Dataset Information

Gene expression responses to mechanical stimulation of mesenchymal stem cells seeded on calcium phosphate cement.

ABSTRACT:

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.

SUBMITTER: Gharibi B

PROVIDER: S-EPMC3807700 | biostudies-literature | 2013 Nov

REPOSITORIES: biostudies-literature

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Publications

Gene expression responses to mechanical stimulation of mesenchymal stem cells seeded on calcium phosphate cement.

Gharibi Borzo B Cama Giuseppe G Capurro Marco M Thompson Ian I Deb Sanjukta S Di Silvio Lucy L Hughes Francis John FJ

Tissue engineering. Part A 20130822 21-22

<h4>Introduction</h4>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.<h4>Methods</h4>Human MSCs were seeded onto CaP scaffolds and subjected to a pulsating compressive force of 5.5± ...[more]

PMID: 23968499

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Project description:Cells in the cardiovascular system are constantly exposed to complex mechanical stimulation due to the pulsatile nature of blood flow and the haemodynamic forces that are key to the regulation of vascular development, remodeling and pathophysiology. Mechanical stretch can also modulate the differentiation of stem cells toward vascular cell lineages (i.e., vascular smooth muscle cells), and represent a critical factor in vascular tissue engineering. Here we report on the development of a microchip platform that can emulate several key aspects of the vascular mechanical environment, such as cyclic stimulation and circumferential strain. This chip consists of an array of microfluidic channels with widths ranging from 20 to 500 micrometers. These channels are covered by suspended deformable membranes, on which cells are cultured and stimulated by cyclic circumferential strain of up to 20% via hydrodynamic actuation of the fluid in the microfluidic channels, thereby mimicking the biomechanical conditions of small blood vessels. We show that human mesenchymal stem cells (MSCs) can be cultured and continuously stimulated by cyclic stretch over a period of 7 days with no evidence of device fatigue or performance degradation. We observed localization and alignment of MSCs when mechanical stretch is larger than 10%, indicating the importance of mechanical stimulation in modulating cellular behavior. We further demonstrated simultaneous detection of proteins in multiple signaling pathways, including SMAD1/SMAD2 and canonical Wnt/?-catenin. This microchip represents a generic and versatile platform for high-throughput and rapid screening of cellular responses, including signal transduction cascades, in response to mechanical cues. The system emulates the physiological conditions of blood vessels and other tissues that are subject to cyclic strain, and may have a wide range of applications in the fields of stem cell mechanobiology, vascular tissue engineering, and other areas of regenerative medicine.

Project description:BackgroundThe use of mesenchymal stem cells (MSCs) and coralline hydroxyapatite (HA) or biphasic calcium phosphate (BCP) as a bone substitute for posterolateral spinal fusion has been reported. However, the genes and molecular signals by which MSCs interact with their surrounding environment require further elucidation.MethodsMSCs were harvested from bone grafting patients and identified by flow cytometry. A composite scaffold was developed using poly(lactide-co-glycolide) (PLGA) copolymer, coralline HA, BCP, and collagen as a carrier matrix for MSCs. The gene expression profiles of MSCs cultured in the scaffolds were measured by microarrays. The alkaline phosphatase (ALP) activity of the MSCs was assessed, and the expression of osteogenic genes and proteins was determined by quantitative polymerase chain reaction (Q-PCR) and Western blotting. Furthermore, we cultured rabbit MSCs in BCP or coralline HA hybrid scaffolds and transplanted these mixtures into rabbits for spinal fusion. We investigated the differences between BCP and coralline HA hybrid scaffolds by dual-energy X-ray absorptiometry (DEXA) and computed tomography (CT).ResultsTested in vitro, the cells were negative for hematopoietic cell markers and positive for MSC markers. There was higher expression of 80 genes and lower of 101 genes of MSCs cultured in BCP hybrid scaffolds. Some of these genes have been shown to play a role in osteogenesis of MSCs. In addition, MSCs cultured in BCP hybrid scaffolds produced more messenger RNA (mRNA) for osteopontin, osteocalcin, Runx2, and leptin receptor (leptin-R) than those cultured in coralline HA hybrid scaffolds. Western blotting showed more Runx2 and leptin-R protein expression in BCP hybrid scaffolds. For in vivo results, 3D reconstructed CT images showed continuous bone bridges and fusion mass incorporated with the transverse processes. Bone mineral content (BMC) values were higher in the BCP hybrid scaffold group than in the coralline HA hybrid scaffold group.ConclusionsThe BCP hybrid scaffold for osteogenesis of MSCs is better than the coralline HA hybrid scaffold by upregulating expression of leptin-R. This was consistent with in vivo data, which indicated that BCP hybrid scaffolds induced more bone formation in a spinal fusion model.

Dataset Information

Gene expression responses to mechanical stimulation of mesenchymal stem cells seeded on calcium phosphate cement.

Introduction

Methods

Results

Conclusion

Publications

Gene expression responses to mechanical stimulation of mesenchymal stem cells seeded on calcium phosphate cement.

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