Height Increase Pages

Friday, December 17, 2010

Increase Height with Hydrostatic Pressure

There are multiple ways to stimulate stem cells and chondrocytes but hydrostatic pressure seems to be the only way discovered so far to cause that initial differentiation of stem cells into chondrocytes.  Which means that things like LIPUS and vibration may be good for augmenting gains induced by Lateral Synovial Joint Loading they cannot replace it as they can't induce that initial differentiation into chondrocytes(they can help during puberty though).  In this study by Hiroki Yokota and P. Zhang, knee drilling diminished the effectiveness of LSJL in inducing bone growth.  They call it intermedullary pressure but there should be pressure generated in the epiphysis too and then the stem cells there can differentiate into chondrocytes.  Hydrostatic pressure like Interstitial Fluid Flow can induce changes in the actin cytoskeleton.

LIPUS and Interstitial fluid flow can induce chondrogenic differentiation but only in the presence of TGF-Beta.  Hydrostatic pressure only needs Type I Collagen to induce chondrogenic differentiation of stem which is far more plentiful in the bone.

It's the hypertrophic phase of chondrogenesis that mainly is involved in the height increase.  This can occur even at a level of one chondrocyte.  One single chondrocyte in your bone can increase height if it hypertrophies.

Step One of height growth though is induction of chondrocyte differentiation of stem cells(can also be achieved by bone deposition on the subchondral plate).  Chondrocyte Hypertrophy requires dexamethasone withdrawl, TGF-Beta Withdrawl, and addition of T3(a thyroid hormone). Which is why a method not requiring TGF-Beta is so beneficial as that would inhibit chondrocyte hypertrophy.

The effect of hydrostatic pressure on three-dimensional chondroinduction of human adipose-derived stem cells.

"Human ASCs underwent primary culture and three passages before being seeded into collagen scaffolds. These constructs were incubated for 1 week in an automated bioreactor using cyclic HP at 0-0.5 MPa, 0.5 Hz, and compared to constructs exposed to atmospheric pressure. In both groups, chondrogenic differentiation medium including transforming growth factor-beta1 was employed[TGF-Beta was there in this group but remember in the Type I Collagen study there was no TGF-Beta]. One, 2, 3, and 4 weeks after incubation, the cell constructs were harvested.
Pericellular and extracellular metachromatic matrix was observed in both groups and increased over 4 weeks, but accumulated at a higher rate in the HP group. Cell number was maintained in the HP group over 4 weeks but decreased after 2 weeks in the atmospheric pressure group. Chondrogenic-specific gene expression of type II and X collagen, aggrecan, and SRY-box9 was increased in the HP group especially after 2 weeks[Type X Collagen increased which is a marker of Hypertrophic differentiation meaning that Hydrostatic Pressure can induce chondrocyte hypertrophy as well as stem cell differentiation into chondrocytes].
Chondrogenic differentiation of ASCs [occurs] in a three-dimensional collagen scaffolds with treatment of a cyclic HP. Cyclic HP was effective in enhancing accumulation of extracellular matrix and expression of genes indicative of chondrogenic differentiation."

"collagen type II and proteoglycans were synthesized only in MSCs in chondrogenic induction using TGF-β3[and not Beta1]."

Atmospheric pressure differs from hydrostatic pressure based on position relative to sea level.  As elevation increases, atmospheric pressure decreases.  Does anybody know of any correlation between elevation and height?  The hydrostatic pressure used in this study was 0-0.5 MPa whereas the hydrostatic pressure in the Type I Collagen study was 1 MPa.  It's unclear about the implications of atmospheric pressure on our goals to increase height with LSJL.

Influence of hydrostatic and distortional stress on chondroinduction. 

"Using C3H10T1/2[murine embryonic fibroblasts] cells as a model, we have investigated the influence of hydrostatic pressure, equibiaxial contraction, and centrifugal pressure on chondroinduction. Cells responded to cyclic hydrostatic compression (5 MPa at 1 Hz)[with BMP-2] and cyclic contractile strain (15% at 1 Hz) by upregulating aggrecan and collagen type II gene expression[This study was 5MPa versus 1 MPa and 0.5MPa]. Centrifugal pressure (4.1 MPa for 30 min) may increase cell proliferation and stimulate proteoglycan and collagen type II production. compression, whether it is distortional or hydrostatic in nature, applied to undifferentiated connective tissue triggers differentiation toward a chondrocyte-like phenotype and production of a less permeable extracellular matrix[LSJL induces Lateral Compression on the epiphysis] which is capable of sustaining increasingly higher hydrostatic fluid pressure for compressive load support." 

"hydrostatic andd istortional compressive stresses play a role in regulating chondrogenic differentiation."

Growth factors TGF-Beeta1 or BMP-2 and high density medium was used for chondroinduction.

"MSC’s from adult bone marrow, which upregulated aggrecan mRNA by 1.6-fold and collagen type II by 3.3-fold in response to 10 MPa hydrostaticpressure applied at 1 Hz for 4 h/day for 14 days in the presence of 10 ng/ml TGF-β3"

"he higher permeability and lower compressive modulus of undifferentiated tissue renders it unableto support such high magnitudes of interstitial fluid pressure. Under compression, the solid matrix wouldbe required to carry the majority of the load and cells would undergo substantial distortion."

So any form of compression like LSJL will lead to stem cells differentiating into chondrocytes.  The problem with most exercises such as say squats is that most of the compressive force is applied to the diaphysis of the bone rather than the epiphysis of the bone. 

Dynamic culturing of cartilage tissue: the significance of hydrostatic pressure.

"Human articular cartilage functions under a wide range of mechanical loads in synovial joints, where hydrostatic pressure (HP) is the prevalent actuating force. We hypothesized that the formation of engineered cartilage can be augmented by applying such physiologic stimuli to chondrogenic cells or stem cells, cultured in hydrogels, using custom-designed hydrostatic pressure bioreactors. To test this hypothesis, we investigated the effects of distinct hydrostatic pressure regimens on cartilage formation in vitro by either human nasal chondrocytes (HNC) or human adipose stem cells (hASCs) encapsulated in gellan gum (GG) hydrogels. To this end, we varied the frequency of low HP, by applying pulsatile (PHP) or steady (SHP) load to HNC-GG constructs over a period of 3 weeks, and evaluated their effects on cartilage tissue engineering outcomes. HNC (10x10<sup>6</sup> cells/mL) were encapsulated in GG hydrogels (1.5%) and cultured in chondrogenic medium under three regimens for 3 weeks: (1) 0.4 MPa Pulsatile HP; (2) 0.4 MPa Steady HP; and (3) Static. Subsequently, we applied the pulsatile regimen to hASC-GG constructs and varied the amplitude of loading, by generating both low (0.4 MPa) and physiologic (5 MPa) HP levels. hASC (10x10<sup>6</sup> cells/mL) were encapsulated in GG hydrogels (1.5%) and cultured in chondrogenic medium under three regimens for 4 weeks: (1) 0.4 MPa Pulsatile HP; (2) 5 MPa Pulsatile HP; and (3) Static. In the HNC study, the best tissue development was achieved by the pulsatile HP regimen, while in the hASC study, greater chondrogenic differentiation and matrix deposition were obtained for physiologic loading[5 MPa], as evidenced by gene expression of aggrecan, collagen type II and sox-9, metachromatic staining of cartilage ECM and immunolocalization of collagens. We thus propose that both HNC and hASC detect and respond to physical forces, thus resembling joint loading[there's fat in the bone so there's likely adiposal stem cells in the epiphyseal bone marrow], by enhancing cartilage tissue development in a frequency- and amplitude-dependant manner."

"In vivo, articular cartilage is exposed to a wide range of static and dynamic mechanical loads, ranging amplitudes of about 5–6 MPa for gait, and as high as 18 MPa for other movements such as running or jumping"<-Thus perhaps the same kind of loads the articular cartilage is exposed to when exposed to epiphyseal stem cells will encourage chondrogenic differentiation.

"the solid components of the extracellular matrix support shear stress, while the incompressible interstitial water is responsible for withstanding compressive loading, by driving out of the tissue. In view of this, 95% of overall applied joint load is supported by interstitial fluid pressurization, so hydrostatic pressure is the prevailing mechanical signal governing normal articular cartilage homeostasis"<-And normal bone can't handle this hydrostatic pressure thus encouraging chondrogenesis.

"Hydrostatic loading does not result in macroscopic deformation of the tissue, once the solid matrix is incompressible, but increase in interstitial fluid flow and/or increased cytoskeleton changes promoted by a
pulsatile loading, might be some of the mechanisms triggering cell response over steady pressurization. Additionally, by increasing loading amplitudes, up to 5 MPa, major mechanotransduction mechanisms should respond proportionally by: 1- increasing integrinmediated responses, majorly by 5ß 1 that perform as primary bridge between the ECM and actin cytoskeleton, and playing an important role on MAPK activation; 2- enhancing molecular conformational changes transduced to the nucleous, and consequently changing accessibility of genomic DNA for transcription; and 3- rising the direct effects on the cell membrane ion pumps and channels – with loading the Na/K pump is inhibited and intracellular Ca2+ waves function as one of the major signal transduction mechanisms"

Effects of intermittent hydrostatic pressure magnitude on the chondrogenesis of MSCs without biochemical agents under 3D co-culture.

"MSCs and primary cultured chondrocytes were separately encapsulated into alginate beads, and the two types of beads were separated by a membrane. For the investigation a computer-controllable bioreactor was designed and used to engage intermittent hydrostatic pressure (IHP). Five different magnitudes (0.20, 0.10, 0.05, 0.02 MPa and no stimulation) of IHP were applied. The stimulation pattern was the same for all groups: 2 h/day for 7 days starting at 24 h after seeding; 2 and 15 min cycles of stimulating and resting, respectively. Biochemical (DNA and GAG contents), histological (Alcian blue), and RT-PCR (Col II, SOX9, AGC) analyses were performed on days 1, 5, 10, and 20. stimulation with higher magnitudes of IHP (≥0.10 MPa)[this is much lower magnitude than in previous studies and much closer to physiological levels we can reach with LSJL] were more effective on the proliferation and differentiation of co-cultured MSCs. "

"The co-culturing technique is based on the fact that stem cells are affected by neighboring cells. Perhaps something secreted by the neighboring cells affects the differentiation of stem cells into the targeted cell type."

"The cell density ratio of MSCs to chondrocytes was 1:2 in this co-culturing system"<-maybe we can get articular chondrocytes to secrete pro-chondrogenic factors.

"the pattern of IHP applied to all groups was 2 and 15 min for stimulating and resting, respectively. IHP was engaged 2 h/day for 7 days, starting 24 h after seeding"

"IHP magnitudes below 0.10 MPa did not provide a lasting effect after IHP treatment was stopped"<-this could be a problem with LSJL.

The key factor in height growth is compression of the epiphysis to increase hydrostatic pressure to induce differentiation towards a chondrogenic phenotype.

Effect of Cytoskeletal Disruption on Mechanotransduction of Hydrostatic Pressure by C3H10T1/2 Murine Fibroblasts.

"Cyclic hydrostatic pressure of physiological magnitude (< 10 MPa) stimulates chondrogenic differentiation of mesenchymal stem cells.  An intact cytoskeleton [is] required for uninhibited mechanotransduction of hydrostatic pressure. Therefore we examined the effects of drugs which selectively interfere with actin and tubulin polymerization on pressure-induced upregulation of aggrecan and col2a1 (type II collagen) mRNA expression. C3H10T1/2 cells were cultured as pellets in either 4microM cytochalasin D or 4microM nocodazole and subjected to 3 days of cyclic hydrostatic compression (1 Hz, 5 MPa, 2 h per day). Phalloidin staining and indirect immunostaining with anti alpha-tubulin antibody confirmed disruption of microfilament and microtubule assemblies, respectively.  Both drugs substantially lowered the basal level of aggrecan and col2a1 mRNA, but that neither drug prevented a pressure-stimulated increase in gene expression relative to the altered basal state. Thus upregulation of macromolecular gene expression by cyclic hydrostatic pressure did not require a completely intact cytoskeleton."

"Cytochalasin D, a drug that inhibits actin-filament polymerization and can affect cell shape and deformation under stress"

"In the absence of cytoskeleton-altering drugs, cyclic hydrostatic pressure caused a 2.26 ± 0.71-fold upregulation of aggrecan mRNA and a 1.72 ± 0.51-fold increase in col2a1 mRNA (Pressure vs. Control)"

"Aggrecan mRNA was downregulated by 0.67 ± 0.19-fold and col2a1 by 0.34 ± 0.11-fold in response to treatment by cytochalasin (CSK Disrupted vs. Control). Nocodazole decreased aggrecan mRNA by 0.35 ± 0.22-fold and col2a1 mRNA by 0.27 ± 0.17-fold (CSK Disrupted vs. Control). However, neither drug significantly diminished the pressure-induced upregulation of either aggrecan or col2a1 above the drug-lowered baseline."

Intermittent Hydrostatic Pressure Enhances Growth Factor-Induced Chondroinduction of Human Adipose-Derived Mesenchymal Stem Cells.

"[We examined the] effects of intermittent HP on chondrogenic differentiation of human adipose-derived mesenchymal stem cells (hASCs) in the presence or absence of chemical chondrogenic medium. Cells were isolated from abdominal fat tissue and confirmed for expression of ASC surface proteins and differentiation potential. Passage 3 pellets were treated with chemical (growth factor), mechanical (HP of 5MPa and 0.5Hz with duration of 4h/day for 7 consecutive days), and combined chemical-mechanical stimuli. Using real-time polymerase chain reaction, the expression of Sox9, collagen II, and aggrecan as three major chondrogenic markers were quantified among three experimental groups and compared to those of stem cells and human cartilage tissue. In comparison to the chemical and mechanical groups, the chemical-mechanical group showed the highest expression for all three chondrogenic genes close to that of cartilage tissue."

"Cell surface protein expression was highly characteristic of MSCs. The isolated hASCs were positive for CD44, CD90, CD105, and CD166 and negative for CD34 and CD45."

"Sox9 mRNA levels were elevated by 2.24- and 3.48-fold in the chemical and chemical–mechanical groups, respectively, while in the native articular cartilage, such expression was 8.75 times bigger"<-LSJL Sox9 elevation was 3 fold.

"addition of TGF-β1, dexamethasone, and ascorbate-2-phosphate resulted in statistically significant elevation of the expression of three factors"

"In the mechanical group, IHP alone was not able to increase Sox9 expression"<-So LSJL involves other factors than IHP in inducing Sox9.

"cartilage is not deformed under physiological range of HP; however, the transporter proteins can be compressed as they experience such loading. As the pressure and the subsequent strain rise, the proteins change their orientations to reach a lower energy state, resulting in change of intracellular ion concentrations. Alterations in intracellular ion concentration lead to modified gene expression and protein synthesis. Hence, specific loads may alter concentration of the ions that affect gene expression and, consequently, protein production"<-The concentrations of Calcium is most likely to be what's effect by deformation of MSCs.

Effect of Hydrostatic Pressure on Bone Regeneration Using Human Mesenchymal Stem Cells.

"hMSCs underwent primary culture for three passages before being seeded into hydroxyapatite (HA) scaffolds. The scaffolds were incubated for 3 weeks in an automated bioprocessor under cyclic HP. Scaffolds exposed to atmospheric pressure (AP) served as the comparator. Osteogenic differentiation medium was employed for both the HP and AP groups. Immediately before and 1, 2, and 3 weeks after incubation, the scaffolds were harvested. 
Cells were only found in the AP scaffold surfaces, whereas in the HP group, they were distributed evenly throughout the scaffolds. Immunohistochemical analysis revealed that the HP group expressed higher levels of osteocalcin (OC), osteopontin (OP), osteonectin (ON), and collagen type 1 (Col1) than the AP group during the 3-week process. Gene expression analysis revealed that the HP group expressed higher levels of ON, Col1, alkaline phosphatase, and integrin β5 than the AP group at the 1-, 2-, and 3-week timepoints. The HP group also expressed higher levels of core-binding factor α-1 (Cbfa1) at the 2- and 3-week timepoints and higher levels of OP and OC at the 1-week timepoint {this is consistent with LSJL}. Their proliferating cell nuclear antigen levels were lower at the 1- and 2-week timepoints{this is also consistent with LSJL}."

Surprisingly, only Col1A1 was expressed above threshold at 49 hours despite LSJL being a primary osteogenic stimulus.  However, OPN and OCN were expressed below threshold at 1 to 2 fold between 1 hour and 2 weeks.

But the expression level patterns of OPN and OCN are consistent with hydrostatic pressure on MSCs.  However, the fold change of COL1 was 6 fold at one week with HP but only approx. 2 fold at one week of LSJL.  OPN was consistent with LSJL with elevated levels at 1 week and lowered levels at 2 weeks.  OCN was not with a continual increase at 2 weeks.

Perhaps this indicates that LSJL does induce hydrostatic pressure but the factors that inhibit hydrostatic pressure are not extrinsic to the cells themselves but rather perhaps to the ECM or the differentiation of stem cells into chondrocytes to inhibit hydrostatic pressure.

"HP in the mammalian bone marrow is 10.7–120 mmHg, which is about a quarter of the systemic blood pressure."

"The porous hydroxyapatite (HA) scaffolds were 5 mm in diameter, 2 mm in height, had 80% porosity, and a pore size of 50–200 μm"

PCNA decreased in expression by HP.

"Integrin β5 is known to promote cellular migration"

Proteomic analysis of chondrocytes exposed to pressure.

"membrane-bound transferrin-like protein p97, a marker of chondrocyte differentiation, was only expressed in chondrocytes under hydrostatic pressure."

The mRNA expression for COL2 was highest in the 250 Pa group.


Evaluation of platelet-rich plasma and hydrostatic pressure regarding cell differentiation in nucleus pulposus tissue engineering.

"This study compares the influence of 3D pellet culture and alginate beads, as well as that of different media compositions, by the addition of human platelet-rich plasma (PRP) or transforming growth factor (TGF-β(1) ) in interaction with hydrostatic pressure on chondrogenic differentiation of human MSCs compared to NP[nucleus pulposus] cells. Gene expression of the chondrogenic markers aggrecan, collagen type 2 and collagen type 1 and Sox9 was considerably lower in cells cultivated with PRP compared to TGF-β(1) . Pellet culture system was found to be more suitable than alginate beads. A positive influence of hydrostatic pressure could only be shown for individual donors.  In comparison to TGF-β(1) , human PRP did not induce adequate chondrogenic differentiation for both culture systems and cell types used. The mixture of growth factors in PRP promoted proliferation rather than chondrogenic differentiation."

The final TGFB1 concencentration was similar in both the PRP and TGFB1 medium.  Medium was either "10 ng/ml recombinant TGF-β1 or 10% pooled human PRP".  The concentration of TGFB1 was 14ng/ml which is higher than the direct TGF-B1 so perhaps compounds in PRP actually inhibit chondrogenesis.  The authors mention an intrinsic osteogenic capacity of PRP.

"MSCs and NP cells showed a considerably greater expression (up to 23-fold) of aggrecan and collagen type 2 in pellet culture compared to alginate beads"

"The Col2:Col1 ratio was higher for NP cells than for MSCs "

Cyclic hydrostatic pressure enhances the chondrogenic phenotype of human mesenchymal progenitor cells differentiated in vitro.

"This study examined the effects of cyclic hydrostatic pressure on human bone marrow-derived mesenchymal progenitor cells undergoing chondrogenic differentiation. Aggregates of bone marrow-derived mesenchymal progenitor cells were cultured in a defined chondrogenic medium and were subjected to cyclic hydrostatic pressure. Aggregates were loaded at various time points: single (day 1 or 3) or multiple (days 1-7). At 14 and 28 days, aggregates were harvested. The aggregates loaded for a single day did not demonstrate significant changes in proteoglycan and collagen contents compared with the non-loaded controls. In contrast, for the multi-day loaded aggregates, statistically significant increases in proteoglycan and collagen contents were found on both day 14 and day 28. Aggregates loaded for seven days were larger and histological staining indicated a greater matrix/cell ratio. hydrostatic pressure enhances the cartilaginous matrix formation of mesenchymal progenitor cells differentiated in vitro."

"application of 2 MPa of hydrostatic pressure to mesenchymal cells in a conduction chamber, implanted in the tibiae of Sprague-Dawley rats was shown to promote cartilage formation"

The pericellular environment regulates cytoskeletal development and the differentiation of mesenchymal stem cells and determines their response to hydrostatic pressure.

"SCs were seeded into 1 %, 2 % or 4 % agarose hydrogels and exposed to cyclic hydrostatic pressure. In a permissive media, the stiffer hydrogels supported an osteogenic phenotype, with little evidence of chondrogenesis observed regardless of the matrix stiffness. In a chondrogenic media, the stiffer gels suppressed cartilage matrix production and gene expression, with the addition of RGDS (an integrin blocker) found to return matrix synthesis to similar levels as in the softer gels. Vinculin, actin and vimentin organisation all adapted within stiffer hydrogels, with the addition of RGDS again preventing these changes. While the stiffer gels inhibited chondrogenesis, they enhanced mechanotransduction of HP. RGDS suppressed the mechanotransduction of HP, suggesting a role for integrin binding as a regulator of both matrix stiffness and HP. Intermediate filaments also appear to play a role in the mechanotransduction of HP, as only vimentin organisation adapted in response to this mechanical stimulus."

"vimentin is critical for chondrogenesis. Adaption of the intermediate filament network as the pericellular matrix becomes denser may play a role in the suppression of a chondrogenic phenotype in MSCs."

3 comments:

  1. I have increased my frequency, duration and now use a vice as well as a dumbbell on my ankles. It seems I have hit a plateau and have not seen any growth past 1/4" or so. I can feel the compression/fluid flow and am wondering if there is something missing in the equation based on this post, possibly because of my age (35)?

    "Hydrostatic pressure only needs Type I Collagen to induce chondrogenic differentiation of stem which is far more plentiful in the bone."

    "So any form of compression like LSJL will lead to stem cells differentiating into chondrocytes."

    "It's the hypertrophic phase of chondrogenesis that mainly is involved in the height increase."

    "Chondrocyte Hypertrophy requires dexamethasone withdrawl, TGF-Beta Withdrawl, and addition of T3(a thyroid hormone)."

    So, lsjl produces dexamethasone withdrawl and TGF-Beta Withdrawl, correct? How about T3(a thyroid hormone)? And, if your body is lacking one of these chondrocyte hypertrophy will not occur resulting in no growth? I am trying to figure out if I can supplement w/ something to stimulate the hypertrophic phase because I believe I am performing lsjl properly.

    Any input?

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  2. From the research it seems that Dexamethasone and TGF-Beta are inhibitory towards chondrocyte hypertrophy. Since LSJL doesn't require TGF-Beta or increase TGF-Beta levels it doesn't inhibit chondrocyte hypertrophy like LIPUS + TGF-Beta might.

    T3 hormone might be something.

    Try LSJL on the fingers like I'm doing on the fingers as described on the forums. Do before and after pics. Once we have some finger proof we'll have more resources and we can leverage that to iron out LSJL issues.

    There are tons of things that could help with the height growth but they could possibly do nothing too(for example sam-E could help is you're not metabolizing Vitamin B12 or Folic Acid properly or Astragalus could help if it involves telomere shortening).

    Get that finger proof and then we can iron out more ways to bust plateaus.

    ReplyDelete
  3. Hi Tyler,

    did you ask Hiroki Yokota abut LSJL in adults? Or have you presented your successful results to get his feedback? I guess it could be interesting to get support from such a scientist and he could also interest in experimenting with adults rather than non-fused rats.

    ReplyDelete