Sunday, September 19, 2010

Grow Taller with Ions

The Sodium-Potassium pump is an enzyme located in the plasma membrane of all cells including chondrocyte cells.  The maximum size achieved by chondrocytes in the hypertrophic zone during normal growth is one of the primary determinants of natural height growth.  If the cells grow too large then they burst.  Hypertrophic chondrocytes die but it is due to lack of nutrients not due to bursting.

Ions like Sodium, Potassium, and Lithium are charged so this may be one mechanism that Pulsed-Electric Magnetic Fields can influence height growth.  If you look at some of the studies listed there, you can see that 20-60% higher cell densities were achieved during the cell expanding phase.  The possible mechanism could be as mentioned earlier due to PEMF stimulating charged ions.

LSJL may help transport these ions into the chondrocyte cells by stimulating fluid flow and increasing pressure. 

A key role for membrane transporter NKCC1 in mediating chondrocyte volume increase in the mammalian growth plate.

"The mechanisms that underlie growth plate chondrocyte volume increase and hence bone lengthening are poorly understood. Many cell types activate the Na-K-Cl cotransporter (NKCC) to bring about volume increase. We hypothesised that NKCC may be responsible for the volume expansion of hypertrophic chondrocytes. Metatarsals/metacarpals from 16 rat pups (P(7)) were incubated in the presence/absence of the specific NKCC inhibitor bumetanide and measurement of whole-bone lengths and histologic analysis of the growth plate were done after 24 hours. Fluorescent NKCC immunohistochemistry was visualised using a confocal laser scanning microscopy on seven rat tibial growth plates (P(7)). Microarray analysis was performed on mRNA isolated from proliferative and hypertrophic zone cells of tibial growth plates from five rats of each of three ages (P(49/53/58)). Exposure to bumetanide resulted in approximately 35% reduction of bone growth in a dose-dependent manner; histologic analysis showed that a reduction in hypertrophic zone height was responsible. Quantification of fluorescence immunohistochemistry revealed a significant (paired Student's t test, p < .05) change in NKCC from the intracellular space of proliferative cells to the cytosolic membrane of hypertrophic zone cells. Further, microarray analysis illustrated an increase in NKCC1 mRNA between proliferative and hypertrophic cells. The increase in NKCC1 mRNA in hypertrophic zone cells, its cellular localization, and reduced bone growth in the presence of the NKCC inhibitor bumetanide implicate NKCC in growth plate hypertrophic chondrocyte volume increase. Further investigation is warranted to determine the regulatory control of NKCC in the mammalian growth plate and the possible detrimental effect on bone growth with chronic exposure to loop diuretics."

The fact that there was only a 35% reduction in bone growth shows that there are other factors(like number of stem cells) but that's still a huge amount. 

The study states that the volume increase in hypertrophic chondrocytes occurs predominantly in the longitudinal direction which is why bones grow longer rather than wider in equal proportion.  "The volume of a typical cell increases from approximately 1000 µm3 in the [proliferative zone] to approximately 15,000 µm3 in the [hypertrophic zone]."  So after a certain point increasing the maximal size attained in chondrocyte hypertrophy while affect height a lot more than increasing the number of cells.

"[A change in the osmotic gradient] can result from a reduction in extracellular osmolarity, an increase in intracellular osmolarity, or a combination of the two."<-we try to change the extracellular osmolarity with hydrostatic pressure via LSJL.  Altering intracellular calcium secretions may alter intracellular osmolarity.

"That for cell swelling to be entirely mediated by a reduction in extracellular osmolarity, these cells would need to be exposed to an approximately 280 mOsmol reduction in osmolarity to approximately 120 mOsmol. Plasma osmolarity therefore would need to be reduced from approximately 300 mOsmol to near 20 mOsmol, which is clearly implausible. Therefore, osmotic gradient–driven water accumulation is more likely to be due to an increase in intracellular osmolarity in the transition of a PZC to an HZC."<-This has been confirmed by the fact that a huge amount of hydrostatic pressure is required to induce chondrogenesis and LSJL only induces a small amount of hydrostatic pressure however LSJL may alter intracellular osmolarity thereby altering intracellular calcium secretions.

"Intracellular osmolarity can be raised by the catabolism of intracellular macromolecules (eg, proteins and/or complex carbohydrates) into osmotically active components (eg, amino acids and/or simple sugars)... The accumulation of organic solutes (eg, simple sugars and amino acids) is responsible for approximately 9% of the osmolytes required"

"The putative presence of transporters other than NKCC1 and/or redundancy in the system to drive HZC volume increase may explain the reduced size of NKCC1 knockout mice over the first 2 weeks, a period of maximal growth rate, but their ability to recover to the size of wild-type animals as they reach maturity.  This ability of NKCC1 knockouts to recover may explain why there are limited reports of skeletal growth retardation in children exposed to loop diuretics (eg, bumetanide and furosemide). "

Chloride might be the key chondrocyte stimulating compound. 

Chloride channels regulate chondrogenesis in chicken mandibular mesenchymal cells.  

"Voltage gated chloride channels (ClCs) play an important role in the regulation of intracellular pH and cell volume homeostasis[So any insights into chloride channels can provide insights into how intracellular pH and cell volume increases height]. Mutations of these genes result in genetic diseases with abnormal bone deformation and body size, indicating that ClCs may have a role in chondrogenesis. In the present study, we isolated chicken mandibular mesenchymal cells (CMMC) from Hamburg-Hamilton (HH) stage 26 chick embryos and induced chondrocyte maturation by using ascorbic acid[Vitamin C] and β-glycerophosphate (AA-BGP). We also determined the effect of the chloride channel inhibitor NPPB [5-nitro-2-(3-phenylpropylamino) benzoic acid] on regulation of growth, differentiation, and gene expression in these cells using MTT and real-time PCR assays. We found that CLCN1 and CLCN3-7 mRNA were expressed in CMMC and NPPB reduced expression of CLCN3{down in LSJL}, CLCN5, and CLCN7 mRNA in these cells. At the same time, NPPB inhibited the growth of the CMMC, but had no effect on the mRNA level of cyclin D1 and cyclin E (P>0.05) with/without AA-BGP treatment. AA-BGP increased markers for early chondrocyte differentiation including type II collagen, aggrecan (P<0.01) and Sox9 (P<0.05), whilst had no effect on the late chondrocyte differentiation marker type X collagen. NPPB antagonized AA-BGP-induced expression of type II collagen and aggrecan (P<0.05). Furthermore, NPPB downregulated type X collagen (P<0.05) with/without AA-BGP treatment. We conclude that abundant chloride channel genes in CMMC play important roles in regulating chondrocyte proliferation and differentiation. Type X collagen might function as a target of chloride channel inhibitors during the differentiation process[Chloride Channel inhibitors downregulated Type X Collagen which is a terminal differentiation marker but since the Chloride Channel inhibitor decreased the growth of the Mesenchymal Cells maybe Type X Collagen is essential for maximizing chondrocyte growth]."

"During embryonic development, chondrogenesis begins with mesenchymal cell recruitment and migration, proliferation, and condensation."<-This is what we try to achieve with LSJL.  Hydrostatic Pressure to recruit  MSCs to the site of the pressure so that the new cartilage acts as a sponge for the pressure.  Then that cartilage undergoes endochondral ossification within the bone and makes a person taller.

"ClC is associated with the volume-activated chloride current and is involved in cell volume regulation"

"endogenous ClC-3 is associated with the volume-activated chloride current and is involved in cell volume regulation. In our study, NPPB suppressed the expression of ClC-3, which may influence chondrocyte swelling (hypertrophy) and further influence the synthesis of type X collagen."<-maximizing the potential of the volume-activated chloride current will help height growth and further evidence that Type X Collagen should not be inhibited but may in fact help maximize growth.

The osmotic sensitivity of rat growth plate chondrocytes in situ; clarifying the mechanisms of hypertrophy. 

"Bone elongation is predominantly driven by the volume expansion of growth plate chondrocytes. This mechanism was initially believed to be "hypertrophy", describing a proportional increase of cell water and organelles. However, morphometrical analysis subsequently assumed the increase to be "swelling", resulting in a disproportionate increase of cell water (osmotically active fraction). Histological approaches were performed on fixed tissue, and for the "swelling" assumption to be valid, the osmotic sensitivity of living cells before and during volume increase should differ. To test this, analysis of images acquired by 2-photon laser scanning microscopy (2PLSM) were used to determine the osmotic sensitivity, and osmotically active/inactive proportions of in situ chondrocytes from 15 living rat growth plates exposed to varying media osmolarities ( approximately 0-580 mOsm). The dimensions of cell volume swelling in hypotonic[hypotonic means that there was less water outside the cell then in cause water to be taken into the cell making it grow larger] media were different to the preferential lengthening seen in vivo, confirming the complexity of directional cell volume increase. Boyle-van't Hoff analysis of cell volume over the range of media osmolarity indicated no significant difference (Student's t-test) in the osmotically inactive fraction, 39.5 +/- 2.9% and 47.0 +/- 4.3% (n = 13) for proliferative and hypertrophic zones, respectively, or the sensitivity of volume to changes in media osmolarity (proliferative 15.5 +/- 0.8 and hypertrophic zone 15.5 +/- 1.2%volume . Osm). The osmotic fractions did not change as chondrocytes progress from proliferative to hypertrophic regions of the growth plate. Our data suggest cell volume increase by hypertrophy may play a greater role in cell enlargement than swelling, and should be re-evaluated as a mechanism responsible for growth plate chondrocyte volume increase and hence bone elongation." 

This suggests that water may not be the primary force in chondrocyte hypertrophy.

"More recently, however, stereological analysis of fixed tissue has shown a disproportionate increase in cell compartments. These observations showed an increase in cell organelle volumes (endoplasmic reticulum, Golgi membranes, and mitochondria) was responsible for ∼15% of the total cell volume increase between proliferative zone chondrocytes (PZC) and hypertrophic zone chondrocytes (HZC), the majority (85%) of volume increase arising from cytoplasm and nucleoplasm expansion"

"The apparent preferential increase of cell cytoplasm and nucleoplasm led to the assumption that cell volume increase was primarily due to “swelling” (fluid accumulation)."<-so swelling is still a part of the cell volume increase

" The osmotically active fraction describes freely mobile water within the cell, and it is this fraction that would increase dramatically if a cell increased in volume by “swelling” alone. Conversely, if a cell showed a general increase in total volume in proportion to its intracellular constituents, “hypertrophy,” then the osmotically active and inactive fractions will increase in proportion. If, as stereological approaches have suggested, cell volume increase occurs primarily due to fluid accumulation rather than hypertrophy, the osmotically inactive/active fractions of the PZC and HZC should differ"<-If much of the size increase is due to hypertrophy that doesn't mean that some is due to swelling.

"Cell volumes increased ∼10-fold, from 1,172 ± 209µm3 in S1 to 10,584 ± 1,315 µm3 in S8"<-this is a pretty large change and illustrates the importance of chondrocyte hypertrophy.

"The increase in volume shown by this PZC was from 768 µm3 in 280 mOsm media to 1,900 µm3 in near 0 mOsm, whereas the HZC volume increased from 6,236 µm3 in 280 mOsm media to 26,878 µm3 in near 0 mOsm media."<-Higher osmolarity meaning a higher concentration of a substance other than water so less hydrostatic pressure. More water still increased swelling it was just the same in just proliferative and hypertrophic zones.

"All cells showed a clear reduction in volume with hyper-osmotic challenge, and hypotonicity increased chondrocyte volume even when media osmolarity reduced from 80 to ∼0 mOsm media, with little apparent restriction to swelling from the surrounding matrix"<-the higher the water concentration(or the lower the concentration of other solutes) the higher the chondrocyte volume.

"GAGs will preferentially attract cations, but matrix/bone restriction to swelling will limit the volume of osmotically obliged water that can follow."<-So GAGs will absorb water.  Since the amount of GAGs increase based on layer this could be why the cells don't change in osmotic sensitivity as it is the level of GAGs that are changing.

"The concentration and the type of GAG have been shown to vary from proliferative to hypertrophic zones"<-So the osmolaric sensitivity varies with the GAGs

"We also cannot discount that the driving force for cell volume increase is by swelling, followed by consolidation through the production of “dry matter.” A PZC increasing in volume by 10-fold through “hypertrophy,” with a concomitant increase in osmotically inactive volume would require ∼3,600 µm3 (assuming a 40% osmotically inactive fraction) increase in “dry matter.” Transport of the components required for synthesis of osmotically inactive structures (e.g., amino acids, simple sugars, etc.) may cause a transient osmotic gradient inducing cell swelling. Indeed, transporter (with receptor) mRNA expression shows a near tripling in the number of relevant up-regulated genes"<-Water may still be involved indirectly.

LSJL may involve ionic transport.

Knee-loading modality drives molecular transport in mouse femur.

"Load-driven interstitial fluid flow and molecular transport[Calcium gate ion channels show the importance of both calcium and barium] play a role in the enhancement of bone formation. In order to evaluate load-driven molecular transport in a lacunocanalicular network, we conducted fluorescence recovery after photobleaching (FRAP) experiments using lacunae stained with uranine (376 Da). Loads were applied to a mouse femur ex vivo with a novel knee-loading modality, where the distal epiphysis was loaded with a sinusoidal force at 2 Hz[40V at 1.7N]. The lacunae in the diaphysis located 25% (approximately 4 mm) proximal to the loading site were photobleached and sequentially imaged, and a time constant for fluorescence recovery was determined both with and without knee loading. The time constant was estimated as the period to recover 63% of fluorescent intensity using a best-fit exponential curve. The results reveal that the applied loads shortened the time constant from 33 +/- 9 s with non-loading control to 25 +/- 11 s with knee loading (p = 0.0014). The strain in the measurement site was <100 microstain along the femoral midshaft, which was an order of magnitude smaller than the minimum effective strain threshold for bone remodeling. molecular transport in cortical bone is enhanced by the loads applied to the epiphysis."

"The observed time constants of fluorescence recovery clearly showed enhanced molecular transport with knee loading"<-All molecules and that's a wide category, some of those molecules are beneficial for height increase.

"The described knee-loading modality may remotely displace fluid from the epiphysis towards the metaphysis and the diaphysis and stimulate molecular transport in cortical bone."<-this displacement increases hydrostatic pressure and is partly what induces chondrogenesis.

"the strain in the femur 25% (∼4 mm) proximal to the distal end of the femur was measured as 26.8 ± 7.8, 43 ± 7.7 and 93.8 ± 8.2 μstrain with 1, 2 and 4 N force, respectively. The best-fit regression line for the force–strain relationship was y = 22.7x + 1.5 with r 2 = 0.99. Note that the background strain level without any loads was ∼10 μstrain."<-So strain increases rapidly as force increases.  To get 1500 micorstain you'd need to use about 66N.  But this did not measure strain directly at the epiphysis.

But an increase in molecular and ionic transport could play a role in the effects of LSJL.

Switch of voltage-gated K+ channel expression in the plasma membrane of chondrogenic cells affects cytosolic Ca2+-oscillations and cartilage formation.

"Using patch-clamp, RT-PCR and Western-blot experiments, we found that chondrogenic cells in primary micromass cell cultures obtained from embryonic chicken limb buds expressed voltage-gated Na(V)1.4, K(V)1.1, K(V)1.3 and K(V)4.1 channels, although K(V)1.3 was not detectable in the plasma membrane. Tetrodotoxin (TTX), the inhibitor of Na(V)1.4 channels, had no effect on cartilage formation[so sodium likely doesn't affect chondrogenesis]. In contrast, presence of 20 mM of the K(+) channel blocker tetraethyl-ammonium (TEA) during the time-window of the final commitment of chondrogenic cells reduced K(V) currents (to 27±3% of control), cell proliferation (thymidine incorporation: to 39±4.4% of control), expression of cartilage-specific genes and consequently, cartilage formation (metachromasia: to 18.0±6.4% of control) and also depolarized the membrane potential (by 9.3±2.1 mV)[potassium plays a large role in chondrogenesis]. High-frequency Ca(2+)-oscillations were also suppressed by 10 mM TEA (confocal microscopy: frequency to 8.5±2.6% of the control). Peak expression of TEA-sensitive K(V)1.1 in the plasma membrane overlapped with this period. Application of TEA to differentiated chondrocytes, mainly expressing the TEA-insensitive K(V)4.1 did not affect cartilage formation.
These data demonstrate that the differentiation and proliferation of chondrogenic cells depend on rapid Ca(2+)-oscillations, which are modulated by K(V)-driven membrane potential changes. K(V)1.1 function seems especially critical during the final commitment period. We show the critical role of voltage-gated cation channels in the differentiation of non-excitable cells with potential therapeutic use."

So altering the K+ channels may be a way to grow taller.

"Differentiating chondrocytes in high density cultures (HDC) displayed rapid and repetitive [Ca2+]i transients"<-So inducing rapid and repetitive [Ca2+]i transients in epiphyseal MSCs may be a way to grow taller.

"With differentiation, the frequency of the transients decreased, (0.163±0.020 Hz on day 1 and 0.089±0.010 Hz on day 3, ANOVA: p<0.01, Holm-Sidak (HS): t=3.719) but their amplitude did not change significantly (expressed as F/F0: 1.98±0.40 on day 1 and 1.64±0.17 on day 3; ANOVA: p>0.05)"<-So the key is to increase the frequency of [Ca2+]i transients.

"The depolarization of the cell membrane by administration of 20 mM KCl on day 2 resulted in a large and transient increase in [Ca2+]i"

"Upon the removal of calcium from the external medium the amplitude and frequency of the repetitive calcium transients were decreased, sometimes completely eliminated"

"chondrogenic mesenchymal cells exhibited unique Ca2+ oscillations in our experiments"<-mimic the Ca2+ oscillations in mesenchymal cells to induce chondrogenesis.

"human mesenchymal stem cells [Ca2+]i oscillations [have] an average frequency of 1 transient in 120 sec were reported"

"cells of HDC exhibited spontaneous oscillations with an extraordinarily high frequency (approx. 5 in 60 sec on day 1)"<-high density culture induces chondrogenesis so our goal should be 5 oscillations per minute.

Frequency is important to LSJL with the optimal frequency being 0.5Hz this could be related to the calcium ion oscillations.

Suppression of mammalian bone growth by membrane transport inhibitors.

"We investigated the role of the Na(+) /H(+) antiporter (NHE1)[regulates pH and cell volume] and anion exchanger (AE2) in bone lengthening and GP chondrocyte hypertrophy in Sprague-Dawley 7-day-old rat (P7) bone rudiments using the inhibitors EIPA (5-(N-ethyl-N-isopropyl)amiloride) and DIDS (4,4-diidothiocyano-2,2-stilbenedisulphonate) respectively. We have also determined cell-associated levels of these transporters along the GP using fluorescent immunohistochemistry (FIHC). Culture of bones with EIPA or DIDS inhibited rudiment growth (50% at approx. 250µM and 25µM respectively). Both decreased the size of the hypertrophic zone (P<0.05) but had no effect on overall length or cell density of the GP. In situ chondrocyte volume in proliferative and hypertrophic zones was decreased (P<0.01) with EIPA but not DIDS. FIHC labelling of NHE1 was relatively high and constant along the GP but declined steeply in the late hypertrophic zone. In contrast, AE2 labelling was relatively low in proliferative zone cells but increased (P<0.05) reaching a maximum in the early hypertrophic zone, before falling rapidly in the late hypertrophic zone suggesting AE2 might regulate the transition phase of chondrocytes between proliferative and hypertrophic zones. The inhibition of bone growth by EIPA may be due to a reduction to chondrocyte volume set-point. However the effect of DIDS could result from inhibition of AE2 and blocking of the transition phase."

"The controlled increase in chondrocyte size and the regulation of the intracellular environment (e.g. ion content, pHi) to permit optimal metabolism must be closely regulated throughout the hypertrophic process mainly by the activity of the membrane transporters in the chondrocyte membrane. It is likely that there is an important role for transporters which regulate the movement of Na+ and anions (e.g. HCO3-) across the cell membrane"

"Increased activity of NHE1 combined with the parallel activation of the anion exchanger isoform AE2 occurs following the shrinking of cells in media of raised osmolarity. This gives rise to the recovery of cell volume through the process of regulatory volume increase (RVI) and also the maintenance of pHi in the face of the osmotic challenge"

"Activation of these transporters without a change in extracellular osmotic pressure, will directly increase cell volume and re-set pHi"<-so increasing NHE1 and AE2 will help you grow taller.

"EIPA significantly reduced the volume of chondrocytes throughout the length of the GP whereas DIDS had no significant effect"

"levels of NHE1 remained high in chondrocytes throughout most of the GP, but declined in late hypertrophic cells whereas AE2 levels demonstrated a biphasic effect"

"DIDS is a relatively non-specific inhibitor of AEs, as it also blocks Cl- channels in a range of cell types [whereas EIPA is a specific inhibitor of NHEs]"

"DIDS has also been reported to block diastrophic dysplasia sulphate transport (DTDST)-mediated SO4 - uptake which is essential for proteoglycan synthesis"

"programmed cell death (‘apoptosis’) in some cell types is associated with a decline in AE2

"the addition of  IGF-1 to isolated chondrocytes stimulates NHE1 potentially resulting in an optimal pHi for the early stages of chondrocyte hypertrophy."

Effect of sodium selenite on growth, insulin-like growth factor-binding proteins and insulin-like growth factor-I in rats.

"30 male Wistar rats were randomized into three groups. Group A was treated with sodium selenite in the drinking water (3.3 mg selenium/l). Group B was ad libitum fed with free access to standard fodder and tap water and group C was pair fed relative to the selenium-treated rats. Serum IGF-I and IGFBPs were determined on days 0, 14 and at the end of the study on day 35. Selenium-treated rats had significantly lower body weights compared with group B rats on day 9 and group C rats on day 14. Tibia length was measured at the end of the study and no difference was observed between groups B and C (3.77 +/- 0.04 cm vs 3.60 +/- 0.02 cm); however, selenium-treated rats had significantly shorter tibia lengths (3.46 +/- 0.03 cm) compared with rats in groups B and C. Selenium treatment induced a significant reduction in circulating IGF-I by the end of the study compared with ad libitum and pair fed rats. Serum subjected to Western ligand blots showed four distinct IGFBP bands with apparent relative molecular weights of 38-47 kDa (doublet) (IGFBP-3), 30 kDa (IGFBP-1 and/or IGFBP-2) and 24 kDa (IGFBP-4)."

Couldn't access full study.

Impaired glycolytic metabolism causes chondrocyte hypertrophy-like changes via promotion of phospho-Smad1/5/8 translocation into nucleus.

"ATP production was dose-dependently decreased by NaF(Sodium Fluoride) in the human chondrocytic cell line HCS-2/8. In addition, both chondrocyte proliferation and differentiation were inhibited, whereas cell death was promoted by treatment with NaF. Combinational treatment with NaF and lactate enhanced translocation of phospho-Smad1/5/8 to the nucleus, as well as gene expression of ALP, VEGF, COL10a1, and MMP13, which were the markers of late mature and hypertrophic chondrocytes. Furthermore, the production of type X collagen and activation of MMP9 were also promoted under the same conditions."

"decreased ATP production by NaF promotes hypertrophy-like changes via activation of phospho-Smad1/5/8 in the presence of lactate."

"cartilage is an avascular tissue and chondrocytes generate energy in the form of adenosine triphosphate (ATP), mostly dependent on anaerobic metabolism of glucose. Therefore, if this metabolism is impaired, intracellular ATP production is profoundly decreased; and the chondrocytes are forced to degenerate, following the pathway to hypertrophy-like changes."

"There was no recovery of the decreased intracellular ATP production when lactate was present"<- Lactate is an end product of glycolosis.

"the gene expression of MCT2 was remarkably increased in HCS-2/8 cells treated with NaF or the NaF combined with lactate in the comparison with that in the untreated ones"

"MCT1 protein was decreased in pellet culture of RCS cells treated with NaF, but MCT2 protein was increased"

"Sodium lactate enhances the BMP signaling but not nuclear factor (NF)-kB signaling under the condition of lowered ATP production in HCS-2/8 cells.  MCT1 contributes to cell death through activation of NF-kB in mouse chondrocytic ATDC5 cells"

"Gene expression of COL10a1, ALP, MMP13, and VEGF, all of which are markers of hypertrophic
chondrocytes, was promoted by treatment with NaF or it in combination with lactate more strongly than by that with PBS or lactate alone"

"the gene expression of AGGRECAN (ACAN), which is a marker of mature chondrocytes, was decreased in the cells treated with NaF or NaF combined with lactate compared with its expression in the presence of PBS or lactate alone."

"the active form of MMP9, which is another marker of hypertrophic chondrocytes, was detected in medium conditioned by HCS-2/8 cells treated with NaF or NaF combined with lactate"

Note that these scientists are looking at articular cartilage not growth plate cartilage where hypertrophy is beneficial.

"stimulation by lactate leads to activation of signal transducers and activators of transcription 3 (STAT3) and extracellular signal-regulated kinase (ERK)1/2 signaling pathways in human mesenchymal stem cells"

A chemical genetics strategy exposes novel modulators of chondrogenesis that act by blocking a potassium channel, Kcnd2, & reveals a potential role for potassium channels in limb development

"[We used] primary cultures of murine limb bud-derived mesenchymal (PLM) cells. Chondroblast differentiation is associated with increased SOX5, 6 and 9 activity; while hypertrophic differentiation is associated with reduced SOX5, 6 and 9 activity. Therefore, a SOX5/6/9-responsive reporter gene was used to follow expression of the chondroblast phenotype. Two compounds identified, Butamben (butyl 4-aminobenzoate; BAB) and Phenazopyridine hydrochloride (PHCl), exhibited strong pro-chondrogenic activity and morphologically similar alcian blue staining. BAB is a member of the benzocaine family of analgesics and functions by inhibiting sodium channel activity. However, BAB has also been shown to have potassium channel-blocking activity. Specifically, BAB inhibits the activity of Kcnd2; which through transcriptional profiling was also found to be down-regulated by bone morphogenetic protein-4 (BMP4). BAB and PHCl may be able to modulate chondrogenesis by acting on potassium channels. To confirm this idea we examined molecular activities of PLM cultures treated with BAB and PHCl at two stages of chondrogenesis: 1. pre-chondrocyte to chondroblast and 2. chondrocyte to hypertrophic chondrocyte. Results confirm, BAB and PHCl increase expression of chondrogenic markers and reduce expression of hypertrophic markers. In addition patch clamp analysis revealed both BAB and PHCl are able to block, at least partially, KCND2 channel activity."

30-45 Mm of BAB was optimal for chondroinduction.

"BAB (10 μM) was found to maximally inhibit [KCN2] channel activity by approximately 30%, and increasing the dose had no further impact on channel activity"

"PHCl is a more effective KCND2 channel blocker than BAB, and at higher concentrations PHCL exhibits > 80 % inhibition of KCND2 channel activity"


"Chondrocytes, the only cell type that produces cartilage, occupy a unique bioelectrical environment. Mechanical load generates bioelectrical stimulation through rapid changes in ionic and water content around cells. A chondrocytes response to changing ionic gradients plays an essential role in regulation of extra cellular matrix synthesis. An abnormal response by the cell may result in abnormal deposition of proteins leading to degeneration of tissue with pathological consequences. Typically, response to ionic and osmotic gradients across cell membranes is by ion movement through channels as a cell reacts to maintain equilibrium. Chondrocytes possess an array of ion channels on par with electrically excitable tissues. Ion [channels are present in] chondrocytes. This proposal will quantify in real time dielectric properties of the membrane and cytoplasm in response to electrochemical changes to cells related to ion channel gene expression. We hypothesize that dielectric response of chondrocytes to changes in environment will be dependent upon ion channel activity. The objectives of this proposal are (1) to document dielectric changes in chondrocytes in real time when exposed to changes in osmolality, pH and ion flow using a novel microfluidic device. (2) Measure gene expression of ion channels in chondrocytes as a response to these changes. (3) Chondrocytes function under hypoxic conditions. We will directly compare dielectric response under normal versus hypoxic conditions and identify ion channels active in chondrocytes under hypoxic conditions. To our knowledge, our results will be the first description of dielectric properties of chondrocytes measured in real time and correlated to ion channel expression under hypoxia and will fundamentally impact cartilage biology unifying biomechanical and bioelectrical events in cartilage through ion channel response."

1 comment:

  1. LSJL should be focused more towards the spine.

    Have you gained any more height?