Preparation method of three-dimensional bone-like tissue used for study of microgravity effect stimulation

A simulating microgravity, three-dimensional technology, applied to animal cells, etc., can solve the problems of not truly reflecting the biological characteristics of cells, bone loss in two-dimensional osteoblast model space, etc.

Inactive Publication Date: 2011-10-05
HARBIN INST OF TECH
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  • Application Information

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Problems solved by technology

[0004] The present invention aims to solve the problem that the current two-dimensional osteoblast model for simulating microgravity effects cannot truly reflect the process of bone loss in space, and cannot truly reflect the intercellular biological characteristics of in vivo tissues. Preparation method of three-dimensional osteoid tissue under microgravity effect

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  • Preparation method of three-dimensional bone-like tissue used for study of microgravity effect stimulation
  • Preparation method of three-dimensional bone-like tissue used for study of microgravity effect stimulation
  • Preparation method of three-dimensional bone-like tissue used for study of microgravity effect stimulation

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specific Embodiment approach 1

[0010] Specific Embodiment 1: This embodiment is used to study the preparation method of three-dimensional osteoid tissue that simulates microgravity effects, and proceeds as follows: 1. Dissolve gelatin in a 37°C incubator, and then dissolve 0.1mol / L PBS Buffer solution or physiological saline with a mass concentration of 0.85% to 0.9% is mixed with sodium alginate, dissolved gelatin and hydroxyapatite to obtain a mixed solution, and the mass concentration of sodium alginate in the mixed solution is 1% to 5%, gelatin The mass concentration of hydroxyapatite is 0.5% to 5%, and the mass concentration of hydroxyapatite is 0.01% to 0.1%; 2. Induction of osteoblasts; 3. Induction of osteoclasts; 4. The induction of osteoblasts and Osteoclasts were mixed with the mixed solution prepared in step 1 to make a cell density of 1×10 6 ~5×10 6 cells / mL mixed cell suspension, use a needle to add the mixed cell suspension dropwise into 25-100mmol / L divalent positive ion solution, and let i...

specific Embodiment approach 2

[0012] Specific embodiment two: the difference between this embodiment and specific embodiment one is that the induction method of osteoblasts in step two is carried out according to the following steps: add 50mmol / L ascorbic acid, 1mol / L ascorbic acid to the α-MEM medium β-glycerophosphate and 1 mmol / L dexamethasone, so that the final concentration of ascorbic acid in the α-MEM medium is 0.05 mmol / L, the final concentration of β-glycerophosphate is 10 mmol / L, and the final concentration of dexamethasone is 1×10 -7 mol / L, the induction medium of MC3T3-E1 cells is obtained, and the induction medium is used to culture MC3T3-E1 cells for 5-10 days to complete the induction of osteoblasts. Others are the same as in the first embodiment.

specific Embodiment approach 3

[0013] Specific embodiment three: the difference between this embodiment and specific embodiment one is: the induction method of osteoclasts in step three is carried out according to the following steps: the mouse monocyte / macrophage cell line RAW264.7 is inoculated in 6-well culture On the plate, use α-MEM full culture medium at 37°C with a volume fraction of 5% CO 2 cultured in a cell culture box, replaced with osteoclast culture medium after 1 day, and then replaced with osteoclast culture medium every 3 days, cultured for 1 to 2 weeks, and the induction of osteoclasts was completed; the α-MEM Containing the HEPES damping fluid that volume fraction is 15% fetal bovine serum (FBS), the penicillin of 100U / mL, the streptomycin of 100mg / L and 0.01mol / L in the whole culture fluid, described osteoclast culture fluid is α-MEM medium containing 30ng / mL macrophage colony-stimulating factor (MCSF) and 50ng / mL osteoclast differentiation factor (RANKL). Others are the same as in the f...

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Abstract

A preparation method of a three-dimensional bone-like tissue used for the study of microgravity effect stimulation relates to a preparation method of a three-dimensional bone-like tissue. The invention is to solve the problem that two-dimensional osteoblast models used for the study of microgravity effect stimulation at present can not truly represent the process of bone loss during spaceflight, and can not truly represent intercellular biological characteristics of in-vivo tissues. The method comprises the following steps of: 1, the mixing of a PBS buffer or normal saline, sodium alginate, gelatin and hydroxyapatite; 2, the induction of osteoblasts; 3, the induction of osteoclasts; 4, the preparation of microspheres for enwrapping the mixed cells; 5, the formation of the three-dimensional bone-like tissue. The invention forms a three-dimensional stent by sodium alginate, and embeds osteoblasts and osteoclasts together in a sodium alginate microsphere; the three-dimensional culture can simulate the growth condition of cells in a physiological state better, which solves the deficiency of existing two-dimensional osteoblast models. The invention is applicable to research fields of space biology, aerospace medicine and tissue engineering.

Description

technical field [0001] The invention relates to a method for preparing a three-dimensional bone-like tissue. Background technique [0002] The outer space environment has the characteristics of microgravity, ultra-low temperature, high vacuum and strong radiation. Among them, microgravity has a significant impact on human physiological functions. Aerospace medical researchers at home and abroad have conducted a large number of medical observations and studies on the influence of microgravity on human body functions, and found that in the space flight environment, the bone mass of astronauts decreases by 1% per month, and it cannot be fully recovered after returning to the earth. Therefore, it is of great significance to study the molecular mechanism of bone loss caused by weightlessness for the protection of astronauts. [0003] At present, most studies use two-dimensional osteoblasts as a model to study the mechanism of bone loss. This model has the following defects: Firs...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/07
Inventor 田维明李钰郑红霞
Owner HARBIN INST OF TECH
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