Application of melatonin in synergy with extracellular matrix biomaterials in the preparation of drugs to promote osteogenic differentiation of mesenchymal stem cells
A technology of biomaterials and mesenchymal stem cells, which is applied in the field of tissue engineering repair and regeneration, can solve the problems of potential protective effects and few mechanisms, and achieve the effect of maintaining fibroblast shape, reducing ROS, and promoting in vitro proliferation
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Embodiment 1
[0046] The preparation of embodiment 1 extracellular matrix biological material
[0047] The preparation steps are:
[0048] S1. Obtaining bone marrow mesenchymal stem cells from human tissue;
[0049] S2. Configure induction culture medium: medium α-MEM80 volume ratio, fetal bovine serum 20% volume ratio, antibiotic 10~200U / mL, adenine 0.25mM, proline 50μg / 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: first wash with phosphate buffer solution of pH 7.4 for 3 times, then add decellularization treatment solution containing TritonX-100 5% volume ratio and ammonia water 4% volume ratio, in 5% CO at 37oC 2 Let stand in the incubator for 5 minutes, wash with pH...
Embodiment 2
[0052] Example 2 Adsorption detection of melatonin by extracellular matrix biological material
[0053] The decellularized extracellular matrix biomaterial cell culture plate in Example 1 and the traditional commercial culture plate without any treatment were incubated in 100uM melatonin phosphate buffer, as 37 ° C 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 Study on proliferation of bone marrow mesenchymal stem cells 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 the culture plates containing extracellular matrix biological materials obtained in Example 1, add basal medium (α-MEM, 10%FBS, 100U / mL penicillin , 100μg / mL streptomycin, 0.25μg / 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. The collected cells were dig...
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