Tuesday, September 6, 2011

Mechanotransduction and Chondrogenesis

Dynamic compressive strain influences chondrogenic gene expression in human periosteal cells: a case study.

"Dynamic compression [selectively enhances] chondrogenic [and] osteogenic differentiation in human periosteal cells from two donors. Donor derived human periosteal cells were expanded in monolayer culture before seeding in 3% (w/v) agarose constructs. Intermittent dynamic compression (1 Hz, 15% strain) was applied to constructs, in the presence or absence of 10 ng/ml TGF-β3, for up to 4 days. The combined effect of TGF-β3 and compressive loading on the expression levels of the Sox-9, Runx-2, ALP, Collagen X, and collagen type I genes was donor dependent. A synergistic effect was noted only in donor two, with peak mRNA expression levels at 24 h, particularly Sox-9 which increased 59.0-fold."

Sox9 increased without any TGF-Beta.

"Runx-2 and Sox-9 gene expressions were highest in constructs treated with TGF-β3 in chondrogenic media and subjected to intermittent compression over 24 h"

"In periosteal cell-constructs derived from donor 1, loading did not have a positive effect on Sox-9 or Runx-2 mRNA levels. In the absence of TGF-β3, periosteal cells responded to mechanical compression through a significant down-regulation of Sox-9 expression at 12 h (1.6-fold ↓) and 48 h (1.7-fold ↓). A similar decrease (1.6-fold) was found in Runx-2 expression after 48 h of loading in constructs treated with TGF-β3."

"stimulated TGF-β signalling pathways [modulates] mechanotransduction, directly or indirectly, by increasing the sensitivity of periosteal cells to loading through the activation of mechanosensitive proteins, such as focal adhesion kinase and paxillin"

"Dynamic compressive loading promoted gene expression and protein production of both TGF-β receptors (TGF--I and II) in rabbit-MSCs in agarose.  TGF--1, phosphorylated by TGF-β, initiates intracellular signal transduction, which mediates chondrogenic differentiation of chondroprogenitor cells and mesenchymal stem cells"

"As the tissue undergoes a compressive load, the pressurization of the fluid phase initially supports the applied load, because water is trapped within the solid matrix of the tissue because of its low permeability"

"In the joint, cartilage is typically exposed to stresses between 3 and 10MPa, with stress as high as 18MPa having been reported in the hip joint. These stresses [are] translated to HP due to fluid phase pressurization"<-MPa's above these levels have been reported to have anti-chondrogenic effects.

"HP has direct effects on cell membrane ion channels. Static HP on isolated bovine chondrocytes for 20s or 10min [resulted in the] sodium–potassium (Na/K) pump [being] substantially inhibited when going from 2.5 to 5MPa"  "[HP] activates Na/hydrogen and stretch-activated calcium (Ca) channels, and triggers release of intracellular Ca stores."<-And this release according to our hypothesis induces chondrogenic differentiation.

"detrimental effects are apparent with loading times exceeding 2h [for loads as low as 15 MPa]."

Induction of chondrogenic phenotype in synovium-derived progenitor cells by intermittent hydrostatic pressure.

"SPCs[synovium derived progenitor cells], bone marrow-derived progenitor cells and skin fibroblasts from rabbits were subjected to IHP ranging from 1.0 to 5.0 MPa. The mRNA expression of proteoglycan core protein (PG), collagen type II and SOX-9 was examined The production of SOX-9 protein and glycosaminoglycan (GAG) by SPCs was analyzed .  Mitogen-activated protein (MAP) kinase inhibitors for c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase (ERK), and the p38 pathway were used.
mRNA expression of PG, collagen type II and SOX-9 was significantly enhanced only in SPCs receiving 5.0 MPa of IHP. The production of SOX-9 protein and GAG by SPCs was also increased by exposure to 5.0 MPa of IHP. These up-regulated expressions were suppressed by pretreatment with an inhibitor of JNK, but not with inhibitors of ERK or p38."

So maybe chondroinduction occurs along the JNK pathway.  "the JNK pathway is involved in signal transduction for IHP and in increased expression of c-Jun"

Mesenchymal Stem Cells: Role of Mechanical Strain in Promoting Apoptosis and Differentiation

"In contrast to necrotic cell death, apoptosis is an ordered process that does not trigger a pronounced inflammatory response in the surrounding tissue, due in part to the maintenance of an intact plasma membrane"

"the apoptotic process occurred as an essential step in normal ontogenesis indicating that apoptosis is an inherently programmed pathway responsible for determining cellular fate and acts as a counterbalance to mitosis thus maintaining homeostasis in the organism"

"Stretch-activated cation channels (SACCs) are understood to be involved in mechanotransduction and the presence of these receptors has been identified on osteocytes where they act as mechanical transducers. Blocking SACCs with gadolinium chloride does not protect against strain-induced apoptosis, indicating that these channels are not involved in the upregulation of apoptotic pathways in MSCs. However, in a separate study investigating the effect of 2.5% continuous strain on MSC differentiation, upregulation of collagen type I is observed. This induction of collagen type I is decreased when SACCs are blocked, highlighting a role for these receptors in the strain-induced expression of bone-related proteins"

"Mechanical stress is also thought to elongate the cell membrane which can result in activation of p38 MAPKs and production of ROS. Both of these signals converge at the level of p53. The p38 MAPK pathway directly phosphorylates p53, while ROS production causes oxidative DNA damage. Indirect activation of p53 by p38 MAPKs may occur by regulation of oxidase activation whereby p53 might be involved in a positive feedback loop by stimulating free radical generation itself. Expression of the pro-apoptotic Bax protein and mitochondrial dysfunction also mediate apoptosis following mechanical stress. Strain-induced apoptosis has also been observed in MSCs as a result of signalling via L-type calcium channels, calpain activity and JNK activation. Application of a continuous 10% strain results in L-type calcium channel activity leading to increased intracellular calcium concentrations which may be responsible for inducing conformational changes in the cysteine protease calpain, leading to its activation. Strain-mediated apoptosis is prevented when cells are strained in the presence of a calpain inhibitor, implicating these proteases in the apoptotic response of MSCs to tensile strain. Calpain has also been associated with JNK activity in response to 10% tensile strain"

"Mechanotransduction signalling pathways and MSC fate decisions in response to mechanical strain. Integrin receptors link the cell membrane to the ECM. Upon receptor ligation, integrins cluster within the membrane and form focal adhesion complexes leading to actin polymerisation via talin, vinculin (VCL) and paxilin (PXN) recruitment. Downstream of these events the key integrin signal mediator, FAK, is activated leading to GTPase Rho, MAPK and ERK 1/2 activation. Stretch-activated cation channels stimulated by mechanical strain activate PI3K and p38 leading to phosphorylation of p53. Application of 2.5% mechanical strain induces MSC differentiation via these pathways. Conversely, apoptotic pathways are upregulated in response to ≥7.5% mechanical strain. Upregulation of p38 and subsequent phosphorylation of p53, in conjunction with cytochrome-c release from the mitochondria, leads to caspase-3 activation via holoenzyme formation of Apaf-1 and caspase-9. Activation of voltage-activated calcium channels increases intracellular calcium concentration, which alters calpain conformation. This calpain activation is associated with p38 phosphorylation leading to cellular apoptosis"

The external mechanical environment can override the influence of local substrate in determining stem cell fate.

"Bone marrow derived mesenchymal stem cells (MSCs) were seeded in agarose and fibrin hydrogels and subjected to dynamic compression in the presence of different concentrations of TGF-β3. Markers of chondrogenic, myogenic and endochondral differentiation were assessed. MSCs embedded within agarose hydrogels adopted a spherical cell morphology, while cells directly adhered to the fibrin matrix and took on a spread morphology. Free-swelling agarose constructs stained positively for chondrogenic markers, with MSCs appearing to progress towards terminal differentiation as indicated by mineral staining. MSC seeded fibrin constructs progressed along an alternative myogenic pathway in long-term free-swelling culture. Dynamic compression suppressed differentiation towards any investigated lineage in both fibrin and agarose hydrogels in the short-term. Given that fibrin clots have been shown to support a chondrogenic phenotype in vivo within mechanically loaded joint defect environments, we next explored the influence of long term (42 days) dynamic compression on MSC differentiation{so you might not get results with LSJL for 42 days}. Mechanical signals generated by this extrinsic loading ultimately governed MSC fate, directing MSCs along a chondrogenic pathway as opposed to the default myogenic phenotype supported within unloaded fibrin clots. In conclusion, this study demonstrates that external cues such as the mechanical environment can override the influence specific substrates, scaffolds or hydrogels have on determining mesenchymal stem cell fate."

The emergence of mechanoregulated endochondral ossification in evolution.

"The emergence of mechanosensitive genes that trigger endochondral ossification in evolution will stabilise in the population and create a variable mechanoregulated response, if the endochondral ossification process enhances fitness for survival.  The fitness of animals in a population is determined by their ability to heal their bones. With the emergence of mechanosensitive genes through evolution enabling skeletal cells to modulate their synthetic activities, novel differentiation pathways such as endochondral ossification could have emerged, which when favoured by natural selection is maintained in a population. Evolutionary forces do not lead to a single optimal mechanoregulated response but that the capacity of endochondral ossification exists with variability in a population."

"low mechanical stimuli promote bone differentiation and high stimuli inhibits differentiation of mesenchymal stem cells to bone cells"

"the fracture callus [is] initially filled with granulation tissue and replaced with bone through endochondral ossification."

"thinner bones have been associated with higher mechanosensitivity and thicker bones with low mechanoresponsiveness"

"Thicker bones would be able to better resist daily loads, but be heavy to carry and less mechanosensitive if subjected to injury. "

 Mechanical regulation of chondrogenesis.

"the effects of dynamic compressive loading alone (that is, in the absence of exogenous growth factors) on MSC chondrogenesis appear to be minimal and transient"  Referring to the study mentioned, growth factors decrease dramatically at the third week.  Supplementary tables with gene expression data are provided that will have to be analyzed.  The study does state however "Despite
the absence of TGF-β3, by week 6, a subset of MSCs in CM- had undergone chondrogenesis to a limited extent, depositing a small amount of type II collagen in the immediate pericellular space"  And gene expression decreases in both the chondrogenic plus and minus mediums at the third week.

"The chondrogenic effects of loading on MSCs in the absence of growth factors also appear to be transient. For example, while loading in the absence of growth factors increased Col2α1 and aggrecan gene expression after 1 and 2 weeks of loading, expression of these chondrogenic markers returned to baseline levels after an additional week of continued loading"

"Joint loading leads to complex tissue strains, including components of compression, tension, and shear, producing direct cellular and nuclear deformation.  indirect biophysical factors are also generated as a result of the exudation of interstitial water and ions from cartilage, including streaming potentials, changes in local pH and osmolarity, and hydrostatic pressure"

" the MSC response to hydrostatic loading does not require a preculture period"

"with TGFβ supplementation,  while 0.1 MPa was sufficient to increase Sox9 expression, upregulation of Col2α1 expression only occurred with loading at 10 MPa"

" in the absence of exogenous TGFβ, hydrostatic pressure increased expression and secretion of TGFβ1, as well as the phosphorylation of Smad2/3 and p38 mitogen-activated protein kinase"

"Mechanical loading of the cartilage layer results in large gradients in hydrostatic pressure, which subsequently induce flow of the interstitial fluid within the extracellular matrix. One way that mechanical loading is predicted to enhance tissue maturation is through this flow-mediated nutrient and growth factor exchange, as well as through physical activation of growth factors. Loading may also influence tissue maturation through direct transduction of fluid shear stress across the cellular membrane."

"the TRPV4 ion channel has been identified as the major sensor of osmolarity in chondrocytes, and activation of this channel leads to an influx of calcium ions."

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