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Application of melatonin synergistic extracellular matrix biomaterial to preparation of medicament for promoting osteoblast differentiation of mesenchymal stem cells

A biomaterial, stem cell technology, applied in the field of tissue engineering repair and regeneration, can solve the problems of potential protection and less mechanism, and achieve the effect of maintaining the shape of fibroblasts, promoting in vitro proliferation, and improving the expression of osteogenic genes

Inactive Publication Date: 2014-04-23
SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are very few reports on the potential protective effect and mechanism of melatonin synergizing with extracellular matrix biomaterials to promote osteogenic differentiation of mesenchymal stem cells

Method used

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  • Application of melatonin synergistic extracellular matrix biomaterial to preparation of medicament for promoting osteoblast differentiation of mesenchymal stem cells
  • Application of melatonin synergistic extracellular matrix biomaterial to preparation of medicament for promoting osteoblast differentiation of mesenchymal stem cells
  • Application of melatonin synergistic extracellular matrix biomaterial to preparation of medicament for promoting osteoblast differentiation of mesenchymal stem cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1 Preparation of extracellular matrix biomaterial

[0047] The preparation steps are:

[0048] S1. Obtain bone marrow mesenchymal stem cells from human tissue;

[0049] S2. Configure induction culture medium: medium α-MEM 80 volume ratio, fetal bovine serum 20% volume ratio, antibiotic 10~200 U / mL, adenine 0.25 mM, proline 50 μg / mL, dexamethasone 100nM;

[0050] S3. According to 20,000 cells / cm 2 Bone marrow mesenchymal stem cells were cultured in vitro on common commercial cell culture plates and induced to secrete extracellular matrix at a density of 5% CO at 37oC 2 Cultivate in the incubator for 7-20 days, and change the medium every other day;

[0051] S4. Decellularization treatment: wash with pH7.4 phosphate buffer for 3 times, then add decellularization treatment solution containing Triton X-100 5% volume ratio and ammonia water 4% volume ratio, at 37oC in 5% CO 2 Let stand in the incubator for 5 minutes, wash with pH7.4 phosphate buffer for 3 times...

Embodiment 2

[0052] Example 2 Adsorption detection of melatonin by extracellular matrix biomaterials

[0053] Incubate the decellularized extracellular matrix biomaterial cell culture plate in Example 1 and the traditional commercial culture plate without any treatment in 100uM melatonin phosphate buffer at 37°C in 5% CO 2 In the incubator, collect the supernatants at 0, 0.5, 1, 2, 4, 6, 12, 24, 48, and 72 hours respectively for detection by high performance liquid chromatography. Adopt the mobile phase of 55 / 45 volume ratio methanol and water, flow velocity is 1ml / min222um ultraviolet detection, supernatant liquid melatonin content and original content ratio are as follows figure 1 shown. The content of melatonin in the supernatant of the TCPS group was 95.4%-97.9%, while that of the biomaterial group was only 71.1%-87.4%. Melanin regulation system.

Embodiment 3

[0054] Example 3 Bone marrow mesenchymal stem cell proliferation and intracellular reactive oxygen species in four culture systems

[0055] P3 generation bone marrow mesenchymal stem cells at 3000cell / cm 2 Inoculate 6-well plates, including traditional commercial culture plates without any treatment and culture plates containing extracellular matrix biological materials prepared in Example 1, add basal medium (α-MEM, 10% FBS, 100 U / mL Penicillin, 100 μg / mL streptomycin, 0.25 μg / mL amphotericin B) or basal medium containing 100uM melatonin, change the medium every other day, and create four culture systems in turn: traditional culture plate system (TCPS), Traditional culture plate and melatonin system (TCPS-MT), extracellular matrix biomaterial system (ECM), melatonin synergistic extracellular matrix biomaterial regulation system (ECM-MT).

[0056] 1) Use 0.25% trypsin to collect the cells in the well plate after 5 days of culture. Collected cells were digested with 125 μg / ...

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Abstract

The invention discloses novel application of melatonin, in particular to application of a melatonin synergistic extracellular matrix biomaterial to preparation of a medicament for promoting osteoblast differentiation of mesenchymal stem cells and application of the melatonin synergistic extracellular matrix biomaterial to preparation of a medicament for treating bone diseases. Proven by experiments, by using the melatonin synergistic extracellular matrix biomaterial system, the in-vitro proliferation of the mesenchymal stem cell can be promoted, a good fibrous shape is kept for the cells, intracellular ROSs (Reactive Oxygen Species) are reduced, the epimatrix calcium deposition is facilitated, the osteogenesis gene expression is enhanced, the promotion of in-vitro osteoblast differentiation by the melatonin synergistic extracellular matrix biomaterial and a mechanism of differentiation are determined, and a basis is laid for the treatment of diseases such as osteoarthritis, bone defect and bone trauma by taking the melatonin synergistic extracellular matrix biomaterial as a repair material.

Description

technical field [0001] The invention belongs to the field of tissue engineering repair and regeneration, and specifically relates to the application of melatonin in synergy with extracellular matrix biomaterials in the preparation of drugs for promoting osteogenesis of mesenchymal stem cells (Osteogenesis) and the preparation of melatonin in synergy with extracellular matrix biomaterials Application of drugs in the treatment of bone diseases. Background technique [0002] Stem cells are a class of cells with self-renewal and differentiation potential, including embryonic stem cells, mesenchymal stem cells and pluripotent induced stem cells, which can be further differentiated into various tissues and constitute various complex body organs, and have been widely used in the field of regenerative medicine and tissue engineering. Mesenchymal stem cells (MSCs) play an important role in development, growth, self-regeneration of tissues and organs, and maintenance of microenviron...

Claims

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Application Information

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IPC IPC(8): A61L27/38A61L27/54
Inventor 龚逸鸿何帆曾易刘小珍蒋庆
Owner SUN YAT SEN UNIV
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