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. 2012 Apr;45(2):158-66.
doi: 10.1111/j.1365-2184.2011.00802.x. Epub 2012 Jan 9.

Mechanical stretch inhibits adipogenesis and stimulates osteogenesis of adipose stem cells

X Yang  1 , X Cai, J Wang, H Tang, Q Yuan, P Gong, Y Lin
Affiliations

Mechanical stretch inhibits adipogenesis and stimulates osteogenesis of adipose stem cells

X Yang et al. Cell Prolif. 2012 Apr.

Abstract

A reciprocal relationships between osteogenesis and adipogenesis has been observed in vitro and in vivo, and mechanical stretch has been believed to be a regulating factor of osteo-adipogenic axis differentiation of mesenchymal stem cells. In this study, rat adipose stem cells (ASCs) were isolated and cultured in adipogenic or normal medium. Their exposure to cyclic mechanical stretch (2000 με, 1 Hz) in the presence of adipogenic medium decreased mRNA and protein level of PPAR-γ, and increased Runx2 mRNA and protein levels as well as Pref-1 mRNA level, compared to static samples. ASCs cultured in normal medium without adipogenic induction did not show any significant change in mRNA expression of PPAR-γ, Runx2, nor Pref-1 irrespective of mechanical loading. Stretching induced phosphorylation of extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) during the induction period. It was concluded that mechanical stretch inhibited adipogenesis and stimulated osteogenesis of these ASCs in the presence of adipogenic medium and that ERK1/2 activation may be involved in the mechanical stress-induced trans-differentiation. VSports手机版.

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Figures

Figure 1
Figure 1
Illustrations of the four‐point bending mechanical loading device. L, distance between two outer pressure points; a, distance between outer and inner pressure points; t, thickness of loading plate; d, distance of pressure point movement; strain(ε) of cells attached to upper side of loading plate was calculated according the following formula: ε = td/a(L − 1.33a). In this experiment, ASCs were exposed to uniaxial cyclic tensile stretch for 2000 με, 1 Hz, which lasted 6 and 2 h.
Figure 2
Figure 2
Mechanical stretch significantly reduced adipogenesis of ASCs. (a) Three days after adipogenic induction, lipid‐filled cells, positive after oil red O staining, were observed under the light microscope. (b) Six hours mechanical stretching significantly reduced numbers of oil droplet‐filled cells from 63% to 41%.
Figure 3
Figure 3
Mechanical stretch inhibited adipogenesis and promoted osteogenesis under adipogenic induction. After adipogenic induction for 72 h and treatment with uniaxial cyclic tensile stretch of 2000 με at 1 Hz, mRNA level of PPAR‐γ reduced to 25% after loading for 6 h (P < 0.01) and 30% after loading for 2 h (P < 0.01) compared to static controls (a); Runx2 transcription increased 7‐fold after 6‐h mechanical loading (P < 0.01) and 4‐fold after 2‐h mechanical loading (b); Pref‐1 significantly increased after 6‐h loading (P < 0.05) (c); otherwise, when ASCs did not undergo adipogenic induction, PPAR‐γ, Runx2 or Pref‐1transcription did not significantly change irrespective of mechanical loading (d–f).
Figure 4
Figure 4
Effects of mechanical stretch on the PPAR‐γ protein expression. Protein level was measured by immunofluorescence staining (a–g) and western blotting (h, i). Images (a–c) show immunofluorescence of PPAR‐γ in cells kept in adipogenic medium for 72 h then mechanically loaded for 6 h, while images (d–e) show PPAR‐γ staining in control groups only kept in adipogenic medium with no mechanical stretch loaded. Images (a) and (d) are DAPI staining of ASC, images (b) and (e) represent PPAR‐γ staining, and (c) and (f) are merged images. PPAR‐γ positive cells reduced from 69% to 19% by 6‐h mechanical loading (g). Images H and I show western blotting results of PPAR‐γ protein level. Six hours and 2 h mechanical stretch significantly reduced protein level of PPAR‐γ compared to cells kept in adipogenic medium without mechanical stretch. ASCs kept in normal medium did not express PPAR‐γ protein. Expression of each protein was normalized to that of GAPDH and noted as fold levels of protein NIC. Graphs are representative of three independent experiments. Values expressed as mean ± SEM; *P < 0.05; **P < 0.01. AM, adipogenic medium group; NIC, the non‐induced control group.
Figure 5
Figure 5
Effects of mechanical stretch on the Runx2 protein expression. Protein level was measured by immunofluorescence staining (a–g) and western blotting (h, i). Images (a–c) show the immunofluorescence staining of PPAR‐γ in control groups retained in adipogenic medium only, with no loaded mechanical stretch, while images (d–e) show PPAR‐γ in cells maintained in adipogenic medium for 72 h then mechanically loaded for 6 h afterwards. Images (a) and (d) are DAPI staining of ASC, images (b) and (e) represent Runx2 staining and (c) and (f) are merged images. Mechanical stretch significantly increased protein level of Runx2 (g). Images (h) and (i) show western blotting of PPAR‐γ protein level. Six hours’ mechanical stretch significantly increased protein level of Runx2 4‐fold compared to cells maintained in adipogenic medium without mechanical stretch (h, i). ASCs kept in normal medium and adipogenic medium without mechanically loaded did not express Runx2 protein (h). Expression of each protein was normalized to that of GAPDH protein and noted as fold changes of protein level of NIC. Graphs representative of three independent experiments. Values expressed as mean ± SEM; *P < 0.05;**P < 0.01. AM, adipogenic medium group; NIC, non‐induced control group.
Figure 6
Figure 6
Effects of mechanical stretch on p‐ERK protein expression. Protein level was measured by immunofluorescence staining (a–g) and western blotting (h, i). Images (a–c) show immunofluorescence of p‐ERK in cells kept in adipogenic medium for 72 h then mechanically loaded for 6 h, while images (d–e) show p‐ERK staining in control groups kept in adipogenic medium only, without mechanical stretch loading. Images (a) and (d) are DAPI staining of ASC, images (b) and (e) represent p‐ERK staining, and (c) and (f) are merged images. Phospho‐ERK positive cells were significantly augmented 3‐fold by 6 h mechanical loading and trans‐located to nuclei (f, g). Images (h) and (i) show western blotting of phosphor ERK protein level. Six hours mechanical stretch significantly increased protein level of p‐ERK 3‐fold compared to cells maintained in adipogenic medium only with no mechanical stretching. ASCs kept in normal medium did not express phospho‐ERK protein. Expression of each protein normalized to that of GAPDH and noted by fold difference of protein level of NIC. Graphs representative of three independent experiments. Values expressed as mean ± SEM; *P < 0.05. AM, adipogenic medium group; NIC, non‐induced control group.
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