CN1746296A - A kind of tissue-engineered bone and manufacture method thereof based on placenta derived mesenchymal stem cell - Google Patents
A kind of tissue-engineered bone and manufacture method thereof based on placenta derived mesenchymal stem cell Download PDFInfo
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- CN1746296A CN1746296A CNA2004100095332A CN200410009533A CN1746296A CN 1746296 A CN1746296 A CN 1746296A CN A2004100095332 A CNA2004100095332 A CN A2004100095332A CN 200410009533 A CN200410009533 A CN 200410009533A CN 1746296 A CN1746296 A CN 1746296A
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Abstract
The present invention relates to a kind of tissue-engineered bone and manufacture method thereof, belong to field of tissue engineering technology based on placenta derived mesenchymal stem cell.Adopt the mescenchymal stem cell compound bio timbering material in placenta source to make up osseous tissue.Constructed tissue-engineered bone can be clogged the damaged lacuna of bone, also can repair bulk or the segmental osseous tissue is damaged, realizes the functional reparation of bone injury, has tangible potential applicability in clinical practice.
Description
Invention field
The invention belongs to field of tissue engineering technology, be specifically related to a kind of tissue-engineered bone and manufacture method thereof based on placenta derived mesenchymal stem cell.
Background of invention
Multiple clinically reason usually causes the osseous tissue lacuna damaged, and as the necrosis of femoral head that aseptic inflammation causes, lacuna that the maxillary sinus tumor post-operation is left over and exodontia tooth are pulled out the tooth socket of back generation etc.These damaged treatments still there is not the ideal method at present.Necrosis of femoral head is waited until cortex of bone or the articulum laggard pedestrian worker's joint replacement that subsides mostly, and the latter can't realize that the physiological of function of joint recovers, and can cause many complication; The damaged often employing that maxillary sinus tumor resection postoperative stays is shifted the musculo cutaneous flap reparation or is not repaired, and the both can cause the change of face shape, makes the patient be difficult to accept, even the sensation that to live is no better than to die is arranged; The tooth socket that stays after the tooth extraction is at present many not to be repaired, normal healing, but in fact often cause ossified level low, and the bone quantity not sufficient is for repairing and treatings such as having a denture made in the future bring difficulty.
Autologous bone transplanting is the damaged optimal path of treatment lacuna bone, but because of limited being restricted of originating.Previously once developed various biocompatible and the good biologic material of mechanics of machinery characteristic in the research and be used for the damaged lacuna filling of bone, but facts have proved that its effect is unsatisfactory, simple repair materials is transplanted the ossified ability of back material, does not reach expected effect.In recent years organizational engineering studies show that, the timbering material of biologically active and cell is combined make up a kind of engineered tissue and be expected to realize damaged physiological reparation.Seed cell wherein can be scleroblast or periosteum cell.But in fact these two kinds of cells all exist the difficulty of drawing materials, vitro culture to lose phenotype easily, can not continuous passage obtain problems such as sufficient amount.Mesenchymal stem cells MSCs can be in external continuous passage, amplification and do not lose its phenotype and multidirectional differentiation capability (comprising to osteoblastic differentiation) in a large number.This specific character is selected its ideal that becomes the bone tissue engineer seed cell.
Yet, from marrow, obtain mescenchymal stem cell and still have tangible deficiency.1) extracts patient's marrow and need cause operation wound, be not easy to be accepted by the patient; 2) extract the marrow amount seldom at every turn, 5-10ml only, and the content of mescenchymal stem cell is extremely low in the marrow, only is 1/100000 of karyocyte, so the cell harvesting amount is minimum; 3) though mescenchymal stem cell can carry out external continuous passage amplification, at present extensive amplification technique is still immature, and cost height, time and effort consuming, uses the needs of patients wait long period from drawing materials; 4) because the mescenchymal stem cell of derived from bone marrow has immunogenicity, so resource can't be shared between the allosome.Above factor shows that mesenchymal stem cells MSCs is not the ideal source of cartilage tissue engineered seed cell.
Current research is found, is had in the placenta tissue and the similar attached cell of mesenchymal stem cells MSCs.Report such as external Jaroscak (2000) separation and Culture from frozen placenta tissue goes out heterogeneous attached cell.Report such as domestic Zhang Yi adopts enzyme digestion to obtain the sustenticular cell of attached cell as hemopoietic stem cell from placenta tissue.Therefore, placenta tissue is expected to become the abundant source of potential of mescenchymal stem cell.The a small amount of that only has at present both at home and abroad fragmentary document to relate to placenta source attached cell is separated, and is mainly used in hematopoiesis support research, and mass-producing separation, purifying and the amplification method of placenta derived mesenchymal stem cell then lacked technical data.The contriver finds out the separation method of the mescenchymal stem cell in placenta source through research practice repeatedly, and confirm that such cell has and derived from bone marrow mescenchymal stem cell similar biological, can be divided into the various kinds of cell type that comprises the chondrocyte under suitable condition.Because placenta tissue is discarded tissue, the source is abundant, the cell harvesting amount is big, so the mescenchymal stem cell in placenta source is expected to become cartilage tissue engineered ideal seed cell.
Summary of the invention
The purpose of this invention is to provide a kind of tissue-engineered bone and manufacture method thereof based on placenta derived mesenchymal stem cell, the mescenchymal stem cell that it is characterized in that originating with placenta is as seed cell, with the compound structure tissue-engineered bone of porous biomaterial.The present invention selects for use placenta derived mesenchymal stem cell as seed cell, and this cell can be taken from depleted placenta tissue behind the healthy parturient childbirth, draws materials easily, can be shared by different crowd.This cell is strong in external easy cultivation, amplification ability, and phenotype is difficult for losing, and breaks up to osteoblasts in vitro easily under certain inductive condition.Technical scheme of the present invention is:
1) separation of placenta derived mesenchymal stem cell, cultivation and amplification
A. the fresh placenta of giving birth to is carried out bloodletting, perfusion immediately, to remove hemocyte and fragment of tissue;
B. continue perfusion, or placental lobules is shredded, use collagenase digesting, the collecting cell composition;
C. adopt immunomagnetic beads absorption method or Percoll density gradient centrifugation to collect mononuclearcell;
D. measure cell doubling time and growth curve;
E. the cell that obtains is carried out freezing preservation;
E. adopt the cell of bio-reactor, comprise that the cell of recovering behind the cryopreservation carries out the mass-producing amplification being obtained.
2) structure of tissue-engineered bone
A. timbering material pre-treatment timbering material can be degradable multiporous bioceramic material, as α-or bata-tricalcium phosphate; Also can be collagen, polymer class.Preferred bata-tricalcium phosphate porous bio-ceramic among the present invention.Prefabricated one-tenth particulate state, or be prefabricated into specified shape, 60gray as required
60The Co irradiation also can be 75% sterilization in alcohol-pickled 10 minutes～1 hour.With serum free medium or PBS damping fluid alcohol is cemented out then to improve wetting ability with preceding usefulness 95% is alcohol-pickled, the sterilization dry gauze blots liquid.The collagenic material of having sterilized can directly be used.
B. the structure of tissue-engineered bone makes 1～2 * 10 with fresh separated or through amplification or through the placenta derived mesenchymal stem cell of freezing preservation
7The cell suspension of/ml drips on timbering material gently, and material overturning gently, makes cell distribution as far as possible evenly.Cell-porous support materials complex body is put into incubator, 37 ℃, 5%CO
2, left standstill under the saturated humidity 6～10 hours, cell is attached in the hole of material and surperficial.Be transferred to then and carry out the 3 D stereo cultivation in the rotary bio-reactor.Speed of rotation 15 ~ 50rpm.
C. the organization engineered cartilage that builds of the preservation of tissue-engineered bone can adopt cryogenic freezing preservation technology to store and transport.Freezing and storing method can be to be the routine preservation technology of cryoprotectant with dimethyl sulfoxide (DMSO) (DMSO), also can be to be that protectant vitrifying does not have ice crystal preservation technology with VS55.
Beneficial effect
The invention provides and be used for the treatment of the damaged tissue-engineered bone of bone.Described bone is damaged can be the necrosis of femoral head that aseptic inflammation causes, the tooth socket that stays after the damaged or exodontia of the big fast lacuna bone that postoperatives such as tumour cause etc., also can be need to fill lacuna to recover form or reservation function, also can be that the big area bone of flat bone such as the damaged or skull of long bone segmental bone is damaged.Tissue-engineered bone of the present invention be mescenchymal stem cell with placenta source as seed cell, cell is taken from the depleted placenta tissue, the source is abundant, harvest yield is big, no immunological rejection.Because this cell can freezingly be preserved, and shares between the crowd, therefore can realize ready access upon use, need not to wait for, thereby make things convenient for clinical application.
Embodiment
The acquisition of embodiment 1 people's placenta derived mesenchymal stem cell
Place aseptic pallet or container after placenta is given birth to immediately, inject through Umbilical artery and contain antithrombotics and antibiotic balanced salt solution, bloodletting, and begin perfusion.Antithrombotics can be the heparin of 1 ~ 100u/mL, and microbiotic can be a mycillin.Collect perfusion liquid, centrifugal acquisition cellular constituent; Or the aseptic clip placental lobules in preliminary perfusion back, with trypsinase or collagenase digesting placenta tissue, obtain cell suspension, again cell suspension is crossed the cell sieve, remove fragment of tissue and hemocyte, the cell that obtains suspends again, with density is the percoll of 1.073g/mL, with 500g centrifugal force centrifugal 30 minutes, and results interface cellular layer, PBS washing back inoculation culture bottle or culture plate, or be directly used in other purpose.Also can separate the mescenchymal stem cell that has certain special marking with the immunomagnetic beads of D7-FIB or NGFR mark.Adopt the phenotype of flow cytometer detection placenta derived mesenchymal stem cell, as CD29, CD34, CD44, CD45, CD105, CD166, HLA-DR etc.Detect the cell cycle simultaneously, determine to be in G
0-G
1The ratio of phase cell.
2 one kinds of manufacture method of embodiment based on the tissue-engineered bone of the mescenchymal stem cell in placenta source
People's placenta derived mesenchymal stem cell of fresh separated or cryopreservation resuscitation is prepared into 1.5～2 * 10
7The cell suspension of/ml is inoculated on the timbering material, makes cell distribution even as far as possible.In a preferred scheme, timbering material adopts cylindric β-TCP.β-TCP porous bio-ceramic material can be by the method and the explained hereafter of last sea cowry Ao Lu house journal, and material is through 60gray
60Be used for tissue-engineered bone behind the Co illumination-based disinfection and make up, in a preferred scheme, its microstructure is: aperture-100～300 μ m, porosity-52% connects footpath-30～100 μ m between the hole.After leaving standstill 6 hours, mixture is moved into RCCS (rotary bio-reactor, Synthecom company), cultivated 48 hours in containing the DMEM of 10%FCS, rotating speed is 30rpm.It is damaged that mixture after the cultivation can be directly used in clinical repair patient bone, but also cryopreservation, as the tissue-engineered bone storehouse.
Claims (10)
1. tissue-engineered bone and manufacture method thereof based on the mescenchymal stem cell in placenta source.The mescenchymal stem cell that obtains with separation and Extraction from placenta tissue is as seed cell, with the compound structure organization engineered cartilage of multiporous biological timbering material.
2. the placenta described in the claim 1 is the placenta of leaving over after healthy puerpera's normal labor or c-section fetus are given birth to.Placenta with give bloodletting immediately after parent and fetus separate, and pour into.Perfusion liquid is aseptic balanced salt solution, wherein contains antithrombotics and microbiotic.
3. can external preservation after the placenta bloodletting described in the claim 2 2-24 hour.Storage temperature can be a room temperature, also can be 4 ℃.
4. the separation of the mescenchymal stem cell in the placenta described in the claim 1 source can be adopted perfusion, promptly continues perfusion and centrifugal collecting cell composition, and it is resuspended to concentrate the back, carries out density gradient centrifugation collecting interface cell layer with the percoll of 1.073g/ml; Also can adopt digestion method, placenta tissue be shredded the back digest with collagenase or pancreatin, the cell suspension that obtains carries out density gradient centrifugation collecting interface cell layer with the percoll of 1.073g/ml.
5. the interface cellular layer described in the claim 3 can be seeded in culturing bottle or the culture dish and cultivate, or compound with microcarrier, places amplification in the bio-reactor; Also can use the immunomagnetic beads isolation technique, separate certain specific cell subsets,, separate cell mass with corresponding phenotype as using the magnetic bead that coupling has NGFR antibody or D7-FIB antibody or Stro-1 antibody or CD105 antibody.
6. the degradable multiporous biologic bracket material described in the claim 1 can be degradable calcium phosphate timbering material, also can be the hydroxyapatite class material of not degrading, and can also be the association of the two.Preferable material is the porous calcium phosphate biological ceramic timbering material among the present invention.
7. the porous bioceramic scaffold material described in the claim 6 is characterized in that it can being that (α-TCP) also can be bata-tricalcium phosphate (β-TCP) to type alpha tricalcium phosphate.Its moiety tricalcium phosphate is identical with the inorganic components of body bone, thereby has excellent biological compatibility and mechanics of machinery characteristic, can provide adapt circumstance and strong support for organizing new life.It is characterized in that having controlled special microstructure, the pass rule, the porosity height has tubulose to connect between the Kong Yukong.This special features of microstructures is that the aperture can be between 100～300 μ m, and porosity can be between 45%～52%, and connecting the footpath between the hole can be between 30～100 μ m.This structure not only can improve the growing into property of tissue and cell, can also be adjusted in intravital degradation rate whereby, so that it is coordinated mutually with the newborn speed of osseous tissue.
8. compound described in the claim 1 can be the cell suspension of the mescenchymal stem cell in placenta source being made high density, is inoculated on the timbering material, forms cell/composite body.Complex body extracorporeal culture certain hour closely attaches cell and material.
9. the extracorporeal culture described in the claim 8 can be that static cultivation was directly used in reparation in 16-48 hour in culturing bottle or culture dish, also can transfer to after static cultivation finishes and continue in the bio-reactor to cultivate 24-48 hour.
10. the described tissue-engineered bone of claim 1, it is characterized in that to be used for the physiological reparation of the various articular cartilage damages that reasons such as wound, inflammation, tumour cause, the treatment that also can be used to improve looks, plastic surgery etc. needs osseous tissue tissue substitute to be provided or to make.
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| CNA2004100095332A CN1746296A (en) | 2004-09-09 | 2004-09-09 | A kind of tissue-engineered bone and manufacture method thereof based on placenta derived mesenchymal stem cell |
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| CNA2004100095332A CN1746296A (en) | 2004-09-09 | 2004-09-09 | A kind of tissue-engineered bone and manufacture method thereof based on placenta derived mesenchymal stem cell |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102989041A (en) * | 2012-10-22 | 2013-03-27 | 北京航空航天大学 | Chitin fiber reinforced collagen base bone tissue engineering scaffold with compounded human mesenchymal stem cells and preparation method |
| CN104096266A (en) * | 2014-07-25 | 2014-10-15 | 中国人民解放军第三军医大学 | Tissue-engineered bone based on entochondrostosis system and construction method thereof |
| CN104188738A (en) * | 2014-08-14 | 2014-12-10 | 卢建熙 | Multi-functional internal bone generator |
| CN107028981A (en) * | 2007-09-19 | 2017-08-11 | 普拉里斯坦有限公司 | Adherent cell from fat or placenta tissue and its purposes in the treatment |
| CN110101914A (en) * | 2019-05-04 | 2019-08-09 | 西北工业大学 | A kind of Prevascularized two-phase artificial bone scaffold and preparation method thereof |
-
2004
- 2004-09-09 CN CNA2004100095332A patent/CN1746296A/en active Pending
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107028981A (en) * | 2007-09-19 | 2017-08-11 | 普拉里斯坦有限公司 | Adherent cell from fat or placenta tissue and its purposes in the treatment |
| CN107028981B (en) * | 2007-09-19 | 2021-04-20 | 普拉里斯坦有限公司 | Adherent cells from adipose or placental tissue and their use in therapy |
| CN102989041A (en) * | 2012-10-22 | 2013-03-27 | 北京航空航天大学 | Chitin fiber reinforced collagen base bone tissue engineering scaffold with compounded human mesenchymal stem cells and preparation method |
| CN104096266A (en) * | 2014-07-25 | 2014-10-15 | 中国人民解放军第三军医大学 | Tissue-engineered bone based on entochondrostosis system and construction method thereof |
| CN104096266B (en) * | 2014-07-25 | 2015-12-02 | 中国人民解放军第三军医大学 | Based on tissue engineered bone and the construction process thereof of entochondrostosis system |
| CN104188738A (en) * | 2014-08-14 | 2014-12-10 | 卢建熙 | Multi-functional internal bone generator |
| CN104188738B (en) * | 2014-08-14 | 2018-04-27 | 卢建熙 | Multifunctional body in-seam generator |
| CN110101914A (en) * | 2019-05-04 | 2019-08-09 | 西北工业大学 | A kind of Prevascularized two-phase artificial bone scaffold and preparation method thereof |
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