CN1923298A - Injection type tissue engineering bone renovation material and construct method thereof - Google Patents
Injection type tissue engineering bone renovation material and construct method thereof Download PDFInfo
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- CN1923298A CN1923298A CN 200510036968 CN200510036968A CN1923298A CN 1923298 A CN1923298 A CN 1923298A CN 200510036968 CN200510036968 CN 200510036968 CN 200510036968 A CN200510036968 A CN 200510036968A CN 1923298 A CN1923298 A CN 1923298A
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Abstract
The invention relates to an injection organism repair material and relative production. Wherein, it comprises carrier support and seed cell adhered on the carrier support to form skeleton three-dimension structure and biological active composite; and it uses the injection chitose-beta-tricalcium phosphate composite as carrier support to couple seed cell; the seed cell is bone marrow substrate stem cell. The test has proved that the chitose-beta-tricalcium phosphate composite has better compatibility with BMSCs.
Description
Technical field
The invention belongs in the biomedical engineering and prepare the artificial organ technical field, specifically relate to a kind of injection type tissue engineering bone renovation material and construction method thereof with Method of Tissue Engineering.
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, progressively solidify, 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.At present, the material that is used for injectable bone mostly is gel-like, mainly comprise fibrin gel, the polyethylene glycol oxide hydrogel, alginate jellies etc., these biomaterials are gel after injecting in the body, do not have biomechanical strength, though certain skeletonization effect is all arranged after the compound seed cell implants, also has suitable distance apart from ideal injectable bone timbering material.
Summary of the invention
The object of the present invention is to provide a kind of injection type tissue engineering bone renovation material, this bone renovating material is that the compound bone marrow stroma stem cell of syringeability material constitutes, can be moulding arbitrarily and compound cells is easy, and degraded and absorbed fully within a certain period of time, the induced osteogenesis effect is obvious, lower cost.
A kind of injection type tissue engineering bone renovation material of the present invention, comprise carrier bracket and seed cell, seed cell is attached on the carrier bracket, formation has the complex of osseous tissue three dimensional structure and physiologically active, with syringeability material chitosan-bata-tricalcium phosphate complex as carrier bracket, the compound seed cell; Described seed cell is a bone marrow stroma stem cell.
Described injectable materials chitosan-bata-tricalcium phosphate is preferably chitosan and bata-tricalcium phosphate in the ratio of 1ml: 0.6g in the external chitosan that is compounded to form-bata-tricalcium phosphate complex.
Described chitosan is preferably handled the chitosan that the back forms organic network structure with citric acid as cross-linking agent.
Described bone marrow stroma stem cell is preferably through the enrichment culture that goes down to posterity to the third generation 10
7The bone marrow stroma stem cell of the order of magnitude.
Another object of the present invention is to provide a kind of construction method of injection type tissue engineering bone renovation material.
The construction method of injection type tissue engineering bone renovation material of the present invention may further comprise the steps:
A. seed cell: extract bone marrow, with full bone marrow culture method separation and Culture bone marrow stroma stem cell, used culture medium is the DMEM culture medium that contains 10% hyclone, cell through the enrichment culture that goes down to posterity to the third generation 10
7The order of magnitude;
B. the preparation of syringeability timbering material: with chitosan and bata-tricalcium phosphate in the ratio of 1ml: 0.6g in the external chitosan-bata-tricalcium phosphate complex that is compounded to form;
C. the processing of seed cell: will be cultured to the third generation 10
7The bone marrow stroma stem cell of the order of magnitude DMEM mixing of 0.3ml;
D. seed cell and chitosan-bata-tricalcium phosphate is compound: chitosan-bata-tricalcium phosphate adds the seed cell of step C immediately after compound 8 minutes, the addition ratio is that chitosan, bata-tricalcium phosphate, DMEM are by 3ml: 1.8g: 0.3ml, fully mixing promptly obtains described injection type tissue engineering bone renovation material.
In the preparation method of described injection type tissue engineering bone renovation material, preferred chitosan is to handle the chitosan that the back forms organic network structure with citric acid as cross-linking agent.
Chitosan of the present invention (chitosan) is the derivant behind the chitin part deacetylation, is natural polymers, has favorable biological degradability, and catabolite has no side effect.(β-TCP) belong to the Ca-P ceramic class has the chemical analysis similar with natural bone, excellent biological compatibility and biological degradability to bata-tricalcium phosphate of the present invention.The present invention is composited both and is comparatively ideal tissue engineered bone syringeability timbering material.
Be glutaraldehyde (Yin Y.Ye F to the chitosan crosslinked cross-linking agent that adopts in the past, Cui J, Zhang F, Li X, Yao K.Preparation and characterization of macroporous chitosan-gelatin/beta-tricalcium phosphatecomposite scaffolds for bone tissue engineering.J Biomed Mater Res A, 2003,67 (3): 844-855.), but glutaraldehyde has been proved cytotoxicity, so adopt the cross-linking agent of citric acid as chitosan among the present invention.Citric acid is the important component part of people's organic matrix, accounts for 70% of whole body, and it plays an important role to sugar, fat, proteinic oxidative metabolism process.Owing to contain carboxyl in the citric acid, can with the hydroxyl generation esterification in the chitosan, unreacted carboxyl will form sat linkage with the amino effect in the chitosan.By this key, make the organic network structure of chitosan crosslinked formation.
It is organic facies that the present invention selects chitosan for use, β-TCP is inorganic phase, after β--TP and chitosan are compound, be dispersed in organic network structure, it is liquid that complex is initially, and by ashamed curing, forms solid-state, porous network structure, and have certain biomechanical strength, thereby prepared the compound injectable materials of a kind of novel organic/inorganic.
The The key factor that injectable bone makes up is a cell with after injection material mixes, cell can be in injection bone three-dimensional rack growth and breeding, whether cell and material have excellent biological compatibility.It is the appropriate proportioning (chitosan of optimizing: β-TCP=1ml: 0.6g) that the present invention adopts 3ml chitosan and 1.8g β-TCP compound, 3ml chitosan and 1.8g β-TCP compound evenly after, be initially liquid, be solidified as solid-stately by ashamed behind 12～15min, and have certain biomechanical strength.On this basis, the present invention has verified that compound BMSCs is as the feasibility of injectable bone and the biocompatibility of material and cell when composite is liquid state.Find to add cultivate in the experiment base unit weight too much (＞0.6ml) will obviously prolong the hardening time of material, so the inventor select the 0.3ml culture medium and centrifugal after the material of BMSCs when making behind the cell suspension with liquid state compound.In the experiment, the observation of inventor under inverted microscope finds that after chitosan-bata-tricalcium phosphate timbering material and the compound cultivation of BMSCs, cell is grown, molecular marker for increased proliferation, and form is normal.Mtt assay is compared ability of cell proliferation unaffected (P＞0.05) after detecting and finding chitosan-bata-tricalcium phosphate timbering material and the compound cultivation of BMSCs with matched group.BMSCs is blended in the liquid material, and behind material forming, the BMSCs of Electron microscope showed material surface and material attach good, and divide, breed, the secretion bone matrix.
The present invention result has by experiment confirmed that the compatibility of chitosan-bata-tricalcium phosphate compound support frame material and BMSCs is good, is a kind of feasible Injectable tissue engineering bone holder material.The injectable tissue engineered bone that makes up with this composite has broad clinical application prospect.
Description of drawings
Fig. 1 is that the 6th day inverted microscope of the compound cultivation of blank group BMSCs observed (* 100) photo.
Fig. 2 is that chitosan-β-TCP and the 6th day inverted microscope of the compound cultivation of BMSCs are observed (* 100) photo.
Fig. 3 is that chitosan-β-TCP solidifies back structure scanning electron microscopic observation (* 5000) photo.
Fig. 4 is chitosan-β-TCP and the 3rd day scanning electron microscopic observation (* 600) of the compound cultivation of BMSCs photo.
Fig. 5 is chitosan-β-TCP and the 6th day scanning electron microscopic observation (* 600) of the compound cultivation of BMSCs photo.
Fig. 6 is chitosan-β-TCP and the 6th day scanning electron microscopic observation (* 2000) of the compound cultivation of BMSCs photo.
The specific embodiment
A kind of injection type tissue engineering bone renovation material of the present invention, comprise carrier bracket and seed cell, seed cell is attached on the carrier bracket, formation has the complex of osseous tissue three dimensional structure and physiologically active, with syringeability material chitosan-bata-tricalcium phosphate complex as carrier bracket, the compound seed cell; Described seed cell is a bone marrow stroma stem cell.
Described injectable materials chitosan-bata-tricalcium phosphate is preferably chitosan and bata-tricalcium phosphate in the ratio of 1ml: 0.6g in the external chitosan that is compounded to form-bata-tricalcium phosphate complex.
Described chitosan is preferably handled the chitosan that the back forms organic network structure with citric acid as cross-linking agent.
Described bone marrow stroma stem cell is preferably through the enrichment culture that goes down to posterity to the third generation 10
7The bone marrow stroma stem cell of the order of magnitude.
Embodiment one: the cultivation of seed cell
Get healthy Chinese QINGSHANYANG (body weight 35.0～40kg of 5 monthly ages, male and female are not limit, provide by Nanfang Medical Univ zoopery center), sterile working, anesthesia back is down with No. 16 capable ilium punctures of medullo-puncture needle, bone marrow 10mL is extracted in the multiple spot puncture, centrifugal and filter the back inoculation with 100 order steel meshes and go in the culture bottle, add the high sugared DMEM culture medium (U.S. Hyclone company) that contains 10% hyclone (U.S. Hyclone company), place CO
2Cultivate in the incubator (U.S. Harris company), changed liquid in per 3 days, at the bottom for the treatment of that primary cell covers with bottle, with 0.25% pancreatin-0.02%DMEM liquid attached cell digestion is separated, add conditioned medium (DMEM+10% hyclone+dexamethasone 10-8mol/L+ sodium 10mmol/L+ ascorbic acid 50mg/L), put CO
2Incubator continue to be cultivated, and changes liquid in per 2～3 days.
Embodiment two: seed cell cell and injectable materials compound
The chitosan that is adopted in the experiment is a liquid material, bata-tricalcium phosphate (β-TCP) is solid-state material (Materials Science and Engineering system of Ji'nan University provides), with two kinds of material mixing evenly after, be initially liquid, be solidified as solid-state behind 12～15min gradually.This experiment adopts β-TCP of 3ml chitosan, 1.8g in external compound, treat solidified forming after, become 4mm * 4mm * 3mm size standby with the aseptic operation blade cuts.Get the 3rd generation BMSCs (planting density 2 * 10
4/ hole) be inoculated in 24 well culture plates, every hole adds culture medium 1ml, and experiment is divided into 2 groups, i.e. material group and blank group.Wherein the every hole of experimental group adds the chitosan-β-TCP composite and the mixing with cells cultivation of 4mm * 4mm * 3mm fritter, and the simple inoculating cell of matched group is handled in different time, is used for MTT and detects.3ml chitosan, 1.8g β-TCP behind external compound 8min, are got the 3rd generation BMSCs (about 1 * 10
7) add 0.3ml DMEM and make cell suspension, with cell suspension and liquid chitosan-β-TCP mixing, be prepared into BMSCs/ chitosan-β-TCP complex.After treating that material solidification is shaped, get small pieces of material and place 6 orifice plates, add complete culture medium, place CO
2Cultivate in the incubator, be used for scanning electron microscopic observation.
Embodiment three: inverted microscope is observed
Day by day use inverted microscope to observe and respectively organize the cell growing state.
The result: cell inoculation is after 4 hours, and cell has part adherent, and adherent fully after 12 hours, cell is polygon, fusiformis.The cell growth is very fast, and cell clone formed in 2～3 days, converged into monolayer after 8～10 days, material group cell growth similar to matched group (as depicted in figs. 1 and 2), and cellular morphology is normal.
Embodiment four: scanning electron microscopic observation
Take out in the 3rd, 6 day biomaterial compound cells, the PBS rinsing, 2% glutaraldehyde is fixed, series dehydration, tert-butyl alcohol displacement, vacuum drying, the laggard line scanning electron microscopic observation of surperficial metal spraying.
Behind injectable tissue engineering material chitosan-β-external combined shaping of TCP, be the 3 D stereo loose structure under the scanning electron microscope, contain a large amount of holes, micropore irregular arrangement, pore size inequality, the mutual traffic of part hole (as shown in Figure 3).When cell and material mixing were cultivated 3 days, the cell that is blended in material surface can be grown at material surface, mostly is irregular similar round (as shown in Figure 4); When cultivating 7 days, cell quantity obviously increases, and cell space is flat, is fusiformis or polygon, and there is granular substance on the cell space surface, and cell connects in flakes, has a large amount of substrate to form, and cell can stride across pore surface or growth (as Fig. 5 and shown in Figure 6) in the space.
Embodiment five: surviving and multiplication capacity after mtt assay analysis BMSCs and the chitosan-β-compound cultivation of TCP composite
Behind the inoculating cell, abandon former culture medium in the 2nd, 4,6,8 day as stated above, add serum-free DMEM 1ml and MTT (5mg/ml) 100 μ l, 37 ℃ of CO
2Hatched in the incubator 4 hours, and after MTT is removed in careful suction, added dimethyl sulfoxide 0.5ml, vibrated 15 minutes, select wavelength 570nm on the microplate reader, measure absorbance value.
With the prolongation of incubation time, each organizes D
570Increase gradually, the highest by the 8th day, chitosan-β-TCP group is in each time point and matched group there are no significant difference (＞0.05) (table 1).
Table 1 liang group mtt assay mensuration absorbance (n=5 x ± s)
Tab1 absorbance determined by MTT assay in two groups(n＝5， x±s)
| | 2 days | 4 | 6 days | 8 days |
| Matched group chitosan-β-TCP group | 0.120±0.027 0.116±0.021 * | 0.404±0.041 0.372±0.051 * | 0.902±0.097 0.878±0.064 * | 1.197±0.074 1.139±0.094 * |
*P＞0.05vs matched group
Statistical method: the The data SPSS10.0 statistical software that surveys carries out the t check of independent sample, the P value less than 0.05 o'clock for significant difference is arranged.
Claims (6)
1, injection type tissue engineering bone renovation material, comprise carrier bracket and seed cell, seed cell is attached on the carrier bracket, formation has the complex of osseous tissue three dimensional structure and physiologically active, it is characterized in that: with syringeability material chitosan-bata-tricalcium phosphate complex as carrier bracket, the compound seed cell; Described seed cell is a bone marrow stroma stem cell.
2, injection type tissue engineering bone renovation material according to claim 1 is characterized in that: described injectable materials chitosan-bata-tricalcium phosphate for chitosan and bata-tricalcium phosphate in the ratio of 1ml: 0.6g in the external chitosan that is compounded to form-bata-tricalcium phosphate complex.
3, injection type tissue engineering bone renovation material according to claim 2 is characterized in that: described chitosan is to handle the chitosan that the back forms organic network structure with citric acid as cross-linking agent.
4, injection type tissue engineering bone renovation material according to claim 1 is characterized in that: described bone marrow stroma stem cell is through the bone marrow stroma stem cell of enrichment culture to the third generation 107 orders of magnitude that go down to posterity.
5, the construction method of injection type tissue engineering bone renovation material as claimed in claim 1 is characterized in that, may further comprise the steps:
A. seed cell: extract bone marrow, with full bone marrow culture method separation and Culture bone marrow stroma stem cell, used culture medium is the DMEM culture medium that contains 10% hyclone, cell through the enrichment culture that goes down to posterity to the third generation 107 orders of magnitude;
B. the preparation of syringeability timbering material: with chitosan and bata-tricalcium phosphate in the ratio of 1ml: 0.6g in the external chitosan-bata-tricalcium phosphate complex that is compounded to form;
C. the processing of seed cell: will be cultured to the DMEM mixing of the bone marrow stroma stem cell of the third generation 107 orders of magnitude with 0.3ml;
D. seed cell and chitosan-bata-tricalcium phosphate is compound: chitosan-bata-tricalcium phosphate adds the seed cell of step C immediately after compound 8 minutes, the addition ratio is that chitosan, bata-tricalcium phosphate, DMEM are by 3ml: 1.8g: 0.3ml, fully mixing promptly obtains described injection type tissue engineering bone renovation material.
6, the preparation method of injection type tissue engineering bone renovation material according to claim 5 is characterized in that: described chitosan is to handle the chitosan that the back forms organic network structure with citric acid as cross-linking agent.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101469309B (en) * | 2007-12-29 | 2012-05-23 | 上海交通大学医学院附属第九人民医院 | Parallel circulating type cell selective filter recombiner |
| US9180094B2 (en) | 2011-10-12 | 2015-11-10 | The Texas A&M University System | High porosity materials, scaffolds, and method of making |
| US10363215B2 (en) | 2013-11-08 | 2019-07-30 | The Texas A&M University System | Porous microparticles with high loading efficiencies |
| CN115702952A (en) * | 2021-08-10 | 2023-02-17 | 上海交通大学医学院附属第九人民医院 | Injectable bone constructed based on injectable hydrogel scaffold material and application thereof |
-
2005
- 2005-09-02 CN CN200510036968A patent/CN100594946C/en not_active Expired - Fee Related
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101469309B (en) * | 2007-12-29 | 2012-05-23 | 上海交通大学医学院附属第九人民医院 | Parallel circulating type cell selective filter recombiner |
| US9180094B2 (en) | 2011-10-12 | 2015-11-10 | The Texas A&M University System | High porosity materials, scaffolds, and method of making |
| US10363215B2 (en) | 2013-11-08 | 2019-07-30 | The Texas A&M University System | Porous microparticles with high loading efficiencies |
| CN115702952A (en) * | 2021-08-10 | 2023-02-17 | 上海交通大学医学院附属第九人民医院 | Injectable bone constructed based on injectable hydrogel scaffold material and application thereof |
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