growth factors；
2）Pulp cells is screened：The second generation pulp cells that step one is obtained is taken, adjustment cell density is 50~200/mL,
37 DEG C, 5%CO2Under the conditions of cultivate 12 hours, the next day change liquid, after cell be expanded to 70~80% converge after can passage, sieved
Pulp cells after choosing.
3. the preparation method of dental pulp is engineered according to claim 1, it is characterised in that the pulp cells polymerization system
It is standby：It is 1 × 10 by the pulp cells adjustment cell density after screening5~5 × 106It is inoculated with after individual/mL, adds pulp cells polymerization
Body nutrient solution, changes liquid, 37 DEG C, 5%CO for every 2~3 days2Under the conditions of cultivate, continuously cultivate 1~3 week, occur in culture dish bottom newborn
White films sample material, forms pulp cells condensate；
The composition of the pulp cells condensate nutrient solution is：Contain 10% in commercial α-MEM nutrient solutions（v/v）Hyclone,
0.2~0.4 mg/mL glutamine, 75~125ng/mL basic fibroblast growth factors, 1 × 10-5~5 × 10-4M snakes
Machine tool element, 1 × 10-7~5 × 10-68- isopentene groups naringenin, 1 × 10 of M-6~5 × 10-5M anhydroicartins, 1 × 10-5
~5 × 10-4M liquorice chalcones, 50~100 μ g/mL vitamin Cs, 1 × 10-4~5 × 10-3M aspirin, 1 × 10-6~5 ×
10-5M rapamycins and 1 × 10-7~5 × 10-5M resveratrols.
4. the preparation method of dental pulp is engineered according to claim 1, it is characterised in that the preparation of the engineering dental pulp：
It is streak by being rolled into after 1~5 pulp cells condensate overlap, reuse biogum and fix, the shape by natural dental pulp is moulding,
It is subsequently adding induction broth to continue to cultivate 1~3 day, that is, obtains engineering dental pulp；
The composition of the induction broth is：Contain in commercial α-MEM nutrient solutions, 10%（v/v）Hyclone, 0.292 mg/
ML glutamine, 75~125ng/mL basic fibroblast growth factors, 5~10ng/mL transforming growth factor-beta 1s, 1 ×
10-8~1 × 10-58- isopentene groups naringenin, 1 × 10 of M-6~5 × 10-4M licochalcone As, 1 × 10-7~1 × 10-5M is excessive
Sheep leaves of pulse plants glycosides, the β-phosphoglycerol sodium of 10~20mM, 5~20nM dexamethasone and 50~100 μ g/mL vitamin Cs.
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