Grow Taller with Aqua Pressure?

Previously, I dismissed Acupuncture(and Aqua Pressure) to increase height.  The videos by Dr. Prakash Verekar did not fully prove height growth and that the height growth may be temporary height due to muscle relaxation or laying down allowing for replenishment of the proteoglycans in the spine.  However, we have learned of the important of water in inducing differentiation of stem cells.  LSJL is based on inducing hydrostatic pressure in the epiphysis.

If water is so important to our theories then couldn't injecting water directly into the epiphysis and cartilage increase hydrostatic pressure?  Well water may diffuse directly immediately but it would still temporarily cause shear strain and hydrostatic pressure.  It would also have to be tested in animals for safety but it should be easier and more cost effective.  All you'd need is a syringe, water, and a Sprague-Dawley rat(plus feeding supplies).  Test out various injection possibilities.  If you don't want to sacrifice the rat you can always just measure it alive.  Just drinking more water wouldn't work because you would be reliant on where your digestive system sends water to.

  In trying to ask scientists about exactly how endochondral ossification increases height, I received an interesting comment from Farshid Guilak.  If endochondral ossification was a merely transformative process from cartilage to bone then limbs would stay the same size!  There must be some part of the process that causes an increase in bone size.  There are two possibilities.  One could be due to the negative charge of the cartilagenous matrix which when turns to a no charge generates force. Sort of like the electron transport chain or the kreb's cycle.  Water is still likely key in this process as the combination of 2 Hydrogens and an Oxygen generates potential energy that can then induce bone formation.

Farshid Guilak presented this possibility: "since cartilage has a lot of water and bone doesn't, so there is the possibility that it could expand durring ossification, or simply replace water and other components with mineral."

With aqua pressure you are changing the typical balance of water in bone which could lead to an adaptation that increases height.

Here's a paper that shows one instance of hydrolysis during endochondral ossification:

Characterization of the phosphatidylinositol-specific phospholipase C-released form of rat osseous plate alkaline phosphatase and its possible significance on endochondral ossification.

"Alkaline phosphatase activity was released up to 100% from the membrane by incubating the rat osseous plate membrane-bound enzyme with phosphatidylinositol-specific phospholipase C[we'll call this PSPC]. The molecular weight of the released enzyme was 145,000 on Sephacryl S-300 gel filtration and 66,000 on PAGE-SDS, suggesting a dimeric structure. Solubilization of the membrane-bound enzyme with phospholipase C did not destroy its ability to hydrolyse PNPP, ATP and pyrophosphate. The hydrolysis of ATP and PNPP by phosphatidylinositol-specific phospholipase C-released enzyme exhibited 'Michaelian' kinetics with K0.5 = 70 and 979 microM, respectively. For pyrophosphate, K0.5 was 128 microM and site-site interactions were observed (n = 1.4). Magnesium ions were stimulatory (K0.5 = 1.5 mM) and zinc ions were a powerful noncompetitive inhibitor (Ki = 6.2 microM) of phosphatidylinositol-specific phospholipase C-released enzyme. Phosphatidylinositol-specific phospholipase C-released alkaline phosphatase was relatively stable at 40 degrees C. However, with increasing temperature from 40-60 degrees C, the enzyme was inactivated rapidly following first order kinetics and thermal inactivation constants varied from 5.08 x 10(-4) min-1 to 0.684 min-1. Treatment of phosphatydilinositol-specific phospholipase C-released alkaline phosphatase with Chellex 100 depleted to 5% its original PNPPase activity. Magnesium (K0.5 = 29.5 microM), manganese (K0.5 = 5 microM) and cobalt ions (K0.5 = 10.1 microM) restored the activity of Chelex-treated enzyme, demonstrating its metalloenzyme nature. The stimulation of Chelex-treated enzyme by calcium ions (K0.5 = 653 microM) was less effective (only 26%) and occurred with site-site interactions (n = 0.7). Zinc ions had no stimulatory effects. The possibility that the soluble form of the enzyme, detected during endochondral ossification, would arise by the hydrolysis of the Pl-anchored form of osseous plate alkaline phosphatase is discussed."

"a membrane-bound and a soluble form of rat osseous plate alkaline phosphatase appear during the ectopic mineralization process"

"soluble enzyme present during endochondral ossification could arise through the hydrolysis of Pl-anchored form of the enzyme by specific anchor-cleaving activities as was reported for liver alkaline phosphatase"

So the osseus plate membrane bound enzyme may become soluble by hydrolysis related properties during endochondral ossification.  So this enzyme may be key to how the changing of bone to cartilage increases height growth.  Hydrolysis(water reactions) are key to making this enzyme soluble and active.

Here's a study that shows the ability of water to induce endochondral ossification:

The in vitro chondrogenic response of limb-bud mesenchyme to a water-soluble fraction prepared from demineralized bone matrix.

"Demineralized adult bone matrix initiates de novo ectopic endochondral ossification 2-3 weeks following its intramuscular implantation into adult animals{this is injection directly into the muscle}. This phenomenon appears to be similar, in some ways, to inductive cell-matrix interactions which regulate cartilage and bone formation during development. In the present study, we used embryonic chick limb-bud mesenchymal-cell cultures to bioassay extracts of demineralized bone matrix for chondrogenic activity. Guanidinium-chloride (4 M) extracts of demineralized bovine bone were dialyzed against buffers of decreasing ionic strength and then cold water. The cold-water-soluble fraction was found to stimulate chondrogenesis in intermediate-density limb-bud cell cultures (2.2 X 10(6) cells per 35-mm dish), as revealed by visual inspection. Further fractionation of this material by anion-exchange, carbohydrate-affinity, and molecular-sieve chromotography produced a semipurified preparation possessing chondrogenic-stimulating activity at doses ranging from 3 to 10 micrograms/ml. The in vitro chondrogenic response of limb-bud mesenchymal cells was dose-dependent, required a minimal initial plating density of 2.08 X 10(5) cells/mm2 of culture dish, and developed gradually over 8-10 days. At an optimal dose of extract, a continuous exposure period of at least 2-3 days was necessary to produce detectable chondrogenic stimulation. In addition, the amount of cartilage formed following an 8-day exposure was markedly influenced by the culture 'age' of the mesenchymal cells (i.e., the time between plating and the start of treatment)."

Cartilage has water and bone doesn't.  The substance that was water soluble was what was effective in inducing chondrogenesis.  Water and hydrolysis are key distinctions between cartilage and bone.  The water levels might influence the microenvironment to induce chondrogenesis.  This is what we are trying to alter with LSJL the water microenvironment.

"adult bone contains substances that stimulate or control the process of  bone repair."

Here's something that explains how water is involved in the change of cartilage to bone:

Mechanisms of cartilage precursor replacement by bone in the mammalian skeleton.

"The initiation of the endochondral process in ontogenesis is preceded by structural and metabolic preparation of the cells of cartilage anlages of the skeleton as well as the satellite cells of the vascular terminations[likely VEGF related] adjoining the cartilage. In the cartilage anlage, due to a progressive specific biosynthesis, the macromolecular chondroitin-protein complexes capable of binding a large amount of water, are accumulated{both chondroitin levels and water levels may affect how much is stored}. As a result the volume of a swelling chondrocyte increases twentyfold or more and a high hydrostatic tension is created within it{the chondrocyte streches due to the water within it}. At the same time the metabolic situation within the cartilage favours a local concentration, near the hypertrophic chondrocytes, of the perivascular cells possessing an increased resorptive function. The resorption of the cartilage partition walls by these cells leads to the opening of swollen chondrocytes[the opening of these chondrocytes could result in water release that push bone apart]. The blood cells and the cells of the blood capillaries themselves are engulfed through the resulting crater into the emptied lacuna. In the lacuna the conditions of stasis are created which promote local redistribution of the supplied cells and reutilization of the cartilage destruction products. The perivascular cells concentrated along the remnants of the cartilaginous matrix are gradually differentiated into preosteoblasts and osteoblasts. The endosteal bony laminae are built on the remnants of the cartilaginous matrix. The perivascular cells of the apical zone of a lacuna retain their clastic function and continue spreading of the endochondral process on the adjacent chondrocytes. In the course of the successive destruction of hypertrophic chondrocytes, the system of vessels is formed in the endochondral centre, according to the sinusoid pattern."

Couldn't get this full study.

So it's likely the release of water by swollen chondrocytes after the reabsorption of tissues near the cartilage that pushes the bones apart and results in a larger size.  You'd need at least a large enough growth plate to build the matrix so they wouldn't swell right away.  Then you'd need sufficient force generated by the release of water to deform(stretch) the bone.

Abundance of evidence points to a huge role in water in endochondral ossification and differentiation of stem cells into chondrocytes.  Therefore it is possible for Aqua Pressure to work.

Increase your height with Osmosis

Osmotic pressure and hydrostatic pressure are in many ways synonymous but we will differentiate the two by studying the effects of fluids on individual cells as osmotic pressures and the effect of fluids of an environment of cells(such as those in the epiphyseal bone marrow) as hydrostatic pressure.  Hydrostatic pressure increase induced by clamping laterally the sides of the bones(thus creating a pressure gradient) is the key factor behind LSJL so it makes sense to study it on a cell to cell level.  Any other factors that increase hydrostatic pressure will also help with gains from LSJL such as lowering your bodyfat percentage(only after puberty when red bone marrow starts to convert to yellow) and flexing your muscles(either with EMS-Dual Channel EMS Unit - EMS 5.0 Muscle Stimulator 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"

"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.

"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."

"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.

"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.

"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.

"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."

Grow Taller by Lowering your Bodyfat Percentage?

We know that stem cell count decreases with bone marrow fat content. Although adipose tissue can differentiate into a chondrogenic phenotype, it may still be worth it to try to lower the amount of yellow bone marrow within the bone.  Fat is not a very dense substance and may lower hydrostatic pressure.  Which is likely to generate more hydrostatic pressure?  Red Bone Marrow and Yellow Bone Marrow.  The change from red bone marrow to yellow bone marrow is one of the marks of the end of the growing phase(but we also know that again yellow bone marrow can change back to red based on nutrition).  Body fat produces leptin which helps with growth but this can be rectified with Leptin supplementation.

Since Hydrostatic Pressure is so key to growing taller with LSJL and the change from Red to Yellow Bone Marrow is one of the markings of growth termination we want to have as much Red Bone Marrow as possible(to increase hydrostatic pressure).  Thus we want to decrease our bodyfat content.  The more Red Bone Marrow we have, the easier it will be to increase hydrostatic pressure.  Thus, why us height seekers should seek to lower fat mass.  A few brief diet tips:

1) If you get on a treadmill, you'll be shocked at how much effort it takes to burn very little amount of calories.  Some forms of food are very dense and you can generate those calories back very quickly.  You must ultimately increase your activity level throughout the entire day or lower caloric consumption.
2) Jawbreakers take an hour to eat and only have 100 or so calories: Jumbo Jawbreakers 2 1/4" Diameter (Packages of 4).

Some notes about fat and insulin:

Many height seekers have noticed a correlation between being big(both fat and muscle wise) and being tall.  Some have also noticed a correlation between being short and small.  Some short people being able to eat anything and seemingly unable to gain weight.  Now it's not a perfect correlation as there are a number of causes of short and tall stature including local growth plate factors.  But, one possible reason for the connection is an increase in insulin sensitivity in big and tall individuals.  Insulin increases cellular proliferation which includes stem cells.  Stem cells have the ability to differentiate into osteoblasts, chondrocytes, and adipocytes.  Chondrocytes being the main way for height to be increased.  Adipocytes of course increasing fat mass.

Now given that insulin increases cellular proliferation it is very important to have your insulin sensitivity be as high as possible.  Cortisol acts as a counter agent to insulin and has been associated with adipose storage in the waist.  Anecdotal experience has shown that individuals who exercise tend to have a more uniform adipose distribution pattern whereas sedentary individuals sometimes have all their fat mass stored in the waist(with seemingly very skinny legs and arms).  This seems to indicate that exercising the muscles of your extremities seems to help increase the insulin sensitivity of the entire limb which would of course increase the insulin sensitivity of your stem cells as well.

This could also indicate that body fat in your extremities could lower insulin sensitivity in those limbs.  This is supported by anecdotal evidence where bodybuilders find that periods of cutting and bulking to be anabolic.  Fat mass could increase insulin resistance which would dampen the anabolic effects of insulin on stem cell proliferation.

On women and losing fat:

Now for women, having a higher body fat percentage than men is a secondary sex characteristic so they might want not to lose fat.  I also know that women don't like being told what to do or what they should look like. Many men including myself prefer women with higher bodyfat percentage.  However, some men prefer lower bodyfat percentage women and growing taller is vital for some careers so a woman might want to grow taller for those careers.  We'll know with time how critical bodyfat is to results with LSJL so it may be best to wait.  Keep all those things in mind when deciding what to do.

Lateral Synovial Joint Loading for the Spine

Hiroki Yokota is moving to a New York University from Purdue.  Maybe the other University poached him so he can work on perfecting the science of LSJL in an upgraded facility.  I asked him if he's performed LSJL on infant rats or on non-long bones and he says he hasn't.  Infant rats would be good to perform the experiment on because infant long bones are almost completely hyaline cartilage thus enabling researchers to see how much of the benefit is based on genes upregulating due to cartilage load versus epiphyseal bone load.  He also has only performed joint loading on long bones.  Performing joint loading on irregular bones would enable us to see how much of a benefit the increase in interstitial fluid flow has in causing shear strain on the periosteum and endosteum.

Here's a full study about LSJL.  It was done slightly before the lengthening of mouse hindlimbs was done with joint loading.  Joint loading increased cortical area.  Now in some cases this is equal to height like the top of the head and the calcaneus.  The problem is that there's no easy way to perform loading on the spine(LIPUS may be a viable option as it can generate waves through several tissues into the bone).  And the vertebrae grow in a similar mechanism as to long bones despite being irregular bones.  They do not have a defined epiphysis but do have large sections of trabecular bone and may have bone marrow(though vertebrae may in fact be poorly vascularized).  The top and bottom and parts of the side do not have periosteum which is how appositional growth occurs thus growth from within the bone via endochondral ossification must be the way to grow taller.  I am testing it on the calcaneus(heel bone) which is the most accessible irregular bone.  I'm doing 1 minute 30 seconds table clamp.

A foam roller might be a good way to cause a shearing force on the periosteum of the spine by rolling against the spinous process and other parts of the spine.  Just causing that shearing force on that part of the periosteum may be enough to increase fluid flow throughout the entire bone including the epiphysis.

Here's a study that shows that growth may continue into adulthood via the vertebrae:

Vertebral height growth predominates over intervertebral disc height growth in adolescents with scoliosis.

<-Meaning it's the bones that grow and the growth is not temporary growth due to increased fluid in the discs

"To determine the relative contributions of the vertebral bodies and intervertebral discs to the increase in spinal length between T5 and L5, over the age range of 7.5-20 years.
The progression of spinal deformity (scoliosis) is associated with skeletal growth, but the relative roles of asymmetrical growth and remodeling of the vertebrae and discs during adolescent growth are unclear.
An existing database of 406 spinal stereoradiographic studies of 188 adolescents with idiopathic scoliosis, aged between 7.5 and 20 years, was used to measure the heights of vertebral bodies and intervertebral discs, and the summation of both (spinal length).
Spinal length was observed to increase from about 250 to 350 mm over this range of ages. Spinal growth was associated with an increase in vertebral height after age 10 years, with minimal if any increase in disc height. The contribution of vertebral and discal height was estimated to be about 17 and 8 mm per year, respectively, at age 7.5 years, but discal height growth was estimated to be effectively zero after age 12 years.
Spinal growth of patients with scoliosis aged between 10 and 20 years occurs almost exclusively by height increases in the vertebrae, not the discs."

"Human vertebrae grow in height by a mechanism similarto that in long bones, by endochondral ossification in
growth plates adjacent to the discs, and they increase in diameter by appositional growth"<-So LSJL will work on the vertebrae

"true values for the increase in disc heights up to age 10 years as well as the apparent continued growth of the spine at age 20 years are not certain."<-Why does the spine continue to grow later than other bones.  Analyzing such properties could lead to new height increase methods.

The article Growth Beyond Skeletal Maturity which is mentioned in this study and may provide some insights into adult height increase was not available.  Here are some of the info mentioned in this study:

"continued growth (about 14 mm increase in sitting height) after skeletal maturity, indicating that the spine continues to grow after cessation of limb growth."

"reported continued spinal growth of about 4 mm per year for 3 years after apparent skeletal maturity. A similar rate of growth after age 17 years was observed in the present study."

A segment found from the paper stated that the discs had no epiphysis.

Here's some pictures of vertebral anatomy(Both taken from svhrad.com):

In the spine the growth plate occurs at the top of the spine in the cartilage plate.  The epiphyseal ring does not apparently result in height growth.

What's interesting about the spine is that some of the chondrocytes are involved in endochondral ossification whereas some contribute to the intervertebral disc.  By what process does that occur?

Chondrocyte moves: clever strategies?

"Chondrocyte movements are herein defined as translocations of the cell body. A brief overview of cell migration in other cell types is presented to set the stage for a discussion of chondrocyte moves; this includes a discussion of the challenges that cells find when moving within tissues. Reports of isolated chondrocyte migration in vitro (isolated cell systems) and ex vivo (cartilage organ cultures) are then summarized, followed by a discussion of recent studies that infer chondrocyte movements in vivo.
Investigators from different laboratories have observed chondrocyte motility in vitro[chondrocytes can move to different areas of the body]. I became interested in the question of whether articular chondrocytes retained their phenotype during their migratory excursions. We devised a simple method to separate migratory and stationary chondrocytes and then showed that migratory chondrocytes synthesized collagen II but not I--consistent with a differentiated phenotype. Our time-lapse video microscopy studies showed that the cells displayed appropriate movement kinetics, albeit with low speed and directionality. Similarly, others have presented data consistent with slow movement of chondrocytes out of cartilage explants. It is important to decipher whether these in vitro movements reflect physiological states and if so, which events are simulated. Examples of in vivo studies that have inferred chondrocyte movements include those describing rotational or gliding movements of chondrocytes in the proliferative zone of the growth plate and its importance in the growth process[so if new stem cells differentiate into chondrocytes they are likely to move into proper growth plate orientation]; and the notion that chondrocytes move from the cartilage endplates to the nucleus pulposus (NP) in the spine of rabbits and rats during development. Such studies are consistent with the hypothesis that chondrocytes exhibit highly controlled and specialized movements during tissue growth and remodeling in vivo. On the other hand, the cartilage explant studies elicit interest in the possibility that matrix injuries resulting in disruption of the collagen network of adult cartilages provide a permissive environment for chondrocyte motility."

So in the LSJL histology slides for instance, if the perceived differentiation was a result of chondrocyte proliferation those cells would likely be within the plate and not outside it.

"Both the extracellular matrix and the growth factor milieu provide motility signals, which the cells coordinate through associations of their signaling receptors, and by coupling of downstream intracellular effectors"<-thus why Hyaluronic Acid and Chondroitin may help increase height.

"the problem of chondrocyte migration is an extremely challenging one, which requires an explanation as to how the cells could overcome the density and pressure of the surrounding matrix to migrate to other sites."<-Maybe chondrocyte migration is just perceived and it's merely a result of a chondrocyte undergoing apoptosis in one location and being generated in another.  It raises the question of how the cartilagenous end plate gives rise to new intervertebral disc tissue.

"positive staining in many of the cells for MT1-MMP, suggesting that this enzyme could be responsible for burrowing a trail in the matrix of the CE[cartilage endplate] to allow cell exodus and colonization of the NP[Nucleus Pulposus]. In addition, many cells expressed the Ki-67 protein marker indicative of cell proliferation and importantly, were surrounded by collagen II. The authors concluded that resting chondrocytes in the endplate are activated to migrate towards the NP."

So it may be matrix degrading enzymes that allow for cartilage migration.  Note that LSJL upregulates some MMP-s which may allow for such canals to be formed.

Do the intervertebral endplates undergo ossification?

The vertebral endplate: disc degeneration, disc regeneration

"The vertebral endplates are critical for maintaining disc function yet like other components of the disc are vulnerable to degeneration[since they are critical for function they cannot fully ossify]. Recent research suggests that the degenerative process can be retarded or reversed."

"At the cranial and caudal ends of each disc are the endplates that separate the vertebral bone from the disc itself and prevent the highly hydrated nucleus from bulging into the adjacent vertebrae. The endplates also absorb the considerable hydrostatic pressure that results from mechanical loading of the spine"<-thus no fusion as the endplates are needed to absorb hydrostatic pressure.  However, how does the endplate repair itself when there is poor vascularization in the spine and thus low access to mesenchymal stem cells.

"The cartilaginous component appears to generate great interest since it persists throughout normal maturation while the adjacent vertebrae undergo ossification. It comprises a gel of hydrated proteoglycan molecules reinforced by a network of collagen fibrils. Unlike the articular cartilage of the synovial joints the collagen fibrils do not connect the endplate directly to the vertebral bone"<-the endplate likely remains as a result of the constant hydrostatic pressure the spine faces.

"A network of microscopic blood vessels penetrates the endplates during development of the growing spine, principally to provide nutrition for the disc, before disappearing around the time of skeletal maturity"<-regrowing these blood vessels may be key for disc height.

"Perhaps surprisingly the endplate can become revascularised after maturity in some species under normal and pathological conditions. In the latter study the revascularisation, presumed to be an attempt at tissue repair, was not able to reverse the inevitable cascade of degeneration caused by annular disruption. The creation of blood vessels in the endplate occurs by activation of the matrix degrading metalloproteinase (MMP) enzymes which are normally maintained in a latent form by tissue inhibitors"<-Thus to regrow those blood vessels it's a matter of increasing MMPs and LSJL highly upregulates MMP-3.

So, even though vertebrae is poorly vascularized, it is possible to revascularize bone with the aid of MMPs.  Thus LSJL should work but the physiology is slightly different than with the legs.  It's hard to load the spine thus LIPUS is recommended.  You'd want to target above and below the spinous process of each disc to try to form new cartilage canals and stimulate chondrogenesis from the cartilage end plate.

Now the studies do say that the typical vertebral bone is undergoing constant hydrostatic pressure so why don't they grow forever?  Remember, that vertebral bones do grow beyond normal time frames.  It's possible that a peak level of hydrostatic pressure is reached at age 20 and thus no more further spinal height growth.  And that anecdotal increases in spinal height are attributed to improvement in posture.

Experiment LIPUS routine

In my research, I've found that LIPUS does in fact generate hydrostatic pressure.  It also generates a rounded cell phenotype even in pre-osteoblastic cells and rounded cells are indicative of a chondrogenic phenotype.  The cells ended up returning to normal after the LIPUS(Ultrasound)-induced treatment stopped but with normal MSCs the cells may stay as differentiated chondrocytes.  The studies also indicated that heat helped encourage chondrogenesis.  Another reason to flex surrounding epiphyseal muscles during LSJL(which generates heat).  One study found that Ultrasound may affect the growth of the Mandibular condyle(the jaw).  That study found possible new cell differentiation up to applying the ultrasound for 10 minutes but only increases in growth rate after 10 minutes but before 20 minutes.

So I definitely think LIPUS is worthwhile to experiment on and I think we have some parameters to go on.

Here's the machine and here's the gel:ReliaMed Portable Ultrasound.  Parker Laboratories Aquasonic Ultrasound Gel .25 Liter Bottle.  You'll have to keep buying new gel as it runs out.

One of the studies found that the most effective was the highest one they used 407 mW per centimeters squared.  The max setting available on the machine is 4.6W or 4600 mW.  The effectiveness of this varies based on the size of your bone.

To perform Ultrasound you apply the gel to the epiphysis of the target bones.  And then move the pad around the epiphysis for around 10 to 20 minutes.  I haven't tried this yet.  I'll be buying an ultrasound soon though so I don't know any complications that can arise from this.

I would do either LIPUS or LSJL because to do both would take too much time.

All application times are 10 minutes.  Since you are doing both inner and outer some of the effects should come through when you're doing the other side of the leg.  You can do it while watching TV.

If you're bald do the top of the head as a control group as this bone is a little bit different.  If not try your heel bone or a tip of your finger.

Inner ankle
Outer ankle
Inner Tibia Epiphysis
Outer Tibia Epiphysis
Inner Femur epiphysis
Outer Femur Epiphysis

For the spine, you have to do one vertebrae at a time and both the tops and bottoms of each vertebrae so I don't think we should try it until we get everything else ironed out.

If you just want to help experiment.  Do one finger of your hand with LSJL and one with LIPUS.  Both for five minutes.

Enhanced Height Increase with FGF?

Previously, one of the factors stimulated by F-spondin was FGF.  Inhibiting F-spondin(and in turn plasmin) resulted in enhancing height increase by 30%.  Since FGF is one of the compounds inhibited by F-spondin, it's possible that FGF's in some way reduce maximal height gain.  By studying the various functions of FGF, we can see if it increases or decreases height growth and whether we can eliminate it from our analysis of how F-spondin inhibits height gain.

Two isoforms of FGFR1 are upregulated by LSJL as well as FGF2 and FGF14.  FGF13 is downregulated by LSJL.

Induction of new bone by basic FGF-loaded porous carbonate apatite implants in femur defects in rats. 

[Carbonate apatite is a form of Calcium Phosphate]

"Thirty-six disc-shaped porous CA implants were inserted into femur defects in 36 Wister rats. Porous CA containing 0, 5, or 50 ng of bFGF was placed in the defect. Bone augmentation was evaluated at 2 and 12 weeks.
At 2 weeks postoperatively, new bone was evident in the defect sites with porous CA containing either 5 or 50 ng of bFGF. The area of regenerated bone was significantly higher with porous CA containing 5 ng of bFGF than with porous CA containing 50 ng. At 12 weeks postoperatively, no differences in new bone formation were seen between porous CA containing 5 and 50 ng bFGF. Porous CA containing bFGF markedly increased the amount of bone newly formed as compared with porous CA without bFGF. Regardless of the implantation period, TRAP-positive cells were visible around the material, indicating that osteoclast-like cells were resorbing porous CA. Bioresorption of the material increased over time: 6.3-7.8% at 2 weeks to 9.1-11.5% at 12 weeks."

"Calcium phosphate ceramics exhibit a high binding affinity for proteins and also provide a cell substratum on which osteogenic cells undergo growth and differentiation "<-can the same be true for chondrocytes?

So too high levels of FGF(10 times higher) inhibits growth rate.  But lower levels 5 ng of FGF benefits growth rate.  It could be that high levels of FGF inhibit cellular differentiation and encourage stem cell proliferation which would have an eventual benefit on height growth.

Actions of fibroblast growth factor-8 in bone cells in vitro. 

"The fibroblast growth factors (FGFs) are a group of at least 25 structurally related peptides. Some FGFs are active in bone, including FGF-1, FGF-2, and FGF-18. FGF-8 is osteogenic in mesenchymal stem cells.FGF-8 was expressed in rat primary osteoblasts and in osteoblastic UMR-106 and MC3T3-E1 cells. Both FGF-8a and FGF-8b potently stimulated the proliferation of osteoblastic cells, whereas they inhibited the formation of mineralized bone nodules in long-term cultures of osteoblasts and reduced the levels of osteoblast differentiation markers, osteocalcin, and bone sialoprotein. FGF-8a induced the phosphorylation of p42/p44 mitogen-activated protein kinase (MAPK) in osteoblastic cells; however, its mitogenic actions were not blocked by either the MAPK kinase (MEK) inhibitor U-0126 or the PI 3-kinase (PI3K) inhibitor LY-294002. FGF-8a, unlike FGF-8b and other members of the family, inhibited osteoclastogenesis in mouse bone marrow cultures, and this was via a receptor activator of NF-kappaB ligand (RANKL)/osteoprotegerin (OPG)-independent manner. FGF-8a did not affect osteoclastogenesis in RAW 264.7 cells (a macrophage cell line devoid of stromal cells) exogenously stimulated by RANKL, nor did it affect mature osteoclast function as assessed in rat calvarial organ cultures and isolated mature osteoclasts. FGF-8 is active in bone cells, stimulating osteoblast proliferation in a MAPK-independent pathway and inhibiting osteoclastogenesis via a RANKL/OPG-independent mechanism." 

FGF-8 affects osteoblasts and osteoclasts. 

"FGF-8 stimulates the expression of core-binding factor a-1 (Cbfa1) in the murine fibroblast-derived cell line C3H10T1/2"

"there is some evidence that ectopic bone and cartilage form in nude mouse tumors of S115 cells known to produce FGF-8"

"FGF-18 has a high homology to FGF-8"

"FGF-2 and FGF-18 stimulate osteoclast function, inducing osteoclastogenesis via receptor activator of NF-κB ligand (RANKL) as well as actively stimulating pit formation"

"FGF-8b and FGF-8e were reported to induce osteoclastic differentiation likely dependent of the RANKL/osteoprotegerin (OPG) pathway"

"FGF-8a stimulated connexin 43 (Cx43) expression in cultured mesenchymal cells from chicken limb buds"

BMP canonical Smad signaling through Smad1 and Smad5 is required for endochondral bone formation. 

"Bone morphogenetic protein (BMP) signaling{which typically use Smad receptors 1/5/8} is required for endochondral bone formation.  Deletion of individual Smads results in viable and fertile mice. Combined loss of Smads 1, 5 and 8, however, results in severe chondrodysplasia[dwarfism]. Smad1/5(CKO) (cartilage-specific knockout) mutant mice are nearly identical to Smad1/5(CKO);Smad8(-/-) mutants, indicating that Smads 1 and 5 have overlapping functions and are more important than Smad8 in cartilage. The Smad1/5(CKO) phenotype is more severe than that of Smad4(CKO) mice. The chondrodysplasia in Smad1/5(CKO) mice is accompanied by imbalances in cross-talk between the BMP, FGF and Ihh/PTHrP pathways. Ihh is a direct target of BMP pathways in chondrocytes and FGF exerts antagonistic effects on Ihh expression." 

"FGFs reduce Bmp4 and Ihh"

FGF has antagonistic effects on Ihh expression.   FGF-8 induces the phosphorylation of MAPK.  Here we see that FGF phosphorylates Smad.  FGF may inhibit height by inhibiting Ihh.  Of course alternatively, optimal levels may be needed to maintain the separate growth plate zones.   Remember that Smad 1/5/8 phosphorylation affects height growth.  Smad phosphorylation activates those genes.  Therefore, perhaps transgenic Smad 1/5/8 expression could encourage height growth.

"[Smad 1/5KO] mutants exhibited an expanded domain of type I collagen-producing cells.  These cells were randomly oriented, unlike the structure of the WT perichondrium, in which they are perpendicular to the growth plate. Type II collagen-producing cells, which are normally restricted to the growth plate, were embedded in the mutant periosteum"

"Apoptosis is normally confined to the hypertrophic zone. Apoptosis was expanded in mutant cartilage"

Smado 1/5KO mice had lower expression of Type II Collagen and Type X Collagen.

"Ucma is a marker for upper (resting) chondrocytes"

"Smad1/5CKO mutants exhibited increased expression of osteocalcin (Bglap1/2)"

"FGF18 treatment reduced C-terminal phosphorylation throughout the growth plate, whereas inhibition of endogenous FGF signaling using the FGF receptor antagonist SU5402 led to activation (C-terminal phosphorylation) of Smad1/5"

FGFR3 promotes synchondrosis closure and fusion of ossification centers through the MAPK pathway. 

"Activating mutations in FGFR3 cause achondroplasia. FGFR3 and MAPK signaling in chondrocytes promote synchondrosis closure and fusion of ossification centers[FGFR1 and FGFR2 are good for height growth whereas FGFR3 is bad for height growth; MAPK signaling chondrocytes is bad.  Note this is not MAPK signaling in osteo- cells]. We observed premature synchondrosis closure in the spine and cranial base in human cases of homozygous achondroplasia and thanatophoric dysplasia as well as in mouse models of achondroplasia. In both species, premature synchondrosis closure was associated with increased bone formation. Chondrocyte-specific activation of Fgfr3 in mice induced premature synchondrosis closure and enhanced osteoblast differentiation around synchondroses. FGF signaling in chondrocytes increases Bmp ligand mRNA expression and decreases Bmp antagonist mRNA expression in a MAPK-dependent manner, suggesting a role for Bmp signaling in the increased bone formation. The enhanced bone formation would accelerate the fusion of ossification centers and limit the endochondral bone growth." 

"Bmp ligands and Bmp antagonists [are] downstream targets of FGF signaling in chondrocytes. BMPs, such as Bmp-2 and Bmp-7, are strongly osteogenic, while their availability to the Bmp receptors is counterbalanced by BMP antagonists, such as Noggin, Gremlin and Chordin. Because BMP enhances chondrocyte hypertrophy, it is possible that increased BMP signaling together with increased FGF signaling in chondrocytes accelerated synchondrosis closure in Fgfr3G374R/+ mice{So too much BMP-2 for instance could be bad for height but it depends on the presence of FGFR3 receptors}. In addition, increased availability of BMPs may have accelerated osteoblast differentiation in the adjacent perichondrium." 

So FGF could be bad for height increase by activating MAPk in the chondrocytes which results in accelerated chondrocyte ossification. 

Proteoglycan desulfation determines the efficiency of chondrocyte autophagy and the extent of FGF signaling during endochondral ossification. 

"Cartilage extracellular matrix (ECM) contains large amounts of proteoglycans made of a protein core decorated by highly sulfated sugar chains, the glycosaminoglycans (GAGs)[LSJL upregulates ECM genes]. GAGs desulfation, a necessary step for their degradation, is exerted by sulfatases that are activated by another enzyme, Sulfatase-Modifying Factor 1 (SUMF1), whose inactivation in humans leads to severe skeletal abnormalities. Despite being expressed in both osteoblasts and chondrocytes Sumf1 does not affect osteoblast differentiation.  In chondrocytes it favors ECM production and autophagy and promotes proliferation and differentiation by limiting FGF signaling[How much Sumf1 promotes proliferation and differentiation may differ on expression levels of FGFR3 versus FGFR1/2]. Proteoglycan desulfation is a critical regulator of chondrogenesis." 

"The extracellular matrix (ECM) secreted by hypertrophic chondrocytes allows vascular invasion, degradation of the calcified ECM, and initiation of osteogenesis." 

"Sumf1, and proteoglycan desulfation, influence FGF signaling in chondrocytes during skeletal development.Sulfatases catalyze desulfation of the GAGs moiety of proteoglycans in the intracellular and extracellular space. These enzymes are substrates of Sumf1 whose only known function is to activate sulfatases. Proteoglycan desulfation regulates several aspects of chondrocyte biology. The block in proteoglycan desulfation caused by Sumf1−/− deletion severely decreases chondrocytes viability by hampering their capacity to generate enough energy through autophagy to survive in their avascular environment. This defect in autophagy is caused, in part, by the engulfment of lysosomes by undigested GAGs that leads to an impairment of the autophagosome–lysosome fusion"  

Proteoglycan desulfation inhibits the ability of GAGs to damage the growth plate tissue.  FGF results in a decrease of chondrocyte proliferation(based on FGFR3 expression). 

Fibroblast growth factor expression in the postnatal growth plate. 

"Fibroblast growth factor (FGF) signaling is essential for endochondral bone formation. In the postnatal growth plate, we quantitated expression of FGFs and FGF receptors (FGFRs) and examined both their spatial and temporal regulation. Tat proximal tibial growth plates and surrounding tissues were microdissected, and specific mRNAs were quantitated. Perichondrium expressed FGFs 1, 2, 6, 7, 9, and 18 and, at lower levels, FGFs 21 and 22. Growth plate expressed FGFs 2, 7, 18, and 22{Note that the growth plate does not generally express more specific FGFs than the perichondrium}. perichondrial FGFs regulate growth plate chondrogenesis{another link between the periosteum and height growth, therefore stimulating the periosteum like for instance mechanical load may affect height growth}. FGFs synthesized by growth plate chondrocytes may be physiologically important because of their proximity to target receptors. In growth plate, we found expression of FGFRs 1, 2, and 3, primarily, but not exclusively, the c isoforms. FGFRs 1 and 3, thought to negatively regulate chondrogenesis, were expressed at greater levels and at later stages of chondrocyte differentiation, with FGFR1 upregulated in the hypertrophic zone and FGFR3 upregulated in both proliferative and hypertrophic zones. FGFRs 2 and 4, putative positive regulators, were expressed at earlier stages of differentiation, with FGFR2 upregulated in the resting zone and FGFR4 in the resting and proliferative zones. FGFRL1, a presumed decoy receptor, was expressed in the resting zone. With increasing age and decreasing growth velocity, FGFR2 and 4 expression was downregulated in proliferative zone. Perichondrial FGF1, FGF7, FGF18, and FGF22 were upregulated. Temporal changes in FGF and FGFR expression may contribute to growth plate senescence and thus help determine the size of the adult skeleton." 

So, the regulation of FGF is complex and the optimal amount of them needed is similarly so.  The best way to grow taller would be to encourage desulfation of GAGs or to supplement with the non-sulfated forms of these compounds(like chondroitin(no sulfate)). 

"In mice, targeted ablation of FGFR2 impairs postnatal long bone growth"

"Activating mutations in FGFR1 cause osteoglophonic dysplasia, [a] short-limbed skeletal dysplasia in humans"

"mice conditionally deleted for FGFR1 in osteo-chondro-progenitor cells display an increased hypertrophic zone size, probably due to a decrease in the rate of cartilage resorption and ossification"

"although ablation of FGFR4 alone produces no apparent growth plate phenotype, double FGFR3/FGFR4-null mice show impaired long bone growth"<-So FGFR4 may be a better form of FGFR3 for height growth.

"dwarfism in mice overexpressing FGF2"

"Mice overexpressing FGF9 develop a skeletal dysplasia involving proximal long bones while FGF9-null mice display disproportionately short proximal skeletal elements"

"Ectopic expression of FGF9 in cranial bones induces [new] endochondral ossification"

"FGF18-null mice display long bone phenotypes similar to but even more severe than FGFR3 knockouts"

FGF may also play a role in cellular senescence.

FGF's detected in the perichondrium but not the growth plate:
FGFR2b
FGF1
FGF5
FGF6
FGF9
FGF10
FGF16
FGF21

FGF's detected in the metaphseal bones but not the growth plate:
FGFR2b
FGF1
FGF9
FGF21
FGF23

"No ligands were detected in growth plate that were not also present in perichondrium, and all overlapping ligands showed similar or greater expression in perichondrium"

"FGF2 expression in proliferative zone chondrocytes remained constant with age while both FGF18 and FGF22 mRNA levels declined"<-so reduction of FGF18&22 could be related to senescence.

"FGFR1c, FGFR2b, FGF1, FGF7, FGF18, FGF22, and FGFR3c mRNA levels all increased in expression with age [in the perichondrium]"

"Local production of FGFs in the growth plate may become increasingly important in the postnatal animal where the distance between the perichondrium and the interior of the growth plate increases."<-so perhaps exogenous FGFs can help increase height when animals no longer have a source of FGFs from the perichondrium.  Perhaps exogenous administration of proteins expressed in the perichondrium but not in the growth plate could help with height.


Inhibition of cellular senescence by developmentally regulated FGF-receptors in mesenchymal stem cells.

"MSCs should express FGF-receptors reflecting their developmental origin and potential. FGFR1 and 2 are expressed by rare mesenchymal progenitors in putative MSC niches in vivo including perichondrium, periosteum and trabecular marrow[So the expression of FGFR1 and FGFR2 is reprentative of a cells ability to differentiate, forcing expression of FGFR1 and FGFR2 may be a way to grow taller]. FGFR1+ cells often appeared as pericytes. These cells display a characteristic MSC phenotype in vitro when expanded with FGF-2, which appears to maintain MSC stemness by inhibiting cellular senescence through a PI3K/AKT-MDM2 pathway and promoting proliferation[FGF-2 may inhibit cellular senescence]. FGFRs may thus be involved in MSCs self-renewal. FGFR1/2 are developmentally-regulated markers of MSCs in vivo and in vitro and are important to maintain MSCs stemness."

"undifferentiated mesenchymal cells are present in murine embryonic perichondrium during endochondral bone formation. These cells were shown to adopt a pericyte identity while migrating from the perichondrium to colonize both the bone collar (cortical bone) and the primary spongiosa (trabecular bone)."

"FGFR1/2 signaling through PI3K/AKT-MDM2 promotes the proliferation of MSCs mainly by inhibiting cellular senescence while maintaining MSC properties."

"FGFR1 could be detected in the marrow cavity of postnatal bones"

"Perichondrial FGFR1/2+ cells were found to be surrounded by FGF-18–expressing cells"

"murine MSCs expanded from whole-bone crushes with FGF-2 express high levels of mRNAs encoding PDGFRα, PDGFRβ, TGFβR1, TGFβR2, and EGFR (HER1/ ErbB1)"

"After chondrocytic differentiation, Sox9+/collagen2+ chondrocytes robustly up-regulated FGFR3 and only a few undifferentiated cells maintained FGFR1 expression at the periphery of the micromass culture"

Check out figure 7 for a great diagram of the role of various FGFs.

Cellular engineering of cells that express FGFR1 and FGFR2 may be a way to grow taller.  In addition, there may be a way to upregulate FGFR1 and FGFR2.

FGFR1 even though it may negatively regulates chondrogenesis it only does so in the hypertrophic zone where it's time to stop chondrogenesis anyways and begin osteogenic differentiation.  Even if you stimulate FGF-1 and FGF-2(and perhaps FGF-4) by stimulating the periosteum and growth then this may not result in taller height growth as the number of receptors is a limiting factor.  Finding ways to upregulate FGFR1(you likely need either FGFR1 or FGFR3 and FGFR1 is preferential to growing taller than FGFR3) and FGFR2 may be a way to grow taller.

Fibroblast growth factor (FGF) 18 signals through FGF receptor 3 to promote chondrogenesis.

"the congenital absence of either FGF18 or FGFR3 resulted in similar expansion of the growth plates of fetal mice and the addition of FGF18 to human articular chondrocytes in culture enhanced proliferation and matrix production. FGF18 signals through FGFR3 to promote cartilage production by chondrocytes. We used the limb buds of FGFR3(+/+) and FGFR3(-/-) embryonic mice as a source of mesenchymal cells to determine how FGF18 signaling affects chondrogenesis. Impaired cartilage nodule formation [occurred] in the FGFR3(-/-) cultures. Potential contributing factors to the phenotype were identified as impaired mitogenic response to FGF18, decreased production of type II collagen and proteoglycan in response to FGF18 stimulation, impaired interactions with the extracellular matrix resulting from altered integrin receptor expression, and altered expression of FGFR1 and FGFR2. FGF18 [is] a selective ligand for FGFR3 in limb bud mesenchymal cells, which suppressed proliferation and promoted their differentiation and production of cartilage matrix."

"FGF2 and FGF18 [are] potential ligands for FGFR1 in hypertrophic chondrocytes, for FGFR3 in resting and proliferating chondrocytes, and for FGFR2 in the perichondrium and periosteum of developing long bones. FGF18 [may signal] selectively through FGFR3 to promote the differentiation of pre-chondrogenic mesenchymal cells to cartilage-producing chondrocytes."

"FGF18 signals through FGFR3 to increase expression of type II collagen and its α1β1 receptor in chondrogenic cells."

Here's a patent involving FGF variants for some height increase applications:

FGF VARIANTS AND METHODS FOR USE THEREOF

"All FGFRs tested so far bind FGF-1 (acidic FGF, aFGF) with moderate to high affinity, demonstrating an apparent redundancy in the FGF system. In contrast to FGFR1 and FGFR2, the third receptor subtype, FGFR3 [binds] to FGF-8, FGF-17 and FGF-18 with high affinity and to FGF-9 with improved selectivity. Specificity may also be achieved by specific proteoglycans expressed in different tissues."

"FGF-9 variants comprising mutations in the loop between the β8 and β9 strands of the polypeptide, previously identified as a conserved receptor binding site, and analogous loops in the other members of the FGF family, unexpectedly provide enhanced receptor subtype specificity."

"The FGF-2 variants are shown to stimulate proliferation of chondrocytes and induce differentiation of neuronal cells and may be used to specifically induce proliferation or differentiation of progenitor cells and embryonic or adult stem cells."

"the FGF variant [to promote growth plate bone growth] is selected from R64M-FGF9 or FGF9-2, and the bioactive agent is a natriuretic peptide. In another currently most preferred embodiment the FGF variant is selected from R64M-FGF9-W144G or FGF9-2-W144G and the bioactive agent is selected from C-type natriuretic peptide (CNP) or an analog thereof."

Fibroblast growth factor receptors in in vitro and in vivo chondrogenesis: relating tissue engineering using adult mesenchymal stem cells to embryonic development.

"mRNA levels of FGFR2 are upregulated in the mesenchymal condensation, whereas FGFR3 is expressed during differentiation and FGFR1 during hypertrophy."

"On embryonic day 12 (E12), N-cadherin was observed in the mesenchymal condensation in the center of the limb bud. Collagen II stained only very weakly in the mesenchymal condensation on E12. No collagen X was seen at this stage. On E12, expression of FGFR1 was lower in the mesenchymal condensation than in the surrounding loose mesenchyme. In contrast, FGFR2 and FGFR3 expression was seen only in the mesenchymal condensation, although the staining for FGFR3 was not very strong"

"no FGFRs are expressed in hyaline articular chondrocytes"

"FGF2 and FGF9 had differential effects when added at days 21–35. While FGF2 inhibited further matrix deposition, FGF9 increased matrix resorption."