Thursday, September 30, 2010

Fracture alters bone electrical potential

If fracture does induce bone electrical potential, maybe another mechanism of inducing bone electrical potential such as LSJL could increase height?

Intraoperative measurement of bone electrical potential: a piece in the puzzle of understanding fracture healing.

"Bone electrical potentials change with the force applied. Also, fracture alters the bone electrical potential, so it becomes more electronegative.
52 patients with a pertrochanteric fracture were included in the study. Bone electrical potentials were measured intraoperatively using a thin Kirschner wire introduced through bone cortex at the selected point and pointed to opposite cortex, not penetrating it. Kirschner wires were connected using clamps to multimeter (YF-78 Multimeter) device. Neutral electrode (inductive rubber) was placed behind ipsilateral gluteus.
Near the fracture site potentials of -199 up to -267 mV were recorded. Mean measured potential of bone plate after fixation was -240 mV. Bone potentials correlated with the subtype of fracture and early mobilisation of patients."

"Strain changes the electrical potential of bone: parts exposed to the compression force develop negative potential, and parts subjected to the tension force positive potential. Negative potential is associated with bone deposition, and positive with bone resorption."

"In the typical electro-potential curve pattern the metaphysis is negative with respect to the epiphysis, and the diaphysis is isopolar or electropositive. Metaphysis reaches a peak in electronegativity two to three centimetres below the epiphysis. In a fractured bone, the entire shaft becomes electronegative; the metaphyseal peak becomes more negative; and a secondary peak of electronegativity, that may exceed the metaphyseal peak, appears over the fracture site"

This was an exploratory study without much definitive results.

how and why scoliosis can affect height

Pathomechanism underlying the onset of scoliosis in a PNX broiler chicken model

"The pinealectomy (PNX)[removal of the pineal gland]{the pineal gland produces melatonin and supplementation with melatonin can reduce scoliosis} in a chicken model consistently induces scoliosis with anatomic features that are similar to human adolescent idiopathic scoliosis (AIS). This experimental study attempted to improve the understanding of the mechanisms underlying the onset of scoliosis in a PNX broiler chicken model.
A histomorphometric study was performed to analyze longitudinal bone growth and cancellous bone remodeling before the development of scoliosis. Static and dynamic parameters in cancellous bone and chondro-osseous junction of the 7th thoracic vertebral body at nine days after hatching were compared between PNX chickens and control chickens with no surgery.
The PNX resulted in a rapid and marked loss of cancellous bone volume (8 ± 1% versus 14 ± 2%, mean ± SD) and profoundly disrupted trabecular structure with increases in dynamic formative parameters, such as mineralizing surface, mineralization apposition rate and adjusted appositional rate. In the chondro-osseous junction, activated osteoclasts phagocitized degenerating chondrocytes, leaving a minimal amount of cartilage matrix and activated osteoblasts, thus losing their scaffolding for bone formation directly covering the hypertrophic zone cells. The osteoid surface and thickness in the chondro-osseous junction were significantly increased in PNX chickens (43 ± 14 % versus 12 ± 6 % and 4 ± 0.2µm versus 3 ± 0.4 µm). In the subjacent cartilage regions being protected from further resorption, abundant labeled cartilage remained with higher cellularity.
Fast-growing birds have a unique paradigm of rapid bone elongation with minimal metaphyseal bone production. A bone-forming surface exists at the front of cartilage ossification in the growth plate. Capillae of hypertrophic chondrocytes become included between the trabeculae of metaphyseal bone, and the overall thickness of the growth plate increases considerably in addition to distal expansion. Our results indicate that the unique mechanism for rapid bone elongation in chickens is more pronounced after PNX. PNX also induces high turnover osteoporosis, which may contribute to the development of scoliosis in the chicken."

Tuesday, September 28, 2010

Aromatase deficiency and tall stature

Lately, we've been linking endocronilogical factors to local growth factors.  HGH may affect DNA Methylation by increasing levels of DNA Methyltransferse, IGF-1 may determine peak chondrocyte hypertrophy, Testosterone inhibits Myostatin, and Estrogen...

Estrogen is more complicated.  The usage of aromatase inhibitors to increase height is controversial in effectiveness.  Normalized levels of estrogen seem essential for maximizing growth.  Growth plate senescence is regulated by DNA Methylation and not estrogen.  Fusion is regulated by estrogen but that does not occur until post senescence.

And yet, there are several cases of aromatase deficient individuals with tall stature.  The number of studies of aromatase deficiency are extremely limited so it's possible that there are normal stature individuals that have aromatase deficiency but are undiagnosed.  The tall stature and aromatase deficiency could share the same cause.

There's also the possibility that because extremely low levels inhibit fusion, it gives an opportunity to wander into the hyaline cartilage growth plate line.  Those stem cells then differentiate into chondrocytes resulting in height growth.  This gets around DNA Methylation as these new stem cells come with methyl counters.  This is the mechanism that LSJL works by too, sending new stem cells into the hyaline cartilage growth plate line.  Stem cells were found to be able to differentiate organically into chondrocytes as well just with Type I collagen(which is a part of all bone) and hydrostatic pressure(which is induced by LSJL).

Alternatively, estrogen has numerous effects on growth and some are positive and negative.  Perhaps, the positive aspects of estrogen inhibition counteract the negative ones. 

GPR30 deficiency causes increased bone mass, mineralization, and growth plate proliferative activity in male mice. 

"Estrogen regulation of the male skeleton was first clearly demonstrated in patients with aromatase deficiency or a mutation in the ERalpha gene. Estrogen action on the skeleton is thought mainly to occur through the action of the nuclear receptors ERalpha and ERbeta. The G-protein coupled receptor GPR30 is a functional ER. GPR30 deficient mouse models have been generated to study the in vivo function of this protein. We have characterized size, body composition, and bone mass in adult male GPR30 knockout (GPR30KO) mice and their wildtype (WT) littermates. GPR30KO mice weighed more and had greater nasal anal length. Both lean mass and percent body fat were increased in the knockout mice. Femur length was greater in GPR30KO mice as was whole body, spine, and femoral areal bone mineral density. GPR30 mice showed increased trabecular bone volume and cortical thickness. Mineralized surface was increased in GPR30KO mice. [There was] greater proliferation in the growth plate of GPR30KO mice. Under osteogenic culture conditions GPR30KO femoral bone marrow cells produced fewer alkaline phosphatase positive colonies in early differentiating osteoblast cultures but showed increased mineralized nodule deposition in mature osteoblast cultures. Serum IGF-I levels were not different." 

Note that other research shows that female mice without the GPR30 gene have reduced growth.  "A small age-dependent decrease in crown-rump and femur lengths, measures of skeletal growth, [occurred] in Gpr30 KO female mice but not males. "

"Estradiol treatment of ovariectomized mice reduced longitudinal skeletal growth, as measured by femur length, and decreased growth plate height in WT not Gpr30 KO mice."

"GPR30 somehow functions to limit matrix mineralization."

"Gpr30 is a Runx2-responsive gene and acts in a promitogenic fashion through a Cdk pathway."

"GPR30 expression in the human has been shown to change with age during puberty, and it is postulated to regulate longitudinal bone growth.  Conceivably, at low doses, when E is postulated to stimulate long bone growth, the conventional ERs are dominant. On the other hand, at higher doses of E, the action of GPR30 may come into play, thereby limiting or terminating long bone growth."

For the first time optimal ranges of estrogen are given in this study(but for female only):


Impact of Estrogen Replacement throughout Childhood on Growth, Pituitary-Gonadal Axis and Bone in a 46,XX Patient with CYP19A1 Deficiency.

"We studied the impact of oral 17β-estradiol treatment, on longitudinal growth, bone age maturation, pituitary gonadotropin feedback, multicystic ovaries and bone mass in the long-term follow-up of a girl compound heterozygote for two point mutations of the CYP19A1 gene. Low doses of 17β-estradiol were needed to achieve normal height velocity and adequate bone age maturation from early childhood on"

"The doses of 17β-estradiol needed during [early childhood] range between 50 and 100 µg."

" In our patient withdrawal of E2 resulted in arrest of bone age maturation and decrease of height velocity"

"in late prepuberty and puberty the patient showed a discordant picture between an already decreasing bone age maturation indicating relative estrogen deficiency, and a rising height velocity indicating sufficient serum estradiol levels."

" when off [estrogen] treatment the patient showed increasing bone age delay and decreasing height velocity"

The girl ended up with slightly above normal predicted height.

Having low estrogen is much better than having high estrogen(which causes apoptosis).  Low estrogen levels may delay senescence by lowering cell proliferation rates and enabling more new stem cells with methyl groups to arrive at the hyaline cartilage growth plate line.  Specific actions of estrogen on various growth stimulators and inhibitors needs to be studied further.

PTN

PTN is upregulated by LSJL.

Effects of pleiotrophin, a heparin-binding growth factor, on human primary and immortalized chondrocytes.

"Pleiotrophin (PTN) is a secreted heparin-binding peptide expressed in mesodermal and neuroectodermal cells during development, but rarely in adult tissues. In fetal and juvenile, but not in mature cartilage, PTN is abundant. PTN is re-expressed in chondrocytes in early stages of osteoarthritis (OA). we investigated the occurrence of PTN receptors in human articular cartilage in situ and PTN effects on human primary and immortalized chondrocytes in vitro.
Of the putative PTN signaling receptors, immortalized and primary chondrocytes (pc) expressed the anaplastic lymphoma kinase (ALK), less the receptor-type protein tyrosine phosphatase zeta/beta (PTPzeta). ALK expression was upregulated upon ligand exposure. PTN stimulation activated the AP-1 (activator protein-1) transcription factor and altered gene expression{so AP-1 transcription factor was likely upregulated in LSJL}. Prolonged stimulation induced PTN mRNA expression slightly, reduced vascular endothelial growth factor (VEGF) mRNA as well as NO production. Whereas mRNA expression of matrix metalloproteinases (MMPs) MMP-1 and MMP-13 was reduced, their inhibitors TIMP-1{upregulated by LSJL} and TIMP-2 were induced. PTN stimulated chondrocyte migration and proliferation.
PTN is an autocrine growth factor in cartilage. PTN may be involved in the clustering and proliferation of chondrocytes observed in the early stages of OA."

"Together with midkine (MK) [PTN] forms a family of heparin-binding proteins that are normally expressed during embryogenesis, but only at low levels in healthy adult tissues."

"Both, PTN and MK bind with high-affinity to extracellular heparan-sulfate proteoglycans as well as to cell surface syndecans, LDL receptor-related protein (LRP), anaplastic lymphoma kinase (ALK), and the receptor-type protein tyrosine phosphatase ζ/β (PTPζ)"

"PTN increased 3H-thymidine incorporation to about 150% of controls in C28/I2"

According to Pleiotrophin regulates the retention and self-renewal of hematopoietic stem cells in the bone marrow vascular niche., PTN is involved in the homing and retention of HSCs.  So the upregulation of HSCs could indicate the presence of more HSCs in LSJL.

BDNF

Central Depletion of Brain-Derived Neurotrophic Factor in Mice Results in High Bone Mass and Metabolic Phenotype.

"BDNF and its receptor are expressed in osteoblasts and chondrocyte. BDNF in vitro has a positive effect on bone; whether central BDNF affects bone mass in vivo is not known. We therefore examined bone mass and energy use in brain-targeted BDNF conditional knockout mice (Bdnf(2lox/2lox)/93). The deletion of BDNF in the brain led to a metabolic phenotype characterized by hyperphagia, obesity, and increased abdominal white adipose tissue. Central BDNF deletion produces a marked skeletal phenotype characterized by increased femur length, elevated whole bone mineral density, and bone mineral content. The skeletal changes are developmentally regulated and appear concurrently with the metabolic phenotype, suggesting that the metabolic and skeletal actions of BDNF are linked. The increased bone development is evident in both the cortical and trabecular regions. Compared with control, Bdnf(2lox/2lox)/93 mice show greater trabecular bone volume (+50% for distal femur; +35% for vertebral body) and midfemoral cortical thickness (+11 to 17%), measured at 3 and 6 months of age. The skeletal and metabolic phenotypes were gender dependent, with female being more affected than male mice. However, uncoupling protein-1 expression in brown fat, a marker of sympathetic tone, was not different between genotypes. Deletion of central BDNF expression in mice results in increased bone mass and white adipose tissue, with no significant changes in sympathetic signaling or peripheral serotonin, associated with hyperphagia, obesity, and leptin resistance."

"BDNF acts on central and peripheral neurons through binding to the product of tyrosine kinase receptor type B/neurotrophic tyrosine kinase, receptor, type 2, a high-affinity tyrosine kinase receptor"

"BDNF levels in bone are not significantly different between the mutant and control mice"

"Bdnf2lox/2lox/93 mice at 3 and 6 months of age had longer femurs ( 6–10%)"

Growth plate thickness was about 10 to 15% greater at six months age.

The problem is that the blood brain barrier is the most selective barrier so anything that would reduce BDNF in the brain would also affect the bone.

BDNF alters ERK/p38 MAPK activity ratios to promote differentiation in growth plate chondrocytes.

"Using primary bovine growth plate chondrocytes and murine ATDC5 cells, we demonstrate that the ERK and p38 pathways have opposing effects on proliferation but are both absolutely required for differentiation. Two factors that promote chondrocyte differentiation, brain-derived neurotrophic factor (BDNF) and C-type natriuretic peptide, increase p38 activity while decreasing, but not completely inhibiting, ERK activity. The attenuation of ERK activity by BDNF occurs via p38-dependent raf-1 inhibition{raf1 is downregulated by LSJL}. The inhibition of raf-1 by p38 is direct, because purified p38 protein inhibits the kinase activity of purified active raf-1 as well as raf-1 immunoprecipitated from chondrocyte lysates. Moreover, IGF-I, which stimulates proliferation, suppresses p38 activation. Unopposed IGF-I promotes high ERK/p38 activity ratios favoring proliferation, whereas BDNF signals a transition to differentiation by decreasing the ERK/p38 activity ratio without completely inhibiting ERK, which involves the direct inhibition of raf-1 by p38."

"The receptor families activating one or more MAPK cascades include receptor tyrosine kinases, cytokine receptors, G protein-coupled receptors, and serine-threonine kinase receptors. The three major MAPK cascades consist of the classic ERK1/2 cascade, which is responsive to mitogens such as IGF-I, and the c-Jun N-terminal kinase (JNK) and p38 MAPK cascades"

"In the case of ERK1/2, the module consists of the MAPK p44 ERK1 and p42 ERK2, the immediate upstream kinases MAPK kinase (MEK)1 and MEK2 that activate ERK1/2 by phosphorylating the dual TEY motif in the ERK kinase domain, and the kinase that activates MEK1/2, raf-1; raf-1, in turn, is activated by the recruitment of the small G protein Ras by Grb/SOS complex formation after receptor tyrosine kinase activation. In the p38 MAPK module, the MAPK kinases 3/6 phosphorylate the TGY motif in the kinase domain of p38, and are themselves activated by upstream kinases, such as MEK kinases 2 and 3. Activation of the p38 pathway often follows cellular stress, such as that caused by UV light or osmotic changes, but the pathway is also activated by a variety of extracellular stimuli"

"brain-derived neurotrophic factor (BDNF), stem cell factor, and growth arrest-specific gene 6 as factors that block IGF-I-stimulated proliferation but enhance differentiation in growth plate chondrocytes and showed that ERK activity is required for chondrocyte proliferation. The three factors, as well as CNP, inhibit chondrocyte proliferation by reducing ERK activity but do not affect IGF-I-dependent phosphorylation of the IGF-I receptor or the immediate downstream signaling molecules Shc1{down in LSJL} and Grb2, suggesting that these factors act on the ERK pathway downstream of the Shc1/Grb2 complex."

"p38 activation by IGF-I plus CNP was 8.1 ± 1.3-fold greater than seen with IGF-I alone."

"IGF-I-stimulated chondrocyte proliferation requires ERK1/2 activation, which is reduced by both BDNF and CNP"

"ERK activity must be high during proliferation but must be low (but not nil) for differentiation to proceed."<-Note this may not be true for initial differentiation of stem cells into chondrocytes.

Monday, September 27, 2010

Increase your Height with S-Adenosyl methionine

Earlier, we discussed how DNA Methylation was involved in growth plate cessation.  Yes, Estrogen handles growth plate fusion but growth plate fusion will not occur until after growth plate cessation.  Then we discussed how folic and folinic acid were involved in producted DNA from damage(And in turn damage to DNA Methyltransferase).  S-Adenosyl Methionine is available for purchase:Nature Made SAM-e Complete 200 mg - 90 Enteric Coated Tablets

Now, S-Adenosyl Methionine has a methyl group that can be donated to different proteins.  If methyl groups were continuously donated to growth plate chondrocytes the body would keep growing!

Exploring the mechanisms behind S-adenosylmethionine (SAMe) in the treatment of osteoarthritis

"Clinical trials have shown reduced pain and stiffness [in osteoarthritis] while in vitro and animal studies have shown SAMe can stimulate the production of cartilage which is critical in reversing the disease process. The author examines many potential mechanisms of action including: reduction of inflammatory mediators; increasing levels of glutathione; direct or indirect signaling of cartilage synthesis or survival; maintenance of DNA methylation. Research into the mechanisms of supplemental SAMe in osteoarthritis is necessary to evaluate the clinical effectiveness and safety of this dietary supplement."

"Osteoarthritic chondrocytes are particularly sensitive to the inflammatory cytokines because they have increased levels of their activating enzymes and receptors. Part of the catabolic role of IL-1 could be due to its ability to up regulate the inducible form of nitric oxide synthase and cyclooxygenases (COX)-2, leading to the increased production of nitric oxide and prostaglandin E2. Nitric oxide can induce apoptosis and decrease proteoglycan synthesis in chondrocytes by interfering with β 1-integrin mediated cell to matrix signal transduction mechanism. Over time, the cartilage would disintegrate, resulting in narrowing of the joint space and damage to the surrounding bone"

"joint space narrowing occurs at < 0.1 mm per year"

"intramuscular injection with 30 mg/kg and 60 mg/kg of SAMe for 12 weeks resulted in an increased number of cells and depth of cartilage in a partial meniscectomy model of osteoarthritis in rabbits compared to placebo"

"positive effects after treatment with both 1 μ g/ml and 10 μ g/ml SAMe, with 10 μ g/ml being the most effective at increasing proteoglycan synthesis and secretion, while 100 μ g/ml had an inhibitory effect"

"SAMe plays a vital role in three biochemical pathways; methylation, transsulfuration and aminopropylation. The loss of a methyl group from SAMe triggers the transsulfuration pathway creating all endogenous sulfur compounds. With the loss of its methyl group to different acceptors (i.e. DNA, proteins, phospholipids, and neurotransmitters) SAMe is converted into s-adenosylhomocysteine, which is hydrolyzed to adenosine and homocysteine. When methionine is needed, homocysteine is remethylated by methyltetrahydrofolate homocysteine methyltransferase with B12 as a cofactor. When methionine is in excess, homocysteine is converted to cystathionine by β cystathionine-synthase with vitamin B6 as a cofactor. Cystathionine is then converted to cysteine, which can act as a reducing agent either alone or as an active part of glutathione. Polyamines are synthesized via the aminopropylation pathway which begins when SAMe is converted to decarboxylated SAMe via SAMe decarboxylase. As an aminopropyl group is transferred to putrescine, methylthioadenosine is formed, which is followed by the production of spermidine and spermine. Methylthioadenosine is then converted back to methionine"

Well, in osteoarthiritis the cartilage is already turning into bone so we don't know if SAMe consumption will result in endochondral ossification of the articular cartilage.  But, SAMe can stimulate the production of cartilage when ordinarily cartilage production is very limited so taking SAMe during development could lead to extra height growth(If you take SAMe you also need to take Folic acid as well to cope with the additional cellular proliferation).  SAMe is produced by the body.

Moderate or supranormal folic acid supplementation does not exert a protective effect for homocysteinemia and methylation markers in growing rats

"Folic acid (FA) deficiency/supplementation effects seem to be dependent on age group and/or physiological status. The aim was to evaluate changes associated with rapid growth in relation to methionine metabolism in rats.
Four groups (n = 10 each) of male Sprague Dawley rats (5 weeks old) were on diets that varied in their FA content: 0 mg FA/kg diet (deficient), 2 mg FA/kg diet (control), 8 mg FA/kg diet (moderate supplementation), 40 mg FA/kg diet (supranormal supplementation). Animals were fed ad libitum for 30 days. Biomarkers of methionine metabolism and antioxidant status were evaluated.
Serum total homocysteine concentration increased (p < 0.01) in FA deficient animals, with no differences between the supplemented groups. The hepatic 'methylation ratio' (S-adenosylmethionine/S-adenosylhomocysteine) of the FA content groups reached similar values, which were significantly higher compared to the deficient group. The brain 'methylation ratio', however, remained unmodified independently of FA content in the diet. FA deficiency induced hepatic DNA hypomethylation, and supranormal FA supplementation exerted the most protective effect (p < 0.01). Serum folate levels increased according to FA dietary level, whereas no differences were seen for vitamin B(12) and vitamin B(6).
FA deficiency compromises methionine metabolism whereas supplementation does not show an additional positive effect compared to the control diet in growing animals"

"The main dietary components that act as methyl groups donors are folates, methionine, vitamin B 12 and choline"

"The methionine cycle is nutritionally regulated by FA, and vitamins B 6 and B 12"

"The so-called methylation ratio (AdoMet/AdoHcy) of the 3 groups whose diet included FA reached similar values, significantly higher compared to the deficient group"

"Serum folate levels are increased, as expected, in accordance with the vitamin supplementation level of the diet. However, serum vitamin B 6 and B 12 in the different groups remained unmodified regardless of dietary FA content"

"neither FA deficiency nor supplementation altered the average weight gain of the animals
throughout the study. These results are comparable to other studies that show neither deficiency nor supplementation of FA in the diet inhibits or improves animal response in relation to growth"

After 200 micrograms/a day of FA, Folic acid is no longer metabolized and floats in the serum.

"high-dose FA supplementation does not seem to induce higher transmethylation activity"

The conclusions seem to differ from the results as they say that supranormal FA supplementation exerts a protective effect and serum folate levels increased with Folic acid supplementation.  But this shows that SAMe supplementation will have an effect independent of Folic acid supplementation as in all the groups the SAMe level in the blood normalized on a particular level.

SAMe hasn't really been tested on healthy adults and it's effects on cell growth but in theory SAMe supplementation should result in some extra height growth as a result of more chondrocytes getting methylating and thus proliferating and differentiating for a longer period.

TRPS1

TRPS1 is upregulated by LSJL.

ROLE OF TRPS1 IN ENDOCHONDRAL BONE FORMATION

"Mutations of the human TRPS1 gene cause a dominantly inherited skeletal dysplasia tricho-rhino-phalangeal syndrome (TRPS). Although the gene was identified several years ago, the molecular and cellular mechanisms underlying TRPS are largely unknown. Trps1 is a GATA-type transcription factor that acts as a transcriptional repressor. Recently, we have demonstrated that disruption of the Trps1 gene in mice results in dramatic elongation of growth plates and delayed replacement of cartilage by bone. These abnormalities are accompanied by increased Indian hedgehog (Ihh) expression and elevated Ihh and BMP signaling. Although BMP and Ihh pathways act in parallel to regulate various aspects of endochondral bone formation, it is unclear how they are integrated and controlled at the transcriptional level. The hypothesis of this proposal is that Trps1 constrains the Ihh-BMP positive feedback loop to assure timely progression of chondrocyte maturation and synchronization of chondrocyte development with perichondrial mineralization. We propose studies aimed at elucidating the Trps1 molecular network and its role in regulation of the cross-talk between differentiating chondrocytes and perichondrium. Specifically, we will focus on understanding the mechanisms of delayed cartilage removal and endochondral ossification in Trps1 mutant mice. To address these questions we propose the following specific aims: 1. To understand the molecular and cellular mechanisms of the growth plate elongation caused by disruption of the Trps1 gene. 2. To determine how Trps1 regulates BMP and hedgehog signaling. The first aim will be accomplished by histological and molecular analyses of abnormalities in the growth plate of the mouse model of TRPS. To achieve the second aim we will employ a combination of electrophoretic mobility shift assay (EMSA), reporter expression assays and analyses of the effect of the Trps1 deficiency and over-expression on BMP and hedgehog signaling in a cellular model of chondrogenesis. Additionally, we will test the Trps1 and Ihh genetic antagonism using Trps1;Ihh double mutant mice. Results of the proposed studies will define molecular mechanisms underlying skeletal dysplasia in tricho-rhino-phalangeal syndrome. Importantly, the results of these studies will directly impact our understanding of the transcriptional control of BMP and hedgehog signaling, that are widely involved in the development of multiple organ systems."

Here's the study noted:

Uncoupling of chondrocyte differentiation and perichondrial mineralization underlies the skeletal dysplasia in tricho-rhino-phalangeal syndrome.

"Patients with TRPS have short stature, hip abnormalities, cone-shaped epiphyses and premature closure of growth plates reflecting defects in endochondral ossification. The TRPS1 gene encodes for the transcription factor TRPS1 that has been demonstrated to repress transcription in vitro. To elucidate the molecular mechanisms underlying skeletal abnormalities in TRPS, we analyzed Trps1 mutant mice (Trps1DeltaGT mice[involves a frame deletion of the binding element of Trps1]). Analyses of growth plates demonstrated delayed chondrocyte differentiation and accelerated mineralization of perichondrium in Trps1 mutant mice. These abnormalities were accompanied by increased Runx2 and Ihh expression and increased Indian hedgehog signaling. We demonstrated that Trps1 physically interacts with Runx2 and represses Runx2-mediated trans-activation. Importantly, generation of Trps1(DeltaGT/+);Runx2(+/-) double heterozygous mice rescued the opposite growth plate phenotypes of single mutants, demonstrating the genetic interaction between Trps1 and Runx2 transcription factors. Collectively, these data suggest that skeletal dysplasia in TRPS is caused by dysregulation of chondrocyte and perichondrium development partially due to loss of Trps1 repression of Runx2."

"Trps1 is highly expressed in regions where Runx2 is downregulated"

"in Trps1ΔGT/ΔGT mice, there is a larger zone of cells co-expressing both Ihh and Col10a1[in addition to expanded growth plates], which indicates an increased number of cells in the transition from prehypertrophic to mature hypertrophic stage."  However height remained largely the same.

"Runx2 expression in perichondrium inhibits chondrocytes proliferation through Fgf18"

The multi zinc-finger protein Trps1 acts as a regulator of histone deacetylation during mitosis.

"TRPS1, the gene mutated in human "Tricho-Rhino-Phalangeal syndrome," encodes a multi zinc-finger nuclear regulator of chondrocyte proliferation and differentiation. Trps1 [controls] mitotic progression in chondrocytes. Loss of Trps1 in mice leads to an increased proportion of cells arrested in mitosis and, subsequently, to chromosome segregation defects. Trps1 acts as regulator of histone deacetylation. Trps1 interacts with two histone deacetylases, Hdac1 and Hdac4, thereby increasing their activity. Loss of Trps1 results in histone H3 hyperacetylation, which is maintained during mitosis. Consequently, chromatin condensation and binding of HP1 is impaired, and Trps1-deficient chondrocytes accumulate in prometaphase. Overexpression of Hdac4 rescues the mitotic defect of Trps1-deficient chondrocytes, identifying Trps1 as an important regulator of chromatin deacetylation during mitosis in chondrocytes. The control of mitosis can be linked to the regulation of chondrocyte differentiation by epigenetic consequences of altered Hdac activity. "

DNA condensation requires histone deacetylases.  Cells with impaired Hdac function are defective.  TRPS1 knockout decreases cellular proliferation.

Addition of Hdac4 didn't affect the proportion of cells in the G2/M phase in the TRPS1+/+ group in contrast to the knockout group where addition of HDAC4 decreased portion of cells in G2/M phase.  Thus meaning that overexpression of TRPS1 may not cause skeletal overgrowth.  Addition of Hdac4 did alter some H3K9 acetylation parameters however.

Saturday, September 25, 2010

Slimechanse grew 1/4" with LSJL

Slimechanse has reported that he's gained 1/4" with LSJL.  LSJL upregulates genes involved in the TGF-Beta, PI3K, ECM, and Wnt pathways.  Wnt is involved in mechanotransduction.  ECM is involved in the production of new extracellular matrix which could explain the large amounts of white in rats post LSJL in the growth platePI3K increases cellular proliferation.  TGF-Beta regulates cellular proliferation and p15 is a part of the TGF-Beta which stops cellular proliferation but overall TGF-Beta is anabolic and it helps cause the differentiation of stem cells into chondrocytes.

Slimechanse has been performing LSJL for a few months and has submitted his ankle picture.
The date on the picture is wrong.  This ankle picture was taken recently. Here's a child's ankle picture:
The right ankle is normal so look at that whereas the right ankle is bruised.  Here's a pic of a normal adult ankle:
In the normal adult ankle, you can see a very hard bone.  No cartilage is on the lateral portion of the ankle.  In the child's bone you can see a softer looking ankle.  In Slime's bone you can see cartilage on the side of the ankle.  There is not supposed to be cartilage on the lateral side of the ankle meaning that LSJL by upregulating TGF-Beta expression caused the differentiation of stem cells into chondrocytes in new areas.  And Slime is 35 years old.

The exact mechanism of LSJL is unclear but it does seem to be successful at achieving growth of new chondrocytes.

Friday, September 24, 2010

Will Alfalfa really help you grow taller?

After discovering encouraging evidence regarding an old height increase staple Milk, I've decided to take another look at Alfalfa.  Alfalfa is reported to be the richest natural source of ipriflavone.   Ipriflavone increases calcitonin levels but as we learned in our research about Calcium, osteoclasts are essential part of endochondral ossification by degrading matrix that prevents cells from growing to their full potential.  Ipriflavone may have similar effects to calcium given that it increases serum Calcium levels.  Calcitonin is important in L-type Calcium Channels.  CGRP is related to Calcitonin and regulates the mechanical response of bone to load.  Alfalfa may help increase mechanosensativity.  There's no need to buy Alfalfa as you can just buy Ipriflavone directly Ipriflavone 300mg 90 Capsules.

TAK-778 induces osteogenesis in ovariectomized rats via an estrogen receptor-dependent pathway. 

"TAK-778, a derivative of ipriflavone, has been shown to induce bone growth both in vitro and in vivo. TAK-778 can enhance osteoblast differentiation of human bone marrow cells via an estrogen receptor (ER)-dependent pathway. We tested if TAK-778 induced osteogenesis via an ER-dependent pathway using an ovariectomized (OVX) rat model. Two weeks after test animals underwent ovariectomy, TAK-778 and/or tamoxifen was administered orally over 3 months. Vehicle-treated and sham-operated rats served as controls. The bone mineral density (BMD) of the lumbar vertebrae and sagittal two-dimensional images of the L3 vertebral body were measured. In addition, bone formation rates (BFR) and serum calcium and osteocalcin levels were measured. TAK-778 significantly increased BMD, serum calcium and osteocalcin levels, and BFR when compared to that of the vehicle-treated group[osteocalcin is a byproduct of osteoblasts so it is used to measure osteoblast levels]. However, tamoxifen, a well-known ER antagonist, clearly inhibited the increase in these parameters induced by TAK-778. Treatment with TAK-778 increased the structure model index, bone volume/tissue volume[volume = length * width * HEIGHT], and trabecular thickness parameters and decreased the trabecular separation/spacing in OVX rats. Tamoxifen suppressed these effects when administered in combination with TAK-778. Taken together, the present study showed that TAK-778 enhanced bone formation in OVX rats and that this effect was dependent on an ER-mediated pathway." 

Now, post fusion you don't need to worry about inhibiting osteoclast activity.  Histological evidence of fusion was established in the paper Histology of Epiphyseal Union in Mammals. Exactly, when fusion occurs is unclear.  X-rays will only tell you if you are not fused and not if you are.  The thin line apparent on most x-rays on bones could be epiphyseal scarring or it could be a hyaline cartilage growth plate line remnant.  X-rays are not three dimensional and parts of the bone fuse at different rates so unfused portions of the bone could be unfused. You should not take extra osteoclast inhibitors unless you are fairly confident that you do not have any leftover cartilage in your bone.  If you aren't trying to grow by a cartilage intermediary like via endochondral ossification then trying to grow by osteoblasts is possible.  If you can get MSCs to differentiate into osteoblasts on the subchondral plate and deposit new bone there you will grow taller. 

Alfalfa can also up mechanosensitivity which will help you grow taller by endochondral ossification.  It's unclear whether the encouraged differentiation of MSCs into osteoblasts will compete with the target goal of differentiating into chondrocytes by means of LSJL.

"TAK-778 stimulates proliferation of uncommitted mesenchymal C3H10T1/2 cells without inducing differentiation"<-so TAK-778 increases stem cell proliferation which is very good.  Maybe the Estrogen agonist can block the induction into an osteoblastic lineage and Alfalfa can solely be used to increase stem cell proliferation

"Cell secretory activities were stimulated by TAK-778. The presence of tamoxifen alone did not affect the synthesis activity of cells."<-The anabolic benefits of Alfalfa can occur even when tamoxifen(an estrogen receptor inhibitor) is inhibiting the differentiation of stem cells into osteoblasts.

So an Estrogen Receptor blocker and Alfalfa in conjunction with LSJL may help you grow taller.  The effects of estrogen receptor blockers will have to be studied further to make sure that the good effects on height growth outweigh the bad.

Ipriflavone modulates IGF-I but is unable to restore bone in rats. 

"Seventy-two, 90 day-old Sprague-Dawley rats were divided into six groups (sham two groups; ovariectomized four groups). Thirty-five days from the date of surgery, one sham and one ovx group were killed to verify the occurrence of bone loss. The remaining four groups were sham, ovx, ovx + ipriflavone (100 mg[sol ]kg body weight per day), or ovx + 17beta-estradiol (10 microg[sol ]kg body weight daily) for a period of 65 days. Ipriflavone was ineffective in restoring bone density and unlike estrogen did not prevent bone resorption as evidenced by increased urinary excretion of hydroxyproline and serum tartrate-resistant acid phosphatase activity. Ipriflavone increased the expression of IGF-I in the femur." 

Increasing IGF-1 expression is very good because IGF-1 determines the maximum size chondrocytes achieve during chondrocyte hypertrophy so maybe Ipriflavone can have positive benefits during puberty.  It's just TAK-778 that inhibits bone resorption which is vital during endochondral ossification.  Normal ipriflavone does not inhibit bone(and chondrocyte matrix) absorption. 

The rats in the control group had a shorter left femur(by .10 cm) than the other groups which is unusual considering the rats were 90 days old.  The estrogen treated group had a shorter left femur length by 0.07 cm then the OVX group and the Ipriflavone treated group.  Maybe Ipriflavone reproduces the good effects of estrogen while taking out the bad.  The rats were controlled for body weight and not length so the control group may have just had denser bones.  But the rats did not have their ovaries removed until after the experiment had begun.


So there is likely an affect on height growth as a result of an ovareictomy.  The estrogen treated group was taller than the control group by 0.03 cm despite having higher estrogen levels than the control group.  So the ovaries may have height reducing effects independent of increasing serum levels of estrogen.


Ipriflavone may help you maintain your bone density if you decide to embark on a regime that inhibits estrogen.

The effect of oral ipriflavone on the rat mandible during growth. 

"The aim of this study was to examine the effect of IF on rat mandibles during the growth stage. Thirty-two 5-week-old Wistar male rats were divided into four groups. The control group was fed a standard diet, group A received a low calcium diet (calcium content 30 per cent of the standard diet) for 6 weeks, and the other two groups were fed a low calcium diet for 3 weeks and then a standard diet without IF (group B) or with IF (group C) for 3 weeks. In addition, distilled water was provided for all groups.  For mandibular length, the control group showed a significantly higher value than groups A and B, while group C demonstrated a significantly higher value than group A. In addition, the control group and group C showed significantly higher values for mandibular ramus height than group A). However, bone mineral density in trabecular bone was significantly higher in the control group than in the other groups and bone mineral density in cortical bone was significantly higher in the control group than groups A, B and C . Bone mineral density in both trabecular and cortical bone was significantly higher in group C than in groups A and B. These results indicate that complete recovery from calcium deficiency to the level of the control group may not be attainable, even though IF enhances calcium absorption to act on bone cells and promote bone construction." 

This study shows that calcium deficiency decreases height.  Now I don't recommend inducing calcium deficiency, I'm just saying that extreme levels of calcium may inhibit osteoclastic activity which is an important part of the height growth process.  Some may point out that ipriflavone is a phytoestrogen, however estrogens are not bad unless in extremely high or low levels.  And ipriflavone does not seem to have the height reducing effects that estrogen may have in too high doses.

Supplementing with ipriflavone during development can increase height by increasing IGF-1 expression.  The usage of TAK-778 is unknown as it inhibits bone resorption which is an important part of the height growth process.

Ipriflavone does seem to have a beneficial effect on chondrocytes which may have a further benefit on LSJL.

Effects of ipriflavone and its metabolites on human articular chondrocytes cultivated in clusters.

"Ipriflavone (IP) is an isoflavone derivative that was suggested to have bone-sparing effects in post-menopausal and senile osteoporosis. A moderate stimulatory effect of IP and its metabolites on proliferation of osteoblastic cells was reported in rat osteoblastic osteosarcoma cell line. We investigated the effects of different concentrations (0, 1, 10 and 100 micrograms/ml) of IP and its metabolites (MET I, II, III and V) on the incorporation of [3H] thymidine and production of proteoglycans (PG) and type II collagen (COL II) by human articular chondrocytes[articular chondrocytes are pretty similar to growth plate chondrocytes except that they don't undergo the final stages of endochondral ossification] during a 12-day period, in a three-dimensional chondrocyte culture model. [3H]thymidine uptake was measured in chondrocyte clusters, and specific PG and COL II radioimmunoassays were performed every 4 days on the culture medium and cell clusters. Incubation with IP or its metabolites did not affect [3H]thymidine uptake regardless of the dose. PG released into the culture medium and PG cluster content rose significantly in presence of IP (1, 10 and 100 micrograms/ml)[Alfalfa increases proteoglycan content which could help you grow taller]. MET I increased PG release in culture medium (10 and 100 micrograms/ml) and PG cluster content (100 micrograms/ml). MET II has no effect on PG production. MET III increased PG in culture medium (100 microgram/ml) but did not influence PG cluster content while MET V (100 micrograms/ml) increased both PG release in culture medium and PG cluster content. COL II release in culture medium and COL II cluster content were significantly increased in presence of IP[Alfalfa increases the release of Type II Collagen which increases growth plate quality] (10 and 100 micrograms/ml), MET III (1, 10 and 100 micrograms/ml) or MET V (100 micrograms/ml). MET I and II did not significantly affect COL II production." 

So it would seem that Ipriflavone could help you grow taller during puberty by increasing your growth plate quality by inducing chondrocytes to secrete more Collagen II and Proteoglycans.  In addition, this would be beneficial if you are already growing taller with LSJL.    Alfalfa also increases stem cell proliferation and IGF-1 expression two anabolic activities which will help you grow taller.  Alfalfa also allows you to inhibit estrogen with less negative consequences. 

Tamoxifen may induce apoptosis in growth plate chondrocytes.  Apoptosis is a necessary part of endochondral ossification so that is not necessarily a bad thing.  This product may have Tamoxifen in it but ingredients can not be validated: Redefine Nutrition Pct Revolution, 60-Count.

Thursday, September 23, 2010

Will Milk help you grow taller?

Milk has been a popularly recommended product due to a perhaps coincidental correlation between the dutch diet and height.  Here's a possible mechanism explaining milk and height growth:

Effect of cow milk consumption on longitudinal height gain in children

"Insulin-like growth factor I is present in much higher concentrations in cow milk than in human milk. It is relatively stable to both heat and acidic conditions; therefore, it survives the conditions of commercial milk processing. Milk whey protein, especially milk basic protein, was reported to promote bone formation and to suppress bone resorption, and daily supplementation with milk basic protein significantly increases bone mineral density independently of dietary intake of minerals and vitamins. Whole-body bone mineral content [elevation] in piglets fed arachidonic acid and that liver arachidonic acid was positively related to plasma insulin-like growth factor I and calcitriol. Furthermore, transforming growth factor ß2 was also well preserved in human milk after holder pasteurization at 56.5 °C . Transforming growth factor ß2 inhibits the differentiation of human adipocyte precursor cells and reduces the activity of the lipogenic enzyme glycero-3-phosphate dehydrogenase. [A correlation was found between avoiding milk and obesity in children]. In our longitudinal study, the change in relative weight in the high-consumption group was lower than that in the low-consumption group."

We are used to hearing about TGF Beta 1 in relation to height growth as the TGF Beta 1 helps to regulation the cell cycle and as we saw in our research on DNA Methylation that is very important in terms of growing taller. When DNA Methylation stops, that may be a signal for the resting zone chondrocytes to stop dividing.  If we can alter DNA Methylation, we can alter height growth.  Transforming growth factor Beta 2 has the less impressive ability to inhibit stem cells into adipose tissue but that does increase the likelihood of stem cells differentiating into chondrocytes and osteoblasts.  IGF-1's role in height growth is increasing the extent of chondrocyte hypertrophy.  Now, how much of an increase of IGF-1 can be achieved with natural mechanisms given negative feedback is unknown.  HGH may play a role in regulating that negative feedback and an increase in IGF-1 without an increase in HGH may not increase height growth.

There have been several studies that have linked milk to an increase in growth.  Recombinant Bovine Growth Hormone injected cows are the ones rumored to have elevated IGF-1 levels and thereby transferring it into humans. 

Short-term effects of replacing milk with cola beverages on insulin-like growth factor-I and insulin-glucose metabolism: a 10 d interventional study in young men. 

"The present study was designed to reflect the trend of replacing milk with carbonated beverages in young men and to study the effects of this replacement on IGF-I, IGF-binding protein 3 (IGFBP-3), IGF-I:IGFBP-3 and glucose-insulin metabolism. A randomised, controlled crossover intervention study, in which eleven men aged 22-29 years were given a low-Ca diet in two 10 d periods with 10 d washout in between. In one period, they drank 2.5 litres of Coca Cola(R) per day and the other period 2.5 litres of semi-skimmed milk. Serum IGF-I, IGFBP-3 (RIA), insulin (fluoro immunoassay) and glucose (Cobas) were determined at baseline and end point of each intervention period. Insulin resistance and beta-cell function were calculated with the homeostasis model assessment. A decrease in serum IGF-I was observed in the cola period compared with the milk period (P < 0.05). No effects of treatment were observed on IGFBP-3, IGF-I:IGFBP-3, insulin, glucose, insulin resistance or beta-cell function. The present study demonstrates that high intake of cola over a 10 d period decreases total IGF-I compared with a high intake of milk, with no effect on glucose-insulin metabolism in adult men." 

Milk consumption and circulating insulin-like growth factor-I level: a systematic literature review. 

"Ten cross-sectional studies showed statistically positive correlation between milk consumption and the circulating IGF-I level. Randomized controlled trials indicated that the circulating IGF-I level was significantly higher in the milk intervention group. After meta-analysis, the weighted mean difference of the circulating IGF-I level was 13.8 ng/ml (95% confidence interval: 6.1-21.5 ng/ml) comparing the intervention group with the control group." 

So Milk increases IGF-1 levels(from cow treated with Growth Hormone) and it doesn't affect Insulin Resistance(a possible negative feedback mechanism).  High serum levels of IGF-1 has been shown to increase mandibular size in rats. 



Dairy intake associates with the IGF rs680 polymorphism to height variation in periadolescent children.


"We screened 795 periadolescent children (424 girls) aged 10-11 years old from the Gene and Diet Attica Investigation (GENDAI) pediatric cohort for the IGF rs680 polymorphism (rs680).
Children homozygous for the common allele (GG) were taller (148.9+/-7.9 cm) compared with those with the A allele (148.1+/-7.9 cm), after adjusting for age, sex and dairy intake (beta+/-s.e.: 2.1+/-0.95, P=0.026). A trend for rs680 x dairy intake interaction was also revealed (P=0.09). Stratification by IGF rs680 genotype revealed positive significant (P=0.014) association between dairy product intake and height in A-allele children adjusted for the same confounders. A daily increase of four dairy servings was associated with a 0.4 cm increase in height[can this still occur at extremes, does this dairy consumption affect only growth rate or adult height?]. On grouping dairy intake into low (1.9+/-0.7 servings per day) and high dairy product consumption (4.4+/-1.5 servings per day), children with the A allele who were high dairy product consumers were taller compared with the low dairy product consumers (148.8+/-7.9 vs 147.4+/-7.7 cm, respectively, P=0.05).
A higher consumption of dairy products is associated with increased height depending on the rs680 IGF2 genotype."

"women with the A/A genotype were shorter than G/G carriers[for IGF2], and a similar trend was observed in men"<-since IGF2 genes are very important to height maybe milk can be important for height for as long as it affects IGF2.

Cow milk consumption, insulin-like growth factor-I, and human biology: a life history approach.
"The literature tends to support milk's role in enhancing growth early in life (prior to age 5 years), but there is less support for this relationship during middle childhood[maybe IGF-1 only increases growth up to a certain point and after age 5 IGF-1 levels are naturally sufficient not to help with height growth?]. Milk has been associated with early menarche and with acceleration of linear growth in adolescence. NHANES data show a positive relationship between milk intake and linear growth in early childhood and adolescence, but not middle childhood, a period of relatively slow growth. IGF-I is a candidate bioactive molecule linking milk consumption to more rapid growth and development.
Routine milk consumption is an evolutionarily novel dietary behavior [may alter] linear growth."

"Cross-species milk consumption and ingestion beyond the typical weaning period may trigger unanticipated life history consequences."<-Maybe humans are only supposed to drink milk during a limited period of time?

"Milk is the only mammalian food that is produced to be consumed by the same species. Of course, it is produced for consumption only by individuals during one life history stage: infancy. After an infant is weaned, it does not consume milk again, and for the vast majority of mammalian species, the ability to digest lactose diminishes with weaning. Each species' milk is tailored with an array of bioactive molecules (e.g., growth hormones and immune proteins) and nutrients that shape the early developmental trajectory of nursing infants, who accrue body size (weight and linear dimensions) while various organ systems mature."<-Humans don't eat amniotic fluid after all. If they did would they grow taller?

"No statistically significant relationship between calcium and growth in height, regardless of whether calcium is supplied as a mineral supplement or as a milk derivative. Children supplemented with calcium did not grow more in height than did those in the control groups."<-It's not really the serum levels of calcium that matter it's the intracellular calcium secretions.

"The casein portion of milk protein, rather than whey protein, is most closely related to rises in circulating IGF-I after milk consumption"

"Diets high in milk and dairy products in childhood are associated with lower IGF-I in adulthood, suggesting that the short- and long-term effects of milk intake may vary by life history stage"

"There are periods of rapid and slow growth, there may be critical periods during which milk may raise IGF-I levels and contribute to growth, and these feed back over the long term to reduce IGF-I production via pituitary secretion of growth hormone"<-Thus you may want to cycle milk consumption so as to avoid reduced pituitary secretions.

"Milk intake has also been found to be positively related to birth length"

"Children who do consume more cow milk during [ages two to five] are taller than those who drink less "

"Milk consumption had no relationship to height in [ages five to eleven] after controlling for energy, or energy, protein, and calcium intake."

Associations between milk and height were found again between ages twelve and seventeen but those studies did not sufficiently account for confounding variables.

Maybe after age five, milk drinkers should start cycling to avoid reductions in the body naturally producing IGF-1 and thereby resulting in no net increase in serum IGF-1 levels.

Things are looking pretty good for Milk(from growth hormone cows) consumption increasing height. Milk consumption increases serum IGF-1 levels and higher serum IGF-1 levels has positive effects on height growth and bone size. And Milk consumption doesn't seem to involve a negative feedback mechanism like insulin resistance at moderate levels.

Regular milk consumption throughout development should result in a small increase in height by increased chondrocyte hypertrophy. Furthermore, once milk consumption is stopped IGF-1 levels reduce to normal so you don't have to worry about faster cancer spread.

Wednesday, September 22, 2010

Increase Height with DNA Methylation?

Previously, in the blog I reported the function of osteoclasts in growing taller.  The use of alendronate inhibited osteoclasts reobsorption capacity and that reduced overall height growth.  What's interesting is that the growth plate was actually larger when osteoclasts were inhibitied.  The authors suggested that this was a result of failure of VEGF to induce chondrocyte apoptosis.  It was also reported previously that growth plate senescence is a result of a decrease in DNA Methylation(Senescence being when a growth plate is inactive).

DNA Methylation is the addition of a Methyl Group to DNA and affects DNA regulation(so transcriptional proteins that stop growth can no longer bind to the gene). DNA Methylation also play a role in cell memory(so cells that were once chondrocytes no longer remember that they're supposed to be chondrocytes).

How does DNA Methylation affect height growth and how does it relate to osteoclasts? Are there any chemicals or substances we can use to alter DNA Methylation within the body?

Expression profile of genes related to osteoclastogenesis in mouse growth plate and articular cartilage.

"Cartilage tissue is broadly classified into transient cartilage (e.g. growth plate, GP) and permanent cartilage (e.g. articular cartilage, AC). The former eventually disappears and is replaced by bone during the endochondral ossification process, whereas the latter retains its permanency. Osteo(chondro)clasts, multinucleated giant cells of the monocyte/macrophage lineage, are selectively induced in the GP during endochondral ossification and play central roles in the resorption of cartilagenous matrices. The aim of this study was to investigate the factors determining the GP-specific recruitment of osteo(chondro)clasts. We especially focused on the expression pattern of the receptor activator of NF-kappaB ligand (RANKL), an essential factor for osteo(chondro)clast differentiation, and on that of epigenetic and transcriptional factors affecting RANKL gene expression. Knee joints of male BALB/c mice aged 8 weeks were dissected and subjected to immunohistochemical analysis using anti-RANKL, Runx2, Dlx5 and Msx2 antibodies. The methylation status of the mouse RANKL gene promoter in both the GP and the AC was analyzed. The expression of BMP-2, -3, -4, -6 and type X collagen mRNA was examined. At the boundary between the calcifying cartilage and the hypertrophic chondrocytes of the GP, RANKL-expressing chondrocytes overlapped those expressing Runx2, Dlx5 and Msx2, near numerous osteo(chondro)clasts. Although similar BMP-2 and -4 expression was observed in chondrocytes in both the GP and the AC as well as in maturing osteoblasts, a rather restricted BMP-6 expression pattern was observed in resting and proliferating chondrocytes in the GP. Mostly non-CpG methylation was scattered in a non-specific manner in chondrocytes in the GP and the AC. Putative Runx2 binding elements are located in the RANKL promoter. Runx2, an essential transcription factor for skeletal development, is a key regulator of RANKL expression in chondrocytes in the GP."

DNA Methylation in growth plates is linked to osteoclasts by RANKL which activates osteoclasts.  BMP-6 is a difference between that chondrocytes of the Articular Cartilage and Growth Plate but if endochondral ossification can be performed safely and effectively within the articular cartilage is unknown.  Osteoclasts may absorb catiligenous matrices which prevent cartilage cells from reaching their full potential.

Growth plate senescence is associated with loss of DNA methylation

"One mechanism that has been proposed to explain replicative senescence involves epigenetic changes in methylation of genomic DNA. Some CG sequences in mammalian genomic DNA are methylated on the cytosine moiety. When DNA is replicated, the new strand is initially not methylated. DNA methyltransferase 1, a maintenance methylase, recognizes the hemimethylated CGs and adds the missing methyl groups. If maintenance methylation is incomplete, methylation levels may gradually decrease with repeated cell replication.  The level of DNA methylation could serve as a cell-cycle counter. The level of DNA methylation can then affect gene expression, in part by altering interaction of transacting elements with the promoter region and in part by altering histone modification, and thus chromatin structure. Such epigenetic changes may contribute to replicative senescence and terminal differentiation in some cell types. Growth plate chondrocytes cultured at high density and exposed to a demethylating agent (5-azacytidine), undergo hypertrophic differentiation. Disruption of PASG (proliferation-associated SNF2-like gene), PASG, which is required for normal maintainance of DNA methylation, results in growth retardation and premature aging. "

So if DNA Methylation does not occur then there is growth retardation but DNA Methylation may serve as a cell-cycle counter to tell growth plates when to stop. So what we have to do is set back the cell-cycle counter some steps to trick it into thinking the number of divisions it's done is less than it currently is.

"Growth plate chondrocytes cultured at high density and briefly exposed to 5-azacytidine, a demethylating agent, differentiate and start expressing markers specific for hypertrophic chondrocytes"<-Since methylation markers increase with differentiation, demethylating chondrocytes may trick them into thinking that they're at an earlier differentiation state.

"We first tested the prediction that cultured growth plate chondrocytes from older animals will undergo fewer cell divisions before undergoing replicative senescence than will chondrocytes from younger animals. Resting zone chondrocytes extracted from fetal, 4-, and 16-week-old male rabbits proliferated for approximately 50 days and underwent three passages (with increasing intervals) before reaching senescence. During this time, chondrocytes from animals of different ages underwent a similar number of population doublings (13.1 ± 1.1 vs 14.6 ± 0.6 vs 14.3 ± 0.8; fetal vs 4 weeks vs 16 weeks respectively). Chondrocytes in primary culture were small, had a round or polygonal shape, and stained for alcian blue and alkaline phosphatase activity, but not for senescence related ß-galactosidase. In contrast, chondrocytes that were becoming senescent were larger, only stained faintly or not at all for alcian blue and alkaline phosphatase activity, but showed an increased senescence-related ß-galactosidase activity"<-Senesent chondrocytes had low alkaline phosphatase level so that may be key for growth. A Senesence B-Galactosidase inhibitor might also increase growth.

"[The] loss of DNA methylation could occur as these resting zone chondrocytes gradually replicate if there is incomplete maintenance methylation of the newly synthesized strand of DNA.  Growth plate senescence is associated with a loss of DNA methylation in rib resting zone chondrocytes. Loss of DNA methylation was observed in the resting zone chondrocytes of the distal ulna. In the distal ulna, loss of methylation with age was observed in the proliferative and hypertrophic zone chondrocytes which are thought to be progeny of the resting zone chondrocytes. Within each age, there was no significant difference in the level of DNA methylation between the different zones of the growth plate. Loss of methylation appears to occur specifically during replication of resting zone chondrocytes but not during the more rapid proliferation of proliferative zone chondrocytes.  There may be complete maintenance methylation in the proliferative zone, but not in the resting zone. Loss of methylation might be responsible for the temporal limits that cause chondrocyte replication to slow with age but not the spatial limits that cause chondrocyte proliferation to slow as the cells descend farther down the chondrocyte columns. The spatial limitation on proliferation may not be controlled by a cell-cycle counter, but instead it may be controlled by a chemical gradient, e.g. parathyroid hormone-related protein."

So you have to make sure those resting zone chondrocytes(stem cells) stay methylated.  I don't know of any good DNA Methylation stimulators.

According to SOX trio decrease in the articular cartilage with the advancement of osteoarthritis, there was no association between age and a reduction of SOX9 methylation promoter regions.

Epigenetic modifiers influence lineage commitment of human bone marrow stromal cells: Differential effects of 5-aza-deoxycytidine and trichostatin A.

"We investigated the effects of the DNA demethylating agent 5-aza-2'-deoxycytidine (5-aza-dC) or the histone deacetylase inhibitor trichostatin A (TSA) on osteogenic and chondrogenic differentiation. Monolayer cultures of HBMSCs were treated for 3 days with the 5-aza-dC or TSA, followed by culture in the absence of modifiers. Cells were subsequently grown in pellet culture to determine matrix production. 5-aza-dC stimulated osteogenic differentiation as evidenced by enhanced alkaline phosphatase activity, increased Runx-2 expression in monolayer, and increased osteoid formation in 3D cell pellets. In pellets cultured in chondrogenic media, TSA enhanced cartilage matrix formation and chondrogenic structure. epigenetic modifiers, as agents, possibly in combination with other factors,  enhance the ability of HBMSCs to form functional bone or cartilage with significant therapeutic implications therein."

In the study TSA increased COL2A1 and Aggrecan levels which are Sox related genes.

So the supplement that encourage demethylation enhanced osteogenic differentiation.  So this indicates that perhaps supplements that encourage DNA methylation may have the reverse effect(chondrogenic differentiation).

Epigenetic regulation in chondrogenesis.

"DNA methylation in CpG-rich promoters correlates with gene silencing. Histone modification including histone acetylation and deacetylation determines the stability of the chromatin structure. Condensed chromatin (heterochromatin), which has a higher-order histone-DNA structure, prevents the access of transcriptional activators to their target genes. The fundamental unit of eukaryotic chromatin consists of 146 bp of DNA wrapped around a histone octamer. Posttranslational modifications of the histone tail and the chromatin remodeling complex disrupt histone-DNA contacts and induce nucleosome mobilization. Histone acetylation of specific lysine residues in the histone tail plays a crucial role in epigenetic regulation. Histone acetylation is a dynamic process regulated by the antagonistic actions of 2 families of enzymes - the histone acetyltransferases (HATs) and the histone deacetylases (HDACs). The balance between histone acetylation and deacetylation serves as a key epigenetic mechanism for transcription factor-dependent gene expression and the developmental process. DNA methylation, histone acetylation modified by HAT and/or HDAC, and transcription factor-associated molecules contribute to a mechanism that can alter chromatin structure, gene expression, and cellular differentiation during chondrogenesis."

"In chondrocyte differentiation, TGF-β stimulation is necessary for MSC-derived primary chondrogenesis. On the other hand, chondrocyte maturation is inhibited by TGF-β. These conflicting effects of TGF-β during chondrogenesis might depend on chromatin structure and/or the epigenetics of each differentiated stage."

"TGF-β-regulated Smad3 activates the Sox9-dependent transcription on the chromatin structure"

"CpG-rich promoters of chondrogenic-related genes, such as Sox9, Runx2, chondromodulin-I, and fibroblast growth factor receptor 3, are hypomethylated during synovium-derived chondrogenesis"<-Hypomethylated means undermethylated so perhaps too much methylation is bad for chondrogenesis.

"The Col2a1 gene is less methylated in chondrocytes than in fibroblasts"

"The demethylation of the Col10a1 promoter correlates with Col10a1 induction during MSC-derived chondrogenesis"

"In chondrogenesis, p300 stimulates transcription factor-mediated chromatin disruption. Coactivator p300 directly associates with the master chondrogenic factor Sox9, and activates Sox9-dependent transcription. Sox9-dependent transactivation is induced by p300-mediated histone acetylation of chromatin"

"p300 potentiates Sox9-dependent transcription on a chromatinized DNA template and is associated with hyperacetylated histones"

"Coactivator Tip60, which mainly acetylates H4, increases Sox9/Sox5-dependent Col2a1 transcription by associating with Sox9 on chromatin"

"HDAC4, which is expressed in prehypertrophic chondrocytes, regulates chondrocyte hypertrophy and endochondral bone formation by interacting with and inhibiting the activity of Runx2"

"Growth factors, cytokines, and nonproteinaceous chemical compounds including dexamethasone, vitamin D3, prostaglandin E2, and ascorbic acid influence gene expression and cellular differentiation during chondrogenesis"

"BMP-2 induces histone hyperacetylation and methylation at the Sox9 gene on chromatin"

So perhaps you actually want less methylation in genes related to chondrogenesis so a supplement like Sam-e wouldn't help increase height.


Genetic and non-genetic influences during pregnancy on infant global and site specific DNA methylation: role for folate gene variants and vitamin B12.

"[Folate metabolism has a] central role in provision of methyl groups for DNA methylation. Global (LUMA) and gene specific (IGF2, ZNT5, IGFBP3) DNA methylation were quantified in 430 infants. Seven polymorphisms in 6 genes (MTHFR, MTRR, FOLH1, CβS, RFC1, SHMT) involved in folate absorption and metabolism were analysed in DNA from both infants and mothers. Red blood cell folate and serum vitamin B(12) concentrations were measured as indices of vitamin status. Relationships between DNA methylation patterns and several covariates viz. sex, gestation length, maternal and infant red cell folate, maternal and infant serum vitamin B(12), maternal age, smoking and genotype were tested. Length of gestation correlated positively with IGF2 methylation[length of gestation means time it took for growth to occur, IGF2 methylation made growth take longer, larger animals have a larger gestation period] and inversely with ZNT5 methylation.  Methylation of the IGFBP3 locus correlated inversely with infant vitamin B(12) concentration[IGFBP3 decreases height], whilst global DNA methylation correlated inversely with maternal vitamin B(12) concentrations. Analysis of common genetic variants in folate pathway genes highlighted several associations including infant MTRR 66G>A genotype with DNA methylation and maternal MTHFR 677C>T genotype with IGF2 methylation. Both environmental and genetic factors involved in one-carbon metabolism influence DNA methylation in infants. Vitamin B(12) status, infant MTRR genotype and maternal MTHFR genotype, may influence the supply of methyl groups for DNA methylation. Gestational length [may determine] infant DNA methylation patterns."

"Reduced methylation at the IGF2 differentially methylated region, H19 DMR, in cord blood DNA has been associated with increased folic acid intake during pregnancy"<-IGF2 methylation could increase height and therefore folic acid intake during pregnancy may be contraindicated for height growth.

"maternal peripheral blood DNA methylation at the IGF2 locus was associated with maternal serum vitamin B12 levels"<-instead you'd want to take vitamin B12.


Bone morphogenetic protein-2 induces chromatin remodeling and modification at the proximal promoter of Sox9 gene.

"BMP-2 [induces] alterations in chromatin organization around the Sox9 core promoter. Nuclease hypersensitive site mapping following BMP-2 stimulation showed an inducible hypersensitive site in the Sox9 proximal promoter. BMP-2 increased the association of the transcription factor NF-Y with histone acetyltransferase p300/CBP.  The binding of the NF-Y-p300 complex to the Sox9 gene proximal promoter along with PCAF and RNA polymerase II. BMP-2 stimulation caused histone hyperacetylation and methylation at the Sox9 gene{But is Sox9 methylation a way to induce height growth or is it a marker that no further stimulation should be applied to Sox9?}. The activation of Sox9 gene transcription by BMP-2 is associated with chromatin remodeling and histone modification."

"BMP-2 regulates the chromatin structure of the Sox9 promoter through the p38 pathway, independent of the Smad pathway"


Epigenetic regulation of mesenchymal stem cells: a focus on osteogenic and adipogenic differentiation.

"The H3K27me3 mark is thought to be critical to the “stemness” of stem cells, as H3K27 demethylation triggers cellular differentiation"

"the ability of HMTs to methylate H3K9 in order to silence transcription often depends on the methylation status of adjacent lysine residues on H3"

"Acetylation of H3K9 (H3K9ac) and acetylation of H4K16 (H4K16ac) are common marks found on euchromatin near genes that are actively being transcribed"

"histone modification-mediated epigenetic alterations in late-passage MSCs may be responsible for a deceased ability to differentiate as cultured MSCs age."

"strong methylation of lineage specification and developmental promoters may restrict MSC differentiation capacity"


Epigenetic Regulation during Fetal Femur Development: DNA Methylation Matters.

" Using human embryonic stem cells, human fetal bone cells (HFBCs), adult chondrocytes and STRO-1(+) marrow stromal cells from human bone marrow, we have examined a spectrum of developmental stages of femur development and the role of DNA methylation therein. Using pyrosequencing methodology we analysed the status of methylation of genes implicated in bone biology; furthermore, we correlated these methylation levels with gene expression levels. During fetal femur development DNA methylation inversely correlates with expression of genes including iNOS (NOS2) and COL9A1{up}, but not catabolic genes including MMP13 and IL1B. Furthermore, significant demethylation was evident in the osteocalcin promoter between the fetal and adult developmental stages. Increased TET1 expression and decreased expression of DNA (cytosine-5-)-methyltransferase 1 (DNMT1) in adult chondrocytes compared to HFBCs could contribute to the loss of methylation observed during fetal development."

"COL9A1 expression levels were significantly correlated with fetal foot length"  Degree of Col9a1 methylation inversely correlated with foot length.

" the degree of [iNos] methylation was inversely correlated to foot length"

"[MMP13] expression significantly correlated with femur length"  Methylation of MMP13 is slightly inversely correlation with foot length.

Foot length is correlated with IL1B Methylation.

DNMT1 expression levels decrease with chondrocyte age.  There is much less of a correlation with age of TET1.

Grow Taller with Tensile Strain

Tensile strain is where an object such as a bone is being stretched.  This is the mechanism being applied by the limbcenter technique(bone stretching).  Now if you pull a pencil apart you're not getting very much tensile strain but if you try to bend the pencil you get much more tensile strain with much less force.  If you bend the pencil equally in both directions you will get equality of tensile strain.  Note that tensile strain is also important in creating gap fractures(a gap fracture is a microfracture induced in an osteon while the osteon is being stretched; new bone is then secreted from an osteocyte within the osteon allowing it to heal in a longer state).  What specific adaptations are induced by tensile strain and how can we use those to grow taller?

Tensile strain and magnetic particle force application do not induce MAP3K8 and IL-1B differential gene expression in a similar manner to fluid shear stress in human mesenchymal stem cells.

"We reported a potentially important role for mitogen-activated protein kinase kinase kinase 8 (MAP3K8) and interleukin-1beta (IL-1B) in MAPK signalling in MSCs exposed to fluid shear stress[fluid shear stress is induced by LSJL]. In this follow-up study, we examined the expression of these genes in MSCs exposed to other types of mechanical force: uniaxial tensile strain (3% cell elongation) and forces generated through the exposure of magnetic particle-labelled MSCs to an oscillating magnetic field (maximum field strength 90 mT). Exposure to both types of mechanical force for 1 h did not significantly alter the gene expression of MAP3K8 or IL-1B over the 24 h period subsequent to force exposure. These data demonstrate that uniaxial tensile strain and magnetic particle-based forces do not induce MAP3K8-related MAPK signalling in the same manner as does fluid flow-induced shear stress. This illustrates divergence in the process of mechanotransduction in mechanically stimulated MSCs"

"[There's a] consistent, significant and marked upregulation (2–50-fold) of the mitogen-activated protein kinase kinase kinase 8 (MAP3K8) and interleukin-1β (IL-1B) genes [in MSCs] in response to fluid shear stress"

MAPK regulates cellular proliferation and differentiation.  So LSJL activation of this is very important.  Now, it's important to note that the scientists only elongated the cells and not the entire bone.  Elongating the entire bone may also increase MAPK growth factors.

How much strain does it take to cause gap fractures?

Microcracking damage and the fracture process in relation to strain rate in human cortical bone tensile failure.[Osteons are the bone units of cortical bone]

"Traumatic failures in-vivo are more likely to be orders of magnitude faster than the quasistatic tests usually employed in-vitro. We have reported recently [The effect of strain rate on the mechanical properties of human cortical bone.] results from tests on specimens of human femoral cortical bone loaded in tension at strain rates (epsilon ) ranging from low (0.08s(-1)) to high (18s(-1)). Across this strain rate range the modulus of elasticity generally increased[modulus of elasticity is the tendency of bone to deform in response to strain; since it increased as strain increased bone became more deformed/lengthened], stress at yield[the yield stress is the point where the bone doesn't return to normal i.e. the bone stays lengthened] and failure and strain at failure decreased for rates higher than 1s(-1), while strain at yield was invariant for most strain rates and only decreased at rates higher than 10s(-1). The results showed that strain rate has a stronger effect on post-yield deformation than on initiation of macroscopic yielding[macroscopic means visible change in length; strain rate is the speed in which the strain is applied; so applying a strain faster was important then more length in the bone]. In general, specimens loaded at high strain rates were brittle, while those loaded at low strain rates were much tougher. Here, a post-test examination of the microcracking damage reveals that microcracking was inversely related to the strain rate. Specimens loaded at low strain rates showed considerable post-yield strain and also much more microcracking[microcracks occurred more when the strain was applied slowly; an increase in bone length may not be due to microcracks]. Partial correlation and regression analysis suggested that the development of post-yield strain was a function of the amount of microcracking incurred (the cause), rather than being a direct result of the strain rate (the excitation). Presumably low strain rates allow time for microcracking to develop, which increases the compliance of the specimen, making them tougher. This behaviour confirms a more general rule that the degree to which bone is brittle or tough depends on the amount of microcracking damage it is able to sustain. More importantly, the key to bone toughness is its ability to avoid a ductile-to-brittle transition for as long as possible during the deformation. The key to bone's brittleness, on the other hand, is the strain and damage localisation early on in the process, which leads to low post-yield strains and low-energy absorption to failure."

So, the faster the bone was increased in length the more likely the bone was to maintain the length post removal.  However, a slower change in length gave bone more time to microcrack(and thus for new bone to form in those gaps) thereby making the bone sturdier in the end.

Tensile strain may also have a positive effect on growth plate chondrocytes.

The effect of mechanical loading on the metabolism of growth plate chondrocytes.

"This study was aimed at evaluating the effect of tensile loadings with various frequencies on the proliferation of growth plate chondrocytes and extracellular matrix synthesis. The chondrocytes obtained from rib growth plate cartilage of 4-week-old male Wistar strain rats were cultured by day 4 and day 11 and used as proliferating and matrix-forming chondrocytes, respectively. Intermittent tensile stresses with different frequencies were applied to each stage chondrocyte. DNA syntheses were examined by measuring the incorporation of [(3)H]thymidine into the cells. Furthermore, the rates of collagen and proteoglycan syntheses were determined by measuring the incorporation of [2,3-(3)H]proline and [(35)S]sulfate into the cells, respectively. At the proliferating stage, intermittent tensions with the frequencies of 30 cycles/min and 150 cycles/min significantly (p < 0.05) upregulated the syntheses of DNA, which indicates the promotion of chondrocyte proliferation. At the matrix-forming stage, collagen, and proteoglycan syntheses also enhanced with increase of the loading frequency. In particular, the intermittent tension with the frequencies of 30 cycles/min and 150 cycles/min increased significantly (p < 0.05 or p < 0.01) both the collagen and proteoglycan syntheses. These results suggest that the proliferation and differentiation of growth plate chondrocytes are regulated by the mechanical loading and that the chondrocyte metabolism enhanced with increase of loading frequency."

A little like muscle growth, size of strain influences proliferation and differentiation(hypertrophy) while frequency of strain increases collagen and proteoglycan synthesis(like muscle nutrition).

Tensile damage and its effects on cortical bone.

Is length increase one of the effects?


"Plexiform bovine bone samples are repeatedly loaded in tension along their longitudinal axis. In order to induce damage in the bone tissue, bone samples are loaded past their yield point. Half of the bone samples from the damaged group were stored in saline to allow for viscoelastic recovery while the others were decalcified. Tensile tests were conducted on these samples to characterize the effects of damage on the mechanical behavior of the organic matrix (decalcified samples) as well as on bone tissue (stored in saline). The ultimate strain of the damaged decalcified bone is 29% higher compared to that of non-damaged decalcified (control) bone. The ultimate stresses as well as the elastic moduli are similar in both decalcified groups. This phenomenon is also observed in other collagenous tissue (tendon and ligament). This may suggest that damage in bone is caused by shear failure of the organic matrix; transverse separation of the collagen molecules or microfibrils from each other. In contrast, there is a trend towards lowered ultimate strains in damaged bone, which is soaked in saline, with respect to control bone samples (not damaged). The damaged bone tissue exhibits a bi-linear behavior in contrast to the mechanical behavior of non-damaged bone. The initial elastic modulus (below 55 MPa) and ultimate strength of damaged bone are similar to that in non-damaged bone."

"Damage in the form of cracks can start and develop by tensile failure of collagen molecules/fibrils and/or mineral, by shear failure between collagen molecules/fibrils, and/or by debonding of the organic matrix from the mineral platelets"<-Do these forms of damage lengthen bone?


"Under excess load, deformation of collagen fibers involves stretching, slippage of laterally adjoining elements and separation (in collagen molecule and/or in collagen fibril levels) and ultimately defibrillation of the fibrils from the overlap regions under shear force transmission"<-Note the usage of the word stretching, the collagen molecules stretch in until there is a slippage of adjoining elements until they separate.

"This behavior seems to be a characteristic of type I collagen as experiments on self assembled type I collagen fibrils indicate that a decrease in the diameter of the collagen microfibrils (thinner microfibrils) increases the toe region (low-strain elastic modulus) while an increase in the length of the collagen microfibrils increases the elastic modulus at higher strains as well as the ultimate strength"<-increase in the length of the collagen microbils likely leads to a height increase

"microcracks (<10 μm) were initiated at 0.4% tensile strain in equine bone and there was a considerable growth in microcrack density when tensile strain reached 0.8%"<-0.4% tensile strain are needed to cause microcracks and thereby height increase.  Calculating 0.4% tensile strain is unknown at this time.

Tensile strain(bone stretching) can enable you to grow taller by stretching collagen microfibrils until the point of breaking.

The importance of the elastic and plastic components of strain in tensile and compressive fatigue of human cortical bone in relation to orthopaedic biomechanics.

"The longevity, success, or failure of an orthopaedic implant is dependent on its osseointegration especially within the initial six months of the initial surgery. The development of strains plays a crucial role in both bone modelling and remodelling. For remodelling, in particular, strains of substantial values are required to activate the osteoblastic and osteoclastic activity for the osseointegration of the implant. Bone, however, is subject to "damage" when strain levels exceed a certain threshold level. Damage is manifested in the form of microcracks; it is linked to increased elastic strain amplitudes and is accompanied by the development of "plastic" (irrecoverable, residual) strains[what we would want is a residual strain such that increases the length of the bone]. Such strains increase the likelihood for the implant to subside or loosen. The present study examines the rates (per cycle) by which these two components of strain (elastic and "plastic") develop during fatigue cycling in two loading modes, tension and compression. The results of this study show that these strain rates depend on the applied stress in both loading modes. It also shows that elastic and plastic strain rates can be linked to each other through simple power law relationships so that one can calculate or predict the latter from the former and vice versa. We anticipate that such basic bone biomechanics data would be of great benefit to both clinicians and bioengineers working in the field of FEA modelling applications and orthopaedic implant surgery."

The difference between stress and strain is that strain actually changes the shape of the object in this case the bone.  To grow taller, strain must occur that causes microcracks not just stress.

Obviously tensile strain is very powerful.  If gradual tensile strain is applied to a bone, microcracks should appear in the osteons and the bone will grow longer one osteon at a time.  The question is:  How do we apply tensile strain to the bone?  If we do the medieval rack, the other types of tissues of the body are likely to fail far sooner than the bone.  And how do we do the vertebrae? 

Lateral Joint Loading does apply a distraction force on the bone(it applys a downward compressive force on the epiphysis and a stretching force along the entire shaft) but is it enough?  Sky's limbcenter method may show promise but he is being cryptic and not forthcoming with the effectiveness of the treatments.  Then there is the problem of loading the arms and upper leg(femur).

Here's an article about changes in genetic expression due to tensile strain:

Effects of Wnt3A and mechanical load on cartilage chondrocyte homeostasis.

"Chondrocytes were pre-stimulated with recombinant Wnt3A for 24 hours prior to the application of tensile strain (7.5%, 1 Hz) for 30 minutes. Activation of Wnt signalling, via β-catenin nuclear translocation and downstream effects including the transcriptional activation of c-jun, c-fos and Lef1, markers of chondrocyte phenotype (type II collagen (col2a1), aggrecan (acan), SOX9) and catabolic genes (MMP3, MMP13, ADAMTS-4, ADAMTS-5) were assessed.
Physiological tensile strain induced col2a1, acan and SOX9 transcription. Load-induced acan and SOX9 expression were repressed in the presence of Wnt3A[Wnt3A encourages cell spreading and cellular condensation is an important preliminary part of chondrogenesis]. Load induced partial β-catenin nuclear translocation[stabilized beta-catenin may inhibit stem cells from undergoing a chondrogenic lineage]; there was an additive effect of load and Wnt3A on β-catenin distribution, with both extensive localisation in the nucleus and cytoplasm. Immediate early response (c-jun) and catabolic genes (MMP3, ADAMTS-4) were up-regulated in Wnt3A stimulated chondrocytes. With load and Wnt3A there was an additive up-regulation of c-fos, MMP3 and ADAMTS-4 transcription, whereas there was a synergistic interplay on c-jun, Lef1 and ADAMTS-5 transcription.
Our data suggest that load and Wnt, in combination, can repress transcription of chondrocyte matrix genes, whilst enhancing expression of catabolic mediators[keep in mind this is tensile strain which is the primary load induced by LSJL]. Future studies will investigate the respective roles of abnormal loading and genetic predisposition in mediating cartilage degeneration."

So tensile strain may inhibit chondrogenesis but that doesn't mean that tensile strain won't play some role in height growth as objects tend to become longer when stretched even bone.  I owned a Stretch Armstrong and you could apply enough stretch such that the arms did not retract.

"In weight-bearing areas of the β-catenin cAct mice the articular cartilage surface was missing"

"After recovery, elevated mRNA levels of c-fos (1.65-fold: P < 0.01) and c-jun (1.5-fold: P < 0.05) were observed in cells subjected to tensile strain. An additive effect of Wnt3A and tensile strain was observed on c-fos transcription after recovery (2.2-fold; P < 0.01 compared to Wnt3A). In contrast, c-jun mRNA levels were significantly elevated in cells irrespective of treatment. Wnt3A treatment independently increased Lef1 mRNA levels after recovery (1.7-fold; P < 0.01), and the synergistic induction of Lef1 transcription remained in cells post-cessation of load (relative to untreated 2.4-fold: P < 0.001 or Wnt3A P < 0.05)."

"Applying a physiological strain induced the transcription of type II collagen (col2a1), aggrecan (acan) and SOX9 (SOX9), which are all markers of the chondrocyte phenotype; the mechano-responsive nature of these matrix genes in chondrocytes has been previously reported. Using this loading regime (in the absence of Wnt3A), β-catenin protein levels, as detected by Western blotting, were unaffected compared to expression in the untreated cells."<-So you don't want Wnt3A if you want to encourage chondrogenesis.  Wnt3A is the main factor to blame and not tensile strain.

"In bone, strain-induced β-catenin translocation (2% strain, 3600 cycles) was shown to result from inhibition of GSK3β activity, which was suggested to be mediated via activation of the Akt pathway"<-phosphorylation of GSK3Beta inhibits it.

How do we get rid of the confounding factors in tensile strain(like muscle fatigue) and effectively apply tensile strain?


Undifferentiated human mesenchymal stem cells (hMSCs) are highly sensitive to mechanical strain: transcriptionally controlled early osteo-chondrogenic response in vitro.

"hMSCs from 10 donors were intermittently stimulated by cyclic tensile strain (CTS) at 3000 mustrain for a period of 3 days. Differential gene expression of strained and unstrained hMSCs was analysed by real-time RT-PCR for several marker genes, including the transcription factors FOS, RUNX2, SOX9, and others. Additionally, alkaline phosphatase activity (ALP) was determined kinetically.
The application of CTS significantly stimulated the expression levels of the early chondrogenic and osteogenic marker genes (SOX9{up in LSJL}, LUM{UP}, DCN; RUNX2, SPARC, SPP1, ALPL); this was accompanied by stimulation of ALP activity (+38%+/-12 standard error of mean, P<0.05). Matrix analysis revealed that the osteo-chondrogenic response followed a coordinated expression pattern, in which FOS{up} was attributed to early osteogenic but not chondrogenic differentiation.
Undifferentiated hMSCs are highly sensitive to mechanical strain with a transcriptionally controlled osteo-chondrogenic differentiation response in vitro."

"Differentiation processes in hMSCs were found to be not synchronized throughout the cell populations, and several pheno- and genotypic investigations suggested that early differentiation may be regulated, at least in vitro, by stochastic[unorganized] mechanisms, while gene expression programs underlying late events in cell differentiation appear to be more fixed"

"no dependences were found between FOS messages of unstrained stem cells and either the chondrogenic or the osteogenic response"

"the activity of AP-1 may be involved in the early stage of MSC chondrogenesis"


Nanoscale Deformation Mechanisms in Bone


We would want the tensile strain to be all in one direction.

"bone deformation is not homogeneous but distributed between a tensile deformation of the fibrils and a shearing in the interfibrillar matrix between them."

"bone consists at the nanometer level of type I collagen molecules (300 nm long, 1.1−1.5 nm wide) interspersed with irregularly shaped nanocrystal platelets of carbonated apatite (3−5 nm thick and a lateral size of  50 nm). A composite of these two constituents forms the mineralized collagen fibril, with typically a diameter of 100 nm. The fibrils are hierarchically organized into lamellae and further on into the compact bone material, which, in combination with a cellular trabecular bone material, forms the organ bone."

"Because the collagen molecules are staggered axially along the fibril, a periodic electron density profile exists along the fibril axis (the D-period ≈ 64−67 nm), with the less dense regions referred to as the “gap” zones. Mineral particles nucleate and form first in the gap zones."

"the mineral density along the fibril axis is a step function (step length ≈ 0.46D^20), which results in a series of Bragg reflections with period 2π/D ≈ 0.094−0.098 nm-1. When a fibril is stretched, the gap zones move apart, and the fibril strain is measured from the percentage shift in D, as measured from the SAXS pattern."

"The typical load−deformation curve of bone showed an initial elastic range up to 0.5−0.6% strain, followed by a slower rate of stress increase with strain following the elastic/inelastic transition (in the postyield regime)."<-So you have to have over 0.7% strain in the longitidunal direction to get a permanent longitudinal increase in bone length.  A 0.7% strain would be a temporary increase of 0.7% in length.

Shear and compression differentially regulate clusters of functionally related temporal transcription patterns in cartilage tissue.

"we subjected intact cartilage explants to 1-24 h of continuous dynamic compression or dynamic shear loading at 0.1 Hz. We then measured the transcription levels of 25 genes known to be involved in cartilage homeostasis using real-time PCR and compared the gene expression profiles obtained from dynamic compression, dynamic shear, and our recent results on static compression amplitude and duration. Using clustering analysis, we determined that transcripts for proteins with similar function had correlated responses to loading. However, the temporal expression patterns were strongly dependent on the type of loading applied. Most matrix proteins were up-regulated by 24 h of dynamic compression or dynamic shear, but down-regulated by 24 h of 50% static compression, suggesting that cyclic matrix deformation is a key stimulator of matrix protein expression. Most matrix proteases were up-regulated by 24 h under all loading types. Transcription factors c-Fos and c-Jun maximally responded within 1 h to all loading types. Pre-incubating cartilage explants with either a chelator of intracellular calcium or an inhibitor of the cyclic AMP pathway demonstrated the involvement of both pathways in transcription induced by dynamic loading."

" Application of a transient, radially unconfined compressive deformation (using displacement or load control) induces an initial build up of hydrostatic pressure within the tissue and concomitant intratissue fluid flow and flow-induced electrical streaming potentials. Mechanical stress relaxation (at constant displacement) or creep relaxation (at constant load) then leads to a new static equilibrium compressed state of the tissue at which fluid exudation has ceased. In contrast, dynamic compression leads to cyclic changes in pressure, deformation, and fluid flow within the tissue. For the case of unconfined dynamic compression of cylindrical cartilage explant disks using impermeable compression platens, theoretical models have predicted frequency-dependent increases in the dynamic amplitude of the hydrostatic pressure and radial strain at the explant center, with radially directed fluid flow velocities greatest at the explant periphery. Dynamic tissue shear in the “simple shear” induces the cyclic matrix strain in a nearly uniform manner throughout the explant disk with minimal fluid flow or increased hydrostatic pressure. Normal joint motion in vivo produces a superposition of all these components of cartilage loading. "


FIGURE 1.
These are for cartilage but they should apply to the bone marrow as well and the growth plate if it exists.

"The expression of COX-2 and most matrix proteases increased by 100–200% by 24 h, although MMP3, MMP9, MMP13, and COX-2 were mainly suppressed during earlier time points"

Dynamic compression:

"Immediate early genes c-Fos and c-Jun and signaling genes mitogen-activated protein kinase-1 (MAPK1), and TNFα were transiently up-regulated by 100–200% after 1 h, returning to control levels after 8 h of loading"

"Sox9, interleukin 1β, and HSP70 were mildly up-regulated only at the 4-h time point"<-this differs from LSJL where Sox9 was significantly upregulated.

LSJL gene expression took place at 49 hours with 1 hour after the third loading.  So genes upregulated at both 1 hour after the time point and 24 hours may be shown in those results.

Genes upregulated at 1 or 24 hours by Dynamic Compression(over 2 fold) also upregulated by LSJL:
Aggrecan
MMP3
c-Fos
c-Jun
ADAMTS4
Col1a1

Col2a1 and Sox9 were upregulated by LSJL but not significantly upregulated by dynamic compression.  0.1Hz of 3% strain was used.  LSJL used 5Hz but far less strain.  The greater frequency may have made the difference.  Or the upregulation of Col2a1 and Sox9 could be due to ectopic chondroinduction of MSCs which is what we're hoping for.

Dynamic Tissue Shear:

"Similar to dynamic compression, c-Fos and c-Jun were transiently up-regulated by 2–3-fold within 1 h in response to dynamic shear, and c-Jun was also up-regulated by 2-fold after 24 h. TGFβ and interleukin 1β were mildly transiently up-regulated at early time points, and ribosomal 6-phosphate was unaffected. MAPK1 was up-regulated ∼150% after 1 and 24 h"

Genes upregulated at 1 or 24 hours by Dynamic Compression(over 2 fold) also up by LSJL:
Aggrecan
Col2a1
COL1A1
ADAMTS4
MMP3
TIMP1
Sox9
c-Fos
c-Jun
PTGS2(aka COX-2)

Static Compression:
Aggrecan
Col1a1
Col2a1
MMP3
ADAMTS4

Co-regulation of LPS and tensile strain downregulating osteogenicity via c-fos expression.

"Cultures of MC3T3-E1 osteoblasts were pre-treated with conditioned medium from RAW264.7 macrophages exposed to 100ng/ml Porphyromonas gingivalis (Pg)-LPS. Conditioned medium was analyzed by ELISA for interleukin-1 beta (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-α). Osteoblasts were then subjected to tensile strain (0.5Hz; 1000μ or 3000μ) for 0min, 5min, 15min, 30min, 1h, 3h, and 6h. The cultures were analyzed for mRNA and protein levels of c-fos. Cells were also analyzed for alkaline phosphatase (ALP) activity.
(Pg)-LPS stimulated the secretion of all three cytokines from RAW264.7 cells in a dose- and time-dependent manner. Medium from (Pg)-LPS stimulated cells induced a 10-fold increase in c-fos expression, which decreased to a 4-fold plateau after 3h. In contrast, ALP activity of control osteoblasts decreased during the first 60min, then recovered over the next 4h. Pretreatment with conditioned medium generated the same initial decrease during tensile strain but prevented the recovery.
Our study showed, for the first time, that the inhibitory effect of inflammation and tensile strain on osteogenicity is associated with the upregulation in c-fos expression{LSJL upregulates c-fos}. In addition, inflammation may reduce the ability of osteoblasts to restore their osteogenic capacity during sustained tensile stress and contribute to periodontium damage."

"LPS can indirectly mediate inflammation-induced bone remodeling through the induction of TNF-α released from macrophages"

"onditioned medium from Pg-LPS-stimulated macrophages contains all three major pro-inflammatory cytokines: TNF-α, IL-1β, and IL-6."