CN104225681A - Tissue-engineered bone and preparation method thereof - Google Patents
Tissue-engineered bone and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a tissue-engineered bone. The tissue-engineered bone is multilayer cell sheet lamination compound with a three-dimensional capillary network, the multilayer cell sheet lamination compound consists of n laminated bone marrow mesenchymal stem cell sheets and vascular endothelial cells among all bone marrow mesenchymal stem cell sheet layers, and n is in a range of 3-8. A preparation method of the tissue-engineered bone comprises steps as follows: firstly, a photosensitive semiconductor structural layer is prepared on the surface of a cell culture dish, so that a photosensitive cell culture dish is obtained; then bone marrow mesenchymal stem cells are cultured with the photosensitive cell culture dish, and the bone marrow mesenchymal stem cell sheets are obtained; finally, the multilayer cell sheet lamination compound consisting of multiple layers of the bone marrow mesenchymal stem cell sheets and the vascular endothelial cells is constructed and cultured to obtain the tissue-engineered bone. The tissue-engineered bone purely consists of homologous cells, so that pollution caused by immunological rejection and stent degradation is reduced, and the tissue-engineered bone is significant in bone defect repair and early vascularization; the method is simple, easy to implement and convenient to popularize.
Description
Technical field
The present invention relates to a kind of tissue engineered bone and preparation method thereof, be specifically related to a kind of tissue engineered bone be made up of multi-layer cellular sheet and preparation method thereof, belong to field of tissue engineering technology.
Background technology
The osseous tissue defect caused due to congenital malformation, wound, tumor etc. and disappearance are clinical common diseases, are therefore the important topic of regenerative medicine to the functional reconstruction reparation of osseous tissue defect always.Traditional Therapeutic Method often adopts autologous or allogenic bone transplantation, but often because supply source is limited or immunological rejection etc. and limit its clinical practice.Tissue engineering technique, as the one be most widely used in current artificial bone substitute materials, is replaced gradually by its biodegradability.Disclosed in CN103285427A " a kind of artificial bone and preparation method thereof ", namely adopt suitable proportion hydroxyapatite, collagen and nano-cellulose synthesis artificial bone, there is good intensity and degradation property.CN103157138A disclosed " a kind of construction method of tissue engineering bone renovation material ", then adopt injectable type chitosan-bata-tricalcium phosphate compound bone marrow stroma stem cell as tissue engineered bone, can random-shaping and degradable absorption, induced osteogenesis effect is obvious.But, the cell adhesion of conventional organization engineering mainly through repair district's periphery cell creep or by corresponding cell suspension titration in material surface or defective region, cell adhesion utilization rate is low, repairing efficiency is long, and its size, shape and location are also difficult to control; Meanwhile, titration cell suspension is often formed and is dispersed in island and repairs district, inapplicable for large stretch of tissue defect; In addition, applying biological bone tissue constructed by traditional organizational project, to implant after host angiogenesis in it slow, be unfavorable for the growth of osseous tissue, reconstruction and timbering material degraded, therefore build the novel tissue Engineering Bone with initial stage vascularization the reparation of osseous tissue and reconstruction are had great importance.
Cell thin culture technique (Tang Z; Akiyama Y; Yamato M, Okano T. Comb-type grafted poly (N-isopropylacrylamide) gel modified surfaces for rapid detachment of cell sheet.Biomaterials (2010); 31 (29): 7435-43. Nagase K; Kobayashi J; Kikuchi A; Akiyama Y; Kanazawa H, Okano T. Thermally-modulated on/off-adsorption materials for pharmaceutical protein purification. Biomaterials. (2011); 32 (2): 619-27.) as a kind of novel tissue engineering method, there is the advantage of its uniqueness.It is by corresponding intermediary material or control hydrophobe-hydrophile conversion bottom attached cell institute grown cultures ware thus control cell adhere to growth in flakes and peel off, after this " cell sheet " multiple-layer stacked or compound criteria, without the need to lapping, itself namely can be used as one " biological support " and implant defect.Compare pancreatin effect obtain cell, its do not destroy intercellular albumen connect and cell with the connection between bottom substrates, cytoactive is better; Meanwhile, the problem of high pollution is caused when cell thin culture technique also can be avoided being reduced in tissue repair because using biodegradation timbering material.CN103097518A and CN103180437A disclosed " manufacture method of cell sheet lamination compound, the cell sheet lamination compound with vasoganglion obtained by the method and Application way thereof ", namely cell sheet is obtained by temperature sensitive Tissue Culture Dish, the superposition of multi-layer cellular sheet is formed cladding cell sheet lamination compound, and its entirety is incubated in the vascular bed that forms containing stream gel or arteriovenous loop, obtain the cardiac muscular tissue containing vasoganglion.But; the condition relative complex of temperature control; higher for equipment requirements; between temperature or change and also affect the activity of cell; accelerating part cell senescence; the people skeletonization class cell more responsive for temperature inapplicable (Fukumori K; Akiyama Y; Yamato M; Kobayashi J; Sakai K, Okano T. Temperatureresponsive glass coverslips with an ultrathin poly (N-isopropylacrylamide) layer. Acta Biomater 2009; 5:470-476).
Summary of the invention
The object of the present invention is to provide a kind of tissue engineered bone be made up of multi-layer cellular sheet with plexus structure and preparation method thereof.
Tissue engineered bone of the present invention is the multi-layer cellular sheet lamination compound with three-dimensional wick vasoganglion, described multi-layer cellular sheet lamination compound is made up of the stacked mesenchymal stem cells MSCs sheet of n sheet and the vascular endothelial cell be filled between each mesenchymal stem cells MSCs sheet, n=3 ~ 8.
Prepare the method for above-mentioned tissue engineered bone, comprise the steps:
1) photosensitive Tissue Culture Dish is prepared
Photoresponse nanoparticulate dispersed is made into the dispersion liquid that mass concentration is 10 ~ 20% in deionized water, the proportions being 0.02 ~ 0.06:6 ~ 9:1 ~ 3 in molar ratio by dispersion liquid and alcohol and organic solvent again becomes precursor solution, precursor solution is dropped to polystyrene culture dish be paved with to it, dry and sterilize, obtaining photosensitive Tissue Culture Dish; Described photoresponse nano-particle is nano-titanium oxide, nano zine oxide or nano-sized iron oxide, and alcohol is methanol or ethanol, and organic solvent is oxolane, chloroform or dichloromethane;
2) mesenchymal stem cells MSCs sheet is cultivated
Get mesenchymal stem cells MSCs, with 5 × 10
4~ 2 × 10
5individual/cm
2density implantation step 1) photosensitive Tissue Culture Dish in, add mesenchymal stem cells MSCs special culture media and cultivate after 4 ~ 10 days, adopt wavelength 320 ~ 450nm, intensity 1 ~ 4mW/cm
2light inject bottom culture dish, expose to mesenchymal stem cells MSCs fall in flakes in photosensitive Tissue Culture Dish, obtain mesenchymal stem cells MSCs sheet;
3) tissue engineered bone is built
By step 2) the mesenchymal stem cells MSCs sheet that obtains is transferred in Tissue Culture Dish, after it is adherent, adds mesenchymal stem cells MSCs special culture media, at 37 DEG C, 5%CO
2and after cultivating 2 ~ 48h under saturated humidity condition, with 5 × 10
4~ 2 × 10
5individual/cm
2density implantable intravascular endotheliocyte thereon, then at 37 DEG C, 5%CO
2and cultivate 2 ~ 48h under saturated humidity condition, after absorbing mesenchymal stem cells MSCs special culture media, said structure shifts a slice mesenchymal stem cells MSCs sheet again, adds mesenchymal stem cells MSCs special culture media, at 37 DEG C, 5%CO
2and cultivate 2 ~ 48h under saturated humidity condition;
Again with 5 × 10 on said structure
4~ 2 × 10
5individual/cm
2density implantable intravascular endotheliocyte thereon, at 37 DEG C, 5%CO
2and cultivate 2 ~ 48h under saturated humidity condition, after absorbing mesenchymal stem cells MSCs special culture media, again shift mesenchymal stem cells MSCs sheet, and add mesenchymal stem cells MSCs special culture media, at 37 DEG C, 5%CO
2and cultivate 2 ~ 48h under saturated humidity condition, repeat above-mentioned process until this structure has n layer mesenchymal stem cells MSCs sheet, n=3 ~ 8, obtain multi-layer cellular sheet lamination compound, osteoblast inducible factor and angioblast inducible factor is added, at 37 DEG C, 5%CO in the mesenchymal stem cells MSCs special culture media in Tissue Culture Dish
2and cultivate 4 ~ 10 days again under saturated humidity condition, obtain tissue engineered bone.
Described osteoblast inducible factor is one or more in ascorbic acid, sodium β-glycerophosphate and dexamethasone, and its addition can be ascorbic acid 50 ~ 100ug/ml, sodium β-glycerophosphate 5 ~ 10mM and dexamethasone 1 × 10 respectively
-8m ~ 1uM; Described angioblast inducible factor is one or both in VEGF and basic fibroblast growth factor, and its addition can be basic fibroblast growth factor 5 ~ 10ng/ml and VEGF 10 ~ 20ng/ml respectively.
Mesenchymal stem cells MSCs involved in the present invention can be differentiation-inducing or be extracted from bone marrow, fatty tissue, peripheral blood, fetal blood or liver by embryo stem cell for directional, and vascular endothelial cell can be differentiation-inducing or be extracted from bone marrow, peripheral blood or Cord blood by embryo stem cell for directional.Because mesenchymal stem cells MSCs source is different; basal medium needed for it is not identical yet; therefore the mesenchymal stem cells MSCs special culture media described in the present invention refers to the culture medium corresponding with the source of mesenchymal stem cells MSCs, specifically can with reference to such as Publication about Document: Small diameter vascular graft engineered using human embryonic stem cell-derived mesenchymal cells(Tissue Eng Part A. 2014 Feb; 20 (3-4): 740-50), Differentiation of human embryonic stem cells into bipotent mesenchymal stem cells(Olivier E, et al., Stem Cells 2006 24:1914-1922) and optimization culture (Pang Yong firm Chen Wen string Cui distance of travel of roc, " the Chinese Reconstructive surgery magazine " 2004(3) 220-224 of human marrow-interstitial stem cell).
Compared with prior art, the present invention has following useful technique effect:
1. the present invention obtains mesenchymal stem cells MSCs sheet by photosensitive cell culture dish, namely photoresponse film surface charging property and group is caused to change by ultraviolet light or radiation of visible light, thus make growth cell thereon obtain cell sheet from the overall desorption of corresponding vessel surface, compared to existing cell thin technology, method of the present invention is more convenient, efficiency is higher, meanwhile, and the phenomenon such as aging, downright bad avoiding that the people skeletonization class cell more responsive to temperature produce because of temperature change.
2. the tissue engineered bone that there is plexus structure and be made up of multilamellar mesenchymal stem cells MSCs sheet lamination compound of the present invention, compared to existing artificial bone, without the need to biomaterial as rack bearing, be made up of homologous cell merely, reduce the pollution that immunologic rejection and scaffold degradation cause, and define the novel tissue Engineering Bone with three-dimensional plexus structure in external, to the reparation of Cranial defect and early stage vascularization significant.
Accompanying drawing explanation
Fig. 1 is the macrograph of multilamellar mesenchymal stem cells MSCs sheet lamination compound
Fig. 2 is inverted microscope figure below of multilamellar mesenchymal stem cells MSCs sheet lamination compound
Wherein a: multi-disc cell sheet neighboring area b: multi-disc cell sheet zone line
Fig. 3 is the specificity fluorescent signature of tissue engineered bone Endothelial Cell
Wherein a: Surface of Vascular Endothelial Cells Specific marker CD31
B: vascular endothelial cell specific markers UEA-I
C: nuclear marker thing.
Detailed description of the invention
Below in conjunction with specific embodiment, the present invention is described further, but implementation of the present invention is not limited thereto.
Embodiment one
1) photosensitive cell culture dish is prepared
Titanium dioxide nanoparticle is dispersed in water and forms the dispersion liquid that mass concentration is 20%, dispersion liquid and methanol and oxolane are made into precursor solution in the ratio of 0.06:9:3; Again above-mentioned precursor solution is pressed 48 μ L/cm
2evenly be added drop-wise in polystyrene culture dish, put into 65 DEG C of baking ovens dry, can obtain that to comprise crystallite dimension be 20 ~ 30nm after drying, thickness is the Tissue Culture Dish of the photoresponse nanophase titania films of 60nm ~ 100nm;
2) mesenchymal stem cells MSCs sheet is cultivated
Get 3 ~ 5 generation human marrow mesenchyme stem cells, with 2 × 10
5individual/cm
2density implanted diameter be 5cm containing in the culture dish of nano-titanium oxide, cultivate after 10 days, employing wavelength 450nm, intensity 4mW/cm
2ultra-vioket radiation 5min, obtain the human marrow mesenchymal stem cell sheet of fall in flakes;
3) tissue engineered bone is built
Adopting novel cell sheet transfer interlock instrument by step 2) obtained monolayer human marrow mesenchymal stem cell sheet is transferred in regular growth culture dish, after it is adherent, add stem cell special culture media (containing 10% hyclone, L-glutaminate 2mmol/L, penicillin 100U/ml, streptomycin 100mg/ml), be placed in 37 DEG C, 5%CO
2after cultivating 48h under saturated humidity condition, with 2 × 10
5individual/cm
2density implant human umbilical vein endothelial cell thereon, under above-mentioned condition of culture, place 2h, absorb after culture medium, with novel cell sheet transfer interlock instrument, second layer human marrow mesenchymal stem cell sheet is transferred on it, quiescent culture 2h under the same terms.Subsequently, then with 2 × 10
5individual/cm
2density implant one deck human umbilical vein endothelial cell thereon, and cultivate 2h under the same terms, finally third layer human marrow mesenchymal stem cell sheet is transferred on it, the multi-layer cellular sheet lamination compound of three layers of human marrow mesenchymal stem cell sheet and double layer sandwich human umbilical vein endothelial cell must be comprised, figure mono-is the macrograph of multi-layer cellular sheet lamination compound, second figure is the inverted microscope figure (a of corresponding multi-layer cellular sheet lamination compound, b represents periphery and the zone line of multi-layer cellular sheet respectively), adhere to good between display multi-layer cellular, have no obvious floating dead cell.Extra interpolation basic fibroblast growth factor 10ng/ml and VEGF 20ng/ml in above-mentioned stem cell special culture media, and add osteoblast inducible factor ascorbic acid 100ug/ml, sodium β-glycerophosphate 10mM and dexamethasone 1uM, namely In vitro culture obtains the tissue engineered bone with plexus structure after 4 days, figure tri-is tissue engineered bone Endothelial Cell specificity fluorescent signature (a, b represents that Surface of Vascular Endothelial Cells Specific marker CD31 and UEA-I dyes respectively), between display lamination compound, vascular endothelial cell forms plexus structure gradually.
The concrete source of the human marrow mesenchymal stem cell used in this example is illustrated:
Obtain young bone grafting patient to agree to, ilium gets bone, 10ml bone marrow fluid is drawn with syringe, the heparin sodium 2500U/ml that syringe includes dilution is about 0.5ml, by adherent for cell suspension be slowly injected into preset equal-volume density be the human lymphocyte separating medium of 1.077kg/L in vitro, make to form interface clearly between the two.With the centrifugal 20min of 2500r/min, milky cloud mononuclear cell layer in the middle of drawing, PBS rinsing, the centrifugal 10min of 1500r/min, abandon supernatant, add human marrow mesenchymal stem cell special culture solution (Jiangsu Celeron the is biological) 10ml containing 10% calf serum, make single cell suspension, be inoculated in floor space for 25cm with suitable density
2in culture bottle, insert 37 DEG C, 5%CO
2cultivate under saturated humidity condition, after 4 days, half amount changes liquid first, and all discarded by not adherent cell, within later every 2 ~ 3 days, full dose changes liquid once.Become after monolayer until cell confluency, with 0.25% trypsin containing 0.02%EDTA) digestion, namely obtain Primary bone marrow mesenchyma stem cell suspension.Treat that cell fusion reaches more than 80%, by 1: 3 or 1: 4 Secondary Culture, get the attached cell that the growth of the 3rd generation reaches 80% fusion, with trypsinization, after counting, get 2 × 10
6individual cell, subpackage 6 is managed, and often pipe adds 10 μ l fluorescent-labeled antibody CD45-FITC, CD34-PE, CD73-PE, CD105-FITC, CD166-PE, separately sets 1 pipe as blank, room temperature lucifuge 30min, repeatedly wash 2-3 time with PBS, add 200 μ l PBS and mix cell, and fix with 1% paraformaldehyde, 4 DEG C of preservations, in 24h, flow cytometer detects, and Normal Human Bone Marrow mescenchymal stem cell high expressed CD73, CD105 and CD166, CD34 and CD45 is then negative.
The concrete source of the human umbilical vein endothelial cell used in this example is illustrated:
Aseptic acquisition neonatal umbilical cord, use normal saline flushing bloodstain immediately, cut off pincers trace and Hemorrhagic location, find out umbilical vein (2 tube chambers less be umbilical artery, what 1 tube chamber was thicker is umbilical vein), umbilical vein is inserted from one section with the syringe of the flat syringe needle of band, fix by vascular forceps, bloodstain in umbilical vein is washed down again with PBS liquid, for preventing the residual blood of umbilical artery to be mixed into, tremulous pulse can be separated a little sterilization toe-in and prick, with the vascular forceps clamp other end, the collagenase liquid injecting 0.1% from the syringe needle rinsed makes it fill, and puts into 37 DEG C of water-baths and hatches 15min.Take out umbilical cord and unclamp vascular forceps release enzyme cell mixture in centrifuge tube, be collected in the lump in centrifuge tube after rinsing umbilical vein with PBS liquid again, 1000r/min, centrifugal 5min, abandon supernatant, add DMEM culture fluid (containing 10% hyclone, L-glutaminate 2mmol/L, penicillin 100U/ml, streptomycin 100mg/ml, basic fibroblast growth factor 10ng/ml, VEGF 20ng/ml), be placed in 37 DEG C, 5%CO
2cultivate under saturated humidity condition, after 24h, change liquid first, within later every 2 ~ 3 days, change liquid once.Treat that cell fusion reaches more than 80%, by 1: 3 or 1: 4 Secondary Culture, get the attached cell that the growth of the 3rd generation reaches 80% fusion, with 0.25% trypsin containing 0.02%EDTA) digestion, after PBS cleaning for several times, add 20 μ l fluorescent-labeled antibody CD31-PE, separately establish blank, row flow cytometry after room temperature lucifuge 30min, normal person's umbilical vein vascular endothelial cells high expressed CD31.
Embodiment two
1) photosensitive cell culture dish is prepared
Zinc oxide nanoparticle is dispersed in water and forms the dispersion liquid that mass concentration is 10%, dispersion liquid and ethanol and chloroform are made into precursor solution in the ratio of 0.02:6:1, then by above-mentioned precursor solution by 25 μ L/cm
2evenly be added drop-wise in polystyrene culture dish, put into 65 DEG C of baking ovens dry, can obtain that to comprise crystallite dimension be 10 ~ 20nm after drying, thickness is the Tissue Culture Dish of the photoresponse Nano zinc oxide film of 40nm ~ 80nm.
2) mesenchymal stem cells MSCs is cultivated
Get the homology human marrow mesenchymal stem cell after cultivating number generation, with 5 × 10
4individual/cm
2density implanted diameter be 5cm containing in the culture dish of nano zine oxide, cultivate after 4 days, employing wavelength 320nm, intensity 1mW/cm
2ultra-vioket radiation 30min, obtain the human marrow mesenchymal stem cell sheet of fall in flakes.
3) tissue engineered bone is built
Novel cell sheet transfer interlock instrument is adopted to be transferred in regular growth culture dish by monolayer homology human marrow mesenchymal stem cell sheet, after it is adherent, add stem cell special culture media (containing 10% hyclone, L-glutaminate 2mmol/L, penicillin 100U/ml, streptomycin 100mg/ml), be placed in 37 DEG C, 5%CO
2after cultivating 2h under saturated humidity condition, with 5 × 10
4individual/cm
2density implant homology human vascular endothelial thereon, 48h is placed under above-mentioned condition of culture, after absorbing culture medium, with novel cell sheet transfer interlock instrument, second layer homology human marrow mesenchymal stem cell sheet is transferred on it, quiescent culture 48h under the same terms.Subsequently, repeat to implant homology human vascular endothelial successively and cultivate and shift human marrow mesenchymal stem cell sheet and cultivate, until this structure has 8 layers of mesenchymal stem cells MSCs sheet, obtain multi-layer cellular sheet lamination compound, extra interpolation 5ng/ml basic fibroblast growth factor and 10ng/ml VEGF in above-mentioned culture medium, and add osteoblast inducible factor ascorbic acid 50ug/ml, sodium β-glycerophosphate 5mM and dexamethasone 1 × 10
-8m, namely In vitro culture obtained tissue engineered bone after 10 days.
The concrete source of the mesenchymal stem cells MSCs used in this example is illustrated:
Human embryo stem cell field planting is incubated on inactivation mouse fibroblast feeder layer, add DMEM/F12 culture fluid (containing 20% serum substitute, 1mM L-glutaminate, 1% non essential amino acid, 100mM beta-mercaptoethanol, 4ng/ml basic fibroblast growth factor), treat that its periphery occurs in clastic from noble cells group, get this scrappy cell sheet and cultivate several weeks in containing the human marrow mesenchymal stem cell special culture solution (Jiangsu Celeron is biological) of 10% calf serum, with comprising pancreatin, the mixed liquor of IV Collagenase Type and neutral protease dissociates, cultivate again, and go out CD45 by selected by flow cytometry apoptosis, CD34 is negative, and SH2, SH3, the homology human marrow mesenchymal stem cell that SH4 is positive.(human embryo stem cell is given in NUS)
The concrete source of the vascular endothelial cell used in this example is illustrated:
Human embryo stem cell field planting is incubated on inactivation mouse fibroblast feeder layer, add DMEM/F12 culture fluid (containing 20% serum substitute, 1mM L-glutaminate, 1% non essential amino acid, 100mM beta-mercaptoethanol, 4ng/ml basic fibroblast growth factor), and urge differentiation after 2 days by 20ng/ml BMP-4, form embryoid body.Subsequently, be colonizated in matrigel surface, add DMEM culture fluid (containing 10% hyclone, L-glutaminate 2mmol/L, penicillin 100U/ml, streptomycin 100mg/ml, basic fibroblast growth factor 5ng/ml, VEGF 10ng/ml) separately urge differentiation more than 10 in the future, take CD31 as the vascular endothelial cell that mark filters out people's embryonic stem cells source.(human embryo stem cell is given in NUS)
Claims (3)
1. a tissue engineered bone, it is characterized in that described tissue engineered bone is the multi-layer cellular sheet lamination compound with three-dimensional wick vasoganglion, described multi-layer cellular sheet lamination compound is made up of the stacked mesenchymal stem cells MSCs sheet of n sheet and the vascular endothelial cell be filled between each mesenchymal stem cells MSCs sheet, n=3 ~ 8.
2. prepare the method for tissue engineered bone as claimed in claim 1, it is characterized in that comprising the steps:
1) photosensitive Tissue Culture Dish is prepared
Photoresponse nanoparticulate dispersed is made into the dispersion liquid that mass concentration is 10 ~ 20% in deionized water, the proportions being 0.02 ~ 0.06:6 ~ 9:1 ~ 3 in molar ratio by dispersion liquid and alcohol and organic solvent again becomes precursor solution, precursor solution is dropped to polystyrene culture dish be paved with to it, dry and sterilize, obtaining photosensitive Tissue Culture Dish; Described photoresponse nano-particle is nano-titanium oxide, nano zine oxide or nano-sized iron oxide, and alcohol is methanol or ethanol, and organic solvent is oxolane, chloroform or dichloromethane;
2) mesenchymal stem cells MSCs sheet is cultivated
Get mesenchymal stem cells MSCs, with 5 × 10
4~ 2 × 10
5individual/cm
2density implantation step 1) photosensitive Tissue Culture Dish in, add mesenchymal stem cells MSCs special culture media and cultivate after 4 ~ 10 days, adopt wavelength 320 ~ 450nm, intensity 1 ~ 4mW/cm
2light inject bottom culture dish, expose to mesenchymal stem cells MSCs fall in flakes in photosensitive Tissue Culture Dish, obtain mesenchymal stem cells MSCs sheet;
3) tissue engineered bone is built
By step 2) the mesenchymal stem cells MSCs sheet that obtains is transferred in Tissue Culture Dish, after it is adherent, adds mesenchymal stem cells MSCs special culture media, at 37 DEG C, 5%CO
2and after cultivating 2 ~ 48h under saturated humidity condition, with 5 × 10
4~ 2 × 10
5individual/cm
2density implantable intravascular endotheliocyte thereon, then at 37 DEG C, 5%CO
2and cultivate 2 ~ 48h under saturated humidity condition, after absorbing mesenchymal stem cells MSCs special culture media, said structure shifts a slice mesenchymal stem cells MSCs sheet again, adds mesenchymal stem cells MSCs special culture media, at 37 DEG C, 5%CO
2and cultivate 2 ~ 48h under saturated humidity condition;
Again with 5 × 10 on said structure
4~ 2 × 10
5individual/cm
2density implantable intravascular endotheliocyte thereon, at 37 DEG C, 5%CO
2and cultivate 2 ~ 48h under saturated humidity condition, after absorbing mesenchymal stem cells MSCs special culture media, again shift mesenchymal stem cells MSCs sheet, and add mesenchymal stem cells MSCs special culture media, at 37 DEG C, 5%CO
2and cultivate 2 ~ 48h under saturated humidity condition, repeat above-mentioned process until this structure has n layer mesenchymal stem cells MSCs sheet, n=3 ~ 8, obtain multi-layer cellular sheet lamination compound, osteoblast inducible factor and angioblast inducible factor is added, at 37 DEG C, 5%CO in the mesenchymal stem cells MSCs special culture media in Tissue Culture Dish
2and cultivate 4 ~ 10 days again under saturated humidity condition, obtain tissue engineered bone.
3. the preparation method of tissue engineered bone according to claim 2, it is characterized in that described osteoblast inducible factor is one or more in ascorbic acid, sodium β-glycerophosphate and dexamethasone, described angioblast inducible factor is one or both in VEGF and basic fibroblast growth factor.
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