This gives us a new theory of the mechanism of action of LSJL. 0.5 Hz is once every 2 seconds. The frequency used in the mouse hindlimbs lengthening study is 5HZ which means once every 0.2seconds. Since the optimal frequency for inducing pressure was 0.5Hz why did they use 5Hz in the limb lengthening study? The frequency of differentiating chondrocytes on day 1 was about 0.163 Hz which is about once every 5 seconds. So the LSJL frequency is well under the frequency of calcium oscillations of day 1 chondrocytes(although you'd expect the need to have more frequent oscillations for stem cells that have not yet begun chondrogenesis).
The degree of hydrostatic pressure typically used to induce chondrogenesis is well over that induced by LSJL thus that LSJL alters calcium oscillations is a more likely mechanism of action.
So this is a bit of a paradigm shift for LSJL which is now less about inducing as much hydrostatic pressure as possible and instead about inducing new fluid flow every 2 seconds. This can be accomplished by either ratcheting the C-class clamp every 2 seconds or by squeezing on the irwin quick grip clamp every 2 seconds.
Here's some ways other than LSJL to alter the flux of calcium into MSCs(the mechanisms involved in altering calcium flux is likely the same as other cells but the frequency is likely greater in MSCs than the other cells):
Mechanically induced calcium signaling in chondrocytes in situ.
"Various physical stimuli can generate an influx of Ca(2+) into the cell, which in turn is thought to trigger a range of metabolic and signaling processes. Calcium signaling was quantified for dynamic loading conditions and at different temperatures. Peak magnitudes of calcium signals were greater and of shorter duration at 37°C than at 21°C. Ca(2+) signals were involved in a greater percentage of cells in the dynamic compared to the relaxation phases of loading. In contrast to the time-delayed signaling observed in isolated chondrocytes seeded in agarose gel, Ca(2+) signaling in situ is virtually instantaneous in response to dynamic loading{thus applying lateral loads on the bone should cause Ca(2+) signaling almost immediately}."
So temperature is one way to increase calcium signaling. There's likely an optimal temperature.
Inhibiting Interleukin-1 may be another way to increase calcium oscillations:
Interleukin-1 inhibits osmotically induced calcium signaling and volume regulation in articular chondrocytes.
"Articular chondrocytes respond to osmotic stress with transient changes in cell volume and the intracellular concentration of calcium ion ([Ca(2+)](i)). interleukin-1 (IL-1), a pro-inflammatory cytokine, influences osmotically induced Ca(2+) signaling.
[We] measure [Ca(2+)](i) and cell volume in response to hypo- or hyper-osmotic stress in isolated porcine chondrocytes, with or without pre-exposure to 10-ng/ml IL-1alpha. Inhibitors of IL-1 (IL-1 receptor antagonist, IL-1Ra), Ca(2+) mobilization (thapsigargin, an inhibitor of Ca-ATPases), and cytoskeletal remodeling (toxin B, an inhibitor of the Rho family of small GTPases) were used to determine the mechanisms involved in increased [Ca(2+)](i), F-actin remodeling, volume adaptation and active volume recovery.
In response to osmotic stress, chondrocytes exhibited transient increases in [Ca(2+)](i), generally followed by decaying oscillations[this is way we have to keep initiating new fluid flow because the oscillations decay]. Pre-exposure to IL-1 significantly inhibited regulatory volume decrease (RVD) following hypo-osmotic swelling and reduced the change in cell volume and the time to peak [Ca(2+)](i) in response to hyper-osmotic stress, but did not affect the peak magnitudes of [Ca(2+)](i) in those cells that did respond. Co-treatment with IL-1Ra, thapsigargin, or toxin B restored these responses to control levels. The effects were associated with alterations in F-actin organization.
IL-1 alters the normal volumetric and Ca(2+) signaling response of chondrocytes to osmotic stress through mechanisms involving F-actin remodeling via small Rho GTPases."
Lithium may inhibit IL-1.
"Joint loading results in deformation of the cartilage layer and associated loss of interstitial water, followed by recovery of this fluid as the load is released "
"As the proteoglycans in cartilage possess large numbers of negatively charged sulfate and carboxyl groups, the loss and gain of water changes the effective “fixed” charge density of the tissue, and thus alters the effective concentration of positively charged counterions (e.g., Na+, K+, Ca2+) in a manner that is directly related to magnitude of tissue dilatation"
"Due to the high permeability of the cell membrane to water either mechanical loading or changes in the extracellular osmolarity result in rapid changes in cell volume"
"The F-actin cytoskeleton, which can serve as a store of Ca2+, is also modulated by osmotic stress in a number of cell types. Dynamic reorganization of the F-actin network after exposure to hypo-osmotic stress has been described in chondrocytes"
"IL-1 increases the production of proteases responsible for matrix degeneration, suppresses matrix biosynthesis, and induces pro-inflammatory mediators, such as nitric oxide and prostaglandins "
"One of the earliest events in the response to IL-1 in chondrocytes is a transient increase in [Ca2+]i by a mechanism which may involve Ca2+ influx from the extracellular space, release from intracellular stores, or mobilization via activation of G-protein coupled receptors. Exposure to IL-1 also stabilizes F-actin in chondrocytic cells by a pathway involving activation of members of the Rho family of small GTPases. Of particular interest are findings showing that the response of chondrocytic cells to mechanical loading is altered by IL-1"
"Control experiments confirmed that perfusion of the chondrocytes with an iso-osmotic solution (340 mOsm) did not elicit changes in [Ca2+]i. Perfusion with either hypo- (240 mOsm) or hyper-osmotic (440 mOsm) medium led to an increase in [Ca2+]i accompanied by oscillations that decreased in magnitude over time"
"Pre-treatment with IL-1 for one hour significantly decreased the percentage of cells responding to osmotic stress. Only 27.3% of IL-1-treated cells displayed [Ca2+]i transients after exposure to hypo-osmotic stress, and none of these cells displayed oscillations "
"The effects of IL-1 on intracellular Ca2+ mobilization and cell volume recovery appear to be due to altered structure and remodeling of the F-actin cytoskeleton that is mediated by Rho family GTPases "
" these findings suggest that one mechanism for inhibition of [Ca2+]i signaling by IL-1 may involve inhibition of Ca2+ transport through a stabilized F-actin in the cell cortex or microvilli, which may then be responsible for inhibiting other Ca2+ mechanisms such as those required for osmotic signaling."
"hypo-osmotically induced Ca2+ signaling appears to involve Ca2+ release from the F-actin network as it is dissociated"
This study states that frequency of loading doesn't matter:
Activation of chondrocytes calcium signalling by dynamic compression is independent of number of cycles.
"Cyclic compression [was applied on] isolated articular chondrocytes cultured in agarose constructs. Cell-agarose constructs were subjected to 1Hz cyclic compression between 0 and 10% gross strain for 1, 10, 100 or 300 cycles. Within unloaded control constructs, a sub-population of approximately 50% of chondrocytes exhibited characteristic spontaneous Ca(2+) transients each lasting approximately 40-60s. Cyclic compression, for only 1 cycle, significantly up-regulated the percentage of cells exhibiting Ca(2+) transients in the subsequent 5min period. Increasing the number of cycles to 10 or 100 had no additional effect. The up-regulated Ca(2+) signalling was maintained for up to 5min before returning to basal levels. By contrast, 300 cycles were followed by Ca(2+) signalling that was not significantly different from that in unloaded controls. However, this response was shown to be due to the increased time following the start of compression. Chondrocyte Ca(2+) signalling is stimulated by dynamic compression, probably mediated by cyclic cell deformation. The overall response appears to be independent of the number of cycles or duration of cyclic compression. The sustained up-regulation of Ca(2+) signalling after 1, 10 or 100 cycles suggests the involvement of an autocrine-paracrine signalling mechanism. the reduced response following 300 cycles indicates a possible receptor desensitisation mechanism. Ca(2+) signalling may be part of a mechanotransduction pathway through which chondrocyte populations can modulate their metabolic activity in response to changing mechanical stimuli."
This study suggests that all you have to do is apply LSJL once every five minutes. Of course this study was in vitro and there are numerous ECM interactions and more chemicals in vivo. And this was with chondrocytes and not stem cells.
"Static compression of chondrocyte–agarose constructs results in cell deformation to an oblate ellipsoid morphology. However, as chondrocytes elaborate extracellular matrix, the level of cell deformation induced by static compression decreases as the matrix forms a relatively stiff pericellular shell. All experiments in the present study were conducted after 24 h in culture, at which time there was insufficient matrix to influence cell deformation."
"although oscillatory fluid flow generates a sustained up-regulation of Ca2+ signalling, the response is not believed to involve an autocrine–paracrine signalling pathway"<-And of course we're using an oscillatory fluid flow mechanism.
So oscillatory changes in calcium secretion represents a better fit for the LSJL mechanism of action than hydrostatic pressure and individuals experimenting with LSJL should keep this in mind.
Here's a patent by Wenhan Chang related to calcium receptors and growth plate development. We should watch this scientist to see if they provide any insights on Calcium signaling and possible ways to alter it to induce chondrogenic differentiation:
"The concentration of Ca2+ in the extracellular fluid ([Ca2+]e) plays a key role in growth plate cartilage development. Raising the [Ca2+]e directly promotes the terminal differentiation and mineralization of cultured mouse growth plate chondrocytes (mGPCs). Like Ca2+, insulin-like growth factor-1 (IGF1) promotes GPC differentiation. High Ca2+ also increases IGF1/IGF1R signaling capacity in mGPCs, suggesting that IGF1R-mediated signaling may be involved in the high [Ca2+]e-induces cell differentiation. The Ca2+-sensing receptor (CaR) is strongly expressed in mGPCs. Altering CaR expression and function profoundly impacts the differentiation of these cells. To determine whether the CaR is responsible for extracellular Ca2+-sensing by cartilage in vivo, we generated mice [CartCaR(-/-)] with tissue-specific knockout of the CaR in chondrocytes by a Cre-lox recombination technique. These mice die in gestation with malformed skeletons. We also developed a tamoxifen-inducible, chondrocyte-specific CaR knockout mouse [(Tam-CartCaR(-/-)]. These mice grow poorly during their postnatal life and have abnormally expanded growth plates with delayed mineralization in their hypertrophic zones. A high [Ca2+]e by activating CaRs stimulates ERK1/2 and PLC activity, increases the intracellular [Ca2+], enhances the expression of and signaling by IGF1 and the IGF1R, and promotes the terminal differentiation of GPCs. AIMs: (1) to determine the role of the CaR at specific stages of growth plate development by inducing CaR gene deletion with Tam at timed intervals before and after birth; (2) to determine whether a high [Ca2+]e alters chondrocyte function by regulating the expression of and signaling by IGF1 and the IGF1R in chondrocytes; (3) to assess the contribution of IGF1 and IGF1R-mediated signaling to the responses of GPCs to changes in the [Ca2+]e; and (4) to identify the high [Ca2+]e-induced signaling responses that are mediated by the CaR and are responsible for changes in the growth, survival, and differentiation of chondrocytes. Chondrocytes from growth plate cartilage are capable of sensing changes in the concentrations of Ca2+ in their surroundings and adjusting their activities accordingly. Changes in the concentrations of Ca2+ near growth plate chondrocytes influence their whole program of gene expression, matrix protein synthesis, and mineral deposition."
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