Mesenchymal stem cells (MSCs) hold great potential for regenerative medicine and tissue-engineering applications. They have multipotent differentiation capabilities and have been shown to differentiate down various lineages, including osteoblasts, adipocytes, chondrocytes, myocytes, and possibly neurons. The majority of approaches to control the MSC fate have been via the use of chemical factors in the form of growth factors within the culture medium. More recently, it has been understood that mechanical forces play a significant role in regulating MSC fate. We and others have shown that mechanical stimuli can control MSC lineage specification. The cytoskeleton is known to play a large role in mechanotransduction, and a growing number of studies are showing that it can also contribute to MSC differentiation. This review analyzes the significant contribution of actin and integrin distribution, and the smaller role of microtubules, in regulating MSC fate. Osteogenic differentiation is more prevalent in MSCs with a stiff, spread actin cytoskeleton and greater numbers of focal adhesions. Both adipogenic differentiation and chondrogenic differentiation are encouraged when MSCs have a spherical morphology associated with a dispersed actin cytoskeleton with few focal adhesions. Different mechanical stimuli can be implemented to alter these cytoskeletal patterns and encourage MSC differentiation to the desired lineage.

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Cytoskeletal and focal adhesion influences on mesenchymal stem cell shape, mechanical properties, and differentiation down osteogenic, adipogenic, and chondrogenic pathways.

Fluorides are present in the environment. Excessive systemic exposure to fluorides can lead to disturbances of bone homeostasis (skeletal fluorosis) and enamel development (dental/enamel fluorosis). The severity of dental fluorosis is also dependent upon fluoride dose and the timing and duration of fluoride exposure. Fluoride's actions on bone cells predominate as anabolic effects both in vitro and in vivo. More recently, fluoride has been shown to induce osteoclastogenesis in mice. Fluorides appear to mediate their actions through the MAPK signaling pathway and can lead to changes in gene expression, cell stress, and cell death. Different strains of inbred mice demonstrate differential physiological responses to ingested fluoride. Genetic studies in mice are capable of identifying and characterizing fluoride-responsive genetic variations. Ultimately, this can lead to the identification of at-risk human populations who are susceptible to the unwanted or potentially adverse effects of fluoride action and to the elucidation of fundamental mechanisms by which fluoride affects biomineralization.

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Fluoride's Effects on the Formation of Teeth and Bones, and the Influence of Genetics

Following myocardial infarction, tissue repair is mediated by the recruitment of monocytes and their subsequent differentiation into macrophages. Recent findings have revealed the dynamic changes in the presence of polarized macrophages with pro-inflammatory (M1) and anti-inflammatory (M2) properties during the early (acute) and late (chronic) stages of cardiac ischemia. Mesenchymal stem cells (MSCs) delivered into the injured myocardium as reparative cells are subjected to the effects of polarized macrophages and the inflammatory milieu. The present study investigated how cytokines and polarized macrophages associated with pro-inflammatory (M1) and anti-inflammatory (M2) responses affect the survival of MSCs. Human MSCs were studied using an in vitro platform with individual and combined M1 and M2 cytokines: IL-1β, IL-6, TNF-α, and IFN-γ (for M1), and IL-10, TGF-β1, TGF-β3, and VEGF (for M2). In addition, polarization molecules (M1: LPS and IFN-γ; M2: IL-4 and IL-13) and common chemokines (SDF-1 and MCP-1) found during inflammation were also studied. Indirect and direct co-cultures were conducted using M1 and M2 polarized human THP-1 monocytes. M2 macrophages and their associated cytokines supported the growth of hMSCs, while M1 macrophages and their associated cytokines inhibited the growth of hMSCs in vitro under certain conditions. These data imply that an anti-inflammatory (M2) environment is more accommodating to the therapeutic hMSCs than a pro-inflammatory (M1) environment at specific concentrations.

Macrophages modulate the viability and growth of human mesenchymal stem cells.

Adipose tissue is a dynamic endocrine organ with a central role in metabolism regulation. Functional differences in adipose tissue seem associated with the regional distribution of fat depots, in particular in subcutaneous and visceral omental pads. Here, we report for the first time the isolation of human adipose-derived adult stem cells from visceral omental and subcutaneous fat (V-ASCs and S-ASCs, respectively) from the same subject. Immunophenotyping shows that plastic culturing selects homogeneous cell populations of V-ASCs and S-ASCs from the corresponding stromal vascular fractions (SVFs), sharing typical markers of mesenchymal stem cells. Electron microscopy and electrophysiological and real-time RT-PCR analyses confirm the mesenchymal stem nature of both V-ASCs and S-ASCs, while no significant differences in a limited pattern of cytokine/chemokine expression can be detected. Similar to S-ASCs, V-ASCs can differentiate in vitro toward adipogenic, osteogenic, chondrogenic, muscular, and neuronal lineages, as demonstrated by histochemical, immunofluorescence, real-time RT-PCR, and electrophysiological analyses, suggesting the multipotency of such adult stem cells. Our data demonstrate that both visceral and subcutaneous adipose tissues are a source of pluripotent stem cells with multigermline potential. However, the visceral rather than the subcutaneous ASC could represent a more appropriate in vitro cell model for investigating the molecular mechanisms implicated in the pathophysiology of metabolic disorders such as obesity.

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Characterization of human adult stem-cell populations isolated from visceral and subcutaneous adipose tissue

Biophysical cues play a key role in directing the lineage commitment of mesenchymal stem cells or multipotent stromal cells (MSCs), but the mechanotransductive mechanisms at play are still not fully understood. This review article first describes the roles of both substrate mechanics (e.g. stiffness and topography) and extrinsic mechanical cues (e.g. fluid flow, compression, hydrostatic pressure, tension) on the differentiation of MSCs. A specific focus is placed on the role of such factors in regulating the osteogenic, chondrogenic, myogenic and adipogenic differentiation of MSCs. Next, the article focuses on the cellular components, specifically integrins, ion channels, focal adhesions and the cytoskeleton, hypothesized to be involved in MSC mechanotransduction. This review aims to illustrate the strides that have been made in elucidating how MSCs sense and respond to their mechanical environment, and also to identify areas where further research is needed.

https://onlinelibrary.wiley.com/doi/pdf/10.1111/joa.12243

Mechanical regulation of mesenchymal stem cell differentiation

Mesenchymal stem cells produce proinflammatory cytokines during their normal growth. Direct or indirect regulation of bone resorption by these cytokines has been reported. However, the effects of osteogenic conditions—chemical and/or mechanical—utilized during in vitro bone tissue engineering on expression of cytokines by hMSCs have not been studied. In this study, we investigated the effects of cyclic tensile strain, culture medium (with and without dexamethasone), and culture duration on the expression of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6), and interleukin-8 (IL-8) by bone marrow derived human mesenchymal stem cells (hMSCs). Human MSCs seeded in three-dimensional Type I collagen matrices were subjected to 0%, 10%, and 12% uniaxial cyclic tensile strains at 1 Hz for 4 h/day for 7 and 14 days in complete growth or dexamethasone-containing osteogenic medium. Viability of hMSCs was maintained irrespective of strain level and media conditions. Expression of either TNF-α or IL-1β was not observed in hMSCs under any of the conditions investigated in this study. Expression of IL-6 was dependent on culture medium. An increase in IL-6 expression was caused by both 10% and 12% strain levels. Both 10% and 12% strain levels caused an increase in IL-8 production by hMSCs that was dependent on the presence of dexamethasone. IL-6 and IL-8 expressions by hMSCs were induced by cyclic tensile strain and osteogenic differentiating media, indicating that IL-6 and IL-8 may be functioning as autocrine signals during osteogenic differentiation of hMSCs. J. Cell. Physiol. 219: 77–83, 2009. © 2008 Wiley-Liss, Inc.

Expression of proinflammatory cytokines by human mesenchymal stem cells in response to cyclic tensile strain.

Genetic background influences fluoride's effects on osteoclastogenesis

Finite element modeling of 3D human mesenchymal stem cell-seeded collagen matrices exposed to tensile strain.

The effects of dietary calcium (Ca) deficiency on skeletal integrity are well characterized in growing and mature mammals; however, less is known about Ca nutrition during the neonatal period. In this study, we examined the effects of neonatal Ca nutrition on bone integrity, endocrine hormones, and mesenchymal stem cell (MSC) activity. Neonatal pigs (24 6 6ho f age) received either a Ca-adequate (1.2 g/100 g) or an ~40% Ca-deficient diet for 18 d. Ca deficiency reduced (P , 0.05) boneflexural strength and bone mineral density without major differences in plasmaindicators of Ca status. There were no meaningful differences in plasma Ca, phosphate (PO4), parathyroid hormone, or 1,25-dihydroxycholecalciferol due to Ca nutrition throughout the study. Calcium deficiency also reduced (P , 0.05) the in vivo proliferation of MSC by ~50%. In vitro studies utilizing homologous sera demonstrated that MSC activity was affected (P , 0.05) by both the Ca status of the pig and the sera as well as by their interaction. The results indicate that neonatal Ca nutrition is crucial for bone integrity and suggest that early-life Ca restriction may have long-term effects on bone integrity via programming of MSC. J. Nutr. 141: 373‐379, 2011.

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T. Wayne Pfeiler

Papers

Expression of proinflammatory cytokines by human mesenchymal stem cells in response to cyclic tensile strain.

Genetic background influences fluoride's effects on osteoclastogenesis

Finite element modeling of 3D human mesenchymal stem cell-seeded collagen matrices exposed to tensile strain.

Dietary Calcium Restriction Affects Mesenchymal Stem Cell Activity and Bone Development in Neonatal Pigs