Cyclic mechanical strain increases production of regulators of bone healing in cultured murine osteoblasts.
BACKGROUND
The adaptive response of bone to mechanical strain, for which angiogenesis is required, is underscored during fracture healing. Vascular endothelial growth factor (VEGF) and transforming growth factor beta-1 (TGF-beta1) are critical regulators of angiogenesis. The purpose of this study was to examine the effect of strain on the production of VEGF and TGF-beta1.


METHODS
MC3T3-E1 mouse osteoblasts underwent cyclic strain (low, 0.1 Hz, or high, 0.2 Hz) for 24 or 48 hours. VEGF and TGF-beta1 protein levels were determined by ELISA, and Northern blot analysis was performed for VEGF mRNA. Alkaline phosphatase (an osteoblast differentiation marker) activity was determined by functional enzymatic assay. All measurements were standardized for cell number by crystal violet colorimetric assay. Statistical significance was determined by t-test, ANOVA, and the Tukey-Kramer test.


RESULTS
Protein production of VEGF and TGF-beta1 was dose-dependently elevated by strain (p < 0.05); alkaline phosphatase did not rise significantly. Northern blot analysis of strained osteoblast cells demonstrated increased VEGF mRNA. Cyclic strain was found to be progressively destructive in a dose-dependent manner, causing 51% and 70% decreases in cell number under low and high strain, respectively (p < 0.01).


CONCLUSIONS
We demonstrated simultaneous, dose-dependent increases in VEGF and TGF-beta1 protein production by osteoblastic cells in response to increasing strain. VEGF mRNA also increased in response to strain. This strain-induced increase in angiogenic cytokines suggests a potential mechanism by which injured bone may recruit a new blood supply. But we also found increasing strain to increase cellular toxicity, suggesting that cyclic mechanical strain may select for a subpopulation of osteoblasts.
Singh
Sunil P
SP
Institute of Reconstructive Plastic Surgery, New York University School of Medicine, New York, NY 10016, USA.
Chang
Edward I
EI
Gossain
Arun K
AK
Mehara
Babak J
BJ
Galiano
Robert D
RD
Jensen
John
J
Longaker
Michael T
MT
Gurtner
Geoffrey C
GC
Saadeh
Pierre B
PB
D000469:Alkaline Phosphatase / Q000096:biosynthesis; D000818:Animals; D015152:Blotting, Northern; D002452:Cell Count; D002478:Cells, Cultured; D003124:Colorimetry; D004195:Disease Models, Animal; D004797:Enzyme-Linked Immunosorbent Assay; D017102:Fracture Healing / Q000235:genetics*; D015870:Gene Expression; D051379:Mice; D010006:Osteoblasts / Q000378:metabolism* / Q000473:pathology; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000235:genetics*; D053773:Transforming Growth Factor beta1 / Q000096:biosynthesis / Q000235:genetics*; D042461:Vascular Endothelial Growth Factor A / Q000096:biosynthesis / Q000235:genetics*
D012333:RNA, Messenger; D053773:Transforming Growth Factor beta1; D042461:Vascular Endothelial Growth Factor A; D000469:Alkaline Phosphatase