Bone cells and their precursors are sensitive to changes in their biomechanical environment. The importance of mechanical stimuli has been observed in bone homeostasis and osteogenesis, but the mechanisms responsible for osteogenic induction in response to mechanical signals are poorly understood. We hypothesized that compressive forces could exert an osteogenic effect on osteoblasts and act in a dose-dependent manner. To test our hypothesis, electrospun poly(epsilon-caprolactone) (PCL) scaffolds were used as a 3-D microenvironment for osteoblast culture. The scaffolds provided a substrate allowing cell exposure to levels of externally applied compressive force. Pre-osteoblasts adhered, proliferated and differentiated in the scaffolds and showed extensive matrix synthesis by scanning electron microscopy (SEM) and increased Young's modulus (136.45+/-9.15 kPa) compared with acellular scaffolds (24.55+/-8.5 kPa). Exposure of cells to 10% compressive strain (11.81+/-0.42 kPa) resulted in a rapid induction of bone morphogenic protein-2 (BMP-2), runt-related transcription factor 2 (Runx2), and MAD homolog 5 (Smad5). These effects further enhanced the expression of genes and proteins required for extracellular matrix (ECM) production, such as alkaline phosphatase (Akp2), collagen type I (Col1a1), osteocalcin/bone gamma carboxyglutamate protein (OC/Bglap), osteonectin/secreted acidic cysteine-rich glycoprotein (ON/Sparc) and osteopontin/secreted phosphoprotein 1 (OPN/Spp1). Exposure of cell-scaffold constructs to 20% compressive strain (30.96+/-2.82 kPa) demonstrated that these signals are not osteogenic. These findings provide the molecular basis for the experimental and clinical observations that appropriate physical activities or microscale compressive loading can enhance fracture healing due in part to the anabolic osteogenic effects.

BackgroundDuring normal physical activities cartilage experiences dynamic compressive forces that are essential to maintain cartilage integrity. However, at non-physiologic levels these signals can induce inflammation and initiate cartilage destruction. Here, by examining the pro-inflammatory signaling networks, we developed a mathematical model to show the magnitude-dependent regulation of chondrocytic responses by compressive forces.Methodology/Principal FindingsChondrocytic cells grown in 3-D scaffolds were subjected to various magnitudes of dynamic compressive strain (DCS), and the regulation of pro-inflammatory gene expression via activation of nuclear factor-kappa B (NF-κB) signaling cascade examined. Experimental evidences provide the existence of a threshold in the magnitude of DCS that regulates the mRNA expression of nitric oxide synthase (NOS2), an inducible pro-inflammatory enzyme. Interestingly, below this threshold, DCS inhibits the interleukin-1β (IL-1β)-induced pro-inflammatory gene expression, with the degree of suppression depending on the magnitude of DCS. This suppression of NOS2 by DCS correlates with the attenuation of the NF-κB signaling pathway as measured by IL-1β-induced phosphorylation of the inhibitor of kappa B (IκB)-α, degradation of IκB-α and IκB-β, and subsequent nuclear translocation of NF-κB p65. A mathematical model developed to understand the complex dynamics of the system predicts two thresholds in the magnitudes of DCS, one for the inhibition of IL-1β-induced expression of NOS2 by DCS at low magnitudes, and second for the DCS-induced expression of NOS2 at higher magnitudes.Conclusions/SignificanceExperimental and computational results indicate that biomechanical signals suppress and induce inflammation at critical thresholds through activation/suppression of the NF-κB signaling pathway. These thresholds arise due to the bistable behavior of the networks originating from the positive feedback loop between NF-κB and its target genes. These findings lay initial groundwork for the identification of the thresholds in physical activities that can differentiate its favorable actions from its unfavorable consequences on joints.

SummaryReplicative senescence has a major impact on function and integrity of cell preparations. This process is reflected by continuous DNA methylation (DNAm) changes at specific CpG dinucleotides in the course of in vitro culture, and such modifications can be used to estimate the state of cellular senescence for quality control of cell preparations. Still, it is unclear how senescence‐associated DNAm changes are regulated and whether they occur simultaneously across a cell population. In this study, we analyzed global DNAm profiles of human mesenchymal stem cells (MSCs) and human umbilical vein endothelial cells (HUVECs) to demonstrate that senescence‐associated DNAm changes are overall similar in these different cell types. Subsequently, an Epigenetic‐Senescence‐Signature, based on six CpGs, was either analyzed by pyrosequencing or by bar‐coded bisulfite amplicon sequencing. There was a good correlation between predicted and real passage numbers in bulk populations of MSCs (R 2 = 0.67) and HUVECs (R 2 = 0.97). However, when we analyzed the Epigenetic‐Senescence‐Signature in subclones of MSCs, the predictions revealed high variation and they were not related to the adipogenic or osteogenic differentiation potential of the subclones. Notably, in clonally derived subpopulations, the DNAm levels of neighboring CpGs differed extensively, indicating that these genomic regions are not synchronously modified during senescence. Taken together, senescence‐associated DNAm changes occur in a highly reproducible manner, but they are not synchronously co‐regulated. They rather appear to be acquired stochastically—potentially evoked by other epigenetic modifications.

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IntroductionThe importance of mechanical signals in normal and inflamed cartilage is well established. Chondrocytes respond to changes in the levels of proinflammatory cytokines and mechanical signals during inflammation. Cytokines like interleukin (IL)-1β suppress homeostatic mechanisms and inhibit cartilage repair and cell proliferation. However, matrix synthesis and chondrocyte (AC) proliferation are upregulated by the physiological levels of mechanical forces. In this study, we investigated intracellular mechanisms underlying reparative actions of mechanical signals during inflammation.MethodsACs isolated from articular cartilage were exposed to low/physiologic levels of dynamic strain in the presence of IL-1β. The cell extracts were probed for differential activation/inhibition of the extracellular signal-regulated kinase 1/2 (ERK1/2) signaling cascade. The regulation of gene transcription was examined by real-time polymerase chain reaction.ResultsMechanoactivation, but not IL-1β treatment, of ACs initiated integrin-linked kinase activation. Mechanical signals induced activation and subsequent C-Raf-mediated activation of MAP kinases (MEK1/2). However, IL-1β activated B-Raf kinase activity. Dynamic strain did not induce B-Raf activation but instead inhibited IL-1β-induced B-Raf activation. Both mechanical signals and IL-1β induced ERK1/2 phosphorylation but discrete gene expression. ERK1/2 activation by mechanical forces induced SRY-related protein-9 (SOX-9), vascular endothelial cell growth factor (VEGF), and c-Myc mRNA expression and AC proliferation. However, IL-1β did not induce SOX-9, VEGF, and c-Myc gene expression and inhibited AC cell proliferation. More importantly, SOX-9, VEGF, and Myc gene transcription and AC proliferation induced by mechanical signals were sustained in the presence of IL-1β.ConclusionsThe findings suggest that mechanical signals may sustain their effects in proinflammatory environments by regulating key molecules in the MAP kinase signaling cascade. Furthermore, the findings point to the potential of mechanosignaling in cartilage repair during inflammation.