Tuesday, November 30, 2010

Super Growth Boosting with IGH-1

IGH-1(LG Sciences I-GH-1 100 Capsules) is one of the herbal remedies that people take with GHenerate on the grow tall forum.  IGH-1 is reported to increase the effectiveness of HGH on height.  Are there any ingredients in IGH-1 that can help us boost our heights?

One of the ingredients is Astragalus Membranaceous: 

The Herbal Formula HT042 Induces Longitudinal Bone Growth in Adolescent Female Rats. 

"The effect of HT042, a blend of three herbal extracts, on longitudinal bone growth was investigated in short- and long-term rat models. In the short-term model, we divided female Sprague-Dawley rats (3 weeks old) into six groups, according to treatment: vehicle, HT042 (100 mg/kg), Phlomis umbrosa[Anti-oxidant may inhibit growth cell killers like TNF-alpha] (100 mg/kg), Astragalus membranaceus (100 mg/kg), and Eleutherococcus senticosus (100 mg/kg) were administered twice daily, and recombinant human growth hormone (rhGH) (1 IU) was subcutaneously injected once daily. Treatments were maintained for 4 days in each case. On day 3, tetracycline (20 mg/kg) was injected intraperitoneally (20 mg/kg) to form the fluorescent band on the growth plates. On days 2-4, 5-bromo-2'-deoxyuridine (BrdU) (50 mg/kg) was injected intraperitoneally to label proliferating cells. On day 5, the tibias were dissected and fixed in 30% sucrose. Dehydrated bone was sectioned at a thickness of 40 μm and observed. The bone growth in groups administered HT042 and rhGH was significantly increased to 433.50 ± 21.61 and 434.49 ± 15.21 μm/day[the groups receiving HT402 had almost as much growth as the rhGH group], respectively, from 410.03 ± 17.4 μm/day (control)[Both methods resulted in an increase in growth rate of about 20 micro meters per day]. The height of the growth plates in the HT042 and rhGH groups was also significantly increased to 556.5 ± 21.1 and 544.2 ± 21.1 μm, respectively, from 518.1 ± 4.1 μm (normal). The number of BrdU-positive cells in chondrocytes of the HT042 and rhGH groups was increased to 389 ± 36 and 627 ± 39 cells/mm(2), respectively, from 264 ± 17 cells/mm(2) (control)[The rhGH group had almost twice as many proliferating cells than the HT402 group so HT402 must increase bone growth rate by a mechanism other than cellular proliferation; both increased cellular proliferation over control]. Insulin-like growth factor-1 and bone morphogenetic protein-2 in the HT042 group were highly expressed in the growth plate. In the long-term rat model, the body weight, nose-tail length, and nose-anus length were measured by microknemometry for 4 weeks. The body weight of the rhGH group was significantly increased. The nose-anus length of the HT042 and rhGH groups was significantly greater at 18.5 ± 0.3 and 18.7 ± 0.3 cm compared to 18.2 ± 0.2 cm (control)[Both methods resulted in growth rate increase]." 

Now increase in growth rate does not always equal increase in height growth.  It depends on how you increase growth rate.  If you are increasing chondrogenic proliferative capacity or peak chondrocyte hypertrophy you will grow taller.  Essentially astragalus membranaceus is a form of astragalus that has a higher concentration of TAT2 which lengthens telomeres(telomere shortening limits cellular proliferation).  

Eleutherocococcus senticosus seems to be another anti-inflammatory(inflammatory agents can kill growth plate cells which is bad for growth). 

Here's another study describing the growth benefits of the herbal supplement HT042:

Skeletal Growth and IGF Levels in Rats after HT042 Treatment.

"HT042, a new herbal prescription[although you can get many of the ingredients of HT042 without a prescription] consisting of Astragalus membranaceus, Phlomis umbrosa and Eleutherococcus senticosus, is used in traditional Korean medicine to stimulate growth in children. This study was conducted to investigate the effects of HT042 on skeletal growth, insulin-like growth factor-1 (IGF-1) and insulin-like growth factor binding protein-3 (IGFBP-3) levels, and oestrogenic activity in female rats. Female Sprague-Dawley rats were divided into control, recombinant human growth hormone (rhGH; 20 µg/kg/day), and HT042 (100 mg/kg/day) groups and treated for 3 weeks. Axial skeletal growth, femur length, and growth plate length were measured every 3 weeks. The serum IGF-1 and IGFBP-3 levels were analysed. Moreover, the oestrogenic activity of the herbal extracts in the immature and ovariectomized rats was tested. The nose-anus, nose-tail, femur and growth-plate lengths were increased significantly in the HT042 group. Both IGF-1 and IGFBP-3 were highly expressed in the hypertrophic zone of the growth plate. The serum IGF-1 levels were increased. Moreover, HT042 had no uterotrophic effects in the rats. Consequently, HT042 promoted longitudinal bone growth by stimulating cell proliferation in the epiphyseal plate and inducing the expression of IGF-1 without an oestrogenic response. HT042 may be helpful in stimulating growth in children with short stature. "

"Short-term treatment with GH increases plasma insulin and IGF-1 concentrations and decreases protein concentrations in urine."

"Korean traditional medicinal herbs have been used to treat children with growth retardation and results were recorded in the traditional medicinal book Dongeuibogam. "

So HT042 stimulated bone growth in length and to a greater degree than GH.  However, as always we can't be sure if this increase in growth rate results in increase adult height or just reaching adult height faster.  GH increased IGF-1 and IGFBP-3 levels higher than HT042 did.

Both GH and HT042 increased the hypertrophic zone size while slightly decreasing the proliferative zone size with GH doing so to a greater degree than HT042.  LSJL on the other hand increased both proliferative and hypertrophic zone.

"the IGF–IGFBP-3 complex increases the half-life of IGF-1 by 12 h compared with the 10 min of free IGF-1"

"E. senticosus induces IGF-1 expression and accelerates bone length in vivo"

Eleutherococcus senticosus extract attenuates LPS-induced iNOS expression through the inhibition of Akt and JNK pathways in murine macrophage. 

"Eleutherococcus senticosus (Araliaceae) is immunological modulator which has been successfully used for anti-inflammatory effectors on anti-rheumatic diseases in oriental medicine. Mitogen-activated protein kinases (MAPKs) and Akt modulate the transcription of many genes involved in the inflammatory process. In this study, we investigated the inhibitory effects of Eleutherococcus senticosus on the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in lipopolysaccharides (LPS)-activated macrophages. Finally, we studied the involvement of MAPKs and Akt signaling in the protective effect of Eleutherococcus senticosus in LPS-activated macrophages. Eleutherococcus senticosus significantly attenuated LPS-induced iNOS expression but not COX-2 expression. In using the standard inhibitors (MAPKs and Akt), our results show that Eleutherococcus senticosus downregulates inflammatory iNOS expression by blocking JNK and Akt activation." 

Now it would seem that Astragalus is the most likely to induce the increase in bone growth because there are tons of anti-inflammatories.  Of course the other two homeopathic medicines could have unknown effects. 

The other ingredients in IGH-1 are mostly growth hormone stimulants: 

Growth hormone isoforms release in response to physiological and pharmacological stimuli. 

"Ten healthy subjects used to performing regular physical activity and eight subjects affected by idiopathic isolated GH deficiency (GHD) were enrolled; 22- and 20-kDa GH secretion and its biological activity were evaluated in response to pharmacological stimuli such as arginine, L-dopa or glucagon in GHD children, while the hormonal response to exercise was studied according to Bruce protocol in healthy subjects. We found a significant increase in 22- and 20-kDa GH level in healthy subjects after monitored physical exercise (MPE; basal 0.28+/-0.12 vs 7.37+/-2.08 ng/ml and basal 0.076+/-0.04 vs 0.18+/-0.05 ng/ml, respectively). Furthermore, the 22-kDa/20-kDa ratio significantly increased in children who had undergone MPE and the GH bioactivity basal mean value also increased significantly after exercise (basal 2.86+/-0.76 vs 7.64+/-1.9 ng/ml). The mean value of 22-kDa GH in GHD patients increased significantly following GH pharmacological stimulation (2.78+/-0.63 ng/ml) when compared with mean basal (0.20+/-0.11 ng/ml) value. In the GHD group the basal concentration of 20-kDa GH significantly increased following GH pharmacological stimulation (0.34+/-0.11 vs 0.72+/-0.2 ng/ml); the 22-kDa/20-kDa ratio significantly increased too. Likewise, GH bioactivity in children with GHD increased significantly after pharmacological stimulation test (basal 2.53+/-0.56 vs 7.33+/-1.26 ng/ml). Both GH isoform concentrations and their biological activity are significantly increased in healthy subjects after submaximal exercise protocol and in GHD children after pharmacological stimuli." 

Elevating growth hormone levels may or may not increase height dependent on Growth Hormone Sensitivity levels and other factors. 

So IGH-1 is a Growth Hormone Booster and a Telomere length increaser.  Increasing these things may increase your height dependent on your sensitivity to growth hormone and the length of the telomeres in your chondrocytes(if they are shorter then that may be limiting cellular proliferation).  

More on HT042:

Effect of HT042, Herbal Formula, on Longitudinal Bone Growth in Spontaneous Dwarf Rats. 

"HT042 is a new herbal prescription consisting of Astragalus membranaceus, Phlomis umbrosa and Eleutherococcus senticosus, which are used in Korean medicine to stimulate growth in children. We investigated the effects of HT042 on the body weight, longitudinal bone growth, and bone length in spontaneous dwarf rats (SDR). Male and female SDRs were divided into three groups: the control group (DW, 10 mL/kg/day), the recombinant human GH group (rhGH; 500 µg/kg/day), and the HT042 (100 mg/kg/day) group. Each group received the respective treatments for 10 days. Body weight was measured on day 10 of treatment. On day 8, tetracycline (20 mg/kg) was injected intraperitoneally into all individuals to form a fluorescent band on the newly synthesized bone. On day 10, femur and tibia lengths were measured. Body weight, longitudinal bone growth, and bone length were not affected in the HT042 group. In contrast, the rhGH group showed significantly increased body weight, longitudinal bone growth, and bone length. In conclusion, HT042 does not act through a GH-like effect to promote longitudinal bone growth. "

"the administration of GH is limited by its poor absorption from the gastrointestinal tract."

"HT042 was reported to increase the longitudinal bone growth, growth plate height, femur length, and nose-anus (N-A) and nose-tail (N-T) lengths in adolescent Sprague-Dawley (SD) rats. HT042 stimulated chondrocyte proliferation and chondrocyte hypertrophy in the growth plate, and directly increased the longitudinal tibia length and the N-A and N-T length in adolescent female rats. The heights of the growth plate and the chondrocyte zone provide direct evidence of longitudinal bone growth. HT042 stimulates cell proliferation and promotes chondrocyte transformation into a bone matrix, where longitudinal bone growth occurs"

"The spontaneous dwarf rat (SDR) is a dwarf strain derived from SD rats. SDR was reported to be a homozygous GH-deficient rat strain that is unable to produce normal GH mRNA because of a splicing abnormality"<-So this will determine if the effects of HT042 are due to GH.

The average longitudinal bone growth in control SDR rats was 164.6+/-14.6 micrometers/day versus 152.5+/-39.4micrometers/day in the SDR + HT042 group.  In contrast, the HT042 formula had a minor statistically insignificant increase in longitudinal bone growth in female rats.  The high variance of longitudinal bone growth in males and the increase in longitudinal bone growth in females could mean that HT042 could have a positive or negative effect on longitudinal bone growth based on genetic factors possibly related to estrogen.  The final bone lengths given are consistent with this.

"The longitudinal bone growth effect of HT042 is induced by local IGF-1 and BMP"

You can get Astragalus Membranaceous super cheap here: Astragalus ( Astragalus membranaceus ) 1 Oz Herb Pharm.

Monday, November 29, 2010

Boost Height with Insulin

Insulin acts through the PI3K pathway to increase cellular proliferation and differentiation.  Leptin, Ghrelin, and Insulin would seem to not synergistic with each other but you can alter these levels by chemical and physical means.  Can altering Insulin levels and Insulin sensitivty(like IRS-1 which maintains the proper rate of bone turnover) boost height? 

Insulin Resistance Secondary to a High-Fat Diet Stimulates Longitudinal Bone Growth and Growth Plate Chondrogenesis in Mice. 

"It is known that overweight children are often more insulin resistant and taller than normal-weight peers. Because it has been hypothesized that insulin is implicated in the obesity-associated growth acceleration, we aimed to determine whether insulin resistance and secondary hyperinsulinemia are the causative mechanisms of such growth acceleration. Three-week-old mice were fed with standard chow or with a high-fat diet without or with daily administration of pioglitazone[a diabetic medication which reduces insulin resistance]. After 6 wk, high-fat mice' body and tibial growth, tibial growth plate height, and serum insulin were all greater than those of standard chow-fed mice. High-fat + pioglitazone mice were shorter, their tibial growth and the growth plate height reduced, and their insulin lower than those of high-fat mice. The addition of insulin to the culture medium of mouse metatarsal bones induced the metatarsal linear growth and increased the metatarsal growth plate height. In addition, insulin stimulated cultured chondrocyte proliferation and differentiation, with both effects being prevented by transfection with a small interfering RNA targeted to the insulin receptor. In conclusion, in high fat-fed mice, insulin resistance is causally related to accelerated skeletal growth. Our in vitro findings suggest that insulin may directly modulate skeletal growth by activating the insulin receptor directly at the growth plate." 

So it may not be Insulin that makes you taller but rather may insulin accelerates fusion via IRS-1 or other receptors.  Now we know that complete absence of IRS-1 will make you shorter but perhaps reducing the sensitivity of IRS-1 to insulin will result in you increasing in height.

"Pioglitazone is a synthetic ligand of the peroxisome-proliferator activated receptor-{gamma}; by activating this receptor, it increases insulin sensitivity in the liver, fat, and skeletal muscle cells"

So Pioglitazone is a way to measure the effect of insulin sensitivity on height growth.  Mice who took a high fat diet with pioglitazone were shorter.

"No difference was found between PIO and control mice with respect to body weight gain, body cumulative growth, tibial growth, and growth plate morphology."

So in normal diet rats there was no effect of PIO meaning that PIO does not have height lowering effects independent of a high fat diet.

"In the high-fat + PIO mice, serum insulin level was significantly lower than that of high-fat micewhereas no significant difference was detected between high-fat + PIO and high-fat mice or between high-fat + PIO and control mice relative to the serum IGF-I, leptin, IGFBP-1, IGFBP-3, testosterone, estradiol, FFA, and β-HB levels"

Insulin is what seems to exert the effects on height rather than related hormones.

"In the high-fat + PIO mice, the expression of phosphorylated-IR was weaker compared with the high-fat mice."

IR stands for insulin receptor.  Phosphorylated means it's inactivated.

"high-fat mice exhibited increased phosphorylated-IR expression"

High fat mice had more inactive insulin receptors in the growth plate.  IRS-1 is an example of an insulin receptor.

Likely what's happening is that the Insulin Receptors compete for insulin with something that increases height.  Insulin resistance allows for more serum levels of insulin available for other types of cells.  IRS-1 deficiency has been shown to be detrimental to height growth so it's unlikely that inactivation of insulin receptors is causing the height growth.


"Our findings of the lack of any stimulatory effect of insulin on growth plate chondrocytes with a targeted silencing of the IR (and the persistent insulin stimulatory effects on chondrocytes with a targeted silencing of the IGF-IR) also support an IR-mediated regulatory role for insulin on skeletal growth and growth plate chondrogenesis."

Insulin does not have a stimulatory effect on chondrocytes apart from insulin receptors.  Maybe the effect of insulin could be on another cell type.

The role of insulin in chondrogenesis. 

"The ATDC5 chondrogenic cell line is typically induced to differentiate by exposure to insulin at high concentration[This can be bad as you want chondrocytes to proliferate for as long as possible] (10 microg/ml, approximately 1600 nM). Differentiation can also be induced by physiological concentrations of insulin-like growth factor-I (IGF-I). Unlike previous reports, we observed a stimulation of differentiation, as measured by collagen X expression and Alcian Blue staining for proteoglycan synthesis, upon exposure to insulin at concentrations (10-50 nM) consistent with signaling via the insulin receptor. Analysis of lysates from proliferating and hypertrophic ATDC5 cells demonstrated that exposure to 50 nM insulin induced tyrosine phosphorylation of insulin receptors but not IGF-I receptors or hybrid receptors. In contrast to the potent effects of IGF-I to stimulate both ATDC5 proliferation and differentiation, insulin was not as potent as IGF-I as a proliferating agent but more selectively a differentiating agent. Consistent with this result, insulin was less potent than IGF-I in inducing activation of the Erk1/Erk2 mitogenic signaling pathway. Furthermore, Erk pathway inhibition did not enhance the differentiating effects of insulin as it does in the case of IGF-I exposure. Extending our observations to fetal rat metatarsal explants, we observed significant stimulation of bone growth by 50 nM insulin. This could be accounted for by a disproportionate stimulatory effect on growth of the hypertrophic zone. The proliferative zone was not significantly affected. Based on our results in both ATDC5 cells and metatarsal explants, we conclude that the insulin functioning through insulin receptor has a dominant effect as an inducer of chondrocyte differentiation." 

This is consistent with the hypothesis that elevated levels of IRS-1 induce premature chondrocyte differentiation.  Organisms Transgenic in IRS-1 have shown enhanced hypertrophy in non-skeletal organs but the effect of transgenic IRS-1 one in bone really has to be studied separately as bone is unique in that premature differentiation of chondrocytes will result in reduced growth potential by shortening the proliferative phase. 

The High Fat +High Insulin Resistance Study was pretty recent so we'll have to watch to see for more details about how to manipulate insulin and insulin resistance for height gain. 

Let's look at some other functions of Insulin within the bone.

Novel functions for insulin in bone.

"The contemporary model assigns IGFs as central regulators of cell proliferation, survival, and organism growth, whereas insulin's action dominates at the level of regulation of fuel accumulation, storage[increased storage of water could encourage chondrogenic differentiation], and energy expenditure."

"leptin, an adipose-tissue-derived hormone that conveys critical information about peripheral energy storage and availability to the brain"<-Leptin can help you grow taller and Leptin also involves energy storage.  Remember that Leptin supplementation helps to reduce the height stunting effects of fasting.

"The skeleton is a highly metabolic tissue and is increasingly recognized as an important player in the coordination of global energy use through its hormonal interactions with other tissues"<-How to use this fact to increase height remains to be seen.

"osteoblasts express functional IR and respond to exogenous insulin by increasing bone anabolic markers, including collagen synthesis, alkaline phosphatase production, and glucose uptake"

No mention is made of chondrocytes, however chondrocytes can benefit from the same nutrient uptake, and this can increase height. 

Here's a study on insulin on human birth length:

Effects of cord serum insulin, IGF-II, IGFBP-2, IL-6 and cortisol concentrations on human birth weight and length: pilot study.

"The IGF system is recognised to be important for fetal growth. We previously described increased Insulin-like growth factor binding protein (IGFBP)-2 cord serum concentrations in intra-uterine growth retardation (IUGR) compared with appropriate for gestational age (AGA) newborns[decrease IGFBP-2 levels to grow taller], and a positive relationship of IGFBP-2 with Interleukin (IL)-6[IL-6 has positive height increasing effects it depends on whether those outweigh it's effects on IGFBP-2]
23 IUGR and 37 AGA subjects were followed up from the beginning of pregnancy, and were of comparable gestational age. Insulin, IGF-II, IGFBP-2, cortisol and IL-6 concentrations were assayed in cord serum at birth, and a multiple regression model was designed and applied to assess which were the significant biochemical determinants of birth size.
Insulin, cortisol, and IL-6, showed similar concentrations in IUGR and AGA as previously described, whereas IGF-II was lower, and IGFBP-2 increased in IUGR compared with AGA. IGF-II serum concentration was found to have a significant positive effect on both birth length (r:(:)0.546; p: 0.001) and weight (r:0.679; p: 0.0001). IGFBP-2 had a near significant negative effect on both birth weight (r:-0.342; p: 0.05) and length (r:-0.372; p:0.03)."

Insulin and cortisol did have a slight positive effect on birth length.

"Insulin increases IGFBP-2, and that IGFBP-2 was an independent predictor of insulin sensitivity."<-this could be why insulin resistance increases height. Lower IGFBP-2 levels.

"[There's] a positive effect of insulin placental concentration on both birth length and weight (unpublished), although the major effect would be mediated through cortisol placental concentration."

The increase in O-GlcNAc protein modification stimulates chondrogenic differentiation both in vitro and in vivo.

"Our aim was to investigate whether insulin-induced chondrocyte hypertrophy occurs through a modification in the amount of O-linked-N-acetylglucosamine (O-GlcNAc) modified proteins and in the expression of the key enzymes of this pathway: O-GlcNAc-transferase (OGT) and O-GlcNAcase (OGA). We also studied if O-GlcNAc accumulation per se, induced by an OGA inhibitor, was able to induce pre-hypertrophic chondrocyte differentiation both in vitro and in vivo. Insulin-induced differentiation of ATDC5 pre-chondrocytes occurred alongside with a gradual increase in the accumulation of O-GlcNac modified proteins (O-GlcNAcylated proteins), as well as an increase in the expression of OGT and OGA. In the absence of insulin, O-GlcNAc accumulation induced by thiamet-G, a specific OGA inhibitor, was able to increase the gene expression of differentiation markers, as well as the activity of MMP-2 and -9, although it was not able to induce proteoglycan accumulation. Thiamet-G also activated pERK, p-JNK and p-p38, and the O-GlcNAcylation of Akt. Thiamet-G administration to C57/bl mice induced a significant expansion in the growth plate height and in the hypertrophic zone height."

"The concentration of UDP-GlcNAc is regulated by glucose levels and is synthesized by the hexosamine biosynthesis pathway (HBP)"

"thiamet-G administration induces a significant increase in endochondral plate height, probably due to an increased length of the hypertrophic zone. However, we did not find this effect to be paralleled by an increase in tibial length. Based on our experiments, we were unable to determine whether a longer treatment with thiamet-G would induce an increase in tibial length."

Boosting Height with GHenerate?

Several people on the grow tall forum have reported height increase with GHenerate(LG Science GHenerate Spray, 4-Ounce-if you want to try please take before and after pics).  GHenerate, which is produced by LGSciences, contains puerarin which is a strong bone growth and PI3K pathway stimulator.  PI3K is a potent anabolic stimulus.  Puerarin stimulates osteoblasts and may increase height if the osteoblasts decide to deposit new bone on the surface of the long bones.  PI3K is also anabolic to chondrocytes but I don't see how a PI3K stimulator alone is making people 1 to 2 inches taller in such a short time frame(the claims are not supported by photographic evidence of any kind, PI3K stimulators should result in thicker bones).  PI3K stimulation can help grow bone during puberty as well...

PI3K/Akt signaling as a key regulatory pathway for chondrocyte terminal differentiation.

"Phosphoinositide-3 kinase (PI3K) and its major downstream molecule, Akt, a serine-threonine kinase, play pivotal roles in [chondrogenesis]. Akt signaling was activated in resting and proliferative chondrocytes but was reduced during terminal differentiation. We adopted two chondrocyte differentiation systems to investigate the roles of PI3K/Akt signaling in chondrogenesis. First, we employed an embryonic forelimb organ culture of transgenic mice expressing an Akt-Mer (a ligand-binding domain of a mutated estrogen receptor) fusion protein whose kinase activity was conditionally activated by treatment with 4-hydroxytamoxifen (4OHT). Activation of Akt signaling in embryonic chondrogenesis enhanced chondrocyte proliferation and inhibited hypertrophic differentiation, presumably due to the suppressed expression of Runx2, a transcription factor critical for chondrocyte terminal differentiation. Conversely, inhibition of PI3K by its inhibitor accelerated terminal hypertrophic differentiation, resulting in a shorter bone. Essentially the same results were obtained in a second line of experiments using human synovial stromal cells (hSSCs), which are mesenchymal progenitor cells isolated from adult joints.  PI3K/Akt signaling is a key regulator in terminal chondrocyte differentiation in both embryonic and adult chondrogenesis."

PI3K regulates Akt which slows down terminal differentiation of chondrocytes.  Increasing PI3K and in turn Akt will result in more height growth by allowing for more proliferative cycles before terminal chondrocyte differentiation.  That would not explain how people are growing post epiphyseal and diaphyseal fusion.  Perhaps PI3K interacts with DNA Methylation in some way allowing for a rebirth of chondrocytes.

Vitamin D receptor expression is associated with PIK3CA and KRAS mutations in colorectal cancer.

"The vitamin D receptor (VDR) is a transcription factor, which plays an important role in cellular differentiation and inhibition of proliferation[VDR reduces height gain]. A link between VDR and the RAS-mitogen-activated protein kinase (MAPK) or phosphatidylinositol 3-kinase (PI3K)-AKT pathway has been suggested. However, the prognostic role of VDR expression or its relationship with PIK3CA or KRAS mutation remains uncertain. Among 619 colorectal cancers in two prospective cohort studies, 233 (38%) tumors showed VDR overexpression by immunohistochemistry. We analyzed for PIK3CA and KRAS mutations and LINE-1 methylation by Pyrosequencing, microsatellite instability (MSI), and DNA methylation (epigenetic changes) in eight CpG island methylator phenotype (CIMP)-specific promoters [CACNA1G, CDKN2A (p16), CRABP1, IGF2, MLH1, NEUROG1, RUNX3, and SOCS1] by MethyLight (real-time PCR). VDR overexpression was significantly associated with KRAS mutation (odds ratio, 1.55; 95% confidence interval, 1.11-2.16) and PIK3CA mutation (odds ratio, 2.17; 95% confidence interval, 1.36-3.47), both of which persisted in multivariate logistic regression analysis. VDR was not independently associated with body mass index, family history of colorectal cancer, tumor location (colon versus rectum), stage, tumor grade, signet ring cells, CIMP, MSI, LINE-1 hypomethylation, BRAF, p53, p21, beta-catenin, or cyclooxygenase-2. VDR expression was not significantly related with patient survival, prognosis, or clinical outcome. In conclusion, VDR overexpression in colorectal cancer is independently associated with PIK3CA and KRAS mutations. Our data support potential interactions between the VDR, RAS-MAPK and PI3K-AKT pathways."

The PI3K pathway may alter the Methylation status of the Vitamin D Receptor allowing for further growth after puberty.

The next ingredient of Ghenerate is a peptide in Marus Alba that mimics the effects of GHRP and Ghrelin(Ghrelin is a growth factor for osteoblasts and chondrocytes).

Ghrelin in plants: what is the function of an appetite hormone in plants?

"The expression of a ghrelin-like peptide hormone that has only been associated with animals, in various plant tissues. Ghrelin, the appetite stimulating hormone, has been identified from a number of different species including humans, rat, pig, mouse, gerbil, eel, goldfish, bullfrog and chicken. The study here was conducted using an immunohistochemistry assay to screen whether plants have any ghrelin immunoreactivity. In this respect, Prunus x domestica L. and Marus alba were examined. Immunohistochemistry results showed that there is a strong human ghrelin immunoreactivity substance in the parenchyma cells of these plants. This was entirely unexpected since this hormone was considered to be present solely in animals. Thus, this study is the first to report the presence of a peptide with ghrelin-like activity in plants, a finding that has only been observed in the animal kingdom. RIA analysis confirmed that these plants contain significant amounts of this substance. Furthermore, reverse-phase HPLC analyses of plant extracts showed an elution characteristic of the peptide identical to that of human ghrelin. In general, fruit from both plants had higher levels of the peptide than the vegetative parts."

This is the only study on the site regarding Marus Alba(could be actually Morus Alba).  We don't know whether consuming exogenous Ghrelin in plant form will result in higher serum levels in humans or whether this Ghrelin will be effective.

My advice skip Ghenerate and save money by buying plain puerarin.

Saturday, November 20, 2010

Add some inches to your height with lactoferrin?

Lactoferrin was recently mentioned on the grow tall forum as a possible mechanism in which height can be increased.  Lactoferrin is present in milk making milk doubly attractive due it's ability to also increase circulating levels of IGF-1.  Even though Lactoferrin is present in milk it's possible that additional supplemental Lactoferrin may increase height further.

Inhibitory effect of lactoferrin on hypertrophic differentiation of ATDC5 mouse chondroprogenitor cells.

"The skeleton is formed by two different mechanisms. In intramembranous ossification, osteoblasts form bone directly, whereas in endochondral ossification, chondrocytes develop a cartilage template, prior to osteoblast-mediated skeletogenesis[both osteoblasts and chondrocytes have the ability to add inches to your height]. Lactoferrin is an iron-binding glycoprotein belonging to the transferrin family. It is known to promote the growth and differentiation of osteoblasts. In this study, we investigated the effects of bovine lactoferrin on the chondrogenic differentiation of ATDC5 chondroprogenitor cells. This mouse embryonic carcinoma-derived clonal cell line provides an in vitro model of chondrogenesis. Lactoferrin treatment of differentiating ATDC5 cells promoted cell proliferation in the initial stage of the differentiation process[So Lactoferrin increases chondrocyte proliferation]. However, lactoferrin treatment resulted in inhibition of hypertrophic differentiation, characterized by suppression of alkaline phosphatase activity[although it's mostly methylation status that affects terminal differentiation and not alkaline phosphatase activity], aggrecan synthesis and N-cadherin expression. This inhibitory effect was accompanied by sustained Sox9 expression, as well as increased Smad2/3 expression and phosphorylation, suggesting that lactoferrin regulates chondrogenic differentiation by up-regulating the Smad2/3-Sox9 signaling pathway[Smad 2/3 phosphorylation is the good kind]."

So lactoferrin could potentially increase height by inhibiting chondrogenic hypertrophic differentiation and keeping chondrocytes in the proliferating phase longer.  You need the chondrocytes to undergo hypertrophic differentiation eventually but what Lactoferrin can do is allow for each chondrocyte to maximally proliferate before undergoing hypertrophy.

"Chondrogenic differentiation is characterized by drastic changes in cell shape, which results in the conversion of a flattened shape to a round or polygonal morphology. Rho GTPase acts as a cell shape regulator by controlling organization, polymerization and de-polymerization of actin stress fibers in chondrocytes, as well as other types of cells. Activated (GTP-bound) RhoA stabilizes actin stress fibers by up-regulating both RhoA kinase (ROCK) and LIM kinase. It has been shown that over-expression of RhoA enhances cell proliferation and inhibits hypertrophic differentiation of ATDC5 cells, while inhibition of ROCK activity results in promotion of hypertrophic differentiation. Thus, RhoA/ROCK signaling inhibits the hypertrophic differentiation of chondrocytes, while promoting the proliferation and initial stage of chondrogenic differentiation. Data from our laboratory demonstrate that lactoferrin enhances collagen gel contractile activity in human fibroblasts by activating Rho and MLCK[Lactoferrin activates Rho activity, although this may be a bad thing as MLCK inhibition has been shown to induce chondrogenesis], which suggests that RhoA/ROCK may be involved in lactoferrin-mediated inhibition of hypertrophic differentiation in ATDC5 cells"<-Now the problem with RhoA is that the formation of actin stress fibers seems to be a cause of chondrogenic de-differentiation.  Thus, too much Lactoferrin may have negative effects on height growth.  But Lactoferrin up to a point may be a great boon for height growth.

"Mesenchymal cells undergo chondrogenic differentiation through a cellular condensation stage. N-cadherin is a calcium-dependent cell adhesion molecule that is required for both pre-cartilage condensation and cartilage nodule formation during chondrogenic differentiation[So not being deficient in calcium is important for height growth]. Inhibition of N-cadherin-mediated cell adhesion results in abrogation of cell condensation and subsequent chondrogenesis[without cellular condensation, there is no chondrogenesis.  The compression of the epiphysis during LSJL helps induce this cellular condensation by reducing the space available]. Here, we observed that lactoferrin antagonized N-cadherin expression in differentiating ATDC5 cells. Although Sox9 was reported to bind specifically to an N-cadherin gene promoter region and to increase the transcription and translation of N-cadherin, Sox9 is not essential for N-cadherin expression in vivo[The negative effects of Lactoferrin on cellular condensation may be counteracted by the increase in Sox9 activity]. This finding suggests that in addition to Sox9, it is possible that lactoferrin controls the expression of other genes required for N-cadherin expression."

Signal transduction and metabolism in chondrocytes is modulated by lactoferrin.

"Activation of granulocytes causes a considerable rise in the concentration of lactoferrin (Lf) in synovial fluid (SF).
Signal transduction was analysed in cultured chondrocytes by immunodetection of mitogen activated protein kinases (MAPK) and analysis of Smad2 translocation to the nucleus. Expression levels of matrix metalloproteinases (MMPs) and of aggrecan were measured by reverse-transcription-PCR. The proteolytic activity of MMPs was ascertained by zymography. Expression of the low-density-lipoprotein-receptor-related-protein-1 (LRP-1), a Lf receptor for signalling, was assayed by immunohistochemistry in cartilage and in cultured chondrocytes by immunoblotting.
We found LRP-1 expressed in dedifferentiated chondrocytes in culture and in cartilage tissue preferentially on the articular surface where it can encounter Lf within SF. Lf stimulated proliferation of chondrocytes, comparable to transforming growth factor-beta1 (TGFbeta1) and activated p38 and the extracellular-signal regulated-kinases 1/2 (ERK1/2) within minutes. Surprisingly, Lf induced nuclear Smad2 translocation, a signal pathway ascribed to TGFbeta receptor activation. Lf significantly increased the levels of catabolic indicators such as MMP1, MMP2, MMP3 and MMP13 and inhibited aggrecan synthesis.
Lf is a robust regulator of chondrocyte metabolism, comparable to TGFbeta1. The catabolic influence together with the proliferative stimulus indicates a function as an early phase cytokine, enhancing MMPs, necessary for degradation of damaged tissue and stimulating proliferation of chondrocytes, necessary for reconstruction."

Again Lactoferrin stimulates chondrocyte proliferation and inhibits chondrocyte hypertrophy as indicated by the inhibition of aggrecans.  Lactoferrin is involved in signal transduction making it of particular interest to LSJL.

Lactoferrin promotes bone growth.

"In vitro, lactoferrin stimulates the proliferation of bone forming cells, osteoblasts, and cartilage cells at physiological concentrations (above 0.1 microg/ml). The magnitude of this effect exceeds that observed in response to other skeletal growth factors such as IGF-1 and TGFbeta. DNA synthesis is also stimulated in a bone organ culture system likely reflecting the proliferation of cells of the osteoblast lineage. Lactoferrin is also a potent osteoblast survival factor. In TUNEL and DNA fragmentation assays, lactoferrin decreased apoptosis, induced by serum withdrawal, by up to 70%. In addition, lactoferrin has powerful effects on bone resorbing cells, osteoclasts, decreasing osteoclast development at concentrations > 1 microg/ml in a murine bone marrow culture system. However, lactoferrin did not alter bone resorption in calvarial organ culture, suggesting that it does not influence mature osteoclast function. In vivo, local injection of lactoferrin in adult mice resulted in increased calvarial bone growth, with significant increases in bone area and dynamic histomorphometric indices of bone formation after only 5 injections. Taken together, these data demonstrate that the naturally-occurring glycoprotein lactoferrin is anabolic to bone in vivo, an effect which is consequent upon its potent proliferative and anti-apoptotic actions in osteoblasts, and its ability to inhibit osteoclastogenesis."

In this study it is suggested that Lactoferrin is more anabolic than TGF-Beta or IGF-1.

Lactoferrin is a potent regulator of bone cell activity and increases bone formation in vivo.

"Lactoferrin is an iron-binding glycoprotein present in epithelial secretions, such as milk, and in the secondary granules of neutrophils. We found it to be present in fractions of milk protein that stimulated osteoblast growth, so we assessed its effects on bone cell function. Lactoferrin produced large, dose-related increases in thymidine incorporation in primary or cell line cultures of human or rat osteoblast-like cells, at physiological concentrations (1-100 microg/ml). Maximal stimulation was 5-fold above control. Lactoferrin also increased osteoblast differentiation and reduced osteoblast apoptosis by up to 50-70%. Similarly, lactoferrin stimulated proliferation of primary chondrocytes. Purified, recombinant, human, or bovine lactoferrins had similar potencies. In mouse bone marrow cultures, osteoclastogenesis was dose-dependently decreased and was completely arrested by lactoferrin, 100 microg/ml, associated with decreased expression of receptor activator of nuclear factor-kappaB ligand. In contrast, lactoferrin had no effect on bone resorption by isolated mature osteoclasts. Lactoferrin was administered over calvariae of adult mice for 5 d. New bone formation, assessed using fluorochrome labels, was increased 4-fold by a 4-mg dose of lactoferrin. Thus, lactoferrin has powerful anabolic, differentiating, and antiapoptotic effects on osteoblasts and inhibits osteoclastogenesis."

So this study gives us some doses five fold above control.  Unlike other bone stimulating mechanisms, lactoferrin does not increase alkaline phosphatase activity and actually inhibits chondrocyte hypertrophy enabling chondrocyte proliferation to occur longer.  

"Comparable effects were found in primary cultures of human osteoblasts treated with bovine lactoferrin (Fig. 1D), and recombinant human lactoferrin was possibly more potent at low concentrations in these cells, increasing thymidine incorporation 2-fold at 10 µg/ml (data not shown). With this exception, the species of origin and method of preparation of the lactoferrin did not substantially impact the extent of the proliferative effect."

Effects of lactoferrin on the differentiation of pluripotent mesenchymal cells.

"We examined the effect of lactoferrin on the differentiation of pluripotent mesenchymal cells using a typical pluripotent mesenchymal cell line, C2C12. Cells were cultured in low-mitogen differentiation medium to induce cell differentiation, with or without the addition of lactoferrin. The cell lineage was determined by alkaline phosphatase (ALPase) activity, mRNA expression of cellular phenotype-specific markers using real-time polymerase chain reaction (PCR), and protein synthesis using Western blotting. The expression of low-density lipoprotein lipase receptor-related proteins (LRPs) 1 and 2, both lactoferrin receptors, was determined by reverse transcription-PCR. ALPase activity increased after the addition of lactoferrin. The mRNA expression of Runx2, osteocalcin, and Sox9 increased markedly as a result of lactoferrin treatment, whereas the expression of MyoD, desmin, and PPARgamma decreased significantly. Western blots showed that lactoferrin stimulation increased Runx2 and Sox9 proteins, whereas it decreased MyoD and PPARgamma synthesis. C2C12 cells expressed the LRP1 lactoferrin receptor. These results indicate that lactoferrin treatment converts the differentiation pathway of C2C12 cells into the osteoblastic and chondroblastic lineage."

More on Lactoferrin and Smad3:

Sustained co-cultivation with human placenta-derived MSCs enhances ALK5/Smad3 signaling in human breast epithelial cells, leading to EMT and differentiation.

"We investigated whether the interactions between mammary epithelial cells and human placenta-derived MSCs (hPMSC) affect the morphology, proliferation, and differentiation of epithelial cells in a co-culture system. We show that after co-culture with hPMSCs, human mammary epithelial cell lines (MCF-10F and HEMC) underwent significant morphologic alterations and a dramatic increase in ductal-alveolar branching, which was accompanied by a decrease or loss of the epithelial marker E-cadherin and a gain of the mesenchymal markers, alpha-SMA and vimentin. MCF-10F and HEMC proliferation was also inhibited in the presence of hPMSCs, and this retardation in growth was due to cell cycle arrest. Furthermore, in MCF-10F and HMEC cells, hPMSCs induced the production of lipid droplets, milk fat globule protein, and milk protein lactoferrin, which are markers of functional mammary differentiation. We also noticed an elevation in ALK5 and phosphorylated Smad3 protein levels upon hPMSC co-culture[so human placental cells increase lactoferrin levels]. Strikingly, the changes in morphology, proliferation, and differentiation were reversed by treatment with ALK5 or Smad3 knockdown in MCF-10F/hPMSC co-cultures. Collectively, our findings suggest that co-cultivation with hPMSCs leads to epithelial to mesenchymal transition (EMT) and differentiation of human breast epithelial cells through the ALK5/Smad3 signaling pathway."

"Lactoferrin [is] an iron-binding milk protein"

According to Differences in whey protein content between cow's milk collected in late pasture and early indoor feeding season from conventional and organic farms in Poland., lactoferrin levels were (Lf, 334.99 [Late Pasture] vs. 188.02 mg [early indoor feeding] L(-1) ).  So Lactoferrin levels in milk are not significantly higher in supplements than milk.

Lactoferring can be absorbed by humans and used by the body.

Bovine lactoferrin can be taken up by the human intestinal lactoferrin receptor and exert bioactivities.

"An intestinal enterocyte model (Caco-2 cells) was used to compare the ability of bovine LF (bLF) purified by our laboratory and CbLF[commercially available bovine-lactoferrin] with hLF[Human Lactoferrin] to resist digestion, bind to the receptor, and exert bioactivities, including cellular proliferation, differentiation, interleukin 18 secretion, and transforming growth factor-β1 expression.
bLf and CbLF, which are partially iron (Fe)-saturated, can bind additional Fe, partially resist digestion either dissolved in phosphate buffered saline or in the presence of infant formula at conditions similar to those of the infant gastrointestinal tract, and bind to Caco-2 cells in a manner similar to hLF. bLF and CbLF, as well as bound Fe, also are internalized by Caco-2 cells, as demonstrated by I and Fe labeling, albeit to somewhat less of an extent than hLF. CbLF promoted cell proliferation and differentiation to an extent similar to that of bLF and hLF, but these effects were not seen when the LF samples were saturated with Fe (holo-LF). Native forms of hLF and bLF significantly increased expression of transforming growth factor-β1, and holo-forms of LFs stimulated interleukin 18 secretion significantly, with the highest results for CbLF.
CbLF is biologically active and is likely to exert several of the bioactivities of hLF if added to infant formula."

"bLF and hLF and bLF purified from fresh cow's milk in the laboratory was predominantly in the apo-form, confirmed with a still high Fe-binding capacity, whereas hLF and purified bLF contained low amounts of LPS. LPS was practically absent in cbLF. Similar to hLF, purified bLF and cbLF were bound to human intestinal epithelial cells and were internalized because Fe was bound to them. At a pH similar to that of the human infant stomach, these bLFs partly resisted proteolytic degradation or were digested to immunoreactive fragments in vitro, whereas hLF was digested completely. Thus, it is possible that cbLF can be used as a supplement for application in products for infants and that it can achieve some of the functions of hLF."<-so milk lactoferrin from cows is best.

Lactoferrin: a biologically active molecule for bone regeneration.

"Lactoferrin, a member of the "Siderophilin" family, is an iron binding glycoprotein. Lactoferrin is produced by various exocrine glands in our body and is abundantly present in milk and colostrums. The uniqueness of lactoferrin as a skeletal regenerative molecule lies in its ability to favorably modulate the responses of the various cell types involved in musculoskeletal regeneration. Lactoferrin exhibits pleiotropic functions and recent studies indicate that lactoferrin promotes the proliferation and differentiation of osteoblast cells and inhibits osteoclast-mediated bone resorption. Human lactoferrin is also known to promote neovascularization. This review aims to summarize the most recent studies on lactoferrin focusing on its anabolic effect to bone tissue and the ability to modulate immune responses with specific focus on osteoimmunology."

On how Lactoferrin works:

Iron-saturated lactoferrin stimulates cell cycle progression through PI3K/Akt pathway.

"A relatively low level of ironsaturated Lf, Lf(Fe(3+)), can stimulate S phase cell cycle entry, and requires Akt activation in MCF-7 cells. Lf(Fe(3+)) immediately induced Akt phosphorylation at Ser473, which subsequently induced the phosphorylation of two G1-checkpoint Cdk inhibitors, p21(Cip/WAF1) and p27(kip1). The Lf(Fe(3+))-induced phosphorylation of Cdk inhibitors impaired their nuclear import behavior, thereby inducing cell cycle progression. However, the treatment of cells with a PI3K inhibitor, LY294002, almost completely blocked Lf(Fe(3+))-stimulated cell cycle progression. LY294002 treatment abrogated Lf(Fe(3+))-induced Akt activation, and prevented the cytoplasmic localization of p27(kip1). Higher levels of p21(Cip/WAF1) were also detected in the cytoplasmic sub-cellular compartment as a measure of cellular response to Lf(Fe(3+)). Consequently, the degree of phosphorylation of retinoblastoma protein was enhanced in response to Lf(Fe(3+)). Therefore, we conclude that Lf(Fe(3+)), as a potential antagonist of Cdk inhibitors, can facilitate the functions of E2F during progression to S phase via the Akt signaling pathway."

"Akt-induced p21 phosphorylation causes accumulation in the cytoplasm, thereby inactivating its inhibitory effects on Cdk2"

"ectopically expressed Lf might phosphorylate the p65 NF-κB subunit via signaling pathways including the TRAFs-NIK-IKK pathway"

Any form of Lactoferrin works not just bovine. It's hard to say exactly how much lactoferrin is optimal since this study was performed on rats but doses for humans seem to be around 250mg.  KAL - Colostrum Lactoferrin 1g/200mg - 60ct Cap .

Lactoferrin seems to be very promising both promoting chondrocyte proliferation and being anabolic to osteoblasts without seeming to inhibit endochondral ossification like some other bone stimulating mechanisms do.  However, the possibility of Lactoferrin to cause chondrocyte de-differentiation may give pause.  Since LSJL did not cause Smad3 signaling, Lactoferrin may help improve LSJL results.

Friday, November 19, 2010

Add inches to your height with Allantoin(or Comfrey)?

Allantoin is one of the components of Comfrey which is of the ingredients of the Adult Height Increase formula.  Comfrey is toxic but Allantoin is not and is a part of many cosmetics.  It increases Extracellular Matrix and Cellular Proliferation.  Can children or even adults supplement with Allantoin to add inches to their height?  Comfrey is available for sale: Starwest Botanicals Organic Comfrey Leaf Cut, 1-pound Bag

Profile of wound healing process induced by allantoin

"The allantoin, 5-ureide-hydantoin [stimulates] cell mitosis as well as promoter of epithelial stimulation"

Epithelial stimulation can increase height possibly.  Endothelial stimulation is certainly important.  Cell mitosis is the big one as it could indicate an increase in cellular proliferation as well.

"greater collagen neoformation"

Type II collagen is a part of bone.  The elastic part of bone.  Increasing collagen content could increase height.

"inhibiting the chemotaxis of inflammatory cells"

Reducing inflammation can increase height in youths by reducing cell death.

"allantoin [is effective] in fibroblastic proliferation and synthesis of extracellular matrix"

FGF has the potential to increase height as well.  Although only certain FGFs.  FGF receptors reduce the bodies levels of FGF and that may be responsible for growth plate senescence.

So Allantoin could possibly increase height by stimulating FGFs(which when reduced by FGF-receptors causes growth plate senescence), reduces inflammation(which kills cells including growth plate chondrocytes but histamine can increase chondrocyte proliferation), and by stimulating cellular mitosis.

Initiation of the bony epiphysis in long bones: chronology of interactions between the vascular system and the chondrocytes., reveals the importance of the chorioallatonoic memberane in the developing epiphysis.

There is no oral form of allantoin at this time so the best bet as of now may be the comfrey tea which may be toxic and has resulted in one hospitalization(well the formula which contains comfrey) in the adult height increase Monster Trial.  Some sources have resulted that allantoin can be absorbed effectively through the skin and that can be further absorbed by the bone.  Lawrens Cucumber Gel 4 oz with Collagen & Allantoin Skin Protector.  If you wanted to experiment you would rub it near the epiphysis' of the target bones.

Comfrey: A Clinical Overview

"The constituents of comfrey root include 0.6–4.7% allantoin; abundant mucilage polysaccharides (about 29%) composed of fructose and glucose units; phenolic acids such as rosmarinic acid (up to 0.2%), chlorogenic acid (0.012%) as well as caffeic acid (0.004%) and α-hydroxy caffeic acid; glycopeptides and amino acids; and triterpene saponins in the form of monodesmosidic and bidesmosidic glycosides based on the aglycones hederagenin (e.g. symphytoxide A), oleanolic acid and lithospermic acid"

"The therapeutic properties of comfrey are based on its antiinflammatory and analgesic effects. Comfrey also stimulates granulation and tissue regeneration, and supports callus formation{these could be related to any height increasing effects}"

"Wound-healing effects have been tested in 40% ethanolic comfrey root extracts and its high molecular weight (MW) fraction (> 1000 kD) in a test model of fibroblasts in a collagen matrix. Both inhibited shrinkage of the collagen matrix"

Thursday, November 18, 2010

I added another half inch of height thanks to LSJL

I am now a hair under 5'10" and my tibial length is now 16 1/4" which is the same as it was on August 31th the last time I did a bone length increase update on the tibia.  It could be that I am evening out the limb length inequality between right and left leg and am growing taller that way.  The limb length inequality caused by the contra-lateral leg adapting to loading of the other leg.  Since I noticed that I am now focusing more on my right leg and as the discrepancy evens out I am growing taller.

So despite not getting a lot of results for a while I preservered. Looks like I just have to keep doing LSJL and grow taller 1/2 an inch or so at a time.  Now I just have to develop a cheap way to perform an Electromagnetic Field Based routine.

Wednesday, November 17, 2010

Adding Height with Vitamin E‏?

Dl-alpha tocopheryl acetate is one of the key ingredients for peak height.  Dl-alpha tocopheryl acetate is also known as Vitamin E.  Peak Height may have some benefits for increasing height but it is really an overpriced vitamin supplement.  It may be possible to increase height with different forms of certain Vitamins.  For example, Cyp27b1 which converts Vitamin D to the active metabolite increases height when it is deactivated.  So ingesting a form of Vitamin D that can't be converted into the active metabolite may increase height.  It could either be that the active metabolite of Vitamin D reduces growth or it could be that the inactive metabolite of Vitamin D increases growth and conversion of Vitamin D into the active metabolite reduces the numbers of the inactive metabolite.  Both of these possible reasons would explain a benefit for a form of Vitamin D that can't be converted into the active metabolite form.

Vitamin E is available as a nutritional supplement: Kirkland Signature Vitamin E 400 IU, 500 Softgels

What effects does Vitamin E have on adding height? 

Vitamin E stimulates trabecular bone formation and alters epiphyseal cartilage morphometry. 

"The effects of dietary vitamin E (VIT E) and lipids on tissue peroxidation and fatty acid composition, epiphyseal growth plate cartilage development, and trabecular bone formation were evaluated in chicks. A 2 x 2 factorial design was followed using two levels (30 and 90 IU/kg of diet) of dl-alpha-tocopheryl acetate and two different dietary lipids. The basal semipurified diet contained one of the following lipid treatments: anhydrous butter oil (40 g/kg) + soybean oil (60 g/kg), [BSO], or soybean oil (100 g/kg), [SBO]. After 14 days of feeding, the level of alpha tocopheryl in plasma was higher and thiobarbituric acid reactive substances (TBARS) were less in plasma and liver of chicks supplemented with 90 IU of VIT E compared with those given 30 IU of VIT E. Body weights and tibiotarsal bone lengths were not affected by the dietary treatments. Saturated fatty acids (14:0, 15:0, 16:0, 17:0, and 18:0) were increased in the tibiotarsal bone of chicks fed the BSO diet. In contrast, total polyunsaturated fatty acids and the ratio unsaturated fatty acids/saturated fatty acids were higher is plasma of chicks fed SBO compared with the values from chicks fed BSO. The thickness of the entire growth plate cartilage and the lower hypertrophic chondrocyte zone was significantly greater in chicks fed 90 IU/kg of VIT E. Kinetic parameters on bone histomorphometry indicated that mineral apposition rate was higher in chicks fed 90 IU of VIT E. The interaction effect between the VIT E and BSO treatments led to the highest trabecular bone formation rate among the groups. VIT E protects against cellular lipid peroxidation in cartilage to sustain normal bone growth and modeling." 

Vitamin E is an anti-oxidant(it scavenges free radicals that can cause damage).  That would indicate that it's only necessary to have sufficient levels of Vitamin E in the diet to grow taller not that excess levels of Vitamin could help you grow taller. 

Synergistic chondroprotective effect of alpha-tocopherol, ascorbic acid, and selenium as well as glucosamine and chondroitin on oxidant induced cell death and inhibition of matrix metalloproteinase-3--studies in cultured chondrocytes. 

"Overproduction of reactive oxygen species and impaired antioxidant defence accompanied by chronic inflammatory processes may impair joint health. Pro-inflammatory cytokines such as interleukin-1beta (IL-1beta) and tumor necrosis factor alpha (TNF-alpha) stimulate the expression of metalloproteinases which degrade the extracellular matrix. Little is known regarding the potential synergistic effects of natural compounds such as alpha-tocopherol (alpha-toc)[Vitamin E], ascorbic acid (AA)[Vitamin C] and selenium (Se) on oxidant induced cell death. Furthermore studies regarding the metalloproteinase-3 inhibitory activity of glucosamine sulfate (GS) and chondroitin sulfate (CS) are scarce. Therefore we have studied the effect of alpha-toc (0.1-2.5 micromol/L), AA (10-50 micromol/L) and Se (1-50 nmol/L) on t-butyl hydroperoxide (t-BHP, 100-500 micromol/L)-induced cell death in SW1353 chondrocytes. Furthermore we have determined the effect of GS and CS alone (100-500 micromol/L each) and in combination on MMP3 mRNA levels and MMP3 secretion in IL-1beta stimulated chondrocytes. A combination of alpha-toc, AA, and Se was more potent in counteracting t-BHP-induced cytotoxicity as compared to the single compounds. Similarly a combination of CS and GS was more effective in inhibiting MMP3 gene expression and secretion than the single components. The inhibition of MMP3 secretion due to GS plus CS was accompanied by a decrease in TNF-alpha production. Combining natural compounds such as alpha-toc, AA, and Se as well as GS and CS seems to be a promising strategy to combat oxidative stress and cytokine induced matrix degradation in chondrocytes." 

Selenium is available as a supplement as well, for cheap: Selenium 200mcg 100 caps. Note that the reason they consider MMP-3 as a bad thing is that they are trying to culture chondrocytes and they don't want a degradation of extracellular matrix then.  MMP-3 is one of the crucial MMPs involved in the formation of cartilage canals.  What's important to note in this article is that Vitamin E, Vitamin C, and Selenium all reduce TNF-Alpha and IL-1.  How much you need is based on the oxidative stress you undergo especially if you smoke. 

Ascorbic acid-induced chondrocyte terminal differentiation: the role of the extracellular matrix and 1,25-dihydroxyvitamin D. 

"Chondrocyte terminal differentiation is associated with cellular hypertrophy increased activity of plasma membrane alkaline phosphatase and the synthesis of collagen type X. The hypertrophic phenotype of cultured chondrocytes can be stimulated by ascorbic acid[Vitamin C] but the underlying mechanisms for this phenotypic change are unclear. As ascorbic acid is central to many hydroxylation reactions, the possibility was examined that its pro-differentiating effects are mediated by its effects on collagen and vitamin D metabolite formation. In vitro studies indicated that ascorbic acid-induced chondrocyte alkaline phosphatase activity was inhibited by the addition of both collagen and proteoglycan synthesis inhibitors. The addition of arginine-glycine-aspartic acid (RGD)-containing peptides also resulted in lower alkaline phosphatase activity. Chicks supplemented with dietary ascorbic acid had higher concentrations of both collagen and proteoglycans within their growth plates but the chondrocyte maturation rate was unaltered. No evidence was obtained to suggest that ascorbic acid-induced collagen production was mediated by lipid peroxidation[like Vitamin E]. In addition, supplementation with dietary ascorbic acid resulted in higher serum 1,25-dihydroxyvitamin D3 concentrations and increased chondrocyte vitamin D receptor number. Ascorbic acid-treated chondrocytes maintained in vitro also had increased vitamin D receptor numbers but chondrocyte receptor affinity for 1,25-dihydroxyvitamin D3 was unaltered. These results indicate that ascorbic acid promotes both chondrocyte matrix production and 1,25-dihydroxyvitamin D3 synthesis, accompanied by upregulation of the vitamin D receptor. Thus, ascorbic acid may be causing amplification of the vitamin D receptor-dependent genomic response to 1,25-dihydroxyvitamin D, resulting in promotion of terminal differentiation. Strong evidence is provided to support the hypothesis that ascorbic acid-induced chondrocyte terminal differentiation is mediated by interactions between integrins and RGD-containing cartilage matrix proteins." 

So Vitamin E doesn't seem to interact with Vitamin C.  These scientists think that the key to elongating the growth phase is by inhibiting Alkaline Phosphatase and Collagen X. LIPUS(and by extensions LSJL) blocked expression of Type X Collagen. This study also indicates that 1,25-dihydroxyvitamin D may result in the terminal differentiation of chondrocytes so now it seems that to increase height we must inhibit the active metabolite of Vitamin D.  We also increase height by increasing the number of peptides containing the RGD group. 

So proper amounts of Vitamin E can increase height by scavenging free radicals.  Cholecalciferol(one of the main ingredients of Peak Height), the inactive form of Vitamin D could increase height if it doesn't convert to the active form.  We'll have to go over those studies to see if it does or doesn't.

Tuesday, November 16, 2010

Lateral Synovial Joint Loading Illustrated Part II: The knees

The knees are harder to get than the ankles, you may not be able to generate as much pressure there.  Just keep practicing.  The exact spot on the epiphysis of the knee doesn't matter as long as you are hitting the epiphysis.  Try to increase the peak fluid flow you generate over time.  I recommend doing it for one minute so you have time to work your way up to more pressure.

Monday, November 15, 2010

Grow Taller with Ultrasound

Previously, ultrasound was spoken about as a way to stimulate interstitial fluid flow in a manner similar to LSJL.  LIPUS also has the ability to stimulate signaling pathways(probably through IFF) and upregulate and down regulate genes.  What if sound was another mechanical signaling pathway like electromagnetic fields and the actin cytoskeleton(which can be modulated by cholesterol)?  Then Sound can be used for us high seekers to grow taller even when other methods have stalled.

The science of ultrasound therapy for fracture healing 

"Transforming growth factor-β (TGF-β) synthesis and adenylyl cyclase activation in osteoblasts are also stimulated by LIPUS." 

Both factors are anabolic. 

"Growth of the calcified portion of the metatarsal diaphysis in LIPUS-treated metatarsal rudiments was noted to triple during the week of treatment as compared to the control, untreated group (530 μm versus 180 μm). LIPUS directly affects osteoblasts and ossifying cartilage, resulting in more active ossification."  

LIPUS increases growth rate in osteoblasts.

"The osteosarcoma cell line, UMR-106, was subjected to 20-min doses of LIPUS. Expression of the immediate early-response genes c-fos and cyclooxygenase-2 (COX-2) were up-regulated by this treatment. Elevated mRNA levels for alkaline phosphatase and osteocalcin were also noted after LIPUS treatment." 

Both Alkaline Phosphatase and osteocalcin are anabolic to bones.

"Prostaglandin E2 (PGE2) production is regulated by COX-2. Both are thought to be vital for fracture healing, and can be affected by treatment with LIPUS. The effect of LIPUS on PGE2 regulation and on COX-2 mRNA expression was recently examined in the mouse osteoblastic cell line, MC3T3-E1. [There was] a three-fold increase in the amount of PGE2 produced by cells exposed to LIPUS for 60 min, as compared to control cells. Up-regulation of COX-2 was also noted after treatment of the cells with LIPUS; this up-regulation peaked at 60 min and subsided by 180 min.
PGE2 stimulates cellular responses by signaling through a cascade of enzymatic reactions. PGE2 binds to a guanine nucleotide-binding (G) protein-coupled receptor (GPCR), a class of heptahelical transmembrane receptors involved in cellular responses to external stimuli as diverse as photons, odorants, hormones (such as glucagon), and other extracellular mediators (such as epinephrine). The GPCRs, upon activation by ligand binding, activate diverse downstream pathways of intracellular signaling, ultimately leading to expression of certain genes associated with intracellular metabolism and with cell growth and proliferation.
If PGE2 production is related to fluid shear (thought to be created by LIPUS), then up-regulation of the downstream effectors of PGE2 should be similarly affected.  [There's an] effect of fluid shear stress on COX-2 gene expression in cultured MC3T3-E1 osteoblasts as well as in primarily cultured osteoblasts from mouse calvaria.  Fluid shear stress increased COX-2 gene expression within 30 min. This [is] under the direct control of extracellular signal-regulated kinase (ERK), one of the mitogen-activated protein (MAP) kinases known to be activated by growth factor receptors and by GPCRs, such as the PGE2 receptor."

So does LIPUS create a separate effect other than fluid shear strain(is it worth doing in addition to LSJL)? 

"At fracture sites, mesenchymal stem cells are differentiated into chondrocytes during the formation of the soft, cartilaginous callus. Mesenchymal stem cells, in primary culture, can be differentiated into chondrocytes by treatment with TGF-β. Aggrecan deposition, indicative of chondrocyte differentiation, was increased (8.7-fold versus 2.3 fold) if LIPUS was added to TGF-β, without increasing total protein content or cellular proliferation. Ultrasound increases chondrocyte differentiation of mesenchymal stem cells treated with TGF-β." 

Remember hydrostatic pressure increases chondrogenic differentiation as well. 

"Chondrocytes can be induced to proliferate and maintain chondrocytic lineage also via treatment with LIPUS. Chondrocytes from neonatal Wistar rats were isolated and cultured. Exposure to LIPUS induced and maintained expression of type II collagen and aggrecan, and blocked expression of type X collagen. It was noted to increase expression of TGF-β. Addition of TGF-β to the nontreated cells stimulated similar results, and blockade of TGF-β by addition of a neutralizing antibody abrogated the results seen after LIPUS stimulation. The mechanism of action of LIPUS likely involves TGF-β, in addition to other mechanisms, and that TGF-β is vital for chondrocyte responsiveness to LIPUS."  

LIPUS seems to have very similar effects to LSJL.  Loading of chondrocytes also increases type II collagen and aggrecan. Blocked expression of Type X Collagen is very good as Type X collagen is associated with fusion. 

"Production of VEGF messenger RNA and protein, NO, and hypoxia-inducible factor-1α (HIF-1α) were all stimulated by LIPUS. Inhibition of NO production reduced the VEGF response to LIPUS. The VEGF and HIF-1α responses to LIPUS are mediated by nitric oxide (NO) production. Integrin receptors, present in focal adhesions, are vital for this signaling cascade"  

Shear strain can also upregulate VEGF.  VEGF, NO, and HIF-1alpha are predicted to be upregulated by LSJL but such upregulation was not observed overthreshold thus maybe LIPUS as use as a supplement to LSJL.

Treatment of human mesenchymal stem cells with pulsed low intensity ultrasound enhances the chondrogenic phenotype in vitro.

"low intensity pulsed ultrasound (LIPUS) [was applied] on human bone marrow-derived mesenchymal stem cells undergoing chondrogenic differentiation. Aggregates of mesenchymal stem cells and mesenchymal stem cells seeded in three dimensional matrices were cultured in a defined chondrogenic medium and subjected to LIPUS for the first 7 days of culture. At 1, 7, 14 and 21 days, samples were harvested. Cell aggregates with daily treatment for 20 minutes showed no significant differences for proteoglycan and collagen content during chondrogenic differentiation. ultrasound application for 40 minutes daily resulted in a statistically significant increase of the proteoglycan and collagen content after 21 days in culture[so duration is important]. Aggregates treated for 20 minutes daily showed decreased expression of chondrogenic genes at all time points. 40 minutes of daily treatment of aggregates resulted in a significant increase of chondrogenic marker genes after an initial decrease at day 7 with time in culture. Ultrasound treated cell-scaffold constructs showed a significant increase of chondrogenic marker gene expression and extracellular matrix deposition. LIPUS can be used to enhance the chondrogenesis of mesenchymal stem cells in cell aggregates and cell-scaffold constructs."

"Pulsed low intensity ultrasound (30 mW/cm2) was applied for 0, 20 and 40 minutes per day, for the first 7 consecutive days of culture"  The specs used were "a carrier frequency of 1.5 MHz and with a 200 µs tone burst repeating at 1.0 kHz"<-That's crazy, that's 1,000 times per second!

"undifferentiated mesenchymal cells and differentiated chondrocytes differ significantly in terms of their responses to biomechanical forces"

"cyclic compressive loading can promote the chondrogenesis of rabbit bone marrow derived mesenchymal stem cells by inducing the synthesis of TGF-β1, which can stimulate the mesenchymal cells to differentiate into chondrocytes"

Low-Intensity Ultrasound (LIUS) as an Innovative Tool for Chondrogenesis of Mesenchymal Stem Cells (MSCs).

"LIUS not only induces chondrogenic differentiation of MSCs but also has diverse additional activities that enhance the viability of MSCs, increase possibly the integrity of the differentiated tissues and delays hypertrophic changes during differentiation. LIUS could be an innovative and versatile tool for chondrogenic differentiation of MSCs and for cartilage tissue engineering."

"preconditioning by LIUS for a short period (e.g., for one week) could program MSCs to differentiate well into chondrogenic lineages or to better maintain chondrogenic phenotypes during subsequent culture in the absence of additional LIUS stimulation."

"other groups used LIUS of pulsed wave at 30 mW/cm2, while we used continuous wave at 200 mW/cm2."

"LIUS signal appears to use the mechanotransduction pathway including integrins, stretch-activated ion channels, and interleukin-4 on the cell surface."

"LIUS could inhibit vascular invasion, as judged by immunohistochemical analysis for pallet/endothelial cell adhesion molecule-1 (PECAM-1) and could reduce the expression of angiogenic factors such as hypoxia inducible factor-1α (HIF-1α) and vascular endothelial growth factor (VEGF) in rabbit MSCs in vitro or in nude mice"

"LIUS induced chondrogenic differentiation of MSCs in the absence of added TGFβ both in vitro and in vivo"

"LIUS induced expression of tissue inhibitor of metalloproteinases-2 (TIMP-2) but showed no effect on the protein level of metalloproteinase-3 (MMP-3) or mRNA levels of MMP-13, and type I and X collagens. TIMP-2 functions as an inhibitor of MMPs and an anti-angiogenic factor that can inhibit vascular invasion following hypertrophy of chondrocytes during endochondral ossification. Type I and X collagens and MMP-13 are also involved positively in the hypertrophic changes of chondrocytes. LIUS might inhibit degradation of extracellular matrix (ECM) proteins and/or hypertrophic changes of differentiated MSCs, thereby enhancing the integrity of cartilage constructs."

"TGFβ is necessary and sufficient to induce chondrogenic differentiation of MSCs in vitro."<-LSJL induces TGF-Beta.

"LIUS [plays an] actively inducing role that directs MSCs to chondrogenic lineages without exogenous growth factors such as TGFβ. LIUS also had diverse additional functions during the chondrogenic differentiation of MSCs in vitro and in vivo. It enhanced (1) viability of MSCs by inhibiting apoptosis of cells, (2) regulated expression of genes involved in the integrity of the differentiated construct, and (3) delayed hypertrophic changes at the late stage of differentiation. "

So LIPUS can increase height but it doesn't seem to do so with a mechanism that's different than LSJL(both induced fluid flow/hydrostatic pressure).  So you should do either one or the other and LSJL is cheaper.  Electromagnetic fields operate differently because of the biophotons stored in intracellular DNA.

Integrin-mediated mechanotransduction pathway of low-intensity continuous ultrasound in human chondrocytes.

"US stimulation of chondrocytes induced phosphorylation of focal adhesion kinase (FAK), Src, p130 Crk-associated substrate (p130Cas), CrkII and extracellular-regulated kinase (Erk)[likely phosphorylated by LSJL]. Pre-incubation with inhibitors of integrin receptors, Src and MAPK/Erk kinase (MEK) reduced US-induced Erk phosphorylation levels, indicating integrins and Src are upstream of Erk in an US-mediated mechanotransduction pathway. US signals through integrin receptors to the MAPK/Erk pathway via a mechanotransduction pathway involving FAK, Src, p130Cas and CrkII."

"Human chondrocytes that were serum-deprived overnight were treated with 5.0 MHz US for 3 min"

"Focal adhesion complexes are formed by the interaction of the cytoplasmic tail of integrins with proteins such as paxillin, talin, vinculin, actin and FAK "

"FAK autophosphorylates at Y397, generating a high-affinity binding site for the Src homology 2 (SH2) domain of Src family kinases"

The effect of ultrasound stimulation on the gene and protein expression of chondrocytes seeded in chitosan scaffolds

"A low-intensity diffuse ultrasound (LIDUS) signal at 5.0 MHz (0.14 mW/cm2) was employed to stimulate bovine chondrocytes seeded in three-dimensional (3D) chitosan-based matrices. While the duration of application was constant at 51 s, US was applied once, twice, four times and eight times/day, and the impacts of US on the biosynthetic activity of chondrocytes and the expression of chondrocyte-specific genes were evaluated. When stimulated with continuous US for predetermined time intervals, chondrocytes had higher levels of type II collagen, aggrecan, L-Sox5 and Sox9{all but Sox5 was up in LSJL} mRNA expression when compared to controls; however, under the same conditions, the expression of MMP-3{up in LSJL} was downregulated. Both Sox5 and Sox9 genes coordinately responded to changes in US stimulation and generally mirrored the response of collagen type II transcript to changes in US stimulation. US stimulation increased the gene expression of cell-surface integrins α5 and β1. The expression of integrins α2 was downregulated by US treatment, suggesting that multiple integrin subunits may be involved in the regulation of chondrocytic function in response to US stimuli. The enhancement in the abundance of the mRNA transcripts upon US stimulation was observed to correlate with the protein expression of collagen type I, collagen type II, and integrins α5 and β1."

4 ultrasound applications per day increased cell count the most.

"indirect mechanical stimulation in the form of an acoustic signal [upregulates] the expression of integrins α5 and β1 and [downregulates] the expression of α2 at the mRNA level"

Low-intensity ultrasound stimulation enhances chondrogenic differentiation in alginate culture of mesenchymal stem cells.

"we investigated the effects of LIUS on the chondrogenesis of rabbit MSCs (rMSCs) in a 3-D alginate culture and on the maintenance of chondrogenic phenotypes after replating them on a monolayer culture. The LIUS treatment of rMSCs increased: (i) the matrix formation; (ii) the expression of chondrogenic markers such as collagen type II, aggrecan, and Sox-9; (iii) the expression of tissue inhibitor of metalloprotease-2 implicated in the integrity of cartilage matrix; and (iv) the capacity to maintain the chondrogenic phenotypes in a monolayer culture. Notably, LIUS effects were clearly shown even without TGF-beta treatment. LIUS treatment could be an efficient and cost-effective method to induce chondrogenic differentiation of MSCs in vitro for cartilage tissue engineering."

"MSCs were isolated from 2-week-old female New Zealand white rabbits"

"LIUS was treated for 10 min every 12 h for 1 or 2 weeks at a frequency of 1 MHz and an intensity of 200 mW/cm2 in a continuous-wave fashion. The intensity of 200 mW/cm2 was selected"

Note though that they were cultured in alginate beads which means the cells had adhesion capabilities.

Effect of therapeutic ultrasound on endochondral ossification.

"Ultrasound was tested on endochondral ossification of in vitro developing metatarsal long bone rudiments of 16- and 17-day-old fetal mice. Bone growth, calcification and resorption following exposure to several doses of pulse-wave (PW) or continuous-wave (CW) ultrasound were examined. PW was applied at intensities between 0.1 W cm-2 and 0.77 W cm-2 (Isatp) and CW intensities were 0.1 W cm-2 or 0.5 W cm-2 (Isata). After 1 week of culture, the metatarsal long bone rudiments were fixed and paraffin sections were prepared for histological evaluation and for measurement of the relative contribution of the various cartilage zones to the total bone length. In contrast to treatment with CW ultrasound, treatment of 16-day-old metatarsal long bone rudiments with PW ultrasound resulted after 4 days of culture in significantly increased longitudinal growth. Histology revealed a significant increased length of the proliferative zone, whereas the length of the hypertrophic cartilage zone was unaltered. This might indicate that proliferation of the cartilage cells is stimulated without influence on cell differentiation."

10% increase in growth rate was observed in 0.77 W cm-2 PW group.

"Doses of 0.1 or 0.5 W cm-2 continuous-wave (CW) ultrasound, applied for 1 or 5 min, had no significant effect on the longitudinal growth of the 16-day-old MT during culture, although the treated samples tended to show decreased growth"

"a dose of 0.77 W cm-2 causes an acoustic pressure amplitude of 276 kPa in the spatial peak in degassed water"

Mechanobiological conditioning of stem cells for cartilage tissue engineering.

"Mechanobiological conditioning (e.g. mechanical compression, hydrostatic pressure, osmotic, shear, ultrasound) [influences chondrogenic differentiation of dedifferentiated chondrocytes. Using an in vitro aggregate culture system enhanced chondrogenesis of mesenchymal progenitor cells, detected by an increased extracellular matrix deposition of collagen and aggrecan, could be shown under repeated cyclic hydrostatic pressure. An increase in chondrogenic differentiation of mesenchymal progenitor cells could be detected, when the cells were loaded in three-dimensional matrices and subjected to cyclic, compressive load or low-intensity pulsed ultrasound."

I do not have access to the full study.

Applications of Exogenous Mesenchymal Stem Cells and Low Intensity Pulsed Ultrasound Enhance Fracture Healing in Rat Model.

"[We] investigate the effects of combined treatment of exogenous mesenchymal stem cells (MSCs) and low intensity pulsed ultrasound (LIPUS) on fracture healing by comparing LIPUS-MSC, MSC and control (CTL) groups. Quantitative callus width/area demonstrated that the MSC-LIPUS group had the best healing, MSC group the second and CTL group the poorest with significant differences among each at different time points. MSC-LIPUS had the highest bone volume/tissue volume. [There was] a significantly faster remodeling in late phase in MSC-LIPUS and MSC groups. Combined treatment of MSCs and LIPUS was beneficial to fracture healing. Regenerative power and homing ability of MSCs were shown by promotion in fracture healing and locally found green fluorescent protein (GFP)-labeled MSCs at fracture calluses."

"three-month-old female Sprague-Dawley (SD) [were used]"

"a metal blade (weighted 500 g) dropping from a height of 35 cm, was used to create the fracture at the midshaft of the femur. The quality of fracture with fracture gap [was] smaller than 0.5 mm and displacement [was] less than 0.5 mm"

" Large amount of cartilaginous tissues bridged the osseous callus in the first week. At week 2, woven bone was observed in MSC-LIPUS and MSC groups with active endochondral ossification at the edge of cartilaginous tissues, while this phenomenon was shown in CTL group at week 3. The area of cartilaginous tissues tended to reduce in both MSC-LIPUS and MSC groups at week 3 and more woven bone replaced the cartilaginous tissues to bridge the fracture site in week 4, particularly in MSC-LIPUS group. There were, however, still large amounts of cartilaginous tissues in CTL group in week 4."

"LIPUS has been proven to increase blood flow around fracture site that might promote angiogenesis, MSCs migration and, hence, fracture healing."It's possible LIPUS could do this to normal bone too.

Combined Treatment of Alendronate and Low-Intensity Pulsed Ultrasound (LIPUS) Increases Bone Mineral Density at the Cancellous Bone Osteotomy Site in Aged Rats: A Preliminary Study.

"9-month-old Sprague-Dawley rats, with a unilateral proximal tibial osteotomy, were treated with alendronate (daily, 1 µg/kg) plus sham-LIPUS, saline plus LIPUS (20 min/day), alendronate plus LIPUS, or saline plus sham-LIPUS as a control for 4 weeks"

"The combined therapy significantly increased BMD at the osteotomy site at 4 weeks compared with the control, without affecting the contralateral, non-osteotomized tibia. Both alendronate and LIPUS alone also exerted a positive, albeit less, effect on BMD in the affected limb. Alendronate and LIPUS cooperate to enhance BMD during metaphyseal bone healing."

LIPUS and Alendronate were applied to both osteomy and non-osteomy bone.

"The ultrasound signals were transferred for 20 min per day with a spatial average-temporal average (SATA) intensity of 30 mW/cm2"

The region of interest was the epiphysis.  It is possible that LIPUS can increase other parameters besides BMD in non-osteomy bone.  BMD was the only parameter that was measured.

Low-intensity ultrasound increases FAK, ERK-1/2, and IRS-1 expression of intact rat bones in a noncumulative manner.

"daily 20-minute LIPUS [was] applied to intact tibias.
FAK, ERK-1/2, and IRS-1 expression and activation [was detected] from the stimulated intact tibias at 7, 14, and 21 days of daily 20-minute LIPUS.
LIPUS increased FAK expression (at 7 days), ERK-1/2 (at 14 days), and IRS-1 (at 7 days), but expression decreased 7 days later, indicating a noncumulative effect of LIPUS. As only FAK expression was detected at 21 days, these observations suggest LIPUS influences nuclear reactions that may be modulated by a major cellular mechanism preferentially inhibiting IRS-1 expression and not FAK expression. Increased ERK-1/2 expression at 14 days suggests the differing mechanisms for promoting ERK-1/2, FAK, and IRS-1 syntheses. IRS-1 expression behaved similarly to FAK expression; therefore, LIPUS may modulate growth factor pathways. LIPUS increased sustained FAK and ERK-1/2 activation, but not IRS-1, suggesting sustained ERK-1/2 activation is not the result of mechanically induced growth factor activation."

"[Male] WH rats, aged 12 weeks [were used]"

ERK 1/2 was phosphorylated at Tyr-204.  p-ERK 1/2 was the same in both control and LIPUS group at 21 days indicating a conditioning response.

"IGF-1 expression may be increased by LIPUS"

12 week old WH mice are still growing.

"Seven days after the augmentation of FAK (at 14 days) and ERK-1/2 (at 21 days) expression, their expression decreased, showing a noncumulative effect of LIPUS on intact bone and suggesting there is a regulatory mechanism in bone cells, similar to negative feedback, that is activated after the augmentation of FAK and ERK-1/2 expression. In healthy bone, this mechanism may have antineoplastic finalities, because FAK is persistently increased in neoplasia"

Mechanical means to improve bone strength: ultrasound and vibration.

"At  intensities of 5 to 300 W/cm2, [ultrasound] is used as a surgical tool to fragment calculi{mineral deposits}"

"LIPUS uses a significantly low intensity and does not yield major temperature changes (typically <1°C). However, even these minor temperature changes are hypothesized to stimulate enzymatic processes involving some sensitive enzymes, such as matrix metalloproteinase 1 and collagenase"

Low-intensity pulsed ultrasound (LIPUS) and cell-to-cell communication in bone marrow stromal cells.

"Single or daily-multiple LIPUS treatment at 1.5MHz, 30mW/cm(2), for 20min was applied to BMSC. BMSC form functional gap junctions and single LIPUS treatment significantly increased the intracellular dye transfer between BMSC. Activated phosphorylation of ERK1/2 and p38 by LIPUS stimulation was diminished when cells were treated with a gap junction inhibitor 18β-glycyrrhetinic acid (18β). We further demonstrated that 18β diminished the significant increase in alkaline phosphatase activity following LIPUS stimulation."

"Gap junctions mediate the intercellular exchange of regulatory ions and small molecules that allow metabolic cooperation between adjacent cells and control cell differentiation and growth. In bone, gap junctions have been suggested to be central to the transmission of biophysical stimuli"

LIPUS increased ALP by 30% and this was diminished by 18B.

The development and validation of a LIPUS system with preliminary observations of ultrasonic effects on human adult stem cells.

" a custom LIPUS system was designed and validated by exploring the effects of using different pulse repetition frequency (PRF) parameters on human adipose derived adult stem cells (hASCs) and bone marrow derived mesenchymal stem cells (hMSCs), two common stem cell sources for creating bone constructs in vitro. Changing the PRF was found to affect cellular response to LIPUS stimulation for both cell types. Proliferation of LIPUS-stimulated cells was found to decrease for hASCs by d 7 for all three groups compared with unstimulated control cells (P = 0.008, 0.011, 0.014 for 1 Hz, 100 Hz and 1 kHz PRF, respectively) and for hMSCs by d 14 (donor 1: P = 0.0005, 0.0002, 0.0003; donor 2: P = 0.0003, 0.0002, 0.0001; for PRFs of 1 Hz, 100 Hz, and 1 kHz, respectively). Additionally, LIPUS was shown to strongly accelerate osteogenic differentiation of hASCs based on amount of calcium accretion normalized by total DNA (P = 0.003, 0.001, 0.003, and 0.032 between control/100 Hz, control/1 kHz, 1 Hz/1 kHz, and 100 Hz/1 kHz pulse repetition frequencies, respectively)."

LIPUS increased calcium accretion per total DNA for hMSCs as well.

Application of low-intensity ultrasound to growing bone in rats.

"he longitudinal growth and bone density of the femur and tibia in young rats was measured after application of an ultrasound transducer emitting 1.5-MHz pulsed ultrasound (30 mW/cm2, SATA) for 20 min/day. After 28 days, no length difference was detected (< or = 2%) compared to the sham-treated leg or to unexposed controls. Also, no significant difference in bone mineral density (BMD) of the femur or tibia was found (< or = 6%). In a repeated experiment in which a periosteal trauma stimulus was created in the femoral diaphysis, the ultrasound also had no effect on growth or BMD."

"Four-week-old Sprague–Dawley male rats (approximately 90 g) [were used]"

Ultrasound was applied to a huge region which could be part of the problem and why there wasn't significant growth.

Ultrasound though seemed to increase femur length but decrease tibia length.

" in fracture experiments, disruption of the cortex may provide better access to activated endosteal and periosteal cells than in the intact limb."

Effects of low-intensity pulsed ultrasound on the differentiation of C2C12 cells.

"Pluripotent mesenchymal cell line C2C12 were cultured in differentiation medium with or without the addition of LIPUS stimulation. The ultrasound signal consisted of 1.5 MHz at an intensity of 70 mW/cm2{more than twice the 30mW/cm2 of LIPUS} for 20 min for all cultures. To verify the cell lineage after LIPUS stimulation, mRNA expression of cellular phenotype-specific markers characterizing osteoblasts (Runx2, Msx2, Dlx5, AJ18), chondroblasts (Sox9), myoblasts (MyoD), and adipocytes (C/EBP, PPARgamma) was studied s. The protein expression of Runx2 and activated phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) and p38 mitogen-activated protein kinase (p38 MAPK) were performed using Western blotting. The mRNA expression of Runx2, Msx2, Dlx5, AJ18, and Sox9 was increased markedly by the LIPUS stimulation, whereas the expression of MyoD, C/EBP, and PPARgamma was drastically decreased. In the Western blot analysis, LIPUS stimulation increased Runx2 protein expression and phosphorylation of ERK1/2 and p38 MAPK. LIPUS stimulation converts the differentiation pathway of C2C12 cells into the osteoblast and/or chondroblast lineage via activated phosphorylation of ERK1/2 and p38 MAPK{LSJL likely phosphorylates these two proteins as well}."

"C2C12 cells are a subclone of C2 myoblasts"

"Dlx5 expression correlates with osteoblast differentiation"

"The maximum expression of Dlx5 occurs in the final stages of osteoblast differentiation in vitro when the extracellular matrix mineralizes, suggesting that Dlx5 may be involved in the differentiation of the osteogenic cells. In contrast, Msx2 is predominantly expressed by proliferating osteoblasts and preosteoblasts, but decreases according to terminal osteoblast differentiation"

"Chick wing bud mesenchymal cell micromass culture allows the study of a variety of developmental mechanisms, ranging from cell adhesion to pattern formation. However, many cells remain in contact with an artificial substratum, which can influence cytoskeletal organisation and differentiation. An ultrasound standing wave trap facilitates the rapid formation of 2-D monolayer cell aggregates with a defined zero time-point, independent from contact with a surface. Aggregates formed rapidly (within 2 min) and intercellular membrane spreading occurred at points of contact. This was associated with an increase in peripheral F-actin within 10 min of cell-cell contact and aggregates had obtained physical integrity by 30 min. The chondrogenic transcription factor Sox9 could be detected in cells in the ultrasound trap within 3 h (ultrasound exposure alone was not responsible for this effect). This approach facilitates the study of the initial cell-cell interactions that occur during condensation formation and demonstrates that a combination of cell shape and cytoskeletal organisation is required for the initiation and maintenance of a differentiated phenotype, which is lost when these phenomena are influenced by contact with an artificial substrate."

"[N-CAM and N-Cadherin while encouraging mesenchymal condensation, their] expression following condensation also inhibits chondrogenesis"  Cadherin 7 may also be key for mesenchymal condensation.

"loss of Sox9 occurs due to reorganisation of the F-actin cytoskeleton"

The pressure generated was 110 kPa.

"During condensation, cells come together and are compacted and take on a rounded morphology, show peripheral actin organisation, form adhesive complexes and gap junctions, then begin to express Sox9"