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Fak silencing impairs osteogenic differentiation of bone mesenchymal stem cells induced by uniaxial mechanical stretch.

ABSTRACT: Background/purpose:Mechanical stretch plays a key role in promoting proliferation and differentiation of bone marrow mesenchymal stem cells (BMSCs) in distraction osteogenesis (DO). A better understanding of how the extracellular biomechanical stimulation is transferred to intracellular signal expression will benefit DO. Focal adhesion kinase (FAK) is a key factor in integrin signaling pathway. However, little is known about the effect of integrin-FAK signaling during the process of stretch induced osteogenic differentiation of BMSCs. Materials and methods:A specific short hairpin RNAs (shRNAs) lentiviral expression vector was used to silence Fak gene and a well-established in vitro uniaxial dynamic stretching device was applied to stimulate DO. Fak silencing was confirmed by fluorescence microscopy and the detection of Fak mRNA and FAK, p-FAK protein expression. Alkaline phosphatase (ALP) activity, expression of osteogenic differentiation markers - runt-related transcription factor 2 (RUNX2/Runx2) and alkaline phosphatase (Alp) together with integrin upstream signal transduction molecules integrin beta-1 (ITGB1/Itgb1) and downstream signal transduction molecules integrin-linked kinase (ILK) were detected after the stretch. Results:The results showed that mechanical stretch in control groups significantly induced the osteogenic differentiation of BMSCs with increased ALP activity, expression of RUNX2/Runx2 and Alp, together with upregulated ITGB1/Itgb1 and ILK, which all vanished in Fak silencing group. Conclusion:Silencing of the Fak gene inhibited the osteogenic differentiation of rat BMSCs induced by in vitro mechanical stretch through integrin signaling pathway.

SUBMITTER: Hu P

PROVIDER: S-EPMC6739265 | biostudies-literature | 2019 Sep

REPOSITORIES: biostudies-literature

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Fak silencing impairs osteogenic differentiation of bone mesenchymal stem cells induced by uniaxial mechanical stretch.

Hu Pei P Zhu Xiaowen X Zhao Chuang C Hu Jing J Luo En E Ye Bin B

Journal of dental sciences 20190320 3

<h4>Background/purpose</h4>Mechanical stretch plays a key role in promoting proliferation and differentiation of bone marrow mesenchymal stem cells (BMSCs) in distraction osteogenesis (DO). A better understanding of how the extracellular biomechanical stimulation is transferred to intracellular signal expression will benefit DO. Focal adhesion kinase (FAK) is a key factor in integrin signaling pathway. However, little is known about the effect of integrin-FAK signaling during the process of stre ...[more]

PMID: 31528249

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Project description:Severe malocclusion can contribute to several serious dental and physical conditions, such as digestive difficulties, periodontal disease, and severe tooth decay. Orthodontic treatment is mainly used to treat malocclusion. Forces in orthodontic tooth results in bone resorption on the pressure side and bone deposition on the tension side. Osteoblasts have been considered as the key component in bone regeneration on the tension side. However, the underlying mechanisms remain unclear. In this study, we focus on how mechanical stretch regulates the osteogenesis during orthodontic treatment. Human jaw bone marrow mesenchymal stem cells (hJBMMSCs) were isolated from healthy adult donors and cultured in regular medium (control) or osteogenic medium (OS). Under OS culture, hJBMMSCs presented osteogenic differentiation potentials, as evidenced by increased mineralization, enhanced calcium deposition, and upregulated expression of osteogenesis markers (ALP, osterix, and Runx). What's more, the OS-induced osteogenesis of hJBMMSCs is associated with the dephosphorylation of IKK, activation of IKBα, and phosphorylation/nucleic accumulation of P65, which all indicated the inhibition of NF-κB activity. Overexpressing P65 in hJBMMSCs, which could constantly activate NF-κB, prevented the osteogenic differentiation in the OS. After that, we applied the Flexcell tension system, which could cause mechanical stretch on cultured hJBMMSCs to mimic the tension forces during tooth movement. Mechanical stretch resulted in 3.5-fold increase of ALP activity and 2.4-fold increase of calcium deposition after 7 days and 21 days treatment, respectively. The expression levels of ALP, Run×2, and Osterix were also significantly upregulated. In the meantime, applying mechanical stretch on OS-cultured hJBMMSCs also dramatically promoted the OS-induced osteogenesis. Both OS and mechanical stretch downregulated NF-κB activity. By overexpressing P65 in hJBMMSCs, neither OS nor mechanical stretch could induce their osteogenesis. These results indicated that, like OS induction, mechanical stretch-facilitated osteogenesis of hJBMMSCs by inhibiting NF-κB in the noninflammatory environments.

Dataset Information

Fak silencing impairs osteogenic differentiation of bone mesenchymal stem cells induced by uniaxial mechanical stretch.

Publications

Fak silencing impairs osteogenic differentiation of bone mesenchymal stem cells induced by uniaxial mechanical stretch.

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