Conditioned media and exosomes from rat adipose-derived mesenchymal stem cells enhance bone regeneration: a study in vitro

doi:10.3877/cma.j.issn.1674-1366.2018.02.006

Abstract

Abstract:

Objective

To investigate the effect of conditioned media and exosomes from rat adipose-derived mesenchymal stem cells (ASCs) on bone regeneration in vitro.

Methods

Primary ASCs and bone marrow-derived mesenchymal stem cells (BMSCs) were isolated, cultured and identified. ASC-derived exosomes were isolated/depleted by ultracentrifugation and identified by transmission electron microscope and particle size analysis. CCK-8 and Transwell assays were used to evaluate cell proliferation and migration, respectively. The osteogenic differentiation in BMSCs were evaluated by qRT-PCR and ALP activity test. The mean values between groups were analyzed by Bonferroni Test in SPSS 20.0.

Results

The ASCs and BMSCs were confirmed. Transmission electron microscope analysis and particle size analysis showed ASC-derived exosomes after osteoinduction with characteristic morphology and size. CCK-8 assay showed significant difference among groups after 7 d culture (F= 157.74, P= 0.001) . Cell proliferation in conditioned media group (A₄₅₀= 2.71±0.15) was significantly higher than that in control (A₄₅₀= 1.95 ± 0.11) , and exosome deleted group (A₄₅₀= 2.28 ± 0.34) . Transwell assay showed significant difference among groups after 24 h culture (F= 46.79, P= 0.001) . Migrating cells in conditioned media group (51.6 ± 1.3) was significantly higher than that in control (28.6±1.4) and exosome deleted group (37.2 ± 2.2) . ALP activity assay showed significant difference among groups after 7 d culture (F= 78.43, P= 0.001) . ALP content in conditioned media group (310 ± 11) was significantly higher than that in control (200 ± 24) and exosome deleted group (125 ± 15) . QPCR showed significant difference among groups (F_ALP= 32.30, P_ALP= 0.001; F_RUNX2= 26.78, P_RUNX2= 0.001) .

Conclusion

ASC-CM and exosomes can enhance the proliferation, migration, and osteogenic differentiation of BMSCs and promote bone regeneration in vitro.

Key words: Mesenchymal stem cells, Exosomes, Culture media, conditioned, Bone and bones, regeneration

Chunhuang Huang, Yuanyuan Ma, Lin Ren, Qingwei Cai, Ruixin Chen, Qiang Fu. Conditioned media and exosomes from rat adipose-derived mesenchymal stem cells enhance bone regeneration: a study in vitro[J]. Chinese Journal of Stomatological Research(Electronic Edition), 2018, 12(02): 101-109.

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Figures/Tables 11

References 30

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基因	引物序列
RUNX2	F:5′-TTCAAGGTTGTAGCCCTCGGAG-3′
?	R:5′-TCAAAGTGAAACTCTTGCCTCGT-3′
ALP	F:5′-AGAACAGAACTGATGTGGAATATGAA-3′
?	R:5′-CAGTGCGGTTCCAGACATAGT-3′
β-actin	F:5′-AGCCATGTACGTAGCCATCCA-3′
?	R:5′-TCTCCGGAGTCCATCACAATG-3′

基因	引物序列
RUNX2	F:5′-TTCAAGGTTGTAGCCCTCGGAG-3′
?	R:5′-TCAAAGTGAAACTCTTGCCTCGT-3′
ALP	F:5′-AGAACAGAACTGATGTGGAATATGAA-3′
?	R:5′-CAGTGCGGTTCCAGACATAGT-3′
β-actin	F:5′-AGCCATGTACGTAGCCATCCA-3′
?	R:5′-TCTCCGGAGTCCATCACAATG-3′

组别	1d	3d	5d	7d
对照组	0.94 ± 0.11	1.35 ± 0.16	1.72 ± 0.07	1.95 ± 0.11
条件培养基组	1.03 ± 0.08	2.00 ± 0.15	2.47 ± 0.21	2.71 ± 0.15
去外泌体组	0.98 ± 0.12	1.56 ± 0.10	2.05 ± 0.11	2.28 ± 0.34
F值	2.87	86.71	106.09	157.74
P值	0.068	0.001	0.001	0.001

组别	1d	3d	5d	7d
对照组	0.94 ± 0.11	1.35 ± 0.16	1.72 ± 0.07	1.95 ± 0.11
条件培养基组	1.03 ± 0.08	2.00 ± 0.15	2.47 ± 0.21	2.71 ± 0.15
去外泌体组	0.98 ± 0.12	1.56 ± 0.10	2.05 ± 0.11	2.28 ± 0.34
F值	2.87	86.71	106.09	157.74
P值	0.068	0.001	0.001	0.001

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