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Method for dynamically constructing ADSCs (adipose-derived stem cells)-chitosan/ gelatin hydrogel engineered cartilage

A hydrogel and chitosan technology, applied in medical science, prosthesis, etc., can solve the problems of unsatisfactory effect and incomplete recovery of cartilage tissue, and achieve the effect of avoiding collision, eliminating impact, and improving mass transfer capacity

Inactive Publication Date: 2014-08-06
DALIAN UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although there are many clinical methods for repairing cartilage defects and trauma, the effect is not satisfactory and the function of cartilage tissue cannot be fully restored.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Embodiment 1: Preparation of 4% chitosan / gelatin hydrogel

[0021] 2% chitosan acetic acid solution and gelatin solution were dissolved in an oven at high temperature and centrifuged to defoam, then mixed slowly and evenly. The mass ratio of gelatin to chitosan in the mixed solution was 3:1. The freeze-dried scaffolds were cross-linked in carbodiimide / hydroxysuccinamide / morpholineethanesulfonic acid (EDC / NHS / MES) cross-linking solution for 6 h, and 0.1 mol / L Na 2 HPO 4 The solution neutralized the acetic acid in the bracket for 2 hours, and washed 4 times with 40% ethanol for 30 minutes each time. After pre-freezing in a -20°C refrigerator, transfer to an ultra-low temperature refrigerator for freezing, and then freeze-dry again to obtain a cross-linked chitosan / gelatin hydrogel.

Embodiment 2

[0022] Example 2: Isolation and culture of adipose stem cells and induction of cartilage

[0023] Adipose-derived stem cells were isolated from subcutaneous normal adipose tissue of patients undergoing surgery using a modified method in our laboratory. Combined digestion with 0.25% trypsin and 0.1% collagenase, suck out the liquid containing mononuclear cells in the lower layer, add high-glucose DMEM containing fetal bovine serum to stop the digestion, repeat the digestion of the remaining adipose tissue 2 to 3 times, add the collected cell pellets to the Resuspend the cells in DMEM with 10% fetal bovine serum, transfer to culture flask, 37°C, 5% CO 2 Incubate in an incubator and change the medium every 3 days.

[0024] Take the 7th passage hADSCs, with 1.0×10 5 cells / mL were inoculated in 24-well plates, incubated in a 37°C incubator for 3 hours, and replaced with chondrogenic induction medium (composition: complete medium, 10ng / mL TGF-β1, 40ng / mL ground Semethasone, 50 μg...

Embodiment 3

[0025] Example 3: Construction of cell-hydrogel complexes

[0026] Move the cross-linked hydrogel to a 24-well plate, soak it in 75% ethanol for three times, then soak it in PBS three times and infiltrate it in DMEM medium, air-dry it, and sterilize it by ultraviolet light, then use a 100 μL micropipette P7-hADSCs with 10 7 The cells / ml density was seeded inside 40 hydrogels, and 20 μL was injected from the center of the front and back sides, and then the cell-hydrogel complex was placed at 37°C, 5% CO 2 Cultivate in the incubator for 2 hours, add 1 mL of chondrogenic medium to the well plate, and continue to cultivate. After culturing for 24 hours, transfer 20 of the complexes to the 75cm 2 The other 20 cell-hydrogel complexes were fixed on the platinum wire on the self-made stainless steel frame, and then cultured in the spinner bottle as the dynamic experimental group. Among them, first add 50mL medium to the spinner bottle, stir intermittently at a rate of 20rpm for 3h, ...

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Abstract

The invention belongs to the field of biotechnology and tissue engineering and relates to a method for dynamically constructing an ADSCs (adipose-derived stem cells)-chitosan / gelatin hydrogel engineered cartilage. The method comprises the steps of preparing 4% chitosan / gelatin hydrogel serving as a scaffold of the tissue engineered cartilage, and preparing a cartilage induction matrix; making a round stainless steel tripod according to the diameter of a spinner bottle and the height of a stirring rod, and fixing an inert metal wire on the round stainless steel tripod; soaking the scaffold into 75% alcohol for at least three times, then soaking the scaffold into a PBS (phosphate buffer solution), infiltrating into a DMEM (Dulbecco's modified eagle medium), drying in air, sterilizing, transferring into a porous plate, inoculating the scaffold with adipose stem cells of which the number of culture generations is not more than eight at the density of 10<5>-10<7>cells / ml, and culturing in a 5% CO2 incubator with a temperature of 37 DEG C for not less than 2h to obtain an ADSCs-hydrogel complex. Compared with the engineered cartilage obtained by traditional static culture, the engineered cartilage constructed by the method has many advantages and lays a foundation for repairing cartilage defects in animals.

Description

technical field [0001] The invention belongs to the fields of biotechnology and tissue engineering, in particular to a method for dynamically and stably constructing tissue engineered cartilage. Background technique [0002] The number of cases of osteochondral defects caused by joint degenerative diseases and trauma is increasing, which brings great pain to many patients. Cartilage has a simple structure without blood vessels, nerves and lymphatic tissue, so its ability to repair itself is limited. Although there are many clinical methods for repairing cartilage defect and trauma, the effect is not ideal, and the function of cartilage tissue cannot be fully restored. The rapid development of cartilage tissue engineering research has brought new hope for the repair of clinical cartilage defects. [0003] Tissue engineering technology is to inoculate in vitro cultured and expanded cells in a biomaterial that has good biocompatibility and can be degraded and absorbed by the ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61L27/38A61L27/26A61L27/24A61L27/20A61L27/52
Inventor 宋克东李文芳刘天庆李丽颖王兆民
Owner DALIAN UNIV OF TECH
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