CN1565644A - Manufacturing method of tissue engineered bone - Google Patents
Manufacturing method of tissue engineered bone Download PDFInfo
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- CN1565644A CN1565644A CNA031479162A CN03147916A CN1565644A CN 1565644 A CN1565644 A CN 1565644A CN A031479162 A CNA031479162 A CN A031479162A CN 03147916 A CN03147916 A CN 03147916A CN 1565644 A CN1565644 A CN 1565644A
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Abstract
The invention concerns a manufacturing method of tissue engineering bone, and belongs to tissue engineering field. It generates bone tissue by employing inorganic biological bracket material(coral-hydroxyapatite) and tissue cell(marrow mesenchymal stem cell). The generated granular bone tissue is filled in the defection lacuna to realize functional restoration of bone injury.
Description
Invention field
The invention belongs to field of tissue engineering technology, be specifically related to a kind of manufacture method of tissue-engineered bone.
Background of invention
Multiple clinically reason usually causes the osseous tissue lacuna damaged, and as the femur head necrosis 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 ideal method at present.Femur head necrosis is waited until cortical bone or the articular surface 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 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 tissue 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 osteoblast or periosteum cell.But in fact these two kinds of cells all exist the difficulty of drawing materials, In 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.
The rise of tissue engineering technique is that the physiological reparation of cartilaginous tissue has brought hope.Domestic and foreign literature reported in succession and adopted this technique construction cartilage, nude mice with have the example of achieving success in the animal bodies such as mice, rat, rabbit, pig of immunity.But the timbering material that these institutes adopt all is organic macromolecules such as polymer or collagen.And can cause during the polymer degradation in vivo acid product to pile up, bring adverse influence for cartilaginous tissue new life; Collagen or gel-like material are then met deformation easily behind the water, and being not enough to new life to cartilage provides and continue strong support.More than two kinds of deficiencies seriously limited cartilage tissue engineered product development and application.
The coral hydroxyapatite artificial bone is to be raw material with specific natural high-quality ocean Corallium Japonicum Kishinouye, adopt patented technology, hydrothermal solution displacement reaction through a series of complexity, mineralogical composition in the Corallium Japonicum Kishinouye is converted to hydroxyapatite and has kept the natural pore structure of Corallium Japonicum Kishinouye, obtain product like physical arrangement and inorganic constituents and the human body bone photo at last, the growth that can be new bone tissue provides natural passage and holds the place.Its product main characteristics is: 1) physical arrangement and chemical composition and human body bone photo are seemingly; 2) have good biocompatibility, bone conductibility and certain osteoinductive; 3) healing time equates with autologous bone transplanting; 4) misery, extra charge and the hematoma brought of no autologous bone transplanting, infection, disease such as hemorrhage; 5) modern crafts are made, and various ways, plurality of specifications such as granule, bulk, abnormity are arranged.Wherein the micro structure of granular material has been simulated the structure of natural osteocomma, is interconnected between the Kong Yukong, has good especially permeability.This natural cavernous structure can promote the quick growth of connective tissue and bone apposition following closely.
Summary of the invention
The manufacture method that the purpose of this invention is to provide a kind of tissue-engineered bone is characterized in that with autologous bone marrow mesenchymal stem cells as seed cell, with the compound structure tissue-engineered bone of Corallium Japonicum Kishinouye-hydroapatite particles.The present invention selects for use autologous bone marrow mesenchymal stem cells as seed cell, this cell can be taken from patient's autologous bone marrow or the blood after medicine is mobilized, wound is little, can not cause the secondary wound, accepted by the patient easily, this cell is strong in external easy cultivation, amplification ability simultaneously, and phenotype is difficult for losing, and breaks up to osteoblasts in vitro easily under certain inductive condition.Timbering material adopts Corallium Japonicum Kishinouye-hydroapatite particles, and the basis-calcium phosphate of this material and hydroxyapatite also are the main inorganic components of natural bone tissue, and therefore having good biocompatibility and Mechanics of Machinery characteristic and bone passes to activity.In addition, prefabricated one-tenth graininess in processing owing to be designed to loose structure, had both improved the mechanical property of material, had increased the growing into property of cell again, helped the formation of freshman bone tissue.
Technical scheme of the present invention is:
1) separation of autologous bone marrow mesenchymal stem cells, cultivation and amplification
Aseptic extraction bone marrow 5～10ml under the local anaesthesia adopts density to separate nucleated cell with the percol of 1073g/l, places culture bottle or culture plate, cultivates in containing the DMEM culture medium of 10～20% hyclones.Cell clone grows and 60～75% trypsinizations with 0.25% when converging go down to posterity at 1: 2.Changed liquid in per 2～3 days later on, went down to posterity in 1: 2 or 1: 3 when cell 65～70% converges.Collect the 8th～15 generation cell be used for making up.
2) pretreatment of Corallium Japonicum Kishinouye-hydroxyapatite timbering material
But Corallium Japonicum Kishinouye-hydroxyapatite material is prefabricated into the graininess of all size, 60gray
60The Co irradiation also can be 75% sterilization in alcohol-pickled 10 minutes～1 hour.With serum-free medium or PBS buffer ethanol is cemented out then to improve hydrophilic with preceding usefulness 95% is alcohol-pickled, sterilize and blot liquid in gauze.
3) structure of tissue-engineered bone
With the 8th～15 generation mesenchymal stem cells MSCs make 1～2 * 10
7The cell suspension of/ml drips on timbering material gently, and material overturning gently, makes cell distribution as far as possible evenly.Cell-composite 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 bioreactor.Rotary speed 15 ~ 50rpm.
4) body is implanted into
To be packed to through the cell-composite body machinery of in vitro cultivation in the damaged lacuna of bone of exposure, with bone mould lobe, musculo cutaneous flap or mucosal flap flap coverage, closely sew up wound.
Beneficial effect
The invention provides and be used for the treatment of the damaged tissue-engineered bone of some lacuna bone.Described bone is damaged can be the femur head necrosis that aseptic inflammation causes, and the tooth socket that stays after the damaged or exodontia of the big fast lacuna bone that postoperatives such as tumor cause etc. need to fill lacuna to recover form or reservation function.Ossified ability is strong in the tissue-engineered bone mechanical strength height that the present invention is constructed, body.Because autologous bone marrow mesenchymal stem cells is drawn materials conveniently, amplification easily, Corallium Japonicum Kishinouye-hydroxyapatite timbering material is convenient to industrialization production, and construction method is easy to operate, and is easy to implement, and therefore, the present invention has broad clinical application prospect.
The specific embodiment
But repair the dog tooth socket with grain structure through engineering approaches bone
Embodiment 1
The separation of dog autologous bone marrow mesenchymal stem cells, cultivation and amplification
Needle tubing with pre-heparinization after 12 adult domesticated dog pentobarbital sodium intravenously administrable anesthesia is extracted bone marrow 5～10ml from the ilium ridge, adopt density to separate nucleated cell with the percol of 1073g/l, 500g, centrifugal 15 minutes of 20～30rpm, the careful white cell ring of drawing at the interface places culture bottle or culture plate, cultivates in containing the DMEM culture medium of 10～20% hyclones.Treat that cell clone grows and 60～75% when converging, the trypsin digestion and cell with 0.25% also goes down to posterity at 1: 2.Changed liquid in per 2～3 days later on, went down to posterity in 1: 2 or 1: 3 when cell 65～70% converges.Collect the 8th～15 generation cell be used for making up.
Embodiment 2
The particulate structure of tissue-engineered bone
With the 8th～15 generation the Os Canitis bone marrow-drived mesenchymal stem make 1～2 * 10
7The cell suspension of/ml drips on the timbering material granule, and material overturning gently, and it is even as far as possible that cell is distributed on all material granule.Cell-composite 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 bioreactor.Rotary speed 15 ~ 50rpm.
Embodiment 3
The particulate body of tissue-engineered bone is implanted into
Pull out A7, B7, C7, D7 after the anesthesia of pentobarbital sodium intravenously administrable, A7 and D7 organize in contrast, directly draw the stitching wound surface over to one's side, do not do filling reparation; B7 and C7 implant corresponding above-mentioned autogenous cell-composite body as experimental group, and each tooth socket is implanted volume to be as the criterion with normally the alveolar bone height is concordant on every side.
The result: postoperative was drawn materials after 3 months, and gross examination of skeletal muscle is found experimental group tooth socket apparent height with the normal teeth land height is approaching on every side, and matched group tooth socket apparent height is starkly lower than alveolar ridge level on every side.The X skiagram shows that the experimental group tooth socket is the high density shadow, and its high point is with alveolar bone is concordant on every side, and the matched group tooth socket only has part high density shadow, and it highly is starkly lower than normal alveolar bone on every side.Histology finds that cambium is an osseous tissue in the experimental group tooth socket, and matched group has a little osseous tissue to form in the tooth socket bottom, and the surface has thicker fibrous connective tissue to cover.This experimental result confirms the tissue-engineered bone grain packing tooth socket that adopts the present invention to make up can effectively keep the having tooth pulled out surface of bone height of back tooth socket, helps the plantation in later stage, repairing and treating such as have a denture made.
Claims (8)
1. the manufacture method of a tissue-engineered bone is characterized in that with the mesenchymal stem cells MSCs being the compound structure tissue-engineered bone of seed cell and graininess Corallium Japonicum Kishinouye-hydroxyapatite.
2. the mesenchymal stem cells MSCs described in the claim 1 is an autologous bone marrow mesenchymal stem cells, adopts density to separate acquisition with the percol of 1073g/l, and cultured and amplified in vitro collecting cell after 5～10 generations is used for making up.The cultivation algebraically defective vision of cell size and deciding.
3. the timbering material described in the claim 1 is Corallium Japonicum Kishinouye-hydroxyapatite porous material.The basis of this material---calcium phosphate and hydroxyapatite also are the main inorganic components of natural bone tissue, therefore have good biocompatibility and Mechanics of Machinery characteristic.
4. the Corallium Japonicum Kishinouye described in the claim 1-hydroxyapatite timbering material has porous micro structure, has both improved the mechanical property of material, has increased the growing into property of cell again, helps the formation of freshman bone tissue.Be prefabricated into the graininess of different size in processing, particulate diameter can be between 1～5mm, and the aperture can be between 200 μ～500 μ, and porosity can be between 35～55%.
5. the construction method described in the claim 1 is the application organizes engineering, and mesenchymal stem cells MSCs and coral hydroxyapatite granule were left standstill earlier 6～8 hours after compound, makes cell climb into material internal and is attached to material cavity inner surface; In reactor, hatched again 48 hours.
6. hatching described in the claim 5, its condition is that complex is mixed with the DMEM culture medium that contains 10～20% hyclones, places reactor, reactor places CO
2In the cell culture incubator, rotary speed is 15～50rpm.
7. the reactor described in the claim 5 can be rotary bioreactor, also can be other similar dynamic solid culture apparatus.
8. can to repair the lacuna bone damaged for the described tissue-engineered bone of claim 1, it can be the tooth socket that stays after the exodontia, or femur head necrosis but articular surface does not subside as yet, can also be go up and the hole tumor resection after the bone lacuna that stays, also can be the bone lacuna that other reasons such as wound or disease cause.Constructed tissue-engineered bone can be clogged the damaged lacuna of these bones, realizes functional therapeutic.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA031479162A CN1565644A (en) | 2003-06-27 | 2003-06-27 | Manufacturing method of tissue engineered bone |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA031479162A CN1565644A (en) | 2003-06-27 | 2003-06-27 | Manufacturing method of tissue engineered bone |
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| CN1565644A true CN1565644A (en) | 2005-01-19 |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2008071074A1 (en) * | 2006-12-13 | 2008-06-19 | Songling Wang | The use of mesenchymal stem cells and the separating and preserving method of stem cells from human tissues |
| CN1990054B (en) * | 2005-12-27 | 2011-09-07 | 上海国睿生命科技有限公司 | Method for constructing cartilage by inducing human bone marrow stroma stem cell in vitro |
| CN101564555B (en) * | 2009-05-27 | 2013-01-23 | 深圳市第二人民医院 | Tissue engineering bone implant and method for constructing the same |
| CN103384535A (en) * | 2010-11-25 | 2013-11-06 | 可乐丽股份有限公司 | Method for producing implant material |
| CN104874023A (en) * | 2015-05-29 | 2015-09-02 | 山东大学齐鲁医院 | Production method of finished-product tissue engineered bone |
| CN106110395A (en) * | 2016-08-18 | 2016-11-16 | 陕西东望科贸有限公司 | A kind of bone repairing support with selfreparing, antibiotic effect and preparation method thereof |
-
2003
- 2003-06-27 CN CNA031479162A patent/CN1565644A/en active Pending
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1990054B (en) * | 2005-12-27 | 2011-09-07 | 上海国睿生命科技有限公司 | Method for constructing cartilage by inducing human bone marrow stroma stem cell in vitro |
| WO2008071074A1 (en) * | 2006-12-13 | 2008-06-19 | Songling Wang | The use of mesenchymal stem cells and the separating and preserving method of stem cells from human tissues |
| CN101564555B (en) * | 2009-05-27 | 2013-01-23 | 深圳市第二人民医院 | Tissue engineering bone implant and method for constructing the same |
| CN103384535A (en) * | 2010-11-25 | 2013-11-06 | 可乐丽股份有限公司 | Method for producing implant material |
| CN103384535B (en) * | 2010-11-25 | 2015-02-11 | 可乐丽股份有限公司 | Method for producing implant material |
| US9782518B2 (en) | 2010-11-25 | 2017-10-10 | Kuraray Co., Ltd. | Method for producing implant material |
| CN104874023A (en) * | 2015-05-29 | 2015-09-02 | 山东大学齐鲁医院 | Production method of finished-product tissue engineered bone |
| CN106110395A (en) * | 2016-08-18 | 2016-11-16 | 陕西东望科贸有限公司 | A kind of bone repairing support with selfreparing, antibiotic effect and preparation method thereof |
| CN106110395B (en) * | 2016-08-18 | 2022-08-02 | 陕西东望科技有限公司 | Bone repair support with self-repairing and antibacterial effects and manufacturing method thereof |
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