CN101642588A - Injectable tissue engineering carrier material and construction method thereof - Google Patents
Injectable tissue engineering carrier material and construction method thereof Download PDFInfo
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- CN101642588A CN101642588A CN200810134885A CN200810134885A CN101642588A CN 101642588 A CN101642588 A CN 101642588A CN 200810134885 A CN200810134885 A CN 200810134885A CN 200810134885 A CN200810134885 A CN 200810134885A CN 101642588 A CN101642588 A CN 101642588A
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- tissue engineering
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Abstract
The invention belongs to the technical field of preparing artificial organs by using a tissue engineering method in a biomedical engineering, and particularly relates to a tissue engineering repairingmaterial and a construction method thereof. The tissue engineering repairing material uses a biodegradable material of which the initial state is liquid state, and a sodium alginate-gelatin blendingsystem as a carrier bracket to which seed cells are attached so as to form a complex with a three-dimensional structure and biological activity. The invention also provides the construction method foran injectable tissue engineering bone repairing material. In vitro and in vivo experiments prove that the sodium alginate-gelatin blending system and osteoplasts have good compatibility, and the tissue engineering repairing material is a practicable injectable tissue engineering bracket material. The injectable tissue engineering bone constructed by the blending system has a wide application prospect.
Description
Technical field
The invention belongs to the technical field for preparing artificial organ in the biomedical engineering with Method of Tissue Engineering, specifically relate to a kind of tissue carrier material and construction method thereof.
Background technology
The Injectable tissue engineering bone is to utilize the degradable biomaterial of original state for liquid state, organic compound with seed cell, be prepared into liquid injectable tissue engineered bone, and in the damaged process of complete filling bone, form gel or solid-state tissue engineered bone repairing bone defect.Injectable bone is easy to moulding, compound convenient with seed cell, somatomedin, and have tissue injury little, do not destroy and repair district's blood for, advantage such as easy to operation, adapted to the requirement of Minimally Invasive Surgical Technology development, therefore just progressively cause showing great attention to of domestic and international many scholars.
Summary of the invention
The object of the present invention is to provide a kind of injection type tissue engineering carrier material, this material constitutes for the syringeability material is combined into osteocyte, can be moulding arbitrarily and compound cells is easy, and degraded and absorbed fully within a certain period of time, bone-forming effect is obvious, lower cost.
Another object of the present invention is to provide a kind of construction method of syringeability organizational project bone material.
A kind of injection type tissue engineering carrier material of the present invention, comprise carrier bracket and seed cell, seed cell is attached on the carrier bracket, formation has the complex of tissue three-dimensional structure and physiologically active, with syringeability material sodium alginate-gelatin cross-blend system as the support carrier, the compound seed cell; Described seed cell is an osteoblast.
Described injectable materials sodium alginate-gelatin cross-blend system is 2: 3 for the mass ratio with sodium alginate and gelatin, and compound concentration is the sodium alginate of 50g/L and the co-mixing system of gelatin.
Described sodium alginate is to use Ca
2+Handle the sodium alginate of organic network structure of back formation as cross-linking agent.
Described osteoblast be bone marrow stroma stem cell through inducing differentiation, enrichment culture to 10 goes down to posterity
7The osteoblast of the order of magnitude.
The construction method of injectable tissue engineering bone renovation material of the present invention may further comprise the steps:
A. seed cell: extract bone marrow, separate bone marrow stroma stem cell with density, used culture medium is the MEM culture fluid of 10% hyclone.The preparation of osteogenic induction cell culture fluid: MEM culture fluid+10%FBS+50mg/LVitC+10
-8Mol/L dexamethasone+10mmol/L sodium.Cell is 10 through the propagation that goes down to posterity
7The order of magnitude.
B. the structure of syringeability timbering material: with MEM is solvent, is 2: 3 with the mass ratio of sodium alginate and gelatin, and compound concentration is the sodium alginate of 50g/L and the co-mixing system of gelatin.
C. seed cell and sodium alginate-gelatin cross-blend system is compound: seed cell is added in the co-mixing system of step B structure, promptly obtain described syringeability tissue engineering bone renovation material.
In the preparation method of described injection type tissue engineering bone renovation material, preferred sodium alginate is to use Ca
2+Handle the sodium alginate of organic network structure of back formation as cross-linking agent.
Sodium alginate is a kind of natural polysaccharide polymer that extracts from Sargassum, its molecular weight height, is line style, has hyperhydrated.Therefore its water soluble salt just has high viscosity when low concentration, is good thickening, suspending agent.Contain a large amount of hydroxyls and carboxyl on its strand, at divalent ion such as Ca
2+Under the condition that exists, will form crosslinked calcium alginate polymer, show as the high gel state of moisture content.A large amount of Cell Biology Experiments prove, the compatibility of itself and cell is fine, has good mechanical strength, is convenient to be shaped.
Gelatin is a kind of denatured protein, is obtained by the connective tissue of animal or the collagen partial hydrolysis in the epidermal tissue, is the abundant natural macromolecular material in source.Gelatin is colourless or faint yellow transparent, translucent thin slice or microgranule, can long term storage under drying regime.Many good physics and chemical property such as possess hydrophilic property is strong, good film-forming property, side-chain radical reactivity height, typical ampholyte feature.Can be dissolved in hot water, be frozen into gelling material after the cooling.Have thermal reversibility, mechanical strength is relatively poor relatively.Simultaneously, be easy to vivo degradation.
Sodium alginate and gelatin all are independent uses in application in the past usually, so all not satisfactory at the aspect of performances such as elasticity, mechanical strength, processing characteristics and moisture content of material.This research is the degradation property and the high-moisture percentage of gelatin, with the good crosslinked forming property of sodium alginate and crosslinked after higher mechanical strength combine, thereby obtained the better composite of a kind of combination property.
The present invention result has by experiment confirmed that sodium alginate-gelatin cross-blend system and the osteoblastic compatibility are good, and zoopery has proved that bone formation performance is definite, is a kind of feasible syringeability engineering carrier timbering material.The syringeability engineering carrier that this composite makes up has broad clinical application prospect.
Description of drawings
Fig. 1 is fine and close island structure for conditioned medium 12d cell, forms calcification tuberosity (inverted microscope 20 * 10);
After cultivating 2w in Fig. 2 gel, can be observed just at splitted cell (inverted microscope 20 * 10);
Show as the resistance projection under the CT during Fig. 3 subcutaneous implantation experimental group 12w;
See that osteoid tissue is arranged in the tuberosity during Fig. 4 subcutaneous implantation experimental group 12w, and have the medullary cavity spline structure to form.(Mallory dyeing 10 * 10);
Fig. 5 skull is repaired experimental group 12w three dimensional CT and is rebuild and see that defect of skull repairs substantially;
Fig. 6 skull is repaired the ripe osseous tissue of experimental group 12w, Haversian system maturation (HE dyeing 10 * 10).
The specific embodiment
Syringeability organizational project repair materials of the present invention comprises carrier bracket and seed cell, and seed cell is attached on the carrier bracket, forms the complex with three dimensional structure and physiologically active.Its characteristic is: as carrier bracket, the compound seed cell: described seed cell is an osteoblast with injectable sodium alginate-gelatin cross-blend system.
Described injectable sodium alginate-gelatin cross-blend system is 2: 3 as the mass ratio with sodium alginate and gelatin, and compound concentration is the sodium alginate of 50g/L and the co-mixing system of gelatin.
Described sodium alginate is to use Ca
2+Handle the sodium alginate of organic network structure of back formation as cross-linking agent.
Described osteoblast be bone marrow stroma stem cell through inducing differentiation, enrichment culture to 10 goes down to posterity
7The osteoblast of the order of magnitude.
Embodiment one: the cultivation of seed cell
The purebred White Rabbit of two monthly ages cleaning level New Zealand (body weight 2.0～2.2kg, plant provides by the prosperous laboratory animal of Haidian District Beijing).After the anesthesia of 5% pentobarbital sodium 0.6ml/kg auricular vein, extract bone marrow fluid 2～3ml with marrow puncture needle.With bone marrow and the equal-volume PBS mixing of getting ready, density gradient centrifugation is drawn the milky cloud nucleated cell layer between Percoll separating medium and D-Hank ' the s liquid.Blow and beat in the cell adding MEM culture medium with gained, regulating cell density is 2 * 10
5Be inoculated in the plastic culture bottle, put 37 ℃, 5%CO
2Cell culture incubator in cultivate, when cell reaches 80～90% when merging, the cultivation of going down to posterity.Cell suspension is moved in the centrifuge tube MEM liquid washing 2 times, the centrifugal 5min of rotating speed 500r/min.Pat centrifugal cell mass sheet and make it loose, add MEM, with 5 * 10
4/ ml is inoculated in the culture plate of disposable culture bottle or experiment usefulness, adds conditioned medium (MEM culture fluid+10%FBS+50mg/LVitC+10
-8Mol/L dexamethasone+10mmol/L sodium) carries out continuous passage and observe and test (Fig. 1).
Embodiment two: seed cell and co-mixing system compound
As solvent, compound concentration is that the mass ratio of the sodium alginate of 50g/L and gelatin is 2: 3 a blend solution with MEM.25 ℃ of constant temperature lower magnetic forces stir 0.5h, leave standstill 0.5h after low-speed centrifugal so that minute bubbles disappear, obtain the red colloidal liquid of transparent powder.With the osteoblast among the embodiment one,, expect the survival rate of blue dyeing check cell with platform, greater than 90% with becoming suspension cell behind 0.25% trypsinization.Mix with sodium alginate-gelatin cross-blend system, the whole density of cell is about 1 * 10
7/ ml injects 0.6%CaCl with co-mixing system with disposable syringe
2In the solution, form gel.Normal saline washing 3 times is after culture fluid washs 1 time.Gel is positioned in the culture dish cultivates, the situation of observation of cell growth under the inverted microscope.Respectively at 1d, draw materials behind the 2w, observe by frozen section HE dyeing and understand the metamorphosis of osteoblast in co-mixing system.
Embodiment three: inverted microscope is observed and HE dyeing
The sodium alginate of external structure-gelatin cross-blend system/osteoblast gel places direct observation finding under the inverted microscope, can see that cell all presents circle.Cultivate 2w in gel after, cell still presents normal circular growth conditions, and can observe just at splitted cell (Fig. 2).After in gel, cultivating 2w, the frozen section HE demonstration of dyeing, Oesteoblast growth is good at sodium alginate-gelatin cross-blend system status, and cell is rounded or oval, and karyon is positioned at cell central authorities.
Embodiment four: dystopy skeletonization in the body of co-mixing system
Sodium alginate-gelatin cross-blend system/osteoblast gel 1ml of embodiment two is implanted the subcutaneous of corresponding osteoblast source laboratory animal with injection system.Check respectively at the injection back capable embedding position of 12w thin-layer CT scanning (bed thickness 0.75mm).
Respectively at drawing materials under the 12w local anaesthesia of injection back, immediately with 10% formaldehyde fixed 10h, tissue slice is made in dehydration step by step after drawing materials, and adopts HE dyeing and the dyeing of Mallory three-color process to observe the bone formation situation.
When sodium alginate-gelatin cross-blend system/osteoblast gel experimental group was injected back 12w, tuberosity was hard like the osseous tissue sample, and specimen outward appearance and section are similar to osseous tissue, need the row decalcification handle before the section.Show as resistance projection (Fig. 3) under the CT.Osteoid tissue is arranged in the tuberosity, and have the medullary cavity spline structure to form, the Mallory trichrome stain mainly is shown as the ripe osseous tissue (Fig. 4) of volume maroon.
Embodiment five: it is damaged that co-mixing system is repaired the skull limit
Laboratory animal is respectively with after the anesthesia of 5% pentobarbital sodium 0.6ml/kg auricular vein, and the manufacturing diameter is that the circular holostrome bone of 1.5cm is damaged on the rabbit headbone with being drilled in slowly.1w after the preparation of animal model, under local anaesthesia, experimental group is implanted with injection system in the laboratory animal that the damaged embodiment of sentencing two makes up the corresponding osteoblast of sodium alginate-gelatin cross-blend system/osteoblast gel source, and the reparation skull limit is damaged.
Check at the descending head CT of anesthesia situation crown position thin slice scan (bed thickness 0.75mm) in postoperative 12w, behind the end of scan, initial data is imported the SIEMENS work station, support descending coronal reconstruction and three-dimensional reconstruction at Syngo software.Method with the auricular vein air embolism is put to death laboratory animal at random, and immediately with 10% formaldehyde fixed 10h, tissue slice is made in dehydration step by step after drawing materials, the formation situation that adopts HE dyeing Mallory trichrome stain to observe osseous tissue.
Postoperative 12w experimental group three-dimensional reconstruction is seen damaged basic reparation (Fig. 5).
Matched group and blank group postoperative 4,8, the CT of 12w shows: along with the propelling of time, damaged area slightly dwindles after the operation, but the edge is still clear.
Defect area and surrounding bone tissue form synostosis during 12w, and the difference of newborn skull and former skull has been difficult to identification.Bone trabecula is thick, is sophisticated laminate shape bone, Haversian system maturation (Fig. 6).
Claims (5)
1, a kind of Injectable tissue engineering repair materials, comprise carrier bracket and seed cell, seed cell is attached on the carrier bracket, formation has the complex of three dimensional structure and physiologically active, its characteristic is: as carrier bracket, the compound seed cell: described seed cell is an osteoblast with injectable sodium alginate-gelatin cross-blend system.
2, Injectable tissue engineering repair materials according to claim 1, it is characterized in that: described injectable sodium alginate-gelatin cross-blend system is 2: 3 for the mass ratio with sodium alginate and gelatin, and compound concentration is the sodium alginate of 50g/L and the co-mixing system of gelatin.
3, Injectable tissue engineering repair materials according to claim 2, it is characterized in that: described sodium alginate is to use Ca
2+Handle the sodium alginate of organic network structure of back formation as cross-linking agent.
4, Injectable tissue engineering repair materials according to claim 1 is characterized in that: described osteoblast be bone marrow stroma stem cell through inducing differentiation, enrichment culture to 10 goes down to posterity
7The osteoblast of the order of magnitude.
5, according to the construction method of any described Injectable tissue engineering repair materials of claim 1～4, it is characterized in that, may further comprise the steps:
A) seed cell: extract bone marrow, separate bone marrow stroma stem cell with density, used culture medium is the MEM culture fluid of 10% hyclone.The preparation of osteogenic induction cell culture fluid: MEM culture fluid+10%FBS+50mg/LVitC+10
-8Mol/L dexamethasone+10mmol/L sodium.Cell is 10 through the propagation that goes down to posterity
7The order of magnitude;
B) structure of syringeability timbering material: with MEM is solvent, is 2: 3 with the mass ratio of sodium alginate and gelatin, and compound concentration is the sodium alginate of 50g/L and the co-mixing system of gelatin;
C) seed cell and sodium alginate-gelatin cross-blend system is compound: seed cell is added in the co-mixing system of step B structure, promptly obtain described syringeability tissue engineering bone renovation material.
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101934090A (en) * | 2010-08-25 | 2011-01-05 | 深圳清华大学研究院 | Injectable bone repairing material and preparation method thereof |
| CN102178984A (en) * | 2011-04-25 | 2011-09-14 | 哈尔滨工业大学 | Injectable gel material of sodium alga acid-protein adhesive used for treating myocardial infarction and preparation method of injectable gel material |
| CN107847646A (en) * | 2015-05-14 | 2018-03-27 | 用于组织工程和细胞为基础的技术和疗法促进协会 | A kind of ureter bracket, method and its purposes |
| CN109010926A (en) * | 2018-08-01 | 2018-12-18 | 北京大学 | A kind of preparation method and its compound system of porous micro rack |
| JP2019512303A (en) * | 2016-03-11 | 2019-05-16 | アソシエーション フォー ジ アドバンスメント オブ ティシュー エンジニアリング アンド セル ベイスト テクノロジーズ アンド セラピーズ（エイ４テック）− アソシアソンＡｓｓｏｃｉａｔｉｏｎ Ｆｏｒ Ｔｈｅ Ａｄｖａｎｃｅｍｅｎｔ Ｏｆ Ｔｉｓｓｕｅ Ｅｎｇｉｎｅｅｒｉｎｇ Ａｎｄ Ｃｅｌｌ Ｂａｓｅｄ Ｔｅｃｈｎｏｌｏｇｉｅｓ ＆ Ｔｈｅｒａｐｉｅｓ （Ａ４Ｔｅｃ） − Ａｓｓｏｃｉａｃａｏ | Biodegradable ureteral stents, methods and uses thereof |
| CN109881300A (en) * | 2019-02-18 | 2019-06-14 | 华侨大学 | A kind of preparation method and applications of injectable type microfibre |
| CN109908404A (en) * | 2019-04-11 | 2019-06-21 | 杭州淡滨尼生物科技有限公司 | Injectable tissue engineering bone renovating material and its construction method |
| CN110478532A (en) * | 2019-08-22 | 2019-11-22 | 上海交通大学医学院附属第九人民医院 | Raw hole aquogel system of a kind of Injectable in-situ and its preparation method and application |
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| CN1853584A (en) * | 2005-04-18 | 2006-11-01 | 南方医院 | Tissue engineering bone containing innervation and its construction |
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Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101934090A (en) * | 2010-08-25 | 2011-01-05 | 深圳清华大学研究院 | Injectable bone repairing material and preparation method thereof |
| CN102178984A (en) * | 2011-04-25 | 2011-09-14 | 哈尔滨工业大学 | Injectable gel material of sodium alga acid-protein adhesive used for treating myocardial infarction and preparation method of injectable gel material |
| CN102178984B (en) * | 2011-04-25 | 2013-09-04 | 哈尔滨工业大学 | Preparation method of injectable gel material of sodium alga acid-protein adhesive used for treating myocardial infarction |
| CN107847646A (en) * | 2015-05-14 | 2018-03-27 | 用于组织工程和细胞为基础的技术和疗法促进协会 | A kind of ureter bracket, method and its purposes |
| JP2019512303A (en) * | 2016-03-11 | 2019-05-16 | アソシエーション フォー ジ アドバンスメント オブ ティシュー エンジニアリング アンド セル ベイスト テクノロジーズ アンド セラピーズ（エイ４テック）− アソシアソンＡｓｓｏｃｉａｔｉｏｎ Ｆｏｒ Ｔｈｅ Ａｄｖａｎｃｅｍｅｎｔ Ｏｆ Ｔｉｓｓｕｅ Ｅｎｇｉｎｅｅｒｉｎｇ Ａｎｄ Ｃｅｌｌ Ｂａｓｅｄ Ｔｅｃｈｎｏｌｏｇｉｅｓ ＆ Ｔｈｅｒａｐｉｅｓ （Ａ４Ｔｅｃ） − Ａｓｓｏｃｉａｃａｏ | Biodegradable ureteral stents, methods and uses thereof |
| CN109010926A (en) * | 2018-08-01 | 2018-12-18 | 北京大学 | A kind of preparation method and its compound system of porous micro rack |
| CN109010926B (en) * | 2018-08-01 | 2019-08-13 | 北京大学 | A kind of preparation method and its compound system of porous micro rack |
| CN109881300A (en) * | 2019-02-18 | 2019-06-14 | 华侨大学 | A kind of preparation method and applications of injectable type microfibre |
| CN109881300B (en) * | 2019-02-18 | 2021-07-30 | 华侨大学 | Preparation method and application of injectable microfiber |
| CN109908404A (en) * | 2019-04-11 | 2019-06-21 | 杭州淡滨尼生物科技有限公司 | Injectable tissue engineering bone renovating material and its construction method |
| CN110478532A (en) * | 2019-08-22 | 2019-11-22 | 上海交通大学医学院附属第九人民医院 | Raw hole aquogel system of a kind of Injectable in-situ and its preparation method and application |
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Application publication date: 20100210 |