Thursday, March 31, 2011

Melatonin's effect on height

Melatonin is known as a sleep compound which has led some to believe that melatonin can be used to increase height by virtue of increasing growth hormone levels.  Growth Hormone has had relatively lackluster benefits on increasing height due to the fact that chondrocytes have a finite proliferative capacity.  Now, IGF-1 may increase stem cell proliferation thereby increasing height that way and increase excretion of extracellular matrix. Growth hormone may help increase height in the top of the head and the heel bone, two bones which grow differently than long bones and two bones that influence height.  However, to grow taller the best way is to get stem cells to differentiate into chondrocytes forming new growth plates.  Melatonin is produced within the bone marrow which contains mesenchymal stem cells.  Can Melatonin help increase height? 

Osteogenic differentiation of rat mesenchymal stem cells from adipose tissue in comparison with bone marrow mesenchymal stem cells: melatonin as a differentiation factor. 

"We assayed and compared the melatonin effect on osteogenic differentiation of BMSC with that of ADSC. Mesenchymal stem cells (MSC) were isolated from the bone marrow and fat of adult rats. Both cell types were cultured in osteogenic medium in the absence and presence of melatonin at physiological concentrations (20-200 pg/ml). After 4 weeks, the expression of osteocalcin gene was analyzed by reverse transcription-PCR, alkaline phosphatase (ALP) activity was assayed and alizarin red S and von Kossa staining were done. Cell viability and apoptosis were also assayed by 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide, a tetrazole (MTT) and flow cytometry, respectively. The osteoblastic differentiation of ADSC as demonstrated by ALP activity was less than that of BMSC. The amount of matrix mineralization showed statistical differences between the two MSC. The incidence of apoptotic cells was higher among ADSC than BMSC. Cell growth reduction is due to a decrease in the number of the cells entering the S phase of the cell cycle. Viable cells were fewer among ADSC than BMSC in control groups.  BMSC have greater osteogenic potential than ADSC and melatonin promotes osteogenic differentiation to BMSC, but has a negative effect on ADSC osteogenic differentiation." 

If Melatonin discourages adipogenic differentiation then chondrogenic differentiation becomes more likely. 

A new guest playing with bone and fat 

"Hypoxia reduces MSC adipogenic differentiation through the hypoxia-inducible factor-1alpha transcription factor. Superoxide dismutase (SOD)-deficient mice show spontaneous adipogenesis. The redox balance in bone marrow may induce differentiation of MSC cells toward osteogenesis or adipogenesis, suggesting a role for oxygen free radicals in these regulatory pathways. Nitric oxide derivatives of linoleic acid are also potent adipogenic agonists in the physiological range, and they can inhibit osteogenesis through PPAR-Y receptors. The free radical scavenging properties of melatonin against oxygen and nitrogen reactive species, and its ability to induce expression of antioxidative enzymes, including SOD, may underline its ability to guide the MSC differentiation toward osteogenesis{Melatonin may alter the oxygen and nitrogen reactive species balance, however some of those are important for height growth but you don't want left over reactive species hanging around and causing potential damage}. The relationship among age, oxidative stress, osteopenia, and adipocyte accumulation in the marrow cavity seems clear. Senescent-accelerated mice (SAM) mice, a murine strain of accelerated senescence and oxidative stress, show osteopenia and high levels of PPAR-Y mRNA. Melatonin prevents the age-dependent oxidative stress and inflammation in SAM mice, suggesting that the significant decay in melatonin production with age may favor the adipogenetic pathway of MSC differentiation."

So essentially melatonin combats oxidants which have the potential to harm DNA and encourages mesenchymal stem cells to differentiate away from adipocytes therefore being more likely to differentiate into chondrocytes. Melatonin is naturally produced by the body, however, melatonin levels decrease with age and perhaps as shown by how much infants sleep maybe very radically. 

"Melatonin [interacts] with cytosolic calcium-calmodulin complex, modifying many of the calcium-calmodulin-dependent enzymes, including the nitric oxide synthase and nitric oxide production{Calcium and Nitric Oxide pathways are important for height growth}. Melatonin [scavenges] free radicals, yielding a series of metabolites with significant physiological activities{Melatonin may take free radicals and turn them into useful metabolites with possible height increasing effects}"

Even though this paper was about Melatonin encouraging osteogenic differentiation, it illustrates the interaction of Melatonin with Calcium and Nitric Oxide pathways both of which are involved in height growth.

Melatonin has an effect on chondrocytes... 

The effect of exogenous melatonin administration on trabecular width, ligament thickness and TGF-beta(1) expression in degenerated intervertebral disk tissue in the rat. 

"Degenerative changes in IVD [intervertebral disc disc] tissue affect the adjacent vertebral structure, resulting in a decreased vertebral trabecular width. It has been suggested that transforming growth factor-beta 1 (TGF-beta(1)) may have a role in the repair of connective tissue, as it occurs in the IVD degeneration process. In this study, we investigated the effects of exogenous melatonin (MEL) administration on vertebral trabecular width, ligament thickness and TGF-beta(1) expression in degenerated IVD tissue. Fifteen adult male Swiss Albino rats were divided randomly into three groups; nonoperated control, operated degeneration, and MEL treatment groups. In the operated degeneration and MEL treatment groups, cuts were made parallel to the end plates in the posterior annulus fibrosus at the fifth and tenth vertebral segments of the tail to induce IVD degeneration. In each group, TGF-beta(1) immunoreactivity and morphometry of vertebral trabecular width and anterior and posterior ligament thickness were evaluated. Histologically, disorganisation and irregularity of collagen fibres was seen in the degenerated (operated) IVD. Increased TGF-beta(1) expression in multinuclear chondrocytes was also observed as was decreased vertebral trabecular width. Importantly, the reduction of trabecular width observed in the operated degenerated group was reversed after MEL administration (p<0.0001). Similarly, TGF-beta(1) expression in multinuclear chondrocytes was dramatically increased after exogenous MEL application. Thus, there was a regression in histopathological changes after MEL treatment, with disk appearances similar to those of the control group. Based on our findings, we suggest that MEL activates the recovery process in the degenerated IVD tissue, possibly by stimulating TGF-beta(1) activity. This is the first report investigating the involvement of the pineal hormone MEL in the repair of rat IVD." 

Melatonin increases TGF-Beta 1 levels which affect the chondrocytes.  TGF Beta is an essential part of chondrogenesis. 

"MEL levels are characteristically high at night and low during the day. Interestingly, MEL enhances synthesis of TGF-β1, as evidenced by preliminary work performed on benign prostate cells"<-More synthesis of TGF-Beta1 is great for LSJL as TGF-Beta1 is a great way to induce chondrogenic differentiation.

"Melatonin has a putative[valid] role in collagen synthesis in human bone cells in vitro. Furthermore, it has been suggested that it plays an important part in the stimulation of synthesis of ECM and related molecules, including TGF-β1, tenascin and fibronectin and also in regulation of ECM construction."<-Melatonin does lots of good stuff that encourages Chondrogenesis.  High levels of Melatonin would seem to increase efficacy of LSJL.  How to raise Melatonin levels though while bypassing the negative feedback loop?

Melatonin enhances cartilage matrix synthesis by porcine articular chondrocytes. 

"The effect of melatonin on cartilage matrix synthesis of articular chondrocytes was evaluated in vitro in a pellet culture system. Melatonin treatment yielded chondrocyte-pellets with a higher expression of chondrogenic markers consisting of collagen II, Sox 9, and aggrecan[so melatonin increases freshly differentiated MSC chondrocyte cell quality] at both the mRNA and protein levels. A hypertrophic marker, collagen X, remained low. up-regulation of internal transforming growth factor beta1 (TGF-beta1) expression was observed in the melatonin-treated cells. "

"Introducing high amounts of TGF-β into a knee joint has adverse effects in the form of marked synovial hyperplasia and chondro-osteophyte formation[chondro-osteophyte is a bone cell within the cartilage, this is good for cartilage within the bone which is what we're trying to induce with LSJL]"<-This is interesting. Hyperplasia equals bigger which is good for the bone and for height.

Sox9 has an impact on chondrogenesis which affects height. So upregulation of Sox9 by melatonin is very good.

Here's some information on the development of Melatonin resistance:

Loss of response to melatonin treatment is associated with slow melatonin metabolism.

"The initial good response to melatonin disappeared within a few weeks after starting treatment, while the good response returned only after considerable dose reduction. We hypothesise that this loss of response is associated with slow melatonin metabolism.
In this study, we determined melatonin clearance in two female (aged 61 and 6 years) and one male (aged 3 years) patients who had chronic insomnia, late melatonin onset and mild ID, and whose sleep quality worsened a few weeks after initial good response to melatonin treatment, suggesting melatonin tolerance. After a 3-week washout period, patients received melatonin 1.0, 0.5 or 0.1 mg, respectively. Salivary melatonin level was measured just before melatonin administration, and 2 and 4 h thereafter. After this melatonin clearance test, melatonin treatment was resumed with a considerably lower dose.
In all patients melatonin concentrations remained >50 pg/mL at 2 and 4 h after melatonin administration. After resuming melatonin treatment sleep problems disappeared. The same procedure was followed in three patients who did not show loss of response to melatonin after 6 months of treatment. In all patients in the control group melatonin concentrations decreased between 2 and 4 h after melatonin administration with a mean of 83%.
Loss of response to melatonin treatment can be caused by slow metabolisation of exogenous melatonin. As melatonin is metabolised in the liver almost exclusively by cytochrome P450 enzyme CYP1A2, this slow melatonin metabolism is probably due to decreased activity/inducibility of CYP1A2[Altering the CYP1A2 enzyme may be a way to increase the response to exogenous melatonin, cycling off Melatonin is the easiest way to increase the CYP1A2 enyzme excitability]. In patients with loss of response to melatonin, a melatonin clearance test should be considered and a considerably dose reduction is advised."

"Melatonin levels in three of the four patients, who received melatonin, were extremely high (>50 pg/mL) after 4 weeks of treatment, while they were very low at baseline."<-Exogenous Melatonin is effective at increasing serum Melatonin levels.

"For CYP1A2, metabolism of most substrates can be described using the Michaelis-Menten equation, demonstrating saturation kinetics. For some substrates in higher concentration the model seems inadequate, suggestive of a two binding sites model, either inhibitory or cooperative. Saturation will fortify the effects of exogenous melatonin in poor metabolisers. Poor metabolisers will initially experience disproportional long lasting higher serum levels after normal doses resulting in effective therapy. During the first stage of saturation they will experience disproportional increase in serum levels after a normal dose, thus still resulting in effective therapy. Finally such a high level will be reached that a next dose will not result in an effective increase of serum level. This could be the explanation for delayed onset of these effects and the huge impact of dosage reduction. The delay in the loss of response to melatonin treatment and the effectiveness of dose reduction with restoration of rhythmicity can be attributed to the saturation phenomenon, with eventually much extended elimination half-life values for melatonin. The proportion of individuals with the slow phenotype narrowly ranges from 12% to 14%[Not an insignificant percentage]"

Another study that shows that exogenous melatonin influences serum melatonin levels:

Endogenous melatonin levels and the fate of exogenous melatonin: age effects.

"This study examines the range of serum melatonin concentrations that occur among young and older adults, and tests the effects of orally administered melatonin on the serum and saliva concentrations of the hormone. Healthy volunteers (20-36 per study), aged 20-73 years, were divided into two groups on the basis of age (29.2 +/- 6.5 and 60 +/- 8.8 years). For study 1: Serum melatonin levels were measured at 15 to 60 min intervals over a 25 h period using a radioimmunoassay. For study 2: serum and saliva melatonin levels were measured before and at intervals after the administration of a 0.3 mg dose of melatonin at 11.00 h[Only 0.3 mg of melatonin was used which is much less than the typical 3 mg used in Melatonin supplements]. The younger subjects had significantly higher peak endogenous melatonin concentrations (+/- SD) and greater inter-individual variability (100.9 +/- 48.6 pg/ml) than the older subjects (34.5 +/- 15.4 pg/ml). Mean melatonin levels following treatment with the hormone tended to be higher and were significantly more variable among the group of older volunteers (254.5 +/- 145.7) than among the younger group (170.2 +/- 22.0 pg/ml)[Older volunteers have higher peak melatonin serum levels, maybe it has to do with the temporarily elevated levels induced by poor Cyp1A2 metabolism]. We conclude that although the peak endogenous serum melatonin levels are lower in elderly adults, the increment in serum melatonin levels induced by a low oral dose of the hormone is greater and more variable among people over 48 years old."

In the young volunteers exogenous Melatonin increased serum levels of Melatonin by 70%. That is quite dramatic.

Melatonin appears to be a good stimulant with possible height increase implications due to its possible enhancement of LSJL induced chondrogenesis and is available for sale:Source Naturals Melatonin 2.5mg, Peppermint, 240 Tablets. 2.5mg of Melatonin may still be too high so here's 1mg: Melatonin 1 mg 120 Caps.  To avoid negative feedback it may be best to take exogenous melatonin intermittently. Or, take a lower mg dosage of Melatonin for a period.

The scavenging properties of Melatonin may be anti-chondrogenic.

Cytoprotective and anti-inflammatory effects of melatonin in hydrogen peroxide-stimulated CHON-001 human chondrocyte cell line and rabbit model of osteoarthritis via the SIRT1 pathway.

"This study examined the effects and underlying mechanism of melatonin in hydrogen peroxide (H(2) O(2) )-stimulated human chondrocytes and rabbit osteoarthritis (OA) model. Melatonin markedly inhibited hydrogen peroxide (H(2) O(2) )-stimulated cytotoxicity, iNOS, and COX-2 protein and mRNA expression, as well as the downstream products, NO and PGE(2)[Both NO and PGE2 may be good for height growth]. Incubation of cells with melatonin decreased H(2) O(2) -induced Sirtuin 1 (SIRT1) mRNA and protein expression. SIRT1 inhibition by sirtinol or Sirt1 siRNA reversed the effects of melatonin on H(2) O(2) -mediated induction of pro-inflammatory cytokines (NO, PGE(2) , TNF-α, IL-1β, and IL-8) and the expression of iNOS, COX-2, and cartilage destruction molecules. Melatonin blocked H(2) O(2) -induced phosphorylation of PI3K/Akt, p38, ERK, JNK, and MAPK, as well as activation of NF-κB[all of these are potentially height increasing proteins and I believe Kinases are activated by being phosphorylated], which was reversed by sirtinol and SIRT1 siRNA."

Geographical distribution of adolescent body height with respect to effective day length in Japan: an ecological analysis.

"The height of Japanese youth raised in the northern region tends to be greater than that of youth raised in the southern region; therefore, a geographical gradient in youth body height exists. This gradient has existed for about 100 years. Consideration of the nutritional improvement, economic growth, and intense migration that has occurred in this period indicates that it is probably the result of environmental rather than nutritional or genetic factors. To identify possible environmental factors, ecological analysis of prefecture-level data on the body size of 8- to 17-year-old youth averaged over a 13-year period (1996 to 2008) and Japanese mesh climatic data on the climatic variables of temperature, solar radiation, and effective day length (duration of photoperiod exceeding the threshold of light intensity) was performed. The geographical distribution of the standardized height of Japanese adolescents was found to be inversely correlated to a great extent with the distribution of effective day length at a light intensity greater than 4000 lx. The results of multiple regression analysis of effective day length, temperature, and weight (as an index of food intake) indicated that a combination of effective day length and weight was statistically significant as predictors of height in early adolescence; however, only effective day length was statistically significant as a predictor of height in late adolescence. Day length may affect height by affecting the secretion of melatonin, a hormone that inhibits sexual and skeletal maturation, which in turn induces increases in height. By affecting melatonin production, regional differences in the duration of the photoperiod may lead to regional differences in height. Exposure to light intensity greater than 4000 lx appears to be the threshold at which light intensity begins to affect the melatonin secretion of humans who spend much of their time indoors."

"[There's a] negative geographical correlation between height and average temperature"

"humans raised in colder climates tend to have larger body sizes than those raised in warmer climates"

Annual mean temperature and annual sun radiation together tended to decrease height.

" Effective day length at 4000 lx was found to be significantly and inversely correlated to a greater extent with height in both sexes, and particularly for 13-year-old males and 12-year-old females"

"Height is negatively correlated to a greater extent with annual mean solar radiation and effective day length than with temperature"

"to delay menses and sexual maturation relatively strong light intensity is required, the distribution of which is strongest between 25–30 degrees of latitude; the light intensity is required in the high latitudes because of lower sun elevation and high cloud cover, and less light intensity is required near the equator because of high cloud cover. These distributions can be explained using the concept of the effective day length, because the distribution of effective day length at a light intensity of more than 1000 lx is almost proportional to the distribution of the amount of solar radiation"

" a light intensity of 50 to 600 lx can induce considerable phase shifts in the human melatonin–circadian rhythm and that phototherapy for treating sleep–wake rhythm disorders is only effective at a light intensity greater than 1000 lx, indicating that a light intensity of 4000 lx is too strong to affect the secretion of melatonin. However, when the modern human lifestyle is taken into consideration–one in which a considerable amount of time is spent indoors–an outdoor light intensity of 4000 lx appears to be an appropriate threshold at which light intensity could begin to affect the melatonin secretion of humans who spend most of their time indoors. In fact, seasonal changes in day length induce seasonal variation in melatonin secretion and physiological changes in the duration of sleep, so that our waking time is earlier in summer and later in winter. This phenomenon is caused by relatively strong daylight that is bright enough to wake individuals who spend most of their time indoors."

"It is postulated that ritalin may adversely affect sleep, appetite, weight and growth of some children with ADHD. Therefore, we aimed to evaluate melatonin supplementation effects on dietary intake, growth and development of children with ADHD treated with ritalin through circadian cycle modification and appetite mechanisms.
After obtaining consent from parents, 50 children aged 7-12 with combined form of AD/HD were randomly divided into two groups based on gender blocks: one received melatonin (3 or 6 mg based on weight) combined with ritalin (1mg/kg) and the other took placebo combined with ritalin (1mg/kg) in a double blind randomized clinical trial. Three-day food record, and standard weight and height of children were evaluated prior to the treatment and 8 weeks after the treatment. Children's appetite and sleep were evaluated in weeks 0, 2, 4 and 8.
Paired sample t-test showed significant changes in sleep latency (23.15±15.25 vs. 17.96±11.66) and total sleep disturbance score (48.84±13.42 vs. 41.30±9.67) before and after melatonin administration, respectively. However, appetite and food intake did not change significantly during the study. Sleep duration and appetite were significantly correlated in melatonin group (Pearson r=0.971). Mean height (138.28±16.24 vs. 141.35±16.78) and weight (36.73±17.82 vs. 38.97±17.93) were significantly increased in melatonin treated children before and after the trial.
Administration of melatonin along with ritalin improves height and weight growth of children. These effects may be attributed to circadian cycle modification, increasing sleep duration and the consequent more growth hormone release during sleep."

So is the increased height due to food consumption or due additional affects of the Melatonin.

"Melatonin stimulates growth hormone release as well as growth hormone responsiveness to growth hormone-releasing hormone (GHRH) secretion."

"melatonin receptor, MT2, was undetectable in some AIS girls. The present study aimed to investigate whether the abnormal MT2 expression in AIS is quantitative or qualitative. Cultured osteoblasts were obtained from 41 AIS girls and nine normal controls. Semi-quantification of protein expression by Western blot and mRNA expression by TaqMan real-time PCR for both MT1 and MT2 were performed. Anthropometric parameters were also compared and correlated with the protein expression and mRNA expression of the receptors. The results showed significantly lower protein and mRNA expression of MT2 in AIS girls compared with that in normal controls (p = 0.02 and p = 0.019, respectively). No differences were found in the expression of MT1. When dichotomizing the AIS girls according to their MT2 expression, the group with low expression was found to have a significantly longer arm span. The results of this study showed for the first time a quantitative change of MT2 in AIS that was also correlated with abnormal arm span as part of abnormal systemic skeletal growth."

"The activation of MT1 or MT2 by melatonin can inhibit adenylate cyclase activity, resulting in the inhibition of forskolin-induced cyclic AMP formation and leads to a decrease in activated protein kinase A and subsequent downstream signaling."

Melatonin enhances chondrogenic differentiation of human mesenchymal stem cells

"the effects of melatonin on human mesenchymal stem cells (MSCs) undergoing chondrogenic differentiation were investigated. Cells were induced along chondrogenic differentiation via high density micromass culture in chondrogenic medium containing vehicle or 50 nM melatonin. Histological study and quantitative analysis of glycosaminoglycan (GAG) showed induced-cartilage tissues to be larger and richer in GAG, collagen type II, and collagen type X in the melatonin group than in the untreated controls. Real time RT-PCR analysis demonstrated that melatonin treatment significantly up-regulated the expression of the genes involved in chondrogenic differentiation, including aggrecan (ACAN), collagen type II (COL2A1), collagen type X (COL10A1), SRY (sex determining region Y)-box 9 (SOX9), runt-related transcription factor 2 (RUNX2), and the potent inducer of chondrogenic differentiation, bone morphogenetic protein 2 (BMP2). And the expression of melatonin membrane receptors (MT) MT1 and MT2 were detected in the chondrogenic-induced-MSCs by immunofluorescence staining. Luzindole, a melatonin receptor antagonist, was found to partially block the ability of melatonin to increase the size and GAG synthesis of the induced-cartilage tissues, as well as to completely reverse the effect of melatonin on the gene expression of ACAN, COL2A1, COL10A1, SOX9 and BMP2 after 7 days of differentiation. These findings demonstrate that melatonin enhances chondrogenic differentiation of human MSCs at least partially through melatonin receptors."

"phosphorylation of smad1/5/8 is positively involved in cartilage development"

So MT1 and MT2 enhance chondrogenesis.

Monday, March 28, 2011

Increase Height by inhibiting Chondrocyte DeDifferentiation

Lateral Synovial Joint Loading involves inducing mesenchymal stem cell differentiation into chondrocytes by a combination of TGF-Beta1 release by osteoblasts and hydrostatic pressure on the stem cells.  Once this differentiation has occurred then these chondrocytes can undergo endochondral ossification.  Chondrocytes seem to function relatively independently until the columnar stage of growth.

However, in cartilage tissue engineering the dedifferentiation of chondrocytes is a problem.   This dedifferentiation can be prevented by housing the chondrocytes in an extracellular matrix.  Chondrocytes themselves can produce this matrix.  And even after growth ceases, some extracellular matrix remains until the bone is fully remodeled and vascularized.  LSJL may induce chondrogenic differentiation but this dedifferentiation that follows must be prevented.

How do we prevent chondrocyte dedifferentiation following LSJL?  So we can successfully grow taller with the LSJL height increase program?

Relation of low-intensity pulsed ultrasound to the cell density of scaffold-free cartilage in a high-density static semi-open culture system.

"We attempted to prevent dedifferentiation and reverse the phenotypic modulations by adjusting the cell density. We investigated whether low-intensity pulsed ultrasound (LIPUS) enhances matrix synthesis of the scaffold-free cartilage construct.
Rat articular chondrocytes multiplied in monolayers were seeded onto the synthetic porous membrane at stepwise cell densities (i.e., 1.0, 2.0, and 4.0 × 10(7) cells/cm(2)) to allow formation of a scaffold-free cartilage construct via cell-cell interaction. The cartilage constructs were then stimulated by LIPUS for 20 min/day. To investigate the effect of LIPUS stimulation on matrix synthesis, expression of mRNA for cartilage matrix molecules was quantified by a real-time reverse transcription-polymerase chain reaction. Synthesis of type II collagen, type I collagen, and proteoglycan was also assessed histologically.
Only the chondrocytes cultured at high cell densities in the 2.0 × 10(7)cells/cm(2) group became concentrated and formed a plate-like construct similar to native articular cartilage by macroscopic and histological assessments[It may be necessary for a certain threshold of chondrocytes to be differentiated into for LSJL to be effective]. Statistical analysis on the matrix gene expression demonstrated that the levels of type II collagen and aggrecan mRNA of the 2.0 × 10(7)cells/cm(2) group were significantly higher than with the other two cell-density groups. Interestingly, the LIPUS application led to a statistically significant enhancement of aggrecan gene expression only in the 2.0 × 10(7) cells/cm(2) group.
The current study presents a semi-open static culture system that facilitates production of the scaffold-free constructs from monolayer-cultured chondrocytes. It suggests that the LIPUS application enhances matrix production in the construct, and its combination with the scaffold-free construct might become a feasible tool for production of implantable constructs of better quality."

Now LIPUS may not be necessary.  Tapping may work as well and also longer durations of LSJL may be needed to result in chondrocytes secreting ECM before they dedifferntiate.

Endoglin differentially regulates TGF-β-induced Smad2/3 and Smad1/5 signalling and its expression correlates with extracellular matrix production and cellular differentiation state in human chondrocytes.

"Transforming growth factor-β (TGF-β) plays a critical role in cartilage homeostasis and deregulation of its signalling is implicated in osteoarthritis (OA). TGF-β isoforms signal through a pair of transmembrane serine/threonine kinases known as the type I and type II TGF-β receptors. Endoglin is a TGF-β co-receptor that binds TGF-β with high affinity in the presence of the type II TGF-β receptor. We have previously shown that endoglin is expressed in human chondrocytes and that it forms a complex with the TGF-β signalling receptors. Our objective was to determine whether endoglin regulates TGF-β/Smad signalling and extracellular matrix (ECM) production in human chondrocytes and whether its expression varies with chondrocyte differentiation state.
Endoglin function was determined by overexpression or antisense morpholino/siRNA knockdown of endoglin in human chondrocytes and measuring TGF-β-induced Smad phosphorylation, transcriptional activity and ECM production. Alterations in endoglin expression levels were determined during subculture-induced dedifferentiation of human chondrocytes and in normal vs OA cartilage samples.
Endoglin enhances TGF-β1-induced Smad1/5 phosphorylation and inhibits TGF-β1-induced Smad2 phosphorylation, Smad3-driven transcriptional activity and ECM production in human chondrocytes[Smad 1/3/5 phosphorylation is bad but Smad 2/3 is good so Endoglin has negative effects]. In addition, the enhancing effect of endoglin siRNA knockdown on TGF-β1-induced Smad3-driven transcription is reversed by ALK1 overexpression. Furthermore, endoglin levels are increased in chondrocytes following subculture-induced dedifferentiation and in OA cartilage as compared to normal cartilage.
 Together, our results suggest that endoglin regulates the balance between TGF-β/ALK1/Smad1/5 and ALK5/Smad2/3 signalling and ECM production in human chondrocytes and that endoglin may represent a marker for chondrocyte phenotype."

Inhibiting Endoglin levels is a way to encourage height growth and discourage chondrocyte dedifferentiation by increasing ECM production.

Inducing Hypoxia may be another way to prevent chondrocyte dedifferentiation.

Lack of oxygen in articular cartilage: consequences for chondrocyte biology.

"Controlling the chondrocytes phenotype remains a major issue for cartilage repair strategies. These cells are crucial for the biomechanical properties and cartilage integrity because they are responsible of the secretion of a specific matrix. But chondrocyte dedifferentiation is frequently observed in cartilage pathology[chondrocyte dedifferentiation occurs normally in the body outside of tissue engineering] as well as in tissue culture, making their study more difficult. Given that normal articular cartilage is hypoxic, chondrocytes have a specific and adapted response to low oxygen environment."

"hypoxia is a strong promoter of matrix deposition by the chondrocytes "<-Hypoxia encourages matrix deposition which helps prevent dedifferentiation.

"Cells can sense surrounding oxygen level through Hypoxia–Inducible transcription Factors (initially named HIF1-) whose expression and function are mainly post-translationally regulated by hydroxylation reactions. Under high oxygen environment, these proteins have a very short half-life (<5 min)[Maybe encouraging matrix deposition by freshly differentiated chondrocytes is possible within this five minutes?]. This is due to specific hydroxylated proline that are recognized by the Von Hippel-Lindau protein (pVHL-containing E3 ubiquitin ligase complex) and that targets them to degradation via the proteasome. Conversely, when oxygen level is low (5–1%), hydroxylation decreases and HIF-1 is prevented from a rapid degradation. Then HIF1- heterodimerizes with the constitutively expressed HIF-1β (also called Aryl hydrocarbon Nuclear Translocator ARNT), translocates into the nucleus, and binds specific consensus sequences (-RCGTG-) on gene promoters "<-Usage of a PHD inhibitor which degrades HIF1 may be another way to enhance HIF1 levels without lowering bone oxygen content by say performing LSJL for a longer duration.

"deletion of pVHL in chondrocytes (which results in HIF-1 and -2 overexpression) increases matrix deposition during growth-plate development"

"As PHDs are the true oxygen sensors of hypoxia, these enzymes may be critical in controlling the chondrocyte phenotype.  All the PHDs are expressed in maturing zone of the mouse growth plate "<-So it's not so much increasing HIF1 but inhibiting PHD expression in the bone.

So increasing cell density, inhibiting of endoglin, and induction of hypoxia are ways to prevent chondrogenic dedifferentiation.  The inhibition of PHD strongly suggests the usage of longer applications of LSJL as PHD levels may be returned to normal after clamp placement is no longer impeding oxygen delivery to the epiphyseal bone marrow.

Thursday, March 24, 2011

What factors can cause lack of results in the LSJL height increase program?

Previously, we discussed possibilities of how to get over various stagnations in the LSJL height increase routine for those who started out gaining height but whose gains slowed down or ceased.  What if you never gained a single cm at all?  It may not be a failure of the routine but rather certain genetic and environmental factors that impair gains.  Some people have reported gains, some have stalled, and others have gotten no gains at all.

LSJL requires some factors to work:

Sufficient Telomere Length for chondrogenic differentiation(can potentially be rectified by Astragalus)
TGF-Beta1 release by the osteoblasts/osteocytes(and possibly the periosteum)
Hydrostatic Pressure on mesenchymal stem cells for chondrogenic differentiation

So there could be issues with the mechanotransduction pathway for TGF-Beta and there could be issues with mesenchymal stem cell proliferation and differentiation.

Too high or too low levels of Barium may affect these pathways.  Remember that Baryta Carb has been used to treat dwarfism in some cases.  Most height increase foods like milk already contain high barium.  So if you are drinking a lot of milk, eating a lot of cereal, hot dog, and nuts.  Your barium levels may be too high and that could be the reason of lack of gains due to closure of calcium voltage gate channels.  No way to know for sure without getting the blood work done.  Potassium supplementation may be able to help with too high barium levels.

Intoxication by large amounts of barium nitrate overcome by early massive K supplementation and oral administration of magnesium sulphate.

"Suicide by ingestion of barium is exceptionally rare. Adverse health effects depend on the solubility of the barium compound. Severe hypokalemia, which generally occurs within 2 hours after ingestion, is the predominating feature of acute barium toxicity, subsequently leading to adverse effects on muscular activity and cardiac automaticity. We report one case of acute poisoning with barium nitrate, a soluble barium compound. A 75-year-old woman was hospitalized after suicidal ingestion of a burrow mole fumigant containing 12.375 g of barium nitrate. About 1 hour post-ingestion, she was only complaining of abdominal pain. The ECG recording demonstrated polymorphic ventricular premature complexes (VPCs). Laboratory data revealed profound hypokalemia (2.1 mmol/L). She made a complete and uneventful recovery after early and massive potassium supplementation combined with oral magnesium sulphate to prevent barium nitrate absorption."

"Toxic effects can occur following ingestion of as little as 200 mg of soluble barium compound."<-So you want to keep barium levels below 200mg probably below 100mg so you have a cushion.  Brazil nuts for instance contain 4mg per gram of nut so you'd have to eat 25g to hit 100mg.

If you think you might be deficient in barium you might want to try a little bit of brazil nut oil: Raw Organic Brazil Nut Oil-250 ml.  Most foods have below 0.002mg/g of Barium so too high of barium is rare unless you eat a very specific diet of food or there's a lot of barium in the drinking water.

If your bone is not increasing in thickness then it means that the mechanotransduction pathways are not working and it could possibly be a problem with barium above or below the optimal range.  If your bone is increasing in thickness then it probably isn't a mechanotransduction problem. It could also be something else like a calcium deficiency   This study states that it's the actin cytoskeleton involved rather than proteins that affect the calcium voltage gate channels.

L-type calcium channel activity in osteoblast cells is regulated by the actin cytoskeleton independent of protein trafficking.

"Voltage-dependent L-type calcium channels (VDCC) play important roles in many cellular processes. The interaction of the actin cytoskeleton with the channel in nonexcitable cells is less well understood. We performed whole-cell patch-clamp surface biotinylation and calcium imaging on different osteoblast cells to determine channel kinetics, amplitude, surface abundance, and intracellular calcium, respectively. Patch-clamp studies showed that actin polymerization by phalloidin increased the peak current density of I (Ca), whereas actin depolymerization by cytochalasin D (CD) significantly decreased the current amplitude[actin polymerization affects the current of Ca]. This result is consistent with calcium imaging, which showed that CD significantly decreased Bay K8644-induced intracellular calcium increase. Surface biotinylation studies showed that CD is not able to affect the surface expression of the pore-forming subunit α(1C). Interestingly, application of CD caused a significantly negative shift in the steady-state inactivation kinetics of I (Ca). There were decreases in the voltage at half-maximal inactivation that changed in a dose-dependent manner. CD also reduced the effect of activated vitamin D(3) (1α,25-D3) on VDCC and intracellular calcium. We conclude that in osteoblasts the actin cytoskeleton affects α(1C) by altering the channel kinetic properties, instead of changing the surface expression, and it is able to regulate 1α,25-D3 signaling through VDCC. Our study provides a new insight into calcium regulation in osteoblasts, which are essential in many physiological functions of this cell."

So, the amount of actin polymerization is what affects Calcium release more than any compound.

"Physiological hormones, such as parathyroid hormone (PTH) and activated vitamin D3, also modulate the calcium homeostasis of bone through voltage-dependent calcium channels[too high levels of calcium can lower PTH]. 1α,25 vitamin D increases the inward barium current through L-type calcium channels at low depolarizing potentials within seconds in a fashion similar to Bay K8644, an L-type calcium channel agonist"<-Barium, PTH, and Vitamin D3 affect the ability of L-type calcium channels

"Calcium channels can regulate mechanical load-induced bone formation. Application of cyclic strain to the substratum resulted in increased incorporation of calcium in Ros 17/2.8 cell cultures, and the response could be diminished in the presence of verapamil, a blocker of voltage-dependent calcium channels"<-If the voltage-dependent calcium channels don't work neither will LSJL or other mechanical stimulation.

Actin polymerisation is good for inducing TGF-Beta the problem is that we want mesenchymal stem cells to not have actin polymerisation as that results in cell movement and not stopping to condense and form new growth plates.

Now the next factor that may be preventing you from getting gains with LSJL is problems with cellular proliferation and differentiation.  Namely with MyostatinMuscle is the primary regulator of myostatin so if you have large muscles you probably don't have a problem with myostatin.  Conversely, if you have small muscles you likely do.  Weight lifting and creatine can inhibit myostatin.

If you have small muscles and didn't get results with LSJL try lifting weights and taking creatine.

As for chonrogenic differentiation, anything that inhibits adipogenic differentiation encourages chondrogenic differentiation.  If you're building up your muscles fine it probably isn't an issue with lack of differentiation but hypoxia may play a role.  Maybe your bone is overvascularized discouraging chondrogenic differentiation.  In that case increase the duration of clamping.

Checklist if you did not gain three months after LSJL:

1) Did my bone grow in thickness?  If not check barium levels and other factors related to the L-type Calcium Voltage Channels

If your bone did grow in thickness then:

2) Start working out and seeing if your muscles hypertrophy.  If not then take creatine and increase testosterone levels which inhibits myostatin.  See Lateral Synovial Joint Loading Supplement Guide for list.

3) Perform LSJL on the fingers to check for local factors or possible intensity issues.  If you can gain length in the fingers with the bar clamp then it is likely a local bone problem or possibly LSJL is not performed with optimal intensity.

4) For Vascularity/Hypoxia there is venous ligation which also increases hydrostatic pressure/fluid flow as well...  But realistically, you can increase duration of clamping.

Tuesday, March 22, 2011

Can you overtrain the bone in a height increase program?

We know that there are several components to the mechanosensativity of the bone and the cells within the bone.  It's important to note that the mechanical sensitivity of the osteogenic bone cells is important as they upregulate TGF-Beta1 which is vital to induce chondrogenic differentiation.  The multiple factors that can affect the mechanosensativity of the relevant cells which include mesenchymal stem cells, chondrocytes, and osteoblasts are:  actin dynamics(actin depolymerisation), neuronal regulation(CGRP and ACTH concentrations), and ion channels(Ca+ which is influenced by CGRP and ACTH, the ion channels may deregulate to lower mechanosensativity).

There are three common modalities of overtraining in muscular performance:

1) Neurological Fatigue.  Caused by lifting very heavy weights.  Your muscles may be fine but your neurons can not properly fire to contract the muscle.

2) Structural Fatigue:  Muscular damage.  There's too much muscular fiber damage for the body to lift the weight.  Your CNS fires for the muscle to lift but nothing happens.

3) Metabolic Fatigue:  Your muscles don't have enough energy.  Glycogen, ATP, Oxygen, etc. levels are too low.  Structurally the muscle can contract and neurologically the synapses can send the signals but there's not enough energy for the muscle to move.

All three of these modalities have to be properly functioning for the muscle to move.  Metabolic Fatigue is the easiest to recover from with recovery being in possible seconds, followed by structural fatigue, and finally neurological fatigue which is the hardest to recover from.

Now in LSJL you are not contracting the bone.  The clamp is providing the stimulus on the bone but there can still be neurological failure.  The neurons involved in inducing chondrogenic differentiation can not fire.  ACTH and CGRP can be too low and need to recover.  Increasing ACTH and CGRP concentrations may help but the only solution to neurological overtraining is rest.

Structural fatigue is not likely to happen in bone for LSJL.  There could be microfractures that may prevent mechanical signaling but since LSJL doesn't involve contracting Type I collagen fibers structural fatigue should be a minor or non-existent issue.

Now metabolic fatigue could play a larger role especially with calcium.  It may be a problem with the voltage gate channels rather than being a problem with low calcium concentration which is easier to fix.  If it is a problem with the voltage gate channels then the most likely solution is rest to give the body time to repair and build new channels.  Barium has the ability to help with the voltage calcium gate channels.  A high barium concentration results in calcium voltage gate closure.  And a refractory period is needed between each pulse.  So unless, you can get blood work done and measure proper barium concentration taking barium as a supplement is not advised at this time.

So aside from possible supplementation options with ACTH, CGRP(which is not available), and Barium the best solution to bone overtraining is rest.

With muscular routines, it's easy to detect overtraining systems as it usually at a global level.  However, we are only training some limb limbs so the overtraining may be harder to detect especially since it's only within the CGRP and ACTH systems.

But, if you are feeling nerve pain then you are likely clamping too hard.  You should be progressively increasing the intensity and duration of clamping to give the body time to adapt and to encourage more adaptations at each stage of increase rather than having large increases in intensity right away.  An increase in load is needed to sufficiently disrupt the actin cytoskeleton to allow for chondrogenesisCH Turner stated that bone needed 24 hours to regain 98% of it's mechanosensativity.  It's unknown whether there's some sort of longitudinal reduction in mechanosensativity to repeated bouts of the same load over time or whether there's a gradual decrease in mechanosensativity over time(for example: the first 24 hours may result in a 98% gain in mechanosensativity whereas the next 24 hours may only result in a 96% gain).  The LSJL scientists performed LSJL on the rats 5 of 7 days a week with the 2 days of rest being non-consecutive.

Here's a paper that provides a model of bone mechanosensativity(osteocyctes and osteoblasts only but that's necessary for TGF-Beta1 and there's probably similarities between osteoblast mechanosensativity and mesenchymal stem cells):

Modeling of biological doses and mechanical effects on bone transduction.

"Shear stress, hormones like parathyroid and mineral elements like calcium mediate the amplitude of stimulus signal[These signals also affect height growth in addition to bone remodeling], which affects the rate of bone remodeling. The current study investigates the theoretical effects of different metabolic doses in stimulus signal level on bone. The model was built considering the osteocyte as the sensing center[no osteocytes in the epiphysis as there is only trabecular bone, but osteocyte signaling in cortical bone close to the epiphysis may be important for the TGF-Beta1] mediated by coupled mechanical shear stress and some biological factors. [Our model] describes the effects of physiological doses variations of calcium, parathyroid hormone, nitric oxide and prostaglandin E2 on the stimulus level sensed by osteocytes in response to applied shear stress generated by interstitial fluid flow. We retained the metabolic factors (parathyroid hormone, nitric oxide and prostaglandin E2) as parameters of bone cell mechanosensitivity because stimulation/inhibition of induced pathways stimulates osteogenic response in vivo."

The scientists state that the strength of the mechanical stimulus is strongly dependent on PGE2 and Nitric Oxide.  A higher fluid viscosity(more shear stress and likely hydrostatic pressure) also increases the strength of results. Shear stress stimulates the NO pathway so there is a connection there.  Damage to the bone canliculus actually made lower levels of shear strain more effective.  So structural damage to the bone is not likely to play a role in overtraining and structural damage to the bone may be beneficial in inducing adaptations. 

Neurological fatigue in bone is not well quantified.  To help prevent neurological fatigue:  Rest from LSJL 2 out of 7 non-consecutive week days and try to avoid using pressure that causes nerve pain.  If bone length stops increasing, take 2 weeks off from LSJL.

Friday, March 11, 2011

I have now grown to a full 5'10"

Previously, my height was hovering at about 5'9 3/4" to a hair under 5'10" now I finally hit a height personal record of 5'10".  Recently, I revealed that I've gone up in clamping to about 1 minute and that seems to have resulted in some success.  I am using a 50lbs dumbell instead of a 75lbs dumbell for 30 seconds, I think that's helped me focus more on getting more compressive force with the dumbell. Here's my limb pics all the way from November.  My tibial measurement was 16 1/4" now it's about 16 1/2".  So my part of the 3 month challenge has been successful so far:

Now I know the pictures are not optimal, that's why I'm working on the finger length videos but I've been hearing that it's not enough of an increase to excite people.

Some people have reported not getting results with LSJL.  I encourage you to post pics of you not getting results for one.  And then do a clamp test on your finger to see if you can gain length.  You can gain a little bit of length there just with pinching but you need to clamp(Bar-clamp is superior to C-class for fingers) for more gains.  If you can gain finger length then you can know if your technique is correct.

I have been wanting to grow taller for over five years so I don't clamp with 100% mental efficiency all the time.  So you don't need perfect technique to grow.  If you don't grow after three months it's okay if you stop.  I realize that people will need an overwhelming amount of proof to believe in a grow taller method.

Sky's Limbcenter Clinic should have been covered by media outlets.  Kojima's clinic should have received western media attention. Sky's Clinic has disappeared despite having promising elements.  Bone stretching is provable on animals by a layman.  Just apply Sky's levers to a Sprague-Dawley rat and measure the increase in bone length.  It would be expensive to get all the tools but it would be doable by a layman.  And you wouldn't get to see the direct effect on the Type I Collagen fibers without specialized equipment.

The LSJL studies should have also gotten more coverage.

Basically, us height seekers have our work cut out for us.  And I'm probably going to have to gain quite a bit of finger length in you tube videos to inspire people's imaginations.  It's probably not going to be enough for a bunch of people to gain a little bit of height.  So if you don't gain height in three months then feel free to take a break for a while until the method is more refined.  But please post before and after pics and do some clamping on your finger as well.

Thursday, March 3, 2011

Factors that affect LSJL Gene Expression

Previously, we found that the benefits of LSJL did not relate to the dynamic loading of chondrocytes.  Under dynamic loading of chondrocytes, the anabolic benefits seemed to be due to the upregulation of aggrecan, Type II collagen, and osteopontin whereas the anabolic benefits of Lateral Joint Loading seem to be due to an upregulation of genes involved in the PI3K pathway and TGF-Beta.  It would make sense for the anabolic response(the height increase) of LSJL be due to loading of the periosteal component(or fibrous capsule) of the bone as that is the part of the bone that is loaded differently in lateral versus vertical loading.

Two potential factors that modulate LSJL gene expression are loading site(more on the cartilage versus the epiphysis) and circulating levels of estrogen(how much estrogen is optimal).

Mechanical loading stimulates rapid changes in periosteal gene expression. 

"External force application increases periosteal bone formation by increasing surface activation and formation rate. In this study, the early tibial periosteal response to external loads was compared between loaded and nonloaded contralateral tibia by examining the results of blot hybridization analyses of total RNA. To study the impact of external load on gene expression, RNA blots were sequentially hybridized to cDNAs encoding the protooncogene c-fos, cytoskeletal protein beta-actin, bone matrix proteins alkaline phosphatase (ALP), osteopontin (Op), and osteocalcin (Oc), and growth factors insulin-like growth factor I (IGF-I) and transforming growth factor-beta (TGF-beta). The rapid yet transient increase in levels of c-fos mRNA seen within 2 hours after load application indirectly suggests that the initial periosteal response to mechanical loading is cell proliferation. This is also supported by the concomitant decline in levels of mRNAs encoding bone matrix proteins ALP, Op, and Oc[Less Alkaline Phosphatase discourages fusion], which are typically produced by mature osteoblasts. Another early periosteal response to mechanical load appeared to be the rapid induction of growth factor synthesis as TGF-beta and IGF-I mRNA levels were increased in the loaded limb with peak levels being observed 4 hours after loading[This increase in TGF-Beta and IGF-1 can have positive benefits on cellular proliferation]. These data indicate that the acute periosteal response to external mechanical loading was a change in the pattern of gene expression which may signal cell proliferation.  Increased periosteal cell proliferation [is] seen both in vivo and in vitro following mechanical loading."

Okay, since dynamic loading of chondrocytes didn't upregulate genes encouraging chondrogenesis like Sox9 but Periosteal loading increased TGF-Beta which encourages chondrogenesis a key part of Lateral Synovial Joint Loading is periosteal shear.  The periosteal shear increases TGF-Beta which encourages a chondrogenic lineage for MSCs.  The benefits seen under LSJL more closely match those of periosteal shear(which is induced by IFF) than that of dynamic compressive loading of chondrocytes.  So it is more likely that it's the fluid flow that causes the periosteal shear strain induces the LSJL benefits(which can induce height gain if the fluid flow increases hydrostatic pressure in the epiphysis) than as a direct result of a compressive loading(which don't seem to match gene expression pathways for LSJL in chondrocyte loading for example).

Therefore, in deciding versus loading the epiphysis versus the cartilage, loading the epiphysis is superior as you still get periosteal shear and you are more likely to cause bone deformation which increases hydrostatic pressure.

Estrogen also regulates the load on bone and this could affect what genes are expressed in lateral synovial joint loading.

Loading-related regulation of gene expression in bone in the contexts of estrogen deficiency, lack of estrogen receptor alpha and disuse.

"Loading-related changes in gene expression in resident cells in the tibia of female mice in the contexts of normality (WT), estrogen deficiency (WT-OVX), absence of estrogen receptor alpha (ERalpha(-/-)) and disuse due to sciatic neurectomy (WT-SN) were established by microarray. Total RNA was extracted from loaded and contra-lateral non-loaded tibiae at selected time points after a single, short period of dynamic loading sufficient to engender an osteogenic response. There were marked changes in the expression of many genes according to context as well as in response to loading within those contexts. In WT mice at 3, 8, 12 and 24 h after loading the expression of 642, 341, 171 and 24 genes, respectively, were differentially regulated compared with contra-lateral bones which were not loaded. Only a few of the genes differentially regulated by loading in the tibiae of WT mice have recognized roles in bone metabolism or have been linked previously to osteogenesis (Opn, Sost, Esr1, Tgfb1, Lrp1, Ostn, Timp, Mmp, Ctgf, Postn and Irs1, BMP and DLX5)[TGF-Beta1, Timp, MMP, IRS-1, and BMPs are compounds that can affect chondrocytes, so the osteogenic response of bone to Estrogen matters even if ER-alpha doesn't have a direct effect of chondrocytes or MSCs]. The canonical pathways showing the greatest loading-related regulation were those involving pyruvate metabolism, mitochondrial dysfunction, calcium-induced apoptosis, glycolysis/gluconeogenesis, aryl hydrocarbon receptor and oxidative phosphorylation. In the tibiae from WT-OVX, ERalpha(-/-) and WT-SN mice, 440, 439 and 987 genes respectively were differentially regulated by context alone compared to WT. The early response to loading in tibiae of WT-OVX mice involved differential regulation compared to their contra-lateral non-loaded pair of fewer genes than in WT, more down-regulation than up-regulation and a later response. This was shared by WT-SN. In tibiae of ERalpha(-/-) mice, the number of genes differentially regulated by loading was markedly reduced at all time points. These data indicate that in resident bone cells, both basal and loading-related gene expression is substantially modified by context. Many of the genes differentially regulated by the earliest loading-related response were primarily involved in energy metabolism and were not specific to bone."

"estrogen receptor (ER), specifically ERα, has been shown to be involved in bone cells' early responses to strain"<-Possibly chondrocytes and stem cells as well

"genetic variation in ERα is associated with different responses to load-bearing exercise"

"In mice when ERα is absent the adaptive response to artificial loading in vivo is attenuated"<-attenuated means reduced.  So lowering Estrogen Receptor-Alpha levels may reduce the effectiveness of joint loading modalities like LSJL.  Note this is Estrogen Receptor-Alpha not Estrogen itself.

"Seven of the 25 genes significantly up-regulated in Zhang and Yokota's LSJL gene expression study were also up-regulated in WT[the normal rats] loaded tibiae in our study. These were mas-related gpr (Mrgpr), tissue inhibitor of metalloproteinase 1 (Timp1), thrombospondin (Thbs), procollagen type III alpha1 (Col3a1), mast cell protease (Mmcp), matrilin 2 (Matn2) and proteoglycan 4 (Prg4)."<-Hyaluronan Synthase and MMP-3 are key differences, thus they are likely key to height growth

"OVX resulted in reduced expression levels of some genes previously associated with bone cells (integrin-alpha 1 (Itga1, − 2.1-fold), Itga4 (− 2.0-fold), heme oxygenase-1 (Hmox1, − 1.8-fold) and transforming growth factor beta 1 (Tgfb1, − 1.9-fold))[Estrogen deficiency resulted in a reduction of TGF-Beta expression] and others (nobox oogenesis homeobox (Nobox, − 7.8-fold) and titin (Ttn, − 2.4-fold)). Expression of the genes for insulin receptor substrate-1 (Irs1), creb-binding protein (Crebbp) and bone morphogenetic protein-4 (Bmp4)[BMP-4 is a capable of inducing chondrogenic differentiation therefore it is good in terms of growing taller] was up-regulated in tibiae from WT-OVX mice (2.6-, 2.1- and 1.6-fold, respectively)."

So in Estrogen deficiency chondrogenic differentiation may be inhibited by the lack of TGF-Beta but on the other hand BMP-4 is present which can also induce chondrogenic differentiation.

For ER-alpha knockout mice:

"striatin (Strn, 10.1-fold), peroxisome proliferative activated receptor gamma coactivator 1 alpha (Ppargc1a, 2.1-fold), myocyte enhancing factor-2a isoform 1 (Mef2a, 1.8-fold), integrin-beta 6 (Itgb6, 2.1-fold), integrin-beta 4 (Itgb4, 1.6-fold), myogenic factor 6 (Myf6, 2.9-fold), connective tissue growth factor (Ctgf, 2.0-fold), Ttn (1.7-fold), osteocrin (Ostn, 1.7-fold), caveolin-1 (Cav1, 1.7-fold), Cav2 (1.7-fold) and Cav3 (1.5-fold)[Caveolin-3 inhibits myostatin so it's upregulation is good for height growth]. Among the genes which were down-regulated were low-density lipoprotein receptor-related protein 5 (Lrp5, − 1.8-fold)[LRP5 is a GSK3 Beta phosphorylater so it's downregulation is not anabolic], acid phosphatase 5, tartrate resistant (Acp5, − 1.7-fold) and Tgfb1 (− 1.8-fold)."

TGF-Beta is downregulated as is LRP5 so it would seem like knocking out ER-Alpha may be bad for height growth.

Sciatic neurectomy refers to removing the sciatic nerve.  The sciatic nerve removal group had the most interesting gene expression patterns.

"Wnt/β-catenin signaling (Lrp1, 1.8-fold), Lrp4 (1.8-fold), Wnt-inducible signaling protein 1 (Wisp1, 1.7-fold), Wisp2 (2.2-fold), secreted frizzled-related protein 1 (Sfrp1, 2.2-fold), Sfrp2 (1.8-fold), Sfrp4 (2.0-fold) and dickkopf-3 (Dkk3, 1.7-fold). Other genes of interest up-regulated in WT-SN mouse tibiae include insulin-like growth factor II receptor[increasing IGF-II receptor is actually a bad thing as it reduces circulating levels of IGF-II] (Igf2r, 2.4-fold), Ctgf (1.7-fold), growth differentiation factor 10 (Gdf10, 1.5-fold), fibroblast growth factor 8 (Fgf8, 1.5-fold), ERα (Esr1, 1.8-fold)[interesting that with no sciatic nerve Estrogen Receptor Alpha is increased], early growth response 1 (Egr1, 2.2-fold)[Egr-1 is also upregulated by LSJL], Myf6 (7.4-fold), periostin (Postn, 2.4-fold), ankyrin repeat domain 1 (Ankrd1, 7.6-fold), Ostn (1.7-fold), osteoglycin (Ogn, 2.5-fold), distal-less homeobox 5 (Dlx5, 1.8-fold), integrin-beta 4 (Itgb4, 1.8-fold), tenascin (Tnn, 2.1-fold) and Bcl-2/E1B 19-kDa interacting protein 3 (Bnip3, 2.1-fold). Five genes coding for matrix metalloproteinases (MMPs) were also up-regulated. A large number of genes involved in adhesion were markedly up-regulated in the tibiae of WT-SN animals including fibronectin 1 (Fn1, 4.0), Postn (2.4-fold), Itgb4 (2.0-fold), Tnn (1.8-fold), fibulin 2 (Fbln2, 3.0-fold), laminin-alpha 4 (Lama4, 2.4-fold), lambin-beta 1, subunit 1 (Lamb1-1, 2.4-fold), glycoprotein 38 (Gp38 (4.0-fold) and thrombospondin 2 (Thbs2, 3.1-fold). Down-regulated genes in this group include histone cluster4, h4 (Hist4h4, −3.6-fold), Tgfb1 (− 2.0-fold), interleukin 16 (Il16, − 1.7-fold)) interleukin receptor, type 2 (Il1r2, − 1.6-fold), interleukin 17 receptor E (Il17re, − 1.6-fold) and interleukin 1 receptor accessory protein (Il1rap, − 2.2-fold)."

So removing the sciatic nerve is bad or neutral for height growth.

More genes altered by estrogen deficiency(24 hours after loading):

"The down-regulated genes included a number involved in adhesion (Lim and senescent cell antigen-like domains 1 (Lims1, − 2.1-fold), Cd44 (− 1.9-fold)[CD-44 degrades hyaluronic acid so it is bad for height growth and a good thing that it is downregulated], Spp1 (− 1.7-fold)), Wnt signaling pathways (frizzled 5 (Fzd5, − 1.6-fold), casein kinase II beta subunit (Csnk1g, − 1.8-fold) and Csnk2b (− 1.5-fold)) and matrix mineralization extracellular matrix protein 2 (Ecm2, − 1.6-fold) and Mmp13 (− 1.5-fold)[MMP-13 can trigger terminal differentiation of chondrocytes so it's downregulation is very good for height growth]"

So loading under Estrogen deficiency is good for height growth.  Estrogen deficiency increase Estrogen Receptor Alpha levels as well so there may be no net change in Estrogen response.

The Estrogen deficient[<-But not totally eliminated] mice had the best gene expression changes for growth but not that the Estrogen Receptor Alpha mice did not express as many "grow tall" genes this could be that Estrogen Receptor-Alpha lowers circulating levels of Estrogen and thus with no ER-alpha more estrogen runs free or Estrogen Receptor-Alpha helps height growth and estrogen deficiency increases ER-alpha levels therefore increasing height growth genes.  You'd have to knockout all the Estrogen receptors to be sure what the cause of the effects are.

"In the experiments reported here, a single 30-s period of dynamic loading engendering strains of physiological magnitude was sufficient to initiate a cascade of events culminating days later in measurable increase in bone formation."<-30 seconds is the loading performed by LSJL, once per day.