Application of vibration stimulation in regulation of in vitro osteogenesis and adipogenic differentiation of bone marrow-derived mesenchymal stem cells

Abstract

The invention discloses application of vibration stimulation in regulation of in vitro osteogenesis and adipogenic differentiation of bone marrow-derived mesenchymal stem cells and belongs to the technical field of cell biotechnology and bone tissue engineering. Osteogenesis differentiation culture and adipogenic differentiation culture are respectively carried out on bone marrow-derived mesenchymal stem cells of people in vitro, vibration stimulation is carried out once a day and every time is 20-45 minutes, and the in vitro osteogenesis differentiation and adipogenic differentiation of bone marrow-derived mesenchymal stem cells are facilitated to be promoted. Vibration stimulation is applied to the bone marrow-derived mesenchymal stem cells, formation of calcium of extracellular matrix and expression of osteogenesis differentiation marker genes of RUNX2 and COL1A1 in mRNA level can be upwards regulated, generation of lipid of epimatrix and expression of adipogenic differentiation marker genes of PPARG2 and CEBPA mRNA can be restrained, and a safe and efficient seed cell source is provided for promoting stem cell osteogenesis differentiation to generate more functional osteoblasts.

Description

technical field

[0001] The invention belongs to the technical fields of cell biotechnology and bone tissue engineering, and in particular relates to the application of high-frequency vibration stimulation in regulating bone marrow mesenchymal stem cell osteogenic differentiation and adipogenic differentiation in vitro. Background technique

[0002] Bone defect repair has always been a problem in orthopedics. The number of people who need to receive bone graft repair due to various reasons such as diseases, natural disasters, accidents and wars is on the rise year by year. Traditional bone repair methods often require sacrificing autologous bone for graft repair, which is likely to cause trauma and functional limitations at the donor site; however, allogeneic and xenogeneic bone are greatly restricted due to safety issues such as immune rejection and cross-infection of viruses. In recent years, the rapid development of bone tissue engineering provides a new way for the repai...

Examples

Embodiment 1

[0023] Effect of high-frequency vibration stimulation on the activity of BM-MSCs

[0024] Bone marrow mesenchymal stem cells were seeded in 96-well plates at 1000 cells / well and cultured in 5% CO2 37oC cell culture incubator, basal medium (α-MEM, 10% FBS, 100 U / mL penicillin, 100 μg / mL streptomycin) culture. After 24 hours, 30 Hz, 750 Hz and 800 Hz, 30 min / day of vibration stimulation were given respectively as the experimental group, and the control group (0 Hz) without vibration. The basal medium was changed every other day.

[0025] After 7 days, cell viability was detected with fluorescein diacetate (FDA). Cells in each group were incubated with 5 μg / mL FDA at 37°C for 10 min, washed twice with PBS, and then photographed under an Olympus IX51 inverted fluorescence microscope. The result is as figure 1 As shown, compared with the control group, the cells in the vibration stimulation group showed obvious spindle-like and fibroblast-like morphology, indicating that the ac...

Embodiment 2

[0027] Effect of high-frequency vibration stimulation on osteogenic differentiation of BM-MSCs

[0028] The cell culture method is the same as in Example 1, and the mesenchymal stem cells are prepared at 3000 cell / cm 2 Inoculate 12-well plates and culture in 5% CO 2 After the cell density reaches 95%, replace with osteogenic induction medium (low glucose DMEM, 10% FBS, 100 U / mL penicillin, 100 μg / mL streptomycin, 2×10 -4 M L-Ascorbic Acid, 10 -7 M Dexamethasone, 10 -2 M β-sodium glycerophosphate), the experimental group was 30, 750 and 800Hz, 30min / day vibration stimulation group, and only the osteogenesis induction group was used as the control.

[0029] (1) Alizarin red staining and quantitative analysis of extracellular matrix calcium deposits after vibration stimulation for 14 days. The cells in the well plate were fixed overnight with 4% paraformaldehyde, washed with PBS and incubated with 1% Alizarin Red (pH=4.3) for 30 min, washed twice with ultrapure water and...

Embodiment 3

[0033] Effect of vibration stimulation on adipogenic differentiation of BM-MSCs

[0034] The cell culture method is the same as in Example 1, and the mesenchymal stem cells are prepared at 3000 cell / cm 2 Inoculate 12-well plates and culture them in a 37oC cell culture incubator with 5% CO2. After the cell density reaches 95%, replace with adipogenic induction medium (high glucose DMEM, 10% FBS, 100 U / mL penicillin, 100 μg / mL streptomycin , 5×10 -4 M Isobutyl methacrylate, 10mg / L insulin, 10 -4 M indomethacin), the experimental groups were 30, 750 and 800 Hz, 30 min / day vibration stimulation groups, and only the adipogenic induction group was used as the control group.

[0035] (1) After 21 days of vibration stimulation, the induced adipocytes were stained with Oil Red O and quantitatively analyzed. The cells in the orifice plate were fixed with 10% formaldehyde, washed and incubated with Oil Red O for 30 min at room temperature, washed with ultrapure water, and photograp...

technical field

[0001] The invention belongs to the technical fields of cell biotechnology and bone tissue engineering, and in particular relates to the application of high-frequency vibration stimulation in regulating bone marrow mesenchymal stem cell osteogenic differentiation and adipogenic differentiation in vitro. Background technique

[0002] Bone defect repair has always been a problem in orthopedics. The number of people who need to receive bone graft repair due to various reasons such as diseases, natural disasters, accidents and wars is on the rise year by year. Traditional bone repair methods often require sacrificing autologous bone for graft repair, which is likely to cause trauma and functional limitations at the donor site; however, allogeneic and xenogeneic bone are greatly restricted due to safety issues such as immune rejection and cross-infection of viruses. In recent years, the rapid development of bone tissue engineering provides a new way for the repai...

Examples

Embodiment 1

[0023] Effect of high-frequency vibration stimulation on the activity of BM-MSCs

[0024] Bone marrow mesenchymal stem cells were seeded in 96-well plates at 1000 cells / well and cultured in 5% CO2 37oC cell culture incubator, basal medium (α-MEM, 10% FBS, 100 U / mL penicillin, 100 μg / mL streptomycin) culture. After 24 hours, 30 Hz, 750 Hz and 800 Hz, 30 min / day of vibration stimulation were given respectively as the experimental group, and the control group (0 Hz) without vibration. The basal medium was changed every other day.

[0025] After 7 days, cell viability was detected with fluorescein diacetate (FDA). Cells in each group were incubated with 5 μg / mL FDA at 37°C for 10 min, washed twice with PBS, and then photographed under an Olympus IX51 inverted fluorescence microscope. The result is as figure 1 As shown, compared with the control group, the cells in the vibration stimulation group showed obvious spindle-like and fibroblast-like morphology, indicating that the ac...

Embodiment 2

[0027] Effect of high-frequency vibration stimulation on osteogenic differentiation of BM-MSCs

[0028] The cell culture method is the same as in Example 1, and the mesenchymal stem cells are prepared at 3000 cell / cm 2 Inoculate 12-well plates and culture in 5% CO 2 After the cell density reaches 95%, replace with osteogenic induction medium (low glucose DMEM, 10% FBS, 100 U / mL penicillin, 100 μg / mL streptomycin, 2×10 -4 M L-Ascorbic Acid, 10 -7 M Dexamethasone, 10 -2 M β-sodium glycerophosphate), the experimental group was 30, 750 and 800Hz, 30min / day vibration stimulation group, and only the osteogenesis induction group was used as the control.

[0029] (1) Alizarin red staining and quantitative analysis of extracellular matrix calcium deposits after vibration stimulation for 14 days. The cells in the well plate were fixed overnight with 4% paraformaldehyde, washed with PBS and incubated with 1% Alizarin Red (pH=4.3) for 30 min, washed twice with ultrapure water and...

Embodiment 3

[0033] Effect of vibration stimulation on adipogenic differentiation of BM-MSCs

[0034] The cell culture method is the same as in Example 1, and the mesenchymal stem cells are prepared at 3000 cell / cm 2 Inoculate 12-well plates and culture them in a 37oC cell culture incubator with 5% CO2. After the cell density reaches 95%, replace with adipogenic induction medium (high glucose DMEM, 10% FBS, 100 U / mL penicillin, 100 μg / mL streptomycin , 5×10 -4 M Isobutyl methacrylate, 10mg / L insulin, 10 -4 M indomethacin), the experimental groups were 30, 750 and 800 Hz, 30 min / day vibration stimulation groups, and only the adipogenic induction group was used as the control group.

[0035] (1) After 21 days of vibration stimulation, the induced adipocytes were stained with Oil Red O and quantitatively analyzed. The cells in the orifice plate were fixed with 10% formaldehyde, washed and incubated with Oil Red O for 30 min at room temperature, washed with ultrapure water, and photograp...

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