or manually).So how do osmotic stresses initiated by LSJL cause mesenchymal cells to differentiate to chondrocytes within bone and start the chain of endochondral ossification?
The effects of osmotic stress on the structure and function of the cell nucleus.
"Osmotic stress is a potent regulator of the normal function of cells that are exposed to osmotically active environments under physiologic or pathologic conditions. Cells alter gene expression and metabolic activity in response to changes in the osmotic environment. In addition to the activation of various osmotically- or volume-activated ion channels, osmotic stress may also act on the genome via a direct biophysical pathway[including genes that affect height]. Changes in extracellular osmolality alter cell volume, and therefore, the concentration of intracellular macromolecules. In turn, intracellular macromolecule concentration is a key physical parameter affecting the spatial organization and pressurization of the nucleus[thus possibly altering the organization in such a way as to differentiate into chondrocytes]. Hyper-osmotic stress shrinks the nucleus and causes it to assume a convoluted shape, whereas hypo-osmotic stress[meaning that the concentration outside the cell increases by a means such as LSJL] swells the nucleus to a size that is limited by stretch of the nuclear lamina and induces a smooth, round shape of the nucleus[Chondrocytes typically have an oval shaped nuclei]. These behaviors are consistent with a model of the nucleus as a charged core/shell structure pressurized by uneven partition of macromolecules between the nucleoplasm and the cytoplasm. These osmotically-induced alterations in the internal structure and arrangement of chromatin, as well as potential changes in the nuclear membrane and pores are hypothesized to influence gene transcription[so again osmotic stress has the ability to influence cellular differentiation] and/or nucleocytoplasmic transport."
"Soft connective tissues such as articular cartilage and intervertebral disk can bear loads of several times body weight because they are osmotically pressurized and hydrated by the presence of large, negatively charged proteoglycans that attract a large number of counter-ions (e.g., Na+, K+, Ca++) that significantly increase the local osmolality"<-This is why stem cells differentiate into chondrocytes in response to hydrostatic pressure because chondrocytes are good at reducing it.
"Although the individual components of cartilaginous tissues are incompressible, mechanical compression can increase the osmotic pressure acting on cells in the tissue due to exudation of the interstitial water and subsequent consolidation of the negatively charged matrix"<-this is what we are doing with LSJL. We are using lateral mechanical compression to increase the osmotic pressure acting on the cells. The reason that axial compression doesn't work such as due to say squats is that the load isn't directly on the bone and there's no pressure gradient.
"Osmotic and mechanical stresses can regulate gene expression via biochemical pathways involving physical connections between the cell and extracellular matrix{extracellular matrix not necessarily being cartilage it can also be the Type I Collagen produced by bone}. Mechanical stresses also act on the genome through a biophysical pathway. Mechanical loads are transferred from extracellular matrix molecules via integrins to the actin cytoskeleton and intermediate filament network, which is in turn connected to the nuclear lamina{thus actin cytoskeleton sensitivity is extremely important for a modality like LSJL and why we load in intervals of every 24-48 hours}. The nuclear lamina binds chromatin directly and via small proteins such as emerin. Therefore, there is a physical connection from the extracellular matrix to the genome along which mechanical stress can be transmitted. Similarly, osmotic stress can also act on the genome via a direct, biophysical pathway."
"The shape and mechanics of the nucleus depend on contractility in the actin cytoskeleton. This dependence is another mechanism by which osmotic stress may act on the nucleus, since hypo-osmotic stress can trigger disassembly of the actin cytoskeleton"-LSJL induces hypo-osmotic stress so we need to how much hypo-osmotic stress we want to induce before it becomes a negative influence? Is dissassembly of the actin cytoskeleton a good or bad thing for differentiation into chondrocytes?
In conclusion, LSJL can influence osmotic(hydrostatic) pressure. Hydrostatic pressure can induce differentiation of cells. Too much osmotic pressure can lead to disassembly of the actin cytoskeleton and it's unclear whether that is a good or bad thing in terms of height growth.
Loading the articular cartilage of the knee may have an effect on the stem cells in the epiphysis as the cartilage can send signals to the stem cells of the epiphysis(remember that the LSJL studies loaded the center of the joint capsule and in turn the articular cartilage).
Functional characterization of TRPV4 as an osmotically sensitive ion channel in porcine articular chondrocytes.
"Transient receptor potential vanilloid 4 (TRPV4) is a Ca(2+)-permeable channel that can be gated by tonicity (osmolarity) and mechanical stimuli{TRPV4 is a channel in chondrocytes that may have the ability to influence other cells via calcium gated ion channels}
In response to TRPV4 agonist/antagonists, osmotic stress, and interleukin-1 (IL-1), changes in Ca(2+) signaling, cell volume, and prostaglandin E(2) (PGE(2)) production were measured in porcine chondrocytes. Exposure to 4alpha-phorbol 12,13-didecanoate (4alphaPDD), a TRPV4 activator, caused Ca(2+) signaling in chondrocytes, which was blocked by the selective TRPV4 antagonist, GSK205. Blocking TRPV4 diminished the chondrocytes' response to hypo-osmotic stress, reducing the fraction of Ca(2+) responsive cells{A TRPV4 mutation could lead to LSJL possibly not working in some people}, the regulatory volume decrease, and PGE(2) production. Ca(2+) signaling was inhibited by removal of extracellular Ca(2+) or depletion of intracellular stores. Specific activation of TRPV4 restored the defective regulatory volume decrease caused by IL-1. Chemical disruption of the primary cilium eliminated Ca(2+) signaling in response to either 4alphaPDD or hypo-osmotic stress.
TRPV4 is present in articular chondrocytes, and chondrocyte response to hypo-osmotic stress is mediated by this channel, which involves both an extracellular Ca(2+) and intracellular Ca(2+) release. TRPV4 may be involved in modulating the production or influence of proinflammatory molecules in response to osmotic stress{This can influence other cells in the body such as the stem cells in the epiphysis}."
"The ECM of cartilage is inherently negatively charged due to the large concentration of the anionic proteoglycan aggrecan which attracts cations to counterbalance the charge. The resulting increase in interstitial osmolarity causes the tissue to imbibe water"<-again why the body tells the stem cells to differentiate in chondrocytes in response to hydrostatic pressure
"Osmotic stimulation of chondrocytes also elicits a cytoplasmic Ca2+-signal originating from both extracellular Ca2+-influx as well as intracellular Ca2+ release from stores"<-these signals could affect other cells in the body
"TRPV4 activation has also been shown to promote chondrogenesis by inducing SOX9 transcription through a Ca2+/calmodulin pathway"<-So loading the articular cartilage could be key for LSJL by inducing SOX9 transcription
"Joint loading can cause changes in the osmotic environment of the chondrocytes as fluid is forced out and reabsorbed into the cartilage"<-If the body lacks this cartilage to absorb like in the epiphysis post puberty the body has to produce new cartilage to absorb the fluid.
"Specific blocking of TRPV4 inhibits the volume-regulatory and Ca2+ signaling responses to hypo-osmotic stress"
Conclusion: a loss of function of TRPV4 may be a reason why LSJL will not work in some people. Although it's likely right now that LSJL is not working in some people for reasons other than a TRPV4 mutation. Also, signaling from the chondrocytes may be a key factor in the effectiveness in LSJL thus it may be worthwhile to directly load the joint capsule rather than each epiphysis individually. However, even loading each epiphysis individually to are still loading the joint capsule as the joint capsule is part of the epiphysis but the largest point of the joint capsule is right in the center between two bones. In the LSJL studies, they directly loaded this part of the joint capsule however that study was done early in development. In that stage the signaling of chondrocytes may play a larger role than epiphyseal compression would play in adults.
Here's some more evidence that fluid flow enhances chondrogenic differentiation of stem cells:
Effect of flow perfusion conditions in the chondrogenic differentiation of bone marrow stromal cells cultured onto starch based biodegradable scaffolds.
"[We] investigate the chondrogenic differentiation of goat bone marrow cells (GBMCs) under flow perfusion culture conditions. For that purpose, GBMCs were seeded into starch-polycaprolactone fiber mesh scaffolds and cultured in a flow perfusion bioreactor for up to 28 days using culture medium supplemented with transforming growth factor-β1. The tissue-engineered constructs were characterized after several end points (7, 14, 21 and 28 days) by histological staining and immunocytochemistry analysis, as well as by glycosaminoglycan and alkaline phosphatase quantification assays. In addition, the expression of typical chondrogenic markers was assessed by real-time reverse-transcription polymerase chain reaction analysis. A flow perfusion microenvironment favors the chondrogenic potential of GBMCs."
Fluid Flow such as that induced by LSJL encourages chondrogenic differentiation of MSCs.
"The addition of TGF-β1 provided the appropriate external signal which, when coupled with endogenous factors, communicated between cells within the condensation, and facilitated chondrogenic differentiation"<-Fluid flow also encourages TGF-B1 release from osteoblasts which gives another stimulation.
"GBMCs delay the proliferation rate until day 28, while they differentiate into the chondrogenic lineage, i.e. pellets display an initial phase of cell proliferation, followed by matrix deposition"<-thus it can take at least 28 days to see results from LSJL. Although if you perform LSJL correctly for 28 days you may see results down the road even if you stop.
Osmolarity regulates chondrogenic differentiation potential of synovial fluid derived mesenchymalprogenitor cells.
"A sub-population of mesenchymal progenitor cells (MPCs) derived from the synovial fluid may be able to affect some degree of cartilage repair both in vivo and in vitro/ex vivo[progenitor cells from the periosteum may be able to induce chondrogenesis as well], however this does not appear to be the case in patients with arthritis. Previously, it has been found that synovial fluid osmolarity is decreased in patients with osteoarthritis (OA) or Rheumatoid arthritis (RA) and these changes in osmolarity has been linked to changes in chondrocyte gene regulation. However, it is yet unknown if changes in osmolarity regulate the gene expression in synovial fluid MPCs[osmolarity may affect the ability of stem cells to undergo chondrogenesis] (sfMPCs), and by extension, chondrogenesis of this cell population. In the present study we have collected synovial fluid samples from normal, OA and RA knee joints, quantified the osmolarity of the fluid and modified the culture/differentiation media to span a range of osmolarities (264-375mOsm). Chondrogenesis was measured with Alcian blue staining of cultures in addition to quantitative PCR (qPCR) using probes to Sox9, ACAN and Col2A1. Overall, sfMPCs from arthritic joints demonstrated decreased chondrogenic potential compared to sfMPCs isolated from normal synovial fluid. Furthermore, the sfMPCs retained increased chondrogenic potential if differentiated under the same osmolarity conditions for which they were initially derived within. The synovial fluid osmolarity regulates the chondrogenic potential of sfMPCs."
"synovial fluid ranging from 404 ± 57 mOsm in normal joints, to 297 ± 16.9 mOsm and 280 ± 7.7 mOsm in OA and RA joints respectively"<-404 mOsm may be the optimal osmolarity for chondrogenesis.
"Osmolarity can trigger gene expression (Sox9 in particular) in these [sfMPCs]"<-So altering osmolarity via LSJL may be able to induce chondrogenesis.
"all sfMPCs tested demonstrated maximal chondrogenic potential at 300 mOsm and this did not appear to differ among patient populations"<-So ideally we would see what leveling of LSJL clamping would achieve an epiphyseal bone marrow osmolarity of 300 mOsm.
Hyperosmolarity regulates SOX9 mRNA posttranscriptionally in human articular chondrocytes.
"The transcription factor SOX9 regulates cartilage extracellular matrix gene expression and is essential for chondrocyte differentiation. Cctivation of p38 MAPK by cycloheximide in human chondrocytes leads to stabilization of SOX9 mRNA. [Does] regulation of p38 MAPK caused by changes in osmotic pressure control SOX9 mRNA levels? Primary human articular chondrocytes isolated from osteoarthritic cartilage at passage 2-4 showed significantly raised SOX9 mRNA levels when exposed to hyperosmotic{meaning that the cells have a higher concentration of compounds other than water} conditions for 5 h. The effect was strongest and most reproducible when actin stress fibers were disrupted by the Rho effector kinase inhibitor Y27632, or by culturing the cells within alginate beads. Freshly isolated chondrocytes, used within 24-48 h of isolation, did not contain actin stress fibers{meaning they did not yet adapt to hyperosmolarity} and upregulated SOX9 mRNA in response to hyperosmolarity in the presence and absence of Y27632. In these freshly isolated chondrocytes, hyperosmolarity led to an increase in the half-life of SOX9 mRNA{this may occur in stem cells in addition to chondrocytes and also chondrogenic differentiation as once you have stem cells differentiating into chondrocytes you want to keep them that way}, which was sensitive to the p38 MAPK inhibitor SB202190. SOX9 protein levels were increased by hyperosmotic culture over 24 h, and, in passaged chondrocytes, the activity of a COL2A1 enhancer driven luciferase assay was upregulated. However, in freshly isolated chondrocytes, COL2A1 mRNA levels were reduced by hyperosmotic conditions and the half-life was decreased. The results showed that the osmotic environment regulated both SOX9 and COL2A1 mRNA posttranscriptionally, but in fresh cells resulted in increased SOX9, but decreased COL2A1."
Hyperosmolarity reduces COL2A1 but Sox9 is way more important than COL2A1.
"p38 MAPK is not the only regulatory process controlling the rate of SOX9 mRNA decay"
Hyperosmolarity regulates SOX9 mRNA posttranscriptionally in human articular chondrocytes.
"The transcription factor SOX9 regulates cartilage extracellular matrix gene expression and is essential for chondrocyte differentiation. Cctivation of p38 MAPK by cycloheximide in human chondrocytes leads to stabilization of SOX9 mRNA. [Does] regulation of p38 MAPK caused by changes in osmotic pressure control SOX9 mRNA levels? Primary human articular chondrocytes isolated from osteoarthritic cartilage at passage 2-4 showed significantly raised SOX9 mRNA levels when exposed to hyperosmotic{meaning that the cells have a higher concentration of compounds other than water} conditions for 5 h. The effect was strongest and most reproducible when actin stress fibers were disrupted by the Rho effector kinase inhibitor Y27632, or by culturing the cells within alginate beads. Freshly isolated chondrocytes, used within 24-48 h of isolation, did not contain actin stress fibers{meaning they did not yet adapt to hyperosmolarity} and upregulated SOX9 mRNA in response to hyperosmolarity in the presence and absence of Y27632. In these freshly isolated chondrocytes, hyperosmolarity led to an increase in the half-life of SOX9 mRNA{this may occur in stem cells in addition to chondrocytes and also chondrogenic differentiation as once you have stem cells differentiating into chondrocytes you want to keep them that way}, which was sensitive to the p38 MAPK inhibitor SB202190. SOX9 protein levels were increased by hyperosmotic culture over 24 h, and, in passaged chondrocytes, the activity of a COL2A1 enhancer driven luciferase assay was upregulated. However, in freshly isolated chondrocytes, COL2A1 mRNA levels were reduced by hyperosmotic conditions and the half-life was decreased. The results showed that the osmotic environment regulated both SOX9 and COL2A1 mRNA posttranscriptionally, but in fresh cells resulted in increased SOX9, but decreased COL2A1."
Hyperosmolarity reduces COL2A1 but Sox9 is way more important than COL2A1.
"p38 MAPK is not the only regulatory process controlling the rate of SOX9 mRNA decay"
"Mice engineered to have constitutive activation[continuously activated] of the p38 MAPK pathway in cartilaginous tissues display a severe dwarfism caused by delayed chondrocyte hypertrophy, which is very similar to that exhibited by mice overexpressing SOX9 "<-So you want Sox9 decay but not too much.
A cell shrinkage artefact in growth plate chondrocytes with common fixative solutions: importance of fixative osmolarity for maintaining morphology.
A cell shrinkage artefact in growth plate chondrocytes with common fixative solutions: importance of fixative osmolarity for maintaining morphology.
"The remarkable increase in chondrocyte volume is a major determinant in the longitudinal growth of mammalian bones. During fixation of growth plates with conventional fixative solutions, there was a marked morphological (shrinkage) artifact, and we postulated that this arose from the hyper-osmotic nature of these solutions[meaning the solution contains more salt than water]. To test this, we fixed proximal tibia growth plates of 7-day-old rat bones in either (a) paraformaldehyde (PFA; 4%), (b) glutaraldehyde (GA; 2%) with PFA (2%) with ruthenium hexamine trichloride (RHT; 0.7%), (c) GA (2%) with RHT (0.7%), or (d) GA (1.3%) with RHT (0.5%) and osmolarity adjusted to a 'physiological' level of approximately 280mOsm. Using conventional histological methods, confocal microscopy, and image analysis on fluorescently-labelled fixed and living chondrocytes, we then quantified the extent of cell shrinkage and volume change. The high osmolarity of conventional fixatives caused a shrinkage artefact to chondrocytes. This was particularly evident when whole bones were fixed, but could be markedly reduced if bones were sagittally bisected prior to fixation. The shrinkage artefact could be avoided by adjusting the osmolarity of the fixatives to the osmotic pressure of normal extracellular fluids ( approximately 280mOsm)[so is low osmolarity of solution relative to the epiphyseal bone marrow better or the optimal level 280mOsm, we know that high osmolarity may be height reducing]."
No osmolarity less than 280 mOsm was used in this study. Note this is the osmolarity of the bone versus the solution. If the bone was hyperosmotic that may give a beneficial effect the solution make the bone a hypoosmotic system. Note that you could dunk your bones into a hypoosmotic solution. Maybe swimming in fresh water will help you grow taller than salt water. Distilled water is a hypotonic solution. Drinking more distilled water might help increase chondrocyte hypertrophy thus making you taller under active growth plates.
"In the whole bone, the large shrinkage artefact suggested that osmotically-induced cell shrinkage occurred before the tissue was fixed. Thus when the bone was placed in the fixative solution, water movement out of the bones to the hyper-osmolar fixative solution occurred rapidly, and before the fixative fully penetrated the bone/cartilage matrix to fix the cells throughout the sample."
Regulation of immature cartilage growth by IGF-I, TGF-β1, BMP-7, and PDGF-AB: role of metabolic balance between fixed charge and collagen network
"Cartilage growth may involve alterations in the balance between the swelling tendency of proteoglycans {thus involving osmosis and water} and the restraining function of the collagen network. Growth factors, including IGF-I, TGF-β1, BMP-7, and PDGF-AB, regulate chondrocyte metabolism and, consequently, may regulate cartilage growth."
"The inclusion of serum, IGF-I, or BMP-7 resulted in expansive tissue growth, stimulation of proteoglycan deposition but not of collagen, and a diminution of tensile integrity. The regulation of cartilage metabolism by TGF-β1 resulted in tissue homeostasis, with maintenance of size, composition, and function. Incubation in basal medium or with PDGF-AB resulted in small volumetric and compositional changes, but a marked decrease in tensile integrity."
"The proteoglycan constituent of the extracellular matrix [of cartilage] provides the tissue with a fixed negative charge that increases the tissue’s propensity to swell and resist compressive loading {like LSJL}
"PDGF-AB stimulated proteoglycan synthesis and decreased the rate of proteoglycan catabolism"
"The volumetric growth of all explants appeared to be predominantly axial, as changes in wet weight can mostly be accounted for by changes in thickness alone. The content of water varied slightly in the S layer, increasing during basal culture or culture with IGF-I, BMP-7, or PDGF-AB"
"only incubation with FBS, IGF-I, and BMP-7 resulted in a total GAG content that was higher than that in explants incubated in basal medium"
"Incubation with FBS, IGF-I, or BMP-7 resulted in expansive cartilage growth {height increase}, characterized by an increase in tissue size"
"larger increases in length were observed in cartilages containing one or more osteogenic zones as compared to that of entirely cartilaginous explants"<-Thus the transformation of cartilage to bone may have size increasing effects. Perhaps the apoptosis stage may contribute to the growth.
"[expansive cartilage] growth may be driven by alterations in the structural organization of the collagen network itself that render it weak and malleable. Such remodeling may involve induction of various proteinases such as collagenase, which can be stimulated by PDGF"
Label-free protein profiling of adipose-derived human stem cells under hyperosmotic treatment.
"Treatment of cells with hyperosmotic media during 2D passaging primes cells for cartilage tissue engineering applications. [We evaluated] the effects of control and hyperosmotic treatment environments on the phenotype of multipotent adipose-derived stem cells (ASCs) cultivated with a chondrogenic growth factor cocktail. The results indicated a complex cellular response to osmotic treatment, with a number of proteins differentially expressed between control and treated cell groups. The roles of some of these proteins have been documented in the literature as characteristic of the physiological states studied, especially aldose reductase (osmotic stress). This protein acted as a positive control in this work, providing independent corroborative validation. Other proteins, including 5'-nucleotidase{up in LSJL as Nt5e} and transgelin{up in LSJL}, have been previously linked to cell differentiation state."
No osmolarity less than 280 mOsm was used in this study. Note this is the osmolarity of the bone versus the solution. If the bone was hyperosmotic that may give a beneficial effect the solution make the bone a hypoosmotic system. Note that you could dunk your bones into a hypoosmotic solution. Maybe swimming in fresh water will help you grow taller than salt water. Distilled water is a hypotonic solution. Drinking more distilled water might help increase chondrocyte hypertrophy thus making you taller under active growth plates.
"In the whole bone, the large shrinkage artefact suggested that osmotically-induced cell shrinkage occurred before the tissue was fixed. Thus when the bone was placed in the fixative solution, water movement out of the bones to the hyper-osmolar fixative solution occurred rapidly, and before the fixative fully penetrated the bone/cartilage matrix to fix the cells throughout the sample."
Regulation of immature cartilage growth by IGF-I, TGF-β1, BMP-7, and PDGF-AB: role of metabolic balance between fixed charge and collagen network
"Cartilage growth may involve alterations in the balance between the swelling tendency of proteoglycans {thus involving osmosis and water} and the restraining function of the collagen network. Growth factors, including IGF-I, TGF-β1, BMP-7, and PDGF-AB, regulate chondrocyte metabolism and, consequently, may regulate cartilage growth."
"The inclusion of serum, IGF-I, or BMP-7 resulted in expansive tissue growth, stimulation of proteoglycan deposition but not of collagen, and a diminution of tensile integrity. The regulation of cartilage metabolism by TGF-β1 resulted in tissue homeostasis, with maintenance of size, composition, and function. Incubation in basal medium or with PDGF-AB resulted in small volumetric and compositional changes, but a marked decrease in tensile integrity."
"The proteoglycan constituent of the extracellular matrix [of cartilage] provides the tissue with a fixed negative charge that increases the tissue’s propensity to swell and resist compressive loading {like LSJL}
"PDGF-AB stimulated proteoglycan synthesis and decreased the rate of proteoglycan catabolism"
"The volumetric growth of all explants appeared to be predominantly axial, as changes in wet weight can mostly be accounted for by changes in thickness alone. The content of water varied slightly in the S layer, increasing during basal culture or culture with IGF-I, BMP-7, or PDGF-AB"
"only incubation with FBS, IGF-I, and BMP-7 resulted in a total GAG content that was higher than that in explants incubated in basal medium"
"Incubation with FBS, IGF-I, or BMP-7 resulted in expansive cartilage growth {height increase}, characterized by an increase in tissue size"
"larger increases in length were observed in cartilages containing one or more osteogenic zones as compared to that of entirely cartilaginous explants"<-Thus the transformation of cartilage to bone may have size increasing effects. Perhaps the apoptosis stage may contribute to the growth.
"[expansive cartilage] growth may be driven by alterations in the structural organization of the collagen network itself that render it weak and malleable. Such remodeling may involve induction of various proteinases such as collagenase, which can be stimulated by PDGF"
Label-free protein profiling of adipose-derived human stem cells under hyperosmotic treatment.
"Treatment of cells with hyperosmotic media during 2D passaging primes cells for cartilage tissue engineering applications. [We evaluated] the effects of control and hyperosmotic treatment environments on the phenotype of multipotent adipose-derived stem cells (ASCs) cultivated with a chondrogenic growth factor cocktail. The results indicated a complex cellular response to osmotic treatment, with a number of proteins differentially expressed between control and treated cell groups. The roles of some of these proteins have been documented in the literature as characteristic of the physiological states studied, especially aldose reductase (osmotic stress). This protein acted as a positive control in this work, providing independent corroborative validation. Other proteins, including 5'-nucleotidase{up in LSJL as Nt5e} and transgelin{up in LSJL}, have been previously linked to cell differentiation state."
"Twice-passaged human ASCs isolated from the abdominal fat of a 42-year-old female patient"
Osmotic medium was 400 mOsm and control medium was 300 mOsm.
G3P was upregulated in some osmotic groups and downregulated in others while a protein labeled as being like G3P was downregulated in LSJL.
Proteins upregulated by Osmotic Treatment also upregulated by LSJL:
Hadha{down}
Downregulated:
Nt5e{up}
Transgelin{up}
"cells in our control group expressed several proteins reliably detected in the chondrocyte proteome[cells in the osmotic group expressed more adiposal proteins]. Six proteins that were most abundantly expressed in cells in the treatment group (ATP synthase subunit alpha, pyruvate kinase isozymes M1/M2 (canonical sequence), AKR1C2, SOD, trifunctional enzyme subunit alpha, transketolase) were also among the most abundant 10% of proteins expressed in adipose tissue (based on spectral counts only),"
"(fibronectin, 5NTD, and nicotinamide N-methyltransferase (NNMT), and P4HA1) were also reliably detected in the chondrocyte proteome"
"Transgelin, which decreased in expression in our treated cells, has been detected in neonatal, but not postnatal, mouse cartilage"<-what does the increase in transgelin expression in LSJL indicate?
Osmolarity determines the in vitro chondrogenic differentiation capacity of progenitor cells via Nuclear Factor of Activated T-cells 5.
Osmolarity determines the in vitro chondrogenic differentiation capacity of progenitor cells via Nuclear Factor of Activated T-cells 5.
"hyperosmolarity facilitates chondrogenic differentiation [of progenitor cells] and Nfat5{down in LSJL} is involved.
ATDC5 cells and human bone marrow stem cells (hBMSCs) were differentiated in the chondrogenic lineage in control and increased osmolarity conditions.
Increasing the osmolarity of differentiation medium with 100 mOsm resulted in significantly increased chondrogenic marker expression (Col2a1{up}, Col10a1{up}, Acan{up}, Sox9{up}, Runx2 and GAGs) during chondrogenic differentiation of the two chondroprogenitors, ATDC5 and hBMSCs. Nfat5 knockdown under both control and increased osmolarity affected chondrogenic differentiation and suppressed the osmolarity-induced chondrogenic induction. Knockdown of Nfat5 in early differentiation significantly decreased early Sox9 expression{so reduction of Nfat5 in LSJL could be a feedback mechanism}, whereas knockdown of Sox9 in early differentiation did not affect early Nfat5 expression."
"The glycosaminoglycan (GAG) side chains of the PGs are sulfated and responsible for a high fixed negative charge density, which attracts mobile cations and water from the ECM-environment. This, together with the quality of the collagen network, determines the osmolarity of the extracellular fluid and provides strength and flexibility to the tissue. The extracellular osmolarity of healthy articular cartilage ranges between 350 and 480 mOsm and is thus markedly higher than that of standard culture medium, which ranges around plasma levels (280 mOsm)[also the level of normal tissue plasma]"
"Nfat5 is a member of the Rel family of transcription factors and mediates transcriptional activation of ion transporters like the sodium/myo-inositol transporter encoded by Slc5a3 and calcium binding proteins like S100a4{up}"
The increase in osmolarity gene expression was not very large 1.4-2.5 fold. Col1a1{up} was downregulated.
Increasing osmolarity by 200 rather than 100mOsm inhibited differentiation. This may be because too high osmolarity exceeds membrane transport capabilities.
"Sox9 expression during chondrogenic differentiation of progenitor cells in vitro is bi-phasic, with a first induction during the first hours of differentiation and a second peak expression later on in differentiation."
Dependence of zonal chondrocyte water transport properties on osmotic environment.
Dependence of zonal chondrocyte water transport properties on osmotic environment.
"The increasing concentration of proteoglycans from the surface to the deep zone of articular cartilage produces a depth-dependent gradient in fixed charge density, and therefore extracellular osmolarity, which may vary with loading conditions, growth and development, or disease. We examine the relationship between in situ variations in osmolarity on chondrocyte water transport properties. Chondrocytes from the depth-dependent zones of cartilage, effectively preconditioned in varying osmolarities, were used to probe this relationship.
First, depth variation in osmolarity of juvenile bovine cartilage under resting and loaded conditions was characterized. Zonal chondrocytes were isolated into two representative "baseline" osmolarities chosen from this analysis to reflect in situ conditions. Osmotic challenge was then used as a tool for determination of water transport properties at each of these baselines. Cell calcium signaling was monitored simultaneously as a preliminary examination of osmotic baseline effects on cell signaling pathways.
Osmotic baseline exhibits a significant effect on the cell membrane hydraulic permeability of certain zonal subpopulations but not on cell water content or incidence of calcium signaling.
Chondrocyte properties can be sensitive to changes in baseline osmolarity, such as those occurring during OA progression (decrease) and de novo tissue synthesis (increase)."
"The osmotic environment in which cartilage tissue cells (chondrocytes) reside is dictated by the local concentrations of proteoglycan (PG) molecules trapped within the extracellular matrix. The highly negatively-charged PG molecules create a fixed charge density (FCD) that attracts positively-charged counter-ions and water according to Donnan osmotic equilibrium, giving rise to local tissue osmolarity"
"The static osmotic baseline varies spatially through the depth of cartilage, due to a gradient in PG molecules, whose content increases from the articular surface to the bony interface."
"prolonged static loading results in significant interstitial fluid volume loss and concomitant increase in FCD "
"Tissue water content was observed to decrease exponentially within the top 20% of the articular layer, from nearly 90% down to 75%, then to hold constant through the remainder of the depth. Fixed charge density increased from 100 mEq/L near the articular surface to 300 mEq/L in the middle zone, rising further to over 400 mEq/L in the lower zone."<-So in endochondral ossification the hypertrophic zone would have about 400 mEw/L and 75% water content.
"Upon physiological loading, as the nominal engineering compressive strain rises from 0 to 25% in 0.5 s, the interstitial osmolarity shows negligible change, due to the negligible fluid loss in the early-time response."
The cell water content of the different chondrocyte layers is almost the same between 300-400 mOsm. But the water membrane permeability decreased greatly between 300-400 mOsm.
"changes in interstitial fluid osmolarity are negligible in the short-term tissue response to loading conditions that reflect physiologic deformational parameters"<-so maybe osmosis related changes in the growth plate are not responsible for the LSJL height increase. However LSJL loading is more dynamic.
"The baseline osmotic environment of chondrocytes increases from ~340 mOsM at the articular surface to ~410 mOsM at the cartilage bone interface, under unloaded conditions; that the depth-varying osmolarity remains nearly unchanged in the short-term response to mechanical loading; and that prolonged static compression may produce an increase of ~80 mOsM near the surface and ~20 mOsm near the bone, as a result of increased localized FCD."
Regulation of SOX9 in normal and osteoarthritic equine articular chondrocytes by hyperosmotic loading.
Regulation of SOX9 in normal and osteoarthritic equine articular chondrocytes by hyperosmotic loading.
"Equine chondrocytes harvested from normal or OA joints were subjected to different osmotic loading patterns as either primary (P0) or passaged (P2) cells. The involvement of MEK-ERK signalling was demonstrated by using pharmacological inhibitors. In addition SOX9 gene stability was determined. Levels of transcripts encoding SOX9, Col2A1 and aggrecan were measured. De novo glycosaminoglycan synthesis of explants was determined with (35)S sulphate during static hyperosmolar loading.
MEK-ERK signalling increases glycosaminoglycans (GAG) synthesis in explants. Static hyperosmotic conditions significantly reduced SOX9 mRNA in normal P2 and OA P0 but not normal P0 chondrocytes. SOX9 mRNA was stabilised by hyperosmotic conditions. Cyclical loading of normal P2 and OA P0 but not normal P0 cells led to an increase in SOX9 gene expression and this was prevented by MEK1/2 inhibition.
The response to osmotic loading of SOX9 mRNA is dependent on the nature of the osmotic stimulation and the chondrocyte phenotype."
"380mOsm control conditions are close to those experienced by healthy chondrocytes in-situ, and 550mOsm represented a hyperosmotic condition"
MECHANICAL AND OSMOTIC SIGNALING IN CHONDROCYTES
"The transient receptor potential vanilloid 4 (TRPV4) ion channel, a non-selective cation channel with a preference for Ca++, has been found to be involved in signal transduction in response to osmotic and mechanical stimuli in mammalian cells. The goals of this study are to characterize the role of TRPV4 in the physiological response of cartilage to mechanical loading. We hypothesize that extracellular Ca++ influx into articular chondrocytes in response to osmotic or mechanical stimuli is critically dependent on TRPV4 ion channels and is significantly modulated by inflammatory, in particular proteolytic signaling. We propose that in vivo, the loss of TRPV4 function in chondrocytes, and the associated loss of osmotic and mechanical sensitivity, will result in an imbalance of cell metabolism that induces cartilage degeneration and osteoarthritis. The specific aims of this study are: 1) To determine the role of TRPV4 in chondrocytes by conducting loss-of-function studies in porcine and murine chondrocytes in vitro, and furthermore, investigation of the modulation of TRPV4 activity by activation of proteinase-activated-receptor 2 (PAR-2), interleukin 1, leptin, or prostaglandin E2; and 2) Using gene-targeted mice, to determine the effects of inducible, cartilage-specific deletion of trpv4 on chondrocyte physiology and subsequent histological, biomechanical, and metabolic changes in their cartilage. Mice will be generated by crossing trpv4lox/lox mice with col2-cre mice, which express a constitutive or inducible CRE transgene in chondrocytes. The effects of trpv4 knockout on articular damage will be determined as a function of (i) aging, and (ii) in a pathophysiologically relevant model of osteoarthritis induced by diet-induced obesity. Chondrocytes in articular cartilage respond to mechanical and osmotic signals arising from joint loading. The Transient Receptor Potential Vanilloid 4 (TRPV4) ion channel, a non-selective cation channel with a preference for Ca++, is a primary transducer of osmotic and mechanical stimuli in mammalian cells. The goals of this study are to characterize the function of TRPV4 in articular chondrocytes in vitro, and to determine its role in an in vivo model of osteoarthritis using a transgenic mouse in which the trpv4 gene is specifically and conditionally deleted from the cartilage."
MECHANICAL AND OSMOTIC SIGNALING IN CHONDROCYTES
"The transient receptor potential vanilloid 4 (TRPV4) ion channel, a non-selective cation channel with a preference for Ca++, has been found to be involved in signal transduction in response to osmotic and mechanical stimuli in mammalian cells. The goals of this study are to characterize the role of TRPV4 in the physiological response of cartilage to mechanical loading. We hypothesize that extracellular Ca++ influx into articular chondrocytes in response to osmotic or mechanical stimuli is critically dependent on TRPV4 ion channels and is significantly modulated by inflammatory, in particular proteolytic signaling. We propose that in vivo, the loss of TRPV4 function in chondrocytes, and the associated loss of osmotic and mechanical sensitivity, will result in an imbalance of cell metabolism that induces cartilage degeneration and osteoarthritis. The specific aims of this study are: 1) To determine the role of TRPV4 in chondrocytes by conducting loss-of-function studies in porcine and murine chondrocytes in vitro, and furthermore, investigation of the modulation of TRPV4 activity by activation of proteinase-activated-receptor 2 (PAR-2), interleukin 1, leptin, or prostaglandin E2; and 2) Using gene-targeted mice, to determine the effects of inducible, cartilage-specific deletion of trpv4 on chondrocyte physiology and subsequent histological, biomechanical, and metabolic changes in their cartilage. Mice will be generated by crossing trpv4lox/lox mice with col2-cre mice, which express a constitutive or inducible CRE transgene in chondrocytes. The effects of trpv4 knockout on articular damage will be determined as a function of (i) aging, and (ii) in a pathophysiologically relevant model of osteoarthritis induced by diet-induced obesity. Chondrocytes in articular cartilage respond to mechanical and osmotic signals arising from joint loading. The Transient Receptor Potential Vanilloid 4 (TRPV4) ion channel, a non-selective cation channel with a preference for Ca++, is a primary transducer of osmotic and mechanical stimuli in mammalian cells. The goals of this study are to characterize the function of TRPV4 in articular chondrocytes in vitro, and to determine its role in an in vivo model of osteoarthritis using a transgenic mouse in which the trpv4 gene is specifically and conditionally deleted from the cartilage."
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