Osteogenic Differentiation of Human Mesenchymal Stem Cells in Collagen Matrices: Effect of Uniaxial Cyclic Tensile Strain on Bone Morphogenetic Protein (BMP-2) mRNA Expression

Sumanasinghe, Ruwan D.; Bernacki, Susan H.; Loboa, Elizabeth G.

doi:10.1089/ten.2006.12.ft-271

Cited by 171 publications

(95 citation statements)

References 32 publications

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“…An interesting, albeit at this point speculative, possibility is that the signals controlling growth of the mesentery are themselves under control of physical forces, creating a feedback loop. There is evidence in other systems that Bmp2 expression can be modulated by tension (34)(35)(36).…”

Section: Discussionmentioning

confidence: 99%

BMP signaling controls buckling forces to modulate looping morphogenesis of the gut

Nerurkar

Mahadevan

Tabin

2017

Proc. Natl. Acad. Sci. U.S.A.

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Looping of the initially straight embryonic gut tube is an essential aspect of intestinal morphogenesis, permitting proper placement of the lengthy small intestine within the confines of the body cavity. The formation of intestinal loops is highly stereotyped within a given species and results from differential-growth–driven mechanical buckling of the gut tube as it elongates against the constraint of a thin, elastic membranous tissue, the dorsal mesentery. Although the physics of this process has been studied, the underlying biology has not. Here, we show that BMP signaling plays a critical role in looping morphogenesis of the avian small intestine. We first exploited differences between chicken and zebra finch gut morphology to identify the BMP pathway as a promising candidate to regulate differential growth in the gut. Next, focusing on the developing chick small intestine, we determined that Bmp2 expressed in the dorsal mesentery establishes differential elongation rates between the gut tube and mesentery, thereby regulating the compressive forces that buckle the gut tube into loops. Consequently, the number and tightness of loops in the chick small intestine can be increased or decreased directly by modulation of BMP activity in the small intestine. In addition to providing insight into the molecular mechanisms underlying intestinal development, our findings provide an example of how biochemical signals act on tissue-level mechanics to drive organogenesis, and suggest a possible mechanism by which they can be modulated to achieve distinct morphologies through evolution.

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Section: Discussionmentioning

confidence: 99%

BMP signaling controls buckling forces to modulate looping morphogenesis of the gut

Nerurkar

Mahadevan

Tabin

2017

Proc. Natl. Acad. Sci. U.S.A.

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“…However, many of these studies have used loads that substantially exceed the physiological or even pathological equivalent loads in vivo and have not been able to closely mimic the bone environment. 13,17,18,32 It is well known that surface stiffness and composition is a key regulator of MSC behavior, with stiff substrates with bone-like properties shown to support osteogenesis. 33,34 Strain measurements in human bones physiologically indicate a strain magnitude of between 0.2% and 0.4%.…”

Section: Discussionmentioning

confidence: 99%

“…Commonly a gel based scaffold or tissue culture plastic has been used as the substrate on which cells were mechanically loaded, and the strain employed has been in the range of 2%-20%. 6,[12][13][14][15][16][17][18][19][20] Neither the scaffold nor the strain of this magnitude represents the true environment or magnitude of physiological or pathological loading of bone in humans in vivo. Further, a number of studies in vivo have been carried out in an attempt to overcome these obstacles, but these are limited to animal models and therefore could be associated with differences in responses to loading and mechanisms involved from one species to another.…”

mentioning

confidence: 99%

Gene Expression Responses to Mechanical Stimulation of Mesenchymal Stem Cells Seeded on Calcium Phosphate Cement

Gharibi

Cama

Capurro

et al. 2013

Tissue Engineering Part A

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Introduction: The aim of the study reported here was to investigate the molecular responses of human mesenchymal stem cells (MSC) to loading with a model that attempts to closely mimic the physiological mechanical loading of bone, using monetite calcium phosphate (CaP) scaffolds to mimic the biomechanical properties of bone and a bioreactor to induce appropriate load and strain. Methods: Human MSCs were seeded onto CaP scaffolds and subjected to a pulsating compressive force of 5.5 -4.5 N at a frequency of 0.1 Hz. Early molecular responses to mechanical loading were assessed by microarray and quantitative reverse transcription-polymerase chain reaction and activation of signal transduction cascades was evaluated by western blotting analysis. Results: The maximum mechanical strain on cell/scaffolds was calculated at around 0.4%. After 2 h of loading, a total of 100 genes were differentially expressed. The largest cluster of genes activated with 2 h stimulation was the regulator of transcription, and it included FOSB. There were also changes in genes involved in cell cycle and regulation of protein kinase cascades. When cells were rested for 6 h after mechanical stimulation, gene expression returned to normal. Further resting for a total of 22 h induced upregulation of 63 totally distinct genes that were mainly involved in cell surface receptor signal transduction and regulation of metabolic and cell division processes. In addition, the osteogenic transcription factor RUNX-2 was upregulated. Twenty-four hours of persistent loading also markedly induced osterix expression. Mechanical loading resulted in upregulation of Erk1/2 phosphorylation and the gene expression study identified a number of possible genes (SPRY2, RIPK1, SPRED2, SERTAD1, TRIB1, and RAPGEF2) that may regulate this process. Conclusion:The results suggest that mechanical loading activates a small number of immediate-early response genes that are mainly associated with transcriptional regulation, which subsequently results in activation of a wider group of genes including those associated with osteoblast proliferation and differentiation. The results provide a valuable insight into molecular events and signal transduction pathways involved in the regulation of MSC osteogenic differentiation in response to a physiological level of mechanical stimulation.

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“…Other studies, as recent as the works by Cho et al (2005), Abdallah et al (2005) and Friedman et al (2006), used ACTB for normalizing the gene expression levels of hBMSCs under osteogenic conditions. Other contemporary publications reported the use of GAPDH for normalization purposes with hBMSCs and osteogenic conditions (Mbalaviele et al 2005;Sumanasinghe et al 2006;Tsukahara et al 2006). Furthermore, none of these studies provided supporting information for the selection of the normalizer gene.…”

Section: Discussionmentioning

confidence: 99%

Housekeeping gene stability influences the quantification of osteogenic markers during stem cell differentiation to the osteogenic lineage

et al. 2010

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Real-time reverse transcription PCR (RT-qPCR) relies on a housekeeping or normalizer gene whose expression remains constant throughout the experiment. RT-qPCR is commonly used for characterization of human bone marrow mesenchymal stem cells (hBMSCs). However, to the best of our knowledge, there are no studies validating the expression stability of the genes used as normalizers during hBMSCs differentiation. This work aimed to study the stability of the housekeeping genes b-actin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and ribosomal protein L13A (RPL13A) during the osteogenic differentiation of hBMSCs. Their stability was evaluated via RT-qPCR in 14 and 20 day differentiation assays to the osteogenic lineage. Different normalization strategies were evaluated to quantify the osteogenic markers collagen type I, bone sialoprotein and osteonectin. Cell differentiation was confirmed via alizarin red staining. The results demonstrated up-regulation of b-actin with maximum fold changes (MFC) of 4.38. GAPDH and RPL13A were not regulated by osteogenic media after 14 days and presented average fold changes lower than 2 in 20 day cultures. RPL13A (MFC \ 2) had a greater stability when normalizing as a function of culture time compared with GAPDH (MFC B 2.2), which resulted in expression patterns of the osteogenic markers more consistent with the observed differentiation process. The results suggest that b-actin regulation could be associated with the morphological changes characteristic of hBMSCs osteogenic differentiation, and provide evidence for the superior performance of RPL13A as a normalizer gene in osteogenic differentiation studies of hBMSCs. This work highlights the importance of validating the normalizer genes used for stem cells characterization via RT-qPCR.

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Osteogenic Differentiation of Human Mesenchymal Stem Cells in Collagen Matrices: Effect of Uniaxial Cyclic Tensile Strain on Bone Morphogenetic Protein (BMP-2) mRNA Expression

Cited by 171 publications

References 32 publications

BMP signaling controls buckling forces to modulate looping morphogenesis of the gut

BMP signaling controls buckling forces to modulate looping morphogenesis of the gut

Gene Expression Responses to Mechanical Stimulation of Mesenchymal Stem Cells Seeded on Calcium Phosphate Cement

Housekeeping gene stability influences the quantification of osteogenic markers during stem cell differentiation to the osteogenic lineage

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