Chondrosarcoma to LSJL gene expression

The study below shows that the genes upregulated in LSJL were very similar to the genes upregulated in chondrogenic human bone marrow mesenchymal stem cells(41.1%).  This provides further evidence that LSJL can cause chondroinduction in aged individuals since the majority 7 out of 8 subjects were over 21 and one was 74.

LSJL gene expression was done on the whole bone of mice whereas this was done in spheroid cultures of MSCs.  And many of the genes could also be expressed by osteoblasts.  However, LSJL upregulates the main chondrocyte differentiation gene Sox9 by over 3-fold whereas LSJL does not upregulate the main osteoblast differentiation gene Runx2 detect-ably over 2-fold.  Interestingly in the below study Sox9 is not upregulated in the chondrogenic normal MSCs.

Since LSJL was done on the whole bone and the area of the osteogenic portion of the bone is larger than the growth plate area you'd expect to have larger osteogenic gene expression than chondrogenic gene expression whereas in LSJL the opposite is observed with the exception of that Col1a1 is expressed at a higher fold than Col2a1. And in turn many of the osteogenic genes would be expected to be expressed in the hypertrophic. You have to take into account as well that growth plates are denser than osteoblasts in the bone marrow or osteocytes in the bone.  You also have to assume that ectopic chondrocyte differentiation is occurring within the bone marrow because our goal with LSJL is to induce ectopic chondrocyte differentiation in the bone marrow to form new growth plates.

However, I think you'd still expect for the osteoblast markers of the entire bone to increase more than the growth plates.

Thus, this study provides further evidence that LSJL can induce mesenchymal chondrocyte differentiation.

A chondrogenic gene expression signature in mesenchymal stem cells is a classifier of conventional central chondrosarcoma.

"Phenotypic and molecular parallels between the development of chondrosarcoma and the differentiation of chondrocytes in normal growth plate suggest that chondrosarcoma may arise from mesenchymal precursor cells driven towards chondrogenesis. We [compare] cartilaginous tumours and mesenchymal stem cells (MSCs). MSCs from eight donors were submitted to chondrogenic differentiation in spheroid cultures. Expression profiles of MSCs at days 0, 7, 14, 28 and 42 of chondrogenesis and of 18 chondrosarcomas with different histological grades were studied using a customized cDNA array. Hierarchical clustering of MSC gene expression during chondrogenesis allowed the classification of samples in a pre-chondrogenic and a chondrogenic cluster corresponding to the phenotypes of early and late differentiation stages. The 74 genes differentially expressed between the two clusters were defined as chondrogenesis-relevant genes. Gene expression profiles of chondrosarcoma were submitted to hierarchical clustering on the basis of these chondrogenesis-relevant genes. This analysis allowed clear distinction between grade I and grade III chondrosarcoma and separated grade II chondrosarcoma into two groups. All grade II chondrosarcomas with occurrence of metastasis were found together with the grade III chondrosarcomas in the pre-chondrogenic cluster."

The ages used for the mesenchymal stem cell samples ranged from 5-74.

"MSCs from eight donors were expanded and submitted to chondrogenic induction in spheroid culture in the presence of TGFβ3"

Genes upregulated during chondrogenic differentiation of MSC spheroids also upregulated during LSJL:

COL10A1

COL2A1

ACAN

H19

COL11A1

FXYD6

HAPLN1

ANGPTL2

MATN3

COL1A1

Osteopontin

BSP

LUM

SERPINA3{as SERPINA3N}

COL12A1

AEBP1

CSPG4

BGN

Acta2

Col3a1

Osteocalcin

41.1% of 51 genes.

Downregulated:

Igfbp6{up}

Timp1{up}

Ninj1

Col14a1{up}

The authors did provide the list of differentially regulated genes in chondrosarcoma but they said that the stages of chondrosarcoma differed based on fold expression of different genes.

Microarray analysis of gene expression in chondrosarcoma cells treated with bee venom.

"a human chondrocyte-like cell line treated with BV[Bee Venom], microarray analysis was performed.

The HTB-94 human chondrosarcoma cells were treated with BV, lipopolysaccharide (LPS), or both. Of the 344 genes profiled in this study, with a cut-off level of 4-fold change in the expression, (1) 35 were downregulated following BV treatment, (2) 16 were upregulated and 7 downregulated following LPS treatment, and (3) 32 were downregulated following co-stimulation of BV and LPS.

Treatment of BV reversed the LPS-induced upregulation of such genes as interleukin-6 (IL-6) receptor, matrix metalloproteinase 15 (MMP-15), tumor necrosis factor (ligand) superfamily-10, caspase-6 and tissue inhibitor of metalloproteinase-1 (TIMP-1)."

Bee venom has both pro- and anti- inflammatory effects.

"Cells were washed with the culture medium and incubated in the culture medium with the following agent(s) for 12 h: (1) vehicle or 10 ng/ml BV (Sigma, USA), (2) vehicle or 1 μg/ml Escherichia coli LPS, (3) 1 μg/ml LPS or 1 μg/ml LPS plus 10 ng/ml BV"

Genes downregulated in the human chondrosarcoma cell line treated with bee venom also downregulated by LSJL:

TIMP1{up}

TNFSF12{up}

TSC1

WISP2{up}

Genes upregulated by LPS treated human chondrosarcoma cell line also upregulated by LSJL:

TIMP1

Genes Downregulated:

Stk4

Genes downregulated in LPS + bee venom human chondrosarcoma treated cell lines also downregulated by LSJL:

CD33

CCNB1
THBS3{up}

"LPS is the major constituent of bacterial endotoxin and serves as an inflammatory agent against chondrocytes at such a concentration as 1 μg/ml"

How will taking Doxycycline affect your height?

There's a lot of contradictory information on doxycycline.  But, it's clear that doxycycline has an effect on chondrocyte hypertrophy with a definite effect on MMP13 and a possible effect on Col10.  A reduction of MMP13 in the growth plates has been linked to one form of dwarfism.  MMP13 is likely biphasic where there's an optimal quantity for growth.  Doxycycline is a commonly prescribed acne medication.  It's clear that doxycycline will affect your height growth but it's not clear how.

Effects of Doxycycline on Mesenchymal Stem Cell Chondrogenesis and Cartilage Repair.

"Ninety hMSC pellets were cultured in chondrogenic media with either 0-, 1- or 2-μg/mL doxycycline. Osteochondral defects (OCD) were created in the trochlear grooves of 24-Sprague-Dawley rats treated with/without oral doxycycline. Rats were sacrificed at 12-weeks.

hMSC pellets with 1-μg/mL=and 2-μg/mL doxycycline had larger areas than pellets without doxycycline. hMSC pellets with 2-μg/mL doxycycline showed reduced mmp-13 mRNA and protein at 21-days. Proteoglycan, DNA contents, and mRNA expressions of chondrogenic genes were similar. For the in vivo study, while the histological scores were similar between the two groups, the gross scores of the OCD repair tissues in doxycycline-treated rats were higher at 12-weeks, reflective of improved repair quality. The doxycycline-treated repairs also showed lower MMP-13 protein.

Doxycycline improves hMSC chondrogenesis and decreases MMP-13{MMP-13 may have beneficial effects on endochondral ossification however} in pellet cultures and within rat OCDs. Doxycycline exerted no negative effect on multiple measures of chondrogenesis and cartilage repair."

"Microfracture (bone marrow stimulation) is commonly used to treat focal cartilage injuries. This technique involves the recruitment of bone marrow derived mesenchymal stem cells (MSCs) to the defect area to participate in cartilage repair. However, the resulting repair does not restore hyaline articular cartilage, but instead results in formation of a mechanically inferior fibrocartilaginous scar tissue"<-Maybe this is why people didn't get taller with microfracture theory?  If microfracture within bones results in the same fibrocartilaginous scar tissue.

"Oral administration of doxycycline has been postulated to have beneficial effects on articular cartilage by inhibiting MMP, specifically MMP-1, -3 (stromelysin), and -13"

"[Comparisons of} joint space narrowing in mildly arthritic knees, [revealed] the doxycycline treatment group had a decreased rate of narrowing by 40% at 16-19 months and 33% at 30-months as compared to placebo"<-This would be a significant reduction in height loss in the mature individuals.

Doxycycline only affected MMP-13 and not Sox9, Aggrecan, Col2a1, Col10a1, and TGFBRII.  Doxycycline had a greater effect on MMP-13 after 21 days in chondrocyte culture at 2 micrograms per millilieter.

"the pellet areas in the 2-μg/mL doxycycline group were larger than those in no doxycycline treatment group."  Since doxycycline does not reduce Col10 levels it's possible that Doxycycline could inhibit MMP-13 without affecting chondrocyte hypertrophy thus having a purely beneficial effect on height.

Influence of doxycycline on the epiphyseal plate cartilage of the rats in experimental osteoarthrosis, induced by iodoacetate.

"In 36 Wistar rats with the iodoacetate-induced experimental osteoarthrosis (OA), effects of doxycycline, given orally, were determined on histochemical reactions of glycosaminoglycans (GAG) in the epiphyseal plate cartilage. The epiphyseal plate of rats with OA was reduced in height (especially the proliferative zone), cell columns were disorganized, many chondrocytes were irregular and polygonal, their nuclei were pycnotic, the intensity of GAG staining was irregular and predominantly reduced, which can be interpreted as signs of degeneration. A concomitant administration of doxycycline in the second group of rats prevented, to some extent, the negative effects of iodoacetate on chondrocytes and led to a more pronounced intensity of GAG reactions in the matrix of the epiphyseal plate."

"in some rats, treated with doxycycline, we observed signs of focal chondrocyte proliferation and of matrix GAG production"

Regulation of cartilage collagenase by doxycycline.

"Chondrocytes were isolated from human OA cartilage and treated with doxycycline. 

We observed significant inhibition of matrix metalloproteinases (MMP-1) and MMP-13 mRNA and protein production by chondrocytes, isolated from OA cartilage, after treatment with doxycycline. The decrease in collagenase protein level paralleled a decrease in mRNA for these enzymes, suggesting a transcriptional/posttranscriptional level of control. In addition, treatment with 10 microg/ml doxycycline resulted in 2.2-fold upregulation of transforming growth factor (TGF-beta3) and a significant decrease of interleukin 1alpha (IL-1alpha), IL-1beta, and IL-6 mRNA. Upregulation of TGF-beta RI and TGF-beta RII was also detected. These cytokines are known to affect collagenase expression and could contribute to inhibition of MMP-1 and MMP-13 production by OA chondrocytes. A decrease in IL-1alpha, IL-1beta, and IL-6 would reduce stimulation of MMP production, while an increase in TGF-B3 would lead to downregulation of local proinflammatory cytokine production as well as of the collagenases themselves.

Our findings show that a decrease in MMP-1 and MMP-13 collagenase production by articular chondrocytes in response to treatment with doxycycline can be explained by a regulatory effect of doxycycline on the production of cytokine and cytokine receptors."

Couldn't get full text.

Differential patterns of response to doxycycline and transforming growth factor beta1 in the down-regulation of collagenases in osteoarthritic and normal human chondrocytes.

"To investigate the ability of doxycycline, transforming growth factor beta1 (TGFbeta1), and phorbol myristate acetate (PMA) to modulate collagenase synthesis in osteoarthritic (OA) chondrocytes.

Levels of fibroblast collagenase (matrix metalloproteinase 1 [MMP-1]), neutrophil collagenase (MMP-8), and collagenase 3 (MMP-13) proteins and messenger RNA (mRNA) were measured in chondrocytes isolated from involved and uninvolved areas of OA cartilage and from normal human chondrocytes, after treatment with doxycycline, TGFbeta1, and PMA.

Chondrocytes isolated from cartilage immediately adjacent to the OA lesion had, on average, 1.8-3.9-fold higher basal levels of MMP mRNA. These cells down-regulated collagenase proteins and mRNA upon incubation with TGFbeta1. In contrast, chondrocytes from areas located more distant from the macroscopic lesion increased MMP-13 mRNA, while MMP-1 and MMP-8 decreased after stimulation with TGFbeta1. Discoordinate regulation was observed after stimulation with PMA, with an increase in MMP-1 and MMP-8 but a decrease in MMP-13. Incubation of OA chondrocytes with doxycycline (1-10 microg/ml), at pharmacologically achievable levels, decreased levels of mRNA of all 3 collagenases, but not G3PDH. In addition, doxycycline inhibited the increase in mRNA for these enzymes in normal chondrocytes stimulated with tumor necrosis factor alpha.

Regulation of MMP-1, MMP-8, and MMP-13 in OA chondrocytes, although mediated by differing pathways, can be decreased by treatment with doxycycline at low concentrations."

"Chondrocytes from normal-appearing cartilage stimulated with TGFb1 showed enhanced synthesis of MMP-13."<-this shows the possible important of MMP-13 to normal processes.

Doxycycline inhibits collagen synthesis by differentiated articular chondrocytes.

"Doxycycline (DOX) profoundly inhibited collagen synthesis by differentiated articular chondrocytes. At 25 microM, the rate of collagen synthesis was suppressed by more than 50% without affecting cell proliferation and general protein synthesis. Steady-state mRNA levels of type II collagen were also reduced, indicating that DOX may have an effect at the transcriptional level of type II collagen. The IC50 value of DOX to downregulate collagen synthesis (17 microM) is close to DOX levels attained in vivo (< 10 microM), and it is more than ten-fold lower than the IC50 values to inhibit the activity of most matrix metalloproteinases (MMPs). As such, these findings support the hypothesis that the reduced severity of OA observed in the dog anterior cruciate ligament model resulting from prophylactic treatment with DOX may involve mechanisms other than MMP inhibition alone. Our findings suggest that prevention of changes in the chondrocyte phenotype may be involved in the beneficial effect of doxycycline in experimental osteoarthritis, for differentiated chondrocytes in early stages of osteoarthritis exhibit elevated collagen synthesis."

"Increased synthesis of type II collagen is a characteristic of early stages of OA"

Doxyclycline inhibits collagen synthesis by bovine chondrocytes cultured in alginate.

"doxycycline exhibits a profound inhibition of collagen synthesis by bovine articular chondrocytes cultured in alginate. At 25 microM doxycycline, collagen synthesis was decreased by 50%; no effect on cell proliferation or general protein synthesis was observed. Messenger RNA levels of type II collagen were also reduced, indicating an effect of doxycycline at the transcriptional level. The concentration of doxycycline needed to downregulate collagen synthesis was > 10-fold lower than that needed to inhibit most of the MMPs. In asmuch as differentiated chondrocytes in the early stages of osteoarthritis exhibit increased collagen synthesis, the beneficial effect of doxycycline in vivo may involve prevention of changes in chondrocyte phenotype."

Doxycycline inhibits type X collagen synthesis in avian hypertrophic chondrocyte cultures. suggests that Doxycycline can inhibit Type X Collagen.

Doxycycline disrupts chondrocyte differentiation and inhibits cartilage matrix degradation.

"The effects of doxycycline were tested in an in vitro system in which the cartilages of embryonic avian tibias are completely degraded.

Tibias were cultured with 5, 20, or 40 microgram/ml doxycycline. Control tibias were cultured without doxycycline. Conditioned media and tissue sections were examined for enzyme activity and matrix loss.

Cartilages were not resorbed in the presence of doxycycline, whereas control cartilages were completely degraded. Collagen degradation was reduced in association with treatment with doxycycline at all doses studied. Higher concentrations of doxycycline reduced collagenase and gelatinase activity and prevented proteoglycan loss, cell death, and deposition of type X collagen in the cartilage matrix; in addition, treatment with doxycycline at higher concentrations caused increases in the length of the hypertrophic region.

The effects of doxycycline extend beyond inhibition of the proteolytic enzymes by stimulating cartilage growth and disrupting the terminal differentiation of chondrocytes."

"in the tibias cultured with 40pg/ml doxycycline, not all of the chondrocytes had undergone hypertrophy. At the proximal ends of the cartilages, there remained a population of small, flattened cells that had not become hypertrophic"

Grow Taller with Sophorae Beans(Quercetin)

Due to abundant evidence that inhibiting Wnt and Beta-Catenin can cause ectopic chondrogenesis and our goal is to form new growth plates.  I am recommending quercetin as a beta-catenin inhibitor.  However, an analysis of the processes altered by axial loading revealed that axial loading may be able to induce ectopic chondrogenesis but just not full integrate them into growth plates.  Wnt and Beta-Catenin are essential for growth plates but mainly in the hypertrophic stage.  Given that the hypertrophic stage is the predominant determinant of adult height you should not take Quercetin if you have active growth plates.

First, you should do LSJL without taking Quercetin and measure height.  Then you cycle off for 1-2 weeks(and all supplements) after 2-3 weeks to allow to regain osteocyte sensitivity which are crucially involved in regulating the growth plates.  You measure bone length increase.  Then you take quercetin on the next LSJL cycle after you have established a baseline to see if Quercetin has any impact on gains.  After the first Quercetin LSJL cycle you can try doubling the dose since higher dosages may further inhibit Beta-Catenin.  Evidence of Quercetin Toxicity has been found after two years but cycling may prevent that.

Quercetin: Jarrow Formulas Quercetin 500mg, 100 Capsules

TGF-Beta and IGF-1 help encourage stem cell differentiation into chondrocytes.  The goal of LSJL is to induce stem cell differentiation into chondrocytes in order to form a kind of mini-growth plate.  Sophorae Beans, although this may be mostly due to Quercetin, increase TGF-Beta and IGF-1 levels.  Of course, we're not sure if it competes with other supplements(if you are taking a particular other supplement will that mean that Sophorae Beans will have no effect?).  Or, if there's a negative feedback mechanism associated with the increase in TGF-Beta and IGF-1 levels.  Do Sophorae Beans(or Quercetin) have to be cycled?

Here's an example of a Sophorae Fructus supplement: FRUCTUS SOPHORAE Gentlex Pill (HUAI JIAO) 250mg X 100 pills per bottle

Isoflavones extracted from Sophorae fructus upregulate IGF-I and TGF-beta and inhibit osteoclastogenesis in rat bone marrow cells.

"Isoflavones have been a central subject in research on the natural phytoestrogens found in Leguminosae. [Isoflavones] can act like estrogen by binding on estrogen receptors on the target cell surface{so when seeing if other supplements have competing effects we want to look at other supplements that act on estrogen receptors particularly other isoflavones, ideally an isaflovone would bind to an estrogen receptor and mimic the pro-height components while blocking the anti-height components of some estrogen compounds.}. When estrogen is no longer produced in the body a remarkable bone remodeling process occurs, and the associated events are regulated by growth factors in the osteoblast lineage. In the present study, we investigated whether isoflavones (Isocal) extracted from Sophorae fructus affect the growth factors IGF-I and TGF-beta that have been known to be related with bone formation. The active control (PIII) effectively enhanced the level of nitric oxide (NO) and growth factors, and thereby inhibited osteoclastogenesis. The most efficient concentration was 10(-8)% within five days, whereas the comparative control (soybean isoflavone) was not as effective even at a lower concentration. In conclusion, the products which contain enriched glucosidic isoflavone and nutrient supplements such as shark cartilage and calcium can be used for osteoporosis therapy by enhancing the production of IGF-I and TGF-beta. Furthermore, the NO produced through endothelial constitutive NO synthase (ecNOS) may play a role in inhibiting bone reabsorption."

So Sophorae Beans can help you grow taller by increasing levels of IGF-1 and TGF-Beta both in active growth plates or if trying to form a new growth plate with LSJL.  This depends of course on you not taking any supplements that compete with the same pathways as Sophorae Beans and the effect of Sophorae Beans not diminishing over time.

Simultaneous determination and pharmacokinetic study of six flavonoids from Fructus Sophorae extract in rat plasma by LC-MS/MS.

"six flavonoids including sophoricoside, genistin, genistein, rutin, quercetin and kaempferol [were elevated] in rat plasma after oral administration of Fructus Sophorae extract"<-you may want to take sophorae without the genistein.

Quercetin can increase Sox9.

The effect of quercetin on expression of SOX9 and subsequent release of type II collagen in spheno-occipital synchondroses of organ-cultured mice.

"A total of 50 one-day-old male BALB/c mice were randomly assigned to the control and experimental groups. Each group was subdivided into five different time points, which were 6, 24, 48, 72, and 168 hours, and each subgroup contained 5 mice. In the experimental group, the spheno-occipital synchondrosis was immersed in the BGJb medium + quercetin dihydrate 1 µM. In the control group, the spheno-occipital synchondrosis was immersed in the BGJb medium. Tissue sections were subjected to immunohistochemical staining for SOX9 and type II collagen expressions.

Quantitative analysis revealed there was a statistically significant increase of 32.31% in the expression of SOX9 between experimental groups and control groups at 24 hours. Furthermore, there was a statistically significant increase of 22.99% in the expression of type II collagen between experimental groups and control groups at 72 hours."

"some minor inhibition of stromelysin expression occurred at 10 uM concentrations of quercetin, this was accompanied by an even larger inhibition of proteoglycan expression." according to The antioxidants curcumin and quercetin inhibit inflammatory processes associated with arthritis., quercetin may have a minimal effect on inflammatory pathways.  And there was no inhibition of proteoglycan at 1 miroM.

quercetin does not inhibit MMP-13 production furthering the theory that it has low effect on inflammatory pathways.

Quercetin is the compound that is most likely to be anabolic and height increasing thus you may want to only take that.

Quercetin may also be a beta-catenin inhibition and beta-catenin inhibition can cause ectopic chondrogenesis.

Isoquercitrin isolated from Hyptis fasciculata reduces glioblastoma cell proliferation and changes beta-catenin cellular localization.

"nuclear beta-catenin staining observed in a subpopulation of untreated Gbm cells was found in the cytoplasm after 100-micromol/l isoquercitrin treatment"

"The flavonoid isoquercitrin (quercetin 3-O-[beta]-D-glucopyranoside), which is typically found in onions, is a quercetin moiety with a glucose molecule on C3 of the flavonoidic nucleus "

"When 100 µmol/l of isoquercitrin was added to Gbm cells, nuclear [beta]-catenin staining dramatically decreased to 4%, while non-nuclear staining increased to 77%"

"The decrease in nuclear [beta]-catenin is consistent with a decrease in Wnt/[beta]-catenin signaling activity."

Comparison of Quercetin Pharmacokinetics Following Oral Supplementation in Humans.

"[What's] the absorption of quercetin aglycone in 18 healthy human subjects administered via the following oral carrier systems: suspension of quercetin (quercetin QU995 powder in Tang(®) and spring water), nutritional bars (First Strike™), and chews (RealFX™ Q-Plus™)?"

Subjects received 500 mg Quercetin.

"The C(max) of quercetin was highest with RealFX™ Q-Plus™ Chews (1051.9 ± 393.1 μg/L) achieved within 3.3 h as compared to that for First Strike™ Bars (698.1 ± 189.5 μg/L in 2.3 h) and Tang(®) suspension (354.4 ± 87.6 μg/L in 4.7 h). "

The molecular weight of quercetin is 302.24.  So in the previous study 30224 ug/L was found to reduce beta-catenin localization to 4%.  

Onions, buck wheat tea, and Apple Sauce were other food sources of Quercetin.

Toxicity and carcinogenicity studies of quercetin, a natural component of foods.

"Quercetin is a naturally occurring chemical found in our daily diet in fruits and vegetables. Toxicity and carcinogenicity studies of quercetin were conducted in male and female F344/N rats, under conditions which allowed comparison to results of approximately 400 previously tested chemicals. The chemical was administered in the feed for 2-years at concentrations of 0, 1000, 10,000, or 40,000 ppm, and the estimated dose delivered was approximately 40-1900 mg/kg/day. There were no treatment-related effects on survival and no treatment-related clinical signs of toxicity. The high-dose groups had reduced body weight gain in comparison to controls during the last half of the study. At interim evaluations at 6 and 15 months, treatment-related toxic lesions were not observed, but at 2 years toxic and neoplastic lesions were seen in the kidney of male rats, including increased severity of chronic nephropathy, hyperplasia, and neoplasia of the renal tubular epithelium. Under the conditions of these 2-year studies quercetin showed carcinogenic activity in the kidney of the male rat, causing primarily benign tumors of the renal tubular epithelium. Quercetin did not cause tumors at other sites. Quercetin is a genotoxic chemical, but the neoplastic response observed in the kidney may be due in part to a combination of nongenotoxic and genotoxic events."<-If you cycle of Quercetin you likely can avoid these effects.

Attenuation of Chondrogenic Transformation in Vascular Smooth Muscle by Dietary Quercetin in the MGP-Deficient Mouse Model.

"Cartilaginous metaplasia of vascular smooth muscle (VSM) is characteristic for arterial calcification in diabetes and uremia and in the background of genetic alterations in matrix Gla protein (MGP){maybe specific removal of MGP can allow for ectopic chondrogesis in the bone?}.
This study aimed to identify the effects of bioflavonoid quercetin on chondrogenic transformation and calcification of VSM in the MGP-null mouse model and upon TGF-β3 stimulation in vitro, and to characterize the associated alterations in cell signaling.
Molecular analysis revealed activation of β-catenin signaling in cartilaginous metaplasia in Mgp-/- aortae in vivo and during chondrogenic transformation of VSMCs in vitro. Quercetin intercepted chondrogenic transformation of VSM and blocked activation of β-catenin both in vivo and in vitro{So quercetin's affect on chondrogenic differentiation may be mediated by it's effects on Beta-Catenin since blocking Beta-Catenin may be pro- and anti-chondrogenic at different points}. Although dietary quercetin drastically attenuated calcifying cartilaginous metaplasia in Mgp-/- animals, approximately one-half of total vascular calcium mineral remained as depositions along elastic lamellae.
Quercetin is potent in preventing VSM chondrogenic transformation caused by diverse stimuli. Combined with the demonstrated efficiency of dietary quercetin in preventing ectopic chondrogenesis in the MGP-null vasculature, these findings indicate a potentially broad therapeutic applicability of this safe for human consumption bioflavonoid in the therapy of cardiovascular conditions linked to cartilaginous metaplasia of VSM. Elastocalcinosis is a major component of MGP-null vascular disease and is controlled by a mechanism different from chondrogenic transformation of VSM and not sensitive to quercetin."

"Taking into account that quercetin inhibits TG2 via direct binding it is possible that modulation of TG2 enzymatic activity is central in chondrogenic metaplasia of VSM and its prevention. "

LSJL gene expression versus trabecular bone

Skeletal site-related variation in human trabecular bone transcriptome and signaling.

"we have compared global gene expression profiles of human trabecular bone from two different skeletal sites that experience vastly different degrees of mechanical loading, namely biopsies from iliac crest and lumbar spinal lamina.

In the lumbar spine, compared to the iliac crest, the majority of the differentially expressed genes showed significantly increased levels of expression; 3406 transcripts were up- whilst 838 were down-regulated. Interestingly, all gene transcripts that have been recently demonstrated to be markers of osteocyte, as well as osteoblast and osteoclast-related genes, were markedly up-regulated in the spine. The transcriptome data is consistent with osteocyte numbers being almost identical at the two anatomical sites, but suggesting a relatively low osteocyte functional activity in the iliac crest. Similarly, osteoblast and osteoclast expression data suggested similar numbers of the cells, but presented with higher activity in the spine than iliac crest. "

The bone biopsy was from trabecular bone.

Genes upregulated in Spinal Lamina versus Iliac Crest(the spinal lamina should receive more loading than the iliac crest);

Col2a1{up in LSJL}

COMP

CILP

MYL1{up}

Pdlim3{up}

Clu

Nebulin{down}

Sox9{up}

Egr1{up}

MMP2{up}

BMP2{up}

Osteopontin{up}

BSP{up}

Cyr61{up}?

Acta2{up}?

Downregulated:

ITGA4{down}

Interesting to see the chondrogenic related Col2a1 and Sox9.

The supplementary data was confusing and I'm not sure which genes were actually upregulated but what's clear is that axial loading does induce chondrogenic genes which is consistent with our pathway analysis that chondroinduction may not be the failure of axial loading and it may be integration of those chondrocytes into a growth plate.

In this case possibly due to lack of Acan and other growth plate proteins.

Differential load-regulated global gene expression in mouse trabecular osteocytes.

"we have used a recently developed model whereby a single caudal mouse vertebra (C5) is subjected to controlled compression loading and further devised a method for the isolation of high quality RNA from trabecular osteocytes. RNA samples from loaded and sham-loaded individual vertebrae where then subjected to gene array analysis following the administration of a single or repetitive loading doses (thrice weekly for 4weeks). Focusing on extracellular genes potentially involved in mediating osteocyte-derived signals to the trabecular surface, we identified sets of genes differentially regulated by either single or multiple loading bouts as well as genes affected by both loading protocols. A comparison with published studies on load-regulated genes in cortical osteocytes revealed that the majority of these genes are specifically activated/silenced in the trabecular bone. Many of these genes could be clustered according to processes directly relevant to the life cycle and activity of osteoblasts and osteoclasts and their progenitors."

"nitric oxide (NO), prostaglandin E2 (PGE2), insulin-like growth factor (IGF-1), dentin matrix protein 1 (DMP-1) and sclerostin [are generated by osteocytes in response to mechanical loading]"

9 week old Female C57BL/6 mice.  All cells were removed other than osteocytes.

"Loading here was at 8 N, as this regimen generates peak strains of 880 με on the dorsal side and 820 με on the ventral side of C5"

Genes upregulated in trabecular osteocytes in response to single loading also up in LSJL:
Adamts4
Adamts1
Aspn
Chi3l3{down}
Olfm4{down}

Genes upregulated in trabecular osteocytes in response to repetitive loading also up in LSJL:
Ptn
Vcan
Aspn
Thbs4
Sema3c
Il33
Thbs2
MMP2
Cyr61
Ntn1
Sod3
Grem2

Down:
Metrnl
Chgb
Lefty2


A significant percentrage of genes were expressed in osteocytes that were also expressed due to Joint Loading and there were no deferentially expressed genes between the two studies.

A portion of LSJL Response is explained by mechanotransduction signals within osteocytes
The LSJL lengthening effect is not solely the result of osteocyte mechanotransduction as axial loading does not typically induce a lengthening response despite sharing many genes with lateral loading.

Confirmation that the LSJL scientists believed it can cause height growth

Here's a grant submitted by Ping Zhang that was proposed to do research on whether LSJL could cause adult height growth!

Load-Driven Bone Lengthening

"The long-term objective of the proposed project is to understand the mechanism of load-driven bone lengthening. Although distraction osteogenesis is effective to treat the patients with limb length discrepancy, this invasive procedure occasionally generates problems such as premature consolidation, delayed union, and infection. In order to investigate a possibility of load-driven non-invasive therapy[like LSJL], we will focus on knee loading a form of joint loading modalities. We address a set of questions:
(1) Does knee loading enhance proliferation of chondrocytes in the growth plate and lengthen the proliferative and hypertrophic zones in the distal femur and the proximal tibia[if knee loading can cause differentiation of stem cells into chondrocytes it can cause new height growth]?
(2) Is the lengthening effect dependent on ages[will it work on adults?]?
(3) Does injection of insulin growth factor-2 (IGF-2) into the growth plate enhance the loading effects?
We hypothesize
 (a) Lateral loads, applied to the knee with appropriate loading conditions, can lengthen both the femur and the tibia through stimulating proliferation of chondrocytes in the growth plate;
(b) Efficacy of load-driven bone lengthening is stronger in the youth than the elderly;
(c) The lengthening effect can be augmented in the elderly by injecting IGF-1 in the growth plate[so LSJL will work even in aged individuals!!!!  However, aged individuals do not have growth plates so maybe he means injecting IGF-1 into the bone marrow?  I will concede that based on some communications with Ping Zhang that he does not have excellent english skills so perhaps he used the wrong terminology].

In order to examine the above hypotheses, three specific aims are proposed using a hindlimb of C57BL/6 mice as a model system.

Evaluation of load-driven bone lengthening under varying mechanical conditions
Comparison of the loading effects among mice with varying ages
Effects of local administration of IGF-2 into the growth plate with and without knee loading.

We will use a custom-made piezoelectric mechanical loader, and conduct bone histomorphometric and gene expression analyses using varying imaging modalities. The proposed study is expected to contribute to developing a non-invasive physical therapy for treatment of patients with limb length discrepancy.

The proposed project will contribute to examining a possibility of non-invasive physical therapy for treatment of patients with limb length discrepancy"

Here's the grant name to track it: Grant 1R03AR055322-01A1.

Here's a link with more info on the project.  It should be noted that a Salubrinal study is included as part of the grant.  So maybe Salubrinal does have an undisclosed effect on lengthening.

Here's Ping Zhang's research information.

Patents for Promoting Heterotopic Ossification or other potential height increasing phenomenon and Possible Adult Growth

Here's a link to the patent.

The three scientists involved are: Benjamin J. Remington (Modesto, CA, US)  David J. Bearss (Modesto, CA, US)  Kavian Shahi (Granite Bay, CA, US)

"The present invention provides an improved technique for spinal fusion involving the administration of an HMG-CoA reductase inhibitor to a fusion. The HMG-CoA reductase inhibitor is preferably delivered to the site by a carrier[This will be virtually impossible to do by homeopathic means]. More preferably, the HMG-CoA reductase inhibitor is delivered to the site by a non-compressible delivery vehicle. The invention is suitable for promoting non-anatomic or heterotopic bone growth between any bony surfaces where bone growth is desired but does not naturally occur."

This means the invention is capable of promoting adult height growth on the longitudinal ends of the bones.  There's evidence though that longitudinal apposition already occurs on long bones but it is too slow and it is likely nullified by osteoclast activity.

One possible HMG-CoA reductase inhibitor listed is lovastatin. But there's no way to mimic the carrier aspect.

"Preferably, the HMG-CoA reductase inhibitors are administered directly to the site of fusion. More preferably, the HMG-CoA reductase inhibitors are administered via a carrier, such as an open cell matrix. The carrier can further comprise of other therapeutic agents such as antibiotics, painkillers, antioxidants, growth factors, and timed release agent."

"the present invention involves the use of HMG-CoA reductase inhibitor to promote bone growth and fusion between any two bones. Bone fusion is effective in treatment post-traumatic, degenerative and/or inflammatory arthritis conditions. In one embodiment, the HMG-CoA reductase inhibitor is used in podiatric surgery, such as to immobilize the metatarsals or the ankle joint. Furthermore, HMG-CoA reductase inhibitors can be utilized in facial plastic and reconstruction surgeries, such as to fix the maxillary and mandibular bones, increase cheekbone morphology, and cranial vault fixation and remodeling (e.g., due to craniosynostosis)."<-the problem is you really don't want to fuse two long bones together.

"Non-compressible carriers such as surgical instrumentations can be used as an adjunct to obtain a solid fusion or provide stability. Surgical instrumentation can be fabricated from titanium and/or alloys of titanium, stainless steel, ceramic materials or rigid polymeric materials. Typical medical instrumentations include, for example, rods, hooks, braided cable, plates, screws, and threaded interbody cages. Instrumentation can decrease the likelihood of non-union by maintaining spinal stability while facilitating the process of fusion. For example, instrumentation can be used to bridge space created by the removal of a spinal element such as an intervertebral disc. Instrumentation can also be used to correct a deformity or as an internal splint to hold the vertebrae together while the bone grafts heal. In a preferred embodiment, an instrument is coated with synthetic apatites, or preferably hydroxyapatite. Unfortunately, even with the use of instrumentation, non-union remains a common problem. "

"The terms "non-anatomic" and "heterotopic" bone growths refer to the generation of bone in regions that naturally do not grow bone."

"The term "HMG-CoA reductase inhibitors" or "statins" as used herein refers to compounds that inhibit the enzyme 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase. HMG-CoA reductase is the principal rate-limiting enzyme involved in cellular cholesterol biosynthesis. The pathway is also responsible for the production of dolichol, ubiquinones, isopentenyl adenine and farnesol. HMG-CoA reductase converts 3-hydroxy-3-methyld-glutaryl CoA (HMG-CoA) to mevalonate."

"HMG-CoA reductase inhibitors, or statins, enhance the production of osteoblasts, the cells that produce new bone and enhance osteoblast differentiation."<-So you could just take Lovastatin and get more osteoblasts depositing bone on the subchondral plate.  The problem is subchondral bone apposition for longitudinal growth is incredibly slow and it's not synergestic with LSJL where you're trying to induce endochondral ossification which is much faster.

"Examples of the HMG-CoA reductase inhibitors of the present invention include, but not limited to, lovastatin, pravastatin, velostatin, simvastatin, fluvastatin, cerivastatin, mevastatin, dalvastatin, fluindostatin, atorvastatin, or a prodrug thereof, a pharmaceutically acceptable salt of any such HMG-CoA reductase inhibitors, or prodrug thereof. Preferable salts include calcium and/or phosphate salts. In a preferred embodiment, the HMG-CoA reductase inhibitor is simvastatin or simvastatin calcium. In another preferred embodiment, the HMG-CoA reductase inhibitor is lovastatin or lovastain calcium."

The problem is that this method does not induce bone growth by endochondral ossification.  Natural appositional longitudinal growth of the fingers is reported to be 0.34mm per decade.

Longitudinal shrinkage in the lower legs occurs at a rate of 2.4mm per year.

It's pretty clear that osteoclasts, osteoblasts, and articular cartilage endochondral ossification does have the ability to add or remove height from bone.  The problem is that you'd need to stimulate osteoblasts to extreme levels(by lovastatin supplementation or other means) to get any noticeable height, the synergy with LSJL is unclear, and it would stimulate bone thickness everywhere so you'd increase bone thickness a great deal with a minor increase in length resulting in acromegaly-like proportions.

The advantage over LSJL, is that you could do it without any exercise and only supplements.

Patent II.

Inventors: E. Skott Greenhalgh (Lower Gwynedd, PA, US)  John-Paul Romano (Chalfont, PA, US)

"An expandable support device for tissue repair is disclosed. The device can be used to repair hard or soft tissue, such as bone. The expandable support device can have interconnected struts. A method of repairing tissue is also disclosed. The expandable support device can be inserted into a damaged bone and radial expanded. The radial expansion of the expandable support device struts can cause the struts to cut mechanically support and/or the bone."

So it's a device that supports the bone and can therefore make you taller but the device would not be natural.

"An expandable support device for repairing damaged bone, the expandable support device having a longitudinal axis, and comprising: a first terminal end comprising a radially external first attachment configuration; a second terminal end comprising a radially external second attachment configuration, and a radially internal attachment configuration; a first strut having a first strut cross-section; a second strut connected to the first strut, wherein the first strut is substantially deformable; and wherein the first strut cross-section is configured to encourage bone growth toward the longitudinal axis. "

"The device can be configured to contract in a longitudinal direction during deployment in a bone."

Here's what the device looks like:

"Any or all elements of the expandable support device and/or deployment tool and/or other devices or apparatuses described herein, can be, have, and/or be completely or partially coated with agents and/or a matrix a matrix for cell ingrowth or used with a fabric, for example a covering (not shown) that acts as a matrix for cell ingrowth. The matrix and/or fabric can be, for example, polyester (e.g., DACRON® from E. I. Du Pont de Nemours and Company, Wilmington, DE), polypropylene, PTFE, ePTFE, nylon, extruded collagen, silicone or combinations there"

The device may have potential to work by generating forces within the bone that stretch it but there have been no studies validating it and we don't know how much force the device can generate.

PatentIII(this is another fixation fixation device that may be useful).

LSJL Pathway Analysis

This was obtained using Partek Pathway software with the excel spreadsheet with list of 2 fold changers.  mm8 of whole mouse genome was selected.  Targeted pathways with relevance to longitudinal bone growth or chondroinduction were selected.  The old pathways generated in the previous gene expression study are included for reference.  The closest thing to a chondroinduction pathway is TGF-Beta.  These pathways can be used to predict phosphorylation and additional gene expression changes.  Green means expression was altered.  Light green means more downregulated whereas darker more redish green means more significantly upregulated.  Most important to note is that LSJL upregulates several genes in cell adhesion which is an important precursor to chondrogenesis(3.3% of genes).  LSJL also upregulates genes involved in cartilage condensation, chondrobast differentiation, and chondrocyte differentiation which are three processes that would be key to formation of new growth plates with p < 0.05 and containing at least 20% of the genes involved in that process expressed at over 2-fold levels.

Comparing this to axial loading genes(Alternative Splicing in Bone Following Mechanical Loading), many processes are shared except that the processes of chondrocyte development, cartilage development in endochondral bone morphogenesis, skeletal system morphogenesis, growth plate cartilage development, and endochondral ossification are significantly altered by LSJL whereas they are not by axial loading(see below for definitions).  It is likely that these 5 processes are part of the reason that LSJL can increase height whereas axial loading does not.  

I don't believe cartilage development in endochondral bone morphogenesis is a key different as the only gene that's present in LSJL and not in axial loading is Col1a1 and that is not a master regulator gene.  But the absence of Col1a1 stimulation in axial loading is odd.  Growth Plate Cartilage Development is a process where axial loading is missing Col9a1 and THBS3.  THBS3 is a gene that could potentially have a big impact.

In Skeletal System Morphogenesis most of the genes are shared except HHIP, Col11a1(Col11a2 is altered by axial loading however), Tgfbr1, and Wwox are altered by LSJL but not by axial loading.  HHIP is a key gene so that is likely the differentiating factor.

In endochondral ossification, Col10a1, Col1a1, and GNAS are three genes altered by LSJL and not by axial loading.

So GNAS, HHIP, and THBS3 may be key genes explaining part of the reason why LSJL can increase height but axial loading does not.

Surprisingly, axial loading significantly stimulates key processes in initial growth plate formation like chondroblast differentiation, cartilage condensation, and chondrocyte differentiation.  Chondrocyte development was very significantly enriched by axial loading but the key gene Sox9 was missing. Therefore, axial loading must fail at a later stage in the process likely at inducing Sox9.  Since the cutoff was much lower for the axial loading study than the LSJL study it's possible that LSJL alters many of the genes by the axial loading study as well.

Axial loading may very well induce the early stages of chondrogenesis and there may in fact be ectopic cartilage(or chondrocyte) formation in response to mechanical loading.  However, that cartilage seems to fail to become a growth plate.  To test this hypothesis we'd need to see if ectopic chondrocytes do form under the bone under Beta-Catenin knockout ectopic chondrocytes occur under the perichondrium(immature periosteum).  If we can find evidence that axial loading can induce ectopic chondrocyte formation in adult bone that would be a step forward in proving LSJL.

Pathways(not all are below) that LSJL upregulates with p < 0.05:
ECM-Receptor
Protein Digestion
Focal Adhesion
Amoebiasis
Mineral Absorption
Hedgehog Signaling
Aminoacyl-tRNA
Caffeine Metabolism
Olfactory Transduction
Homologous Recombination

Gene Ontology(calculated using Partek) for Chondroinduction Related Gene Sets:

The Enrichment Score is Next to the data.  The higher the enrichment score the better.  Multiples of genes are intentional.  Bolded means that p < 0.05.  % is the precent of genes in gene group that are present.

Positive Regulation of Cartilage Development(4.33):<-"Any process that increases the rate, frequency, or extent of cartilage development, the process whose specific outcome is the progression of the cartilage over time, from its formation to the mature structure."
Cyr61
Smad1{down}
Sox9
Sox9

Chondrocyte Differentiation(3.98):<-"The process in which a chondroblast acquires specialized structural and/or functional features of a chondrocyte." 20%
Col2a1
Fgfr1
Fgfr1
Mef2d{down}
Osr1{down}

Cartilage Condensation(8.77):<-"The condensation of mesenchymal cells that have been committed to differentiate into chondrocytes." 35.29%
Acan
Barx2
Barx2
BMPR1B
Col11a1
Col2a1
Sox9
Sox9

Full Gene List.

Chondroblast Differentiation(4.17):<-"The process in which a mesenchymal cell, acquires specialized structural and/or functional features of a chondroblast." 50%
Cyr61
FGF2

Full Gene List.  RARA downregulation is key, all others need to be upregulated.  FGF-4 downregulation may also be important.

Cell Adhesion Molecule Binding(9.04):<-"Interacting selectively and non-covalently with a cell adhesion molecule."
Adam8{down}
Anp32a{down}
Cadm1{down}
CD226{down}
CD226{down}
FGFR1
FGFR1
PVRL3
Sdcbp
Vcam1{down}

Positive regulation of Chondrocyte Differentiation(1.69):<-"Any process that activates or increases the frequency, rate or extent of chondrocyte differentiation. "
Gli3
Gli3
Sox9
Sox9

Regulation of Cell Shape(2.30):<-Chondrocytes are round shaped.  "Any process that modulates the surface configuration of a cell."
Epb4.1
Fgd4{down}
Gna13{down}
IL6
KDR
PDPN
PDPN

Bone morphogenesis(1.50):<-"The process in which bones are generated and organized. "
Cdx1{down}
Pax1

Endochondral Bone Morphogenesis(no enrichment score unfortunately):<-"The process in which bones are generated and organized as a result of the conversion of initial cartilaginous anlage into bone. "

Skeletal system morphogenesis(4.88):<-"The process in which the anatomical structures of the skeleton are generated and organized."
Col11a1
Col11a1
Col2a1
HHIP
MMP2
TGFBR1{down}
WWOX{down}

Cartilage Development(6.72):<-"The process whose specific outcome is the progression of the cartilage over time, from its formation to the mature structure. Cartilage is a connective tissue dominated by extracellular matrix containing collagen type II and large amounts of proteoglycan, particularly chondroitin sulfate."
BMPR1B
COL10A1
COL11A1
COL2A1
COL9A1
EDN1
GNAS{down}
PRRX1
PRRX1
PRRX2
SOX9
SOX9

Cartilage development involved in Endochondral bone morphogenesis(3.03):<-"The process whose specific outcome is the progression of the cartilage that will provide a scaffold for mineralization of endochondral bones. "
Col1a1
Col2a1

Growth Plate Cartilage Development(3.03):<-"The process whose specific outcome is the progression of the cartilage that will provide a scaffold for mineralization of endochondral bones as they elongate or grow. "
Col9a1
THBS3

Endochondral Ossification(6.54):<-"Replacement ossification wherein bone tissue replaces cartilage. "
Col10a1
Col1a1
GNAS
GNAS{Down}
MEF2D{down}

Stem Cell Differentiation(0.39):<-"The process in which a relatively unspecialized cell acquires specialized features of a stem cell. A stem cell is a cell that retains the ability to divide and proliferate throughout life to provide progenitor cells that can differentiate into specialized cells. "
Osr1{down}

Cell Adhesion Molecule Binding(9.04):<-"Interacting selectively and non-covalently with a cell adhesion molecule. "
Adam8{down}
Anp32a{down}
Cadm1{down}
CD226{down}
CD226{down}
Fgfr1
Fgfr1
Pvrl3
Sdcbp
Vcam1{down}

Actin Cytoskeleton Organization(4.43):<-"A process that is carried out at the cellular level which results in the assembly, arrangement of constituent parts, or disassembly of cytoskeletal structures comprising actin filaments and their associated proteins."  Reorganization of the actin cytoskeleton is essential for chondrogenic differentiation
Diap3{down}
Enah{down}
Epb4.1
Fhod1{down}
Fhod3
Fscn1
Kras{down}
Pfn2
Shc1{down}
Tesk2{down}
Wasf2{down}

Skeletal System Development(6.87):<-"The process whose specific outcome is the progression of the skeleton over time, from its formation to the mature structure. The skeleton is the bony framework of the body in vertebrates (endoskeleton) or the hard outer envelope of insects (exoskeleton or dermoskeleton)."
BMPR1B
COL1A1
COL2A1
COL5A2
EDN1
GNAS
GNAS{DOWN}
HEXA{DOWN}
HOXD10
PAX1
SOX9
SOX9
TGFBR1{DOWN}
WDR5{DOWN}

Positive Regulation of Cell Migration(9.41): Stem Cell Migration is required to form new growth plates.  "Any process that activates or increases the frequency, rate or extent of cell migration."
Adam8{DOWN}
C3AR1
ccl24
Cdh13
Col18a1
Col18a1
Col1a1
Cyr61
EDN1
EGFR
GRB7{DOWN}
HBEGF
IRS1{DOWN}
KDR
MAPK8{DOWN}
PDPN
PDPN
TRIP6{DOWN}

Chondrocyte Development(3.80):<-"The process whose specific outcome is the progression of a chondrocyte over time, from its commitment to its mature state. Chondrocyte development does not include the steps involved in committing a chondroblast to a chondrocyte fate."
Acan
Col11a1
Sox9
Sox9

Homophilic Cell Adhesion(8.52)<-Required for prechondrogenic mesenchymal condensation.
Chondroblast differentiation is the strongest proof of chondroinduction by LSJL.  "The attachment of an adhesion molecule in one cell to an identical molecule in an adjacent cell. "
Cadm1{down}
Cadm3
Cdh10
Cdh11{down}
Cdh13
Cdh15{down}
Chl1
Dsc2
Dsg4{down}
Frem2
Pvrl3
Robo2

Tissue Remodeling(8.36)<-To make room for new growth plates..."The reorganization or renovation of existing tissues. This process can either change the characteristics of a tissue such as in blood vessel remodeling, or result in the dynamic equilibrium of a tissue such as in bone remodeling." 57.14%
CSPG4
CSPG4
LIPA{down}
MMP14
MMP2

Other possibly related pathways(genes not enumerated with p < 0.01, possible involvement with endochondral ossification, and enrichment score >1):

Reponse to Zinc Ion("Any process that results in a change in state or activity of a cell or an organism (in terms of movement, secretion, enzyme production, gene expression, etc.) as a result of a zinc ion stimulus. ")

Inflammatory Response("The immediate defensive reaction (by vertebrate tissue) to infection or injury caused by chemical or physical agents. The process is characterized by local vasodilation, extravasation of plasma into intercellular spaces and accumulation of white blood cells and macrophages. ")

Ras Protein Signal Transduction("A series of molecular signals within the cell that are mediated by a member of the Ras superfamily of proteins switching to a GTP-bound active state. ")

PI3K Cascade("A series of reactions, mediated by the intracellular phosphatidylinositol 3-kinase (PI3K). PI3K cascades lie downstream of many cell surface receptor linked signaling pathways and regulate numerous cellular functions. ")

Wnt Receptor Signaling Pathway("The series of molecular signals initiated by binding of a Wnt protein to a frizzled family receptor on the surface of the target cell and ending with a change in cell state. ")

Centriole Replication("The cell cycle process in which a daughter centriole is formed perpendicular to an existing centriole. An immature centriole contains a ninefold radially symmetric array of single microtubules; mature centrioles consist of a radial array of nine microtubule triplets, doublets, or singlets depending upon the species and cell type. ")

transcription from RNA polymerase II promoter("The synthesis of RNA from a DNA template by RNA polymerase II, originating at an RNA polymerase II promoter. Includes transcription of messenger RNA (mRNA) and certain small nuclear RNAs (snRNAs). ")

Protein autophosphorylation("The phosphorylation by a protein of one or more of its own amino acid residues, or residues on an identical protein. ")

Endocytosis("A vesicle-mediated transport process in which cells take up external materials or membrane constituents by the invagination of a small region of the plasma membrane to form a new membrane-bounded vesicle. ")

Apoptosis("A programmed cell death process which begins when a cell receives an internal (e.g. DNA damage) or external signal (e.g. an extracellular death ligand), and proceeds through a series of biochemical events (signaling pathways) which typically lead to rounding-up of the cell, retraction of pseudopodes, reduction of cellular volume (pyknosis), chromatin condensation, nuclear fragmentation (karyorrhexis), plasma membrane blebbing and fragmentation of the cell into apoptotic bodies. The process ends when the cell has died. The process is divided into a signaling pathway phase and into an execution phase, which is triggered by the former. ")

Multicellular organism growth("The increase in size or mass of an entire multicellular organism, as opposed to cell growth. ")

Positive regulation of endothelial cell proliferation("Any process that activates or increases the rate or extent of endothelial cell proliferation. ")

Positive regulation of mesenchymal cell proliferation

negative regulation of transcription from RNA polymerase II promoter

positive regulation of cell proliferation

negative regulation of cell proliferation

negative regulation of cell adhesion

negative regulation of apoptosis

Positive regulation of inflammatory response("Any process that activates or increases the frequency, rate or extent of the inflammatory response. ")


positive regulation of transcription from RNA polymerase II promoter

positive regulation of angiogenesis

positive regulation of cell differentiation

peptide hormone processing(endocrine hormones)("The generation of a mature peptide hormone by posttranslational processing of a prohormone.")

Collagen fibril organization("The generation of a mature peptide hormone by posttranslational processing of a prohormone.")

Alpha-tubulin binding("Interacting selectively and non-covalently with the microtubule constituent protein alpha-tubulin. ")

Beta-tubulin binding

Integrin binding

protein C-terminis binding("Interacting selectively and non-covalently with a protein C-terminus, the end of any peptide chain at which the 1-carboxy function of a constituent amino acid is not attached in peptide linkage to another amino-acid residue. ")

histone acetylase binding

histone deacetylase binding

fibronectin binding

misfolded protein binding

epidermal growth factor receptor binding

neurexin family protein binding

platelet-derived growth factor binding

PDZ domain binding

Heparin Binding

Hyaluronic Acid binding

Calcium ion binding

Magnesium ion binding

UDP-N-acetylmuramate dehydrogenase activity("Catalysis of the reaction: UDP-N-acetylmuramate + NADP+ = UDP-N-acetyl-3-O-(1-carboxyvinyl)-D-glucosamine + NADPH + H+. ") NADPH is required for chondrogenesis.

Endocytic vesicle("A membrane-bounded intracellular vesicle formed by invagination of the plasma membrane around an extracellular substance. Endocytic vesicles fuse with early endosomes to deliver the cargo for further sorting. ")

Spliceosomal complex("Any of a series of ribonucleoprotein complexes that contain RNA and small nuclear ribonucleoproteins (snRNPs), and are formed sequentially during the splicing of a messenger RNA primary transcript to excise an intron. ")

Cellular response to calcium ion

Nitric Oxide Mediated Signal Transduction

Peptidyl-tyrosine dephosphorylation("The removal of phosphoric residues from peptidyl-O-phospho-tyrosine to form peptidyl-tyrosine.")

Gene Expression analysis from Life Map Discovery:(Genes that are uporegulated in LSJL and also that cell lineage.  Bold are selective markers.  - means that the cell type is characterized by a lack of that protein.

The apical ectodermal ridge (AER) is a structure that forms from the ectodermal cells at the distal end of each limb bud and acts as a major signaling center to ensure proper development of a limb.


Zeugopod growth plate is used.  The zeugopod growth plates are located at both ends of the long bones (ulna, radius, tibia and fibula):

Aldh1a3{down}
Angptl1
Bmp2
Car12
Col10a1
Epyc
Fgf2
Fgfr1
Kctd4
MMP14
Smad1{down}
Smpd3{down}
Sox9
Bsp
Zcchc5

Progress Zone cells: Progress zone cells are rapidly proliferating, undifferentiated mesenchyme cells, situated directly underneath the Apical Ectodermal Ridge.

Hoxd10
Bmp2
Fgfr1

Prechondrocytic mesenchymal cells:  Prechondrocytic mesenchymal cells are mesenchymal cells ready to acquire osteochondral fate. They produce an extracellular matrix rich in hyaluronan.

Col1a1
-Sox9
Lin28b
Pkia
Slitrk6
Tax1bp1{down}

Mesenchymal condensate cells:  Mesenchymal condensate cells are mesenchymal cells that aggregate at future sites of bone formation at the initiation of chondrogenesis. The aggregation step is essential for further osteochondral differentiation.

Acan
Col11a1
Col2a1
Sox9
Bmpr1b
Bmp2
Fgf2
Fgfr1
Gli3
Thbs2
Aspn
Brpf3{down}
Ccng2{down}
Cd9{down}
Cops2{down}
Ebf3{down}
Egfr
Flnc
Gas1
Hapln1
Htra1
Jun
Lum
Matn3
Moxd1
Myl1
Osr1{down}
Sstr4{down}
Sulf1
Thbs4
Vcan
Zcchc5
1110012J17Rik

Zeugopod prechondrocytes: Prechondrocytes are resting, non-proliferative cells.

Acan
Col11a1
Col9a1
Sox9
Fgf2
Thbs2

Zeugopod epiphyseal end chondrocytes: Chondrocytes are highly proliferative columnar cells.

Acan
Col2a1
Sox9
Matn3
Hapln1
Bmp2
Gli3
Lepre1
Tgfbr1 {down}
Arsi
Cgref1
D10Bwg1379e
Dock8 {down}
Epha3
Epyc
Fry {down}
Matn2
Plekha6 {down}
Smpd3{down}
Tcf7 {down}
Thbs4

Prehypertrophic Chondrocytes: Prehypertrophic chondrocytes are chondrocytes at maturation, which no longer proliferate.
Col10a1
Fgfr1

Hypertrophic Chondrocytes: Hypertrophic chondrocytes are non-proliferative cells, larger in size than prehypertrophic chondrocytes and synthesize a characteristic cartilogenous matrix.

Col10a1
Bsp
Sox9
Bmp2
Fgf2
Fgfr1
Smad1 {down}

Terminal Chondrocytes: Terminal chondrocytes undergo apoptosis and are replaced by bone cells. They have an osteoblast-like phenotype.

MMP14
Bsp

Grow Taller with Heparin & Perlecan Sulfate?

Heparin sulfate is available for sale: Monoject Heparin PreFill I.V. Flush Syringes.  I couldn't find an oral supplement.  Just gels and needles (see above). Here's the gel supplement: Heparin Oinment 25 g.

I'm not sure if you can inject the syringe directly into the epiphyseal bone marrow(the target is likely the knee cartilage) or if the ointment can result in heparin transport to the bone marrow but heparin is an intriguing possibility that may be growth promoting.  And it's not clear whether the heparin is sulfated.

There are a number of questions regarding the possible use but I think their potential availability and there chondro-promoting affects makes them worthwhile for presentation if anyone has more information.

Chondrogenesis on sulfonate-coated hydrogels is regulated by their mechanical properties.

"sulfur-containing acidic groups induce chondrogenesis in vitro and in vivo. Mechanical properties of cell substrates largely influence cell differentiation. Mechanical properties of sulfonate-coated hydrogels [influence] chondrogenesis of mesenchymal stem cells (MSCs). Sulfonate-coated polyacrylamide gels (S-PAAm gels) which have the elastic modulus, E, of about 1, 15 and 150kPa, were used in this study. MSCs cultured on the high stiffness S-PAAm gels (E=∼150kPa) spread out with strong expression of stress fibers, while MSCs cultured on the low stiffness S-PAAm gels (E=∼1kPa) had round shapes with less stress fibers but more cortical actins. Even in the absence of differentiation supplements, the lower stiffness S-PAAm gels led to the higher mRNA levels of chondrogenic markers such as Col2a1, Agc and Sox9 and the lower mRNA levels of an undifferentiation marker Sca1, indicating that the mechanical properties of S-PAAm gels strongly influence chondrogenesis{decrease bone stiffness to grow taller?}. Blebbistatin which blocks myosin II-mediated mechanical sensing suppressed chondrogenesis induced by the low stiffness S-PAAm gels. soft S-PAAm gels effectively drive MSC chondrogenesis even in the absence of soluble differentiation factors and thus suggests that sulfonate-containing hydrogels with low stiffness could be a powerful tool for cartilage regeneration."

"sulfated proteoglycans interact with several growth factors"

"heparan sulfate stimulated cells of a murine fibroblast line C3H10T1/2 to differentiate into chondrocytes in vitro"

"the sulfate-bearing domain of perlecan was responsible for the in vitro aggregation and chondrogenic activation of cultured C3H10T1/2 cells"  Here's a product that contains "perlecan activators": Sirtuin Phytohormone Replenishing Cream 50ml/1.7oz.  Again it's a gel.  Sulfation level of perlecan is unclear and whether any perlecan arrives in the bone marrow is unclear as well.

" the addition of heparan sulfate, heparin, or dermatan sulfate increased the formation of chondrogenic cell aggregates along with increasing sulfate incorporation"

"hydrogels composed of sulfonic acid polymers promotes prechondrogenic cell line ATDC5 into chondrocyte in vitro and induces cartilage regeneration in vivo"

"sulfonate-coated hydrogels with low stiffness effectively direct MSC chondrogenesis even in the absence of differentiation supplements."

"MSCs cultured on the low stiffness gels formed the tightly-packed aggregation, whereas MSCs on the high stiffness gels were spread out and then proliferated rapidly to become confluent within 1 week"<-maybe we can lower the stiffness of bone in some way?

"the cellular shapes and cytoskeletal structures in MSCs depended strongly on the gel stiffness"

"disorganization of cytoskeletal architectures promoted chondrogenesis"<-LSJL alters genes involved in actin cytoskeleton organization.

"The structural changes of cytoskeletal architecture can affect nuclear signaling by distorting the nucleus, focal adhesion signaling by mechanically inducing changes in focal adhesion, and receptor-mediated signaling by changing the spatial distribution of signaling intermediaries relative to the plasma membrane"

Phenol red may hurt children's ability to grow taller

This site lists high phenol red containing foods(well all high phenol foods of which phenol red is a subset).  The food list includes many foods of which would be almost impossible to avoid all of them.  If you replace milk with colostrum that will take phenol red levels down a little.


Phenol Red Inhibits Chondrogenic Differentiation and Affects Osteogenic Differentiation of Human Mesenchymal Stem Cells in Vitro.

"The purpose with this study was to investigate the effect of phenol red (PR) on chondrogenic and osteogenic differentiation of human mesenchymal stem cells (hMSCs). hMSCs were differentiated into chondrogenic and osteogenic directions in DMEM with and without PR for 2, 7, 14, 21, and 28 days. Gene expression of chondrogenic and osteogenic markers were analyzed by RT-qPCR. The presence of proteoglycans was visualized histologically. Osteogenic matrix deposition and mineralization were examined measuring the alkaline phophatase activity and calcium deposition. During chondrogenic differentiation PR decreased sox9, collagen type 2, aggrecan on day 14 and 21 (P < 0.05), and proteoglycan synthesis on day 21 and 28. Collagen type 10 was decreased on day 21 (P < 0.05). During osteogenic differentiation PR increased alkaline phosphatase on day 7 while decreased on day 21 (P < 0.05). PR increased collagen type 1 on day 7, 14, and day 21 (P < 0.05). The alkaline phosphatase activity was increased after 2, 7, and 14 days (P < 0.05). The deposition of calcium was decreased on day 21 (P < 0.05)."

Now this doesn't necessarily mean that phenol red decreases height as Sox9 and extracellular matrix components decrease during the hypertrophic and ossification stage and phenol red may be accelerating endochondral ossification while not affecting adult height.  However Col10 a hypertrophic stage marker decreases and proteoglycan synthesis decreases as well indicating that phenol red may actually decrease adult height rather than just accelerating it.  However, we can't say for sure without a longitudinal study with phenol red supplementation versus control.

b and e have phenol red whereas c and f do not and you can see the size difference.  Although it's possible that phenol red just slows down growth and eventually phenol red treated chondrocytes grow to normal levels.

"Chondrogenesis was induced in pellet cultures in polypropylene tubes, 2.5 × 105 cells/pellet, by addition of 100 nM Dexamethasone, 50 μg/ml L-Ascorbic Acid-2 phosphate, 40 μg/ml L-Proline, 1 mM sodium pyruvate, 1:100 ITS™+premix, and 10 ng/ml TGFβ3"

It's also possible that the effects of phenol red were specific to this medium and in the human body or with different medium phenol red would have no catabolic or even an anabolic effect.  Phenol red has estrogen dependent and independent effects.

"Bone marrow derived human hMSCs, male donor, 22 years, 7.39 population doublings (PDs) were seeded with 6.000 cells/cm2 in Mesenchymal Stem Cell Growth Medium "<-It's also possible that results would be different with a different donor.

Specific dosages of phenol red were not mentioned so we don't know how much phenol red is needed to take effect.  In the medium there is no where for phenol red to go but in the body it can be removed so that is another consideration.  But phenol red is still worth looking into.

New LSJL and IGF-2 related study by Zhang and Yokota

Lengthening of mouse hind limbs with local administration of insulin-like growth factor 2

"C57/BL/6 mice (∼ 8 weeks old) were used in this study, and the mice were separated into two groups: an IGF2-treated group and a placebo group. In the IGF2-treated group, IGF2 was locally administered into the distal epiphysis of the left femur, and the right femur was used as a contralateral control. In the placebo group, saline was administered to the left femur as a vehicle control. The left and right tibiae, without any direct intervention, were employed as negative controls. The dosage of IGF2 was 100 μg/kg/day for 5 consecutive days, and bone samples were harvested on Day 14. Microcomputed tomography images did not show any anomaly at the IGF2 or saline injection sites.
In comparison with the vehicle control as well as the contralateral control, the results revealed that IGF2 significantly lengthened the treated femur, with an elevation of bone mineral density (BMD) as well as bone mineral content (BMC). The increase in the femoral length of the IGF2-treated left limb was 1.6% (p < 0.05) to the vehicle control, and 1.7% (p < 0.05) to the contralateral control. However, the length, BMD, and BMC of the tibiae were not affected by administration of IGF2 or saline. Western blotting analysis demonstrated that this administration of IGF2 upregulated phosphorylation of an extracellular signal-regulated kinase in the treated femur."

LSJL knee loading was actually more effective than IGF2 lengthening both the tibia and femur and by a greater amount.  The fact that LSJL lengthened both is actually mentioned as a downside as they are developing this as a method to treat limb length discrepencies.  With LSJL there was an increase in the contralateral limb but only the local limb was increased with IGF2.

"IGF2 activates transforming growth factor beta (TGFb) and extracellular signal-regulated kinase (ERK) signaling"<-both of these are likely activated by LSJL as well.  An increase in ERK1/2-p was validated by this study.

"Both IGF1 and IGF2 bind to IGF receptor 1, but only IGF receptor 2 can be bound by IGF2 and not by IGF1. They are reported to be able to recruit primary osteoblastic cells, and regulate proliferation and differentiation of chondrocytes in the growth plate."

"a C28/I2 chondrocyte cell line that administration of IGF2 (5 hours at 10–100 ng/ml) activates TGFb signaling"

It would suck if they abondoned LSJL for IGF-2. Maybe someone other than me can email Ping Zhang and ask if the results of this study means that they're not researching their lateral joint loading method? And we still don't know if IGF2 results in adult height increase than an increase in height velocity.  The scientists allude to testing IGF2 on mature skeletons in the future.

Look at the growth plate in figure 2 C, you can see that the growth plate is open at the end at the computer screen's left whereas it is fused at the end of all other growth plates.  Therefore IGF-2 may have an anti-fusion effect.  Could this effect occur in the adult plate?  Unfortunately there are no before pictures.  The IGF2 bone is much more porous than the other bones.

The scientists state that 8 week old mice are equivalent to twelve year olds.