Wednesday, December 28, 2011

Grow Taller with Ascorbic Acid(Vitamin C)

Previously, we talked about the potential of vitamins augmenting potential height growth methods.  Now, we find a study that shows the ability of Ascorbic Acid to directly induce chondrogenic differentation.

The cell line used in the study is ATDC5 which is a rat cell line so human chondrocyte cells might have different results.

The mechanism of ascorbic acid-induced differentiation of ATDC5 chondrogenic cells.

"The ATDC5 cell line exhibits a multistep process of chondrogenic differentiation analogous to that observed during endochondral bone formation.  Insulin at high concentrations [can induce ATDC differentiation]. spontaneous differentiation of ATDC5 cells maintained in ascorbic acid-containing α-MEM [occurred]{so it has to be Vitamin C containing MEM alpha, MEM stands for Minimum Essentail Medium a listing of what the contains can be found here, none of the compounds would be anything particularly unusual to find in the body except for something like phenol red which is used more as a measurement tool}. A comparison of the differentiation events in response to high-dose insulin vs. ascorbic acid showed similar expression patterns of key genes, including collagen II, Runx2, Sox9, Indian hedgehog, and collagen X{Col2, Sox9, and Col10 are upregulated by LSJL}.  In contrast to high-dose insulin, which downregulates both IGF-I and insulin receptors, there were only minimal changes in the abundance of these receptors during ascorbic acid-induced differentiation[so ascorbic acid is better than Insulin as it doesn't impact IGF-1 and insulin receptors]. ascorbic acid exposure was associated with ERK activation[so this leads us to believe that activating ERK increases chondrogenic differentiation and may help us to grow taller], and ERK inhibition attenuated ascorbic acid-induced differentiation{so ERK enhances Vitamin C-induced chondrogenic differentiation}. ERK activation [has an inhibitory effect] during IGF-I-induced differentiation{ERK impairs IGF-1 induced chondrogenic differentiation}. Inhibition of collagen formation with a proline analog markedly attenuated the differentiating effect of ascorbic acid on ATDC5 cells. When plates were conditioned with ATDC5 cells exposed to ascorbic acid, ATDC5 cells were able to differentiate in the absence of ascorbic acid. matrix formation early in the differentiation process is essential for ascorbic acid-induced ATDC5 differentiation[so maybe ascorbic acid plays a role in ECM interaction?]. ascorbic acid can promote the differentiation of ATDC5 cells by promoting the formation of collagenous matrix and that matrix formation mediates activation of the ERK signaling pathway{so forming the cartilage matrix is key to early height growth}, which promotes the differentiation program."

"At the growth plate, ascorbic acid deficiency can result in decreased chondrocyte proliferation, impaired matrix synthesis, and a reduction in osteoblast cell number"

"ERK activation can be mediated by the cross-linking of extracellular matrix with integrins. In human chondrocytes, the interaction of collagen II with the β1-integrin receptor has been shown to activate ERK" <-So injecting type II collagen into the bone and increasing serum levels of the Beta1-integrin receptor may be one way to grow taller.

"Given that ascorbic acid is required for the formation of collagen triple helices, the ability of ascorbic acid to induce ATDC5 cell differentiation [may depend] on its ability to promote synthesis of collagen matrix."<-So amounts of ascorbic acid greater than that required to form collagen triple helices would be useless.

"Ascorbic acid stimulates procollagen hydroxylation and processing and is required for collagen fibril assembly and collagen secretion"<-collagen hydroxylation is the addition of hydroxyl groups to the amino acids proline or lysine.  Supranormal levels of ascorbic acid, proline, and lysine could stimulation supranormal collagen fibril assembly and collagen secretion.

"The rate-limiting step in this overall process is the hydroxylation and secretion of unprocessed procollagen chains that accumulate in the endoplasmic reticulum of ascorbic acid-deficient cells"<-so you have to have enough Vitamin C such that no cell is definition to maximize height growth.

"collagen II hydrogels could stimulate the differentiation of bone marrow mesenchymal stem cells in the absence of growth factors."<-Thus injecting type II collagen hydrogels into your bone marrow could make you taller.

Since the compounds of Vitamin C and the other compounds of the medium are so readily available in the body it's likely to be a problem with the stem cells themselves that's inhibiting chondrogenic differentiation.

Thus study was done on adult stem cells:

Effects of osteogenic differentiation inducers on in vitro expanded adult mesenchymal stromalcells.

"MSC were isolated from the bone marrow of donors (46-73 years of age) undergoing total hip replacement, and expanded in vitro. At confluence, MSC were cultured under four different conditions: a-MEM plus serum (basal medium or C1), basal medium plus ascorbate (C2), basal medium plus ascorbate and dexamethasone (C3), or basal medium plus ascorbate, dexamethasone and ß-glycerophosphate (C4).
MSC proliferation and the number of colony forming units were increased by ascorbic acid, whereas dexamethasone enhanced the proportion of ALP-positive CFU and was critical for mineral deposition. Runx-2 and type I collagen gene expression decreased along with additive-induced MSC differentiation[we don't want Runx-2 and type I collagen as that is osteogenic while we want chondrogenic so we would not want Beta-glyerophosphate(dexamethasone has been found to have potential height decreasing effects)], i.e. from C1 to C4, while ALP and osteocalcin were differently regulated."

Chondrogenic markers were not measured in this study.  However, using a different cell line produced very different results in regards to Vitamin C.

Here's a study that shows that age doesn't affect stem cell differentiation however:

Human mesenchymal stem cell proliferation and osteogenic differentiation during long-term ex vivo cultivation is not age dependent.

"Ex vivo cultures of primary human MSCs [were taken] from patients in different age groups. Fifteen patients (8 men/7 women) comprised three age groups: (I) <50 years, (II) 50-65 years, and (III) >65 years. MSCs harvested from bone marrow derived from routine surgical procedures were isolated and cultured in standard medium over eight passages. Osteogenic differentiation was induced by dexamethasone (10 nM), ascorbic acid (300 μM), and β-glycerophosphate (3.5 mM). Osteogenic differentiation capacity of MSCs was quantified by alkaline phosphatase (ALP) activity, [levels of] the surface markers CD9, CD90, CD54, CD166, CD105, CD44, and CD73, [and] Coll I and II, Cbfa 1, ALP, OC, BSP1, and GAPDH gene [levels] characterized the phenotypic changes during monolayer expansion. In vitro chondrogenic differentiation was analyzed. Progenitor cells could be expanded in the long term from all bone marrow donations. MSCs showed no significant difference between the age groups. The surface antigen CD166 was predominantly found in all cell cultures independently of differentiation stage. Comparison of expanded and differentiated MSCs within a single age group showed that undifferentiated MSCs had higher CD44 levels.  The highest ALP activity was found in probands of the age group >65 years. we observed a tendency toward male-specific ALP increase during differentiation. Osteogenic marker gene expression in MSCs was detected [and] no significant expression differences were detected between the three donor age groups. Micromass culture of MSCs resulted histologically and immunohistologically in a chondrogenic phenotype[This page lists the micromass protocol for MSCs to generate chondrocytes].  Cultivation leads to a reduced osteogenic differentiation capacity regardless of age. Donor age does not affect osteogenic differentiation potential."

"Expanded MSCs were released by trypsin treatment, counted and resuspended at a density of 1 × 106. The medium was changed to 500 μl DMEM with 10% FCS, 1% antibiotic mix (penicillin/streptomycin), 37.5 μg/ml (100 μmol/l) ascorbate-2 phosphate, and 10–7 mol/l dexamethasone. Pellet cultures were incubated with 10 ng/ml recombinant human transforming growth factor-β3 during chondrogenesis. After 3 weeks the samples were used."

"Pellet cultures of MSCs resulted in the formation of dense nodules consistent with chondrogenic differentiation."<-So forming a pellet culture is most important in inducing chondrogenesis. Can hydrostatic pressure like that induced by LSJL result in the MSC formation of pellet cultures?

"CD9{down in LSJL}, a mediator of tetraspanin protein-driven cellular adhesion, invasion, and fusion, was verified in mesenchymal cells during expansion "

So Vitamin C enhances both chondrogenic and osteogenic differentiation. Age does not seem to affect whether differentiation is headed towards an osteogenic or chondrogenic path but rather how the stem cells are cultured. Hydrostatic pressure may change the culture within the bone marrow to be more chondrogenic.

Ascorbate-enhanced chondrogenesis of ATDC5 cells.

"The ATDC5 cell line exhibits the multistep chondrogenic differentiation observed during endochondral bone formation. However, it takes up to two months to complete the process of cell expansion, insulin addition to promote differentiation and further changes in culture conditions effectively to induce hypertrophy.  By adding ascorbate, the prechondrogenic proliferation phase was shortened from 21 to 7 days, with production of cartilaginous nodules during the chondrogenic phase, after insulin addition, that were greater in number and larger in size.  [There was] much greater matrix elaboration and the mRNA expression of sox9, aggrecan and collagen type II were all significantly increased earlier and to a much higher degree when compared with controls. [Hypertrophic indicators] Col10a1, Runx2 and Mmp13 were all induced within 7-10 days. addition of ascorbate to ATDC5 cultures shortened the prechondrogenic proliferation phase, produced earlier chondrogenic differentiation, heightened gene expression and robust hypertrophic differentiation."

Increasing serum levels of Vitamin C, proline, and lysine may increase height but there may be negative feed mechanisms and points of diminishing returns.

Effects of both vitamin C and mechanical stimulation on improving the mechanical characteristics of regenerated cartilage.

"[We studied] vitamin C (VC) and mechanical stimulation on development of the extracellular matrix (ECM) and improvement in mechanical properties of a chondrocyte-agarose construct in a regenerating tissue disease model of hyaline cartilage. We used primary bovine chondrocytes and two types of VC, ascorbic acid (AsA) as an acidic form and ascorbic acid 2-phosphate (A2P) as a non-acidic form, and applied uniaxial compressive strain to the tissue model using a purpose-built bioreactor. When added to the medium in free-swelling culture conditions, A2P downregulated development of ECM and suppressed improvement of the tangent modulus more than AsA.  Application of mechanical stimulation to the construct both increased the tangent modulus more than the free-swelling group containing A2P and enhanced the ECM network of inner tissue to levels nearly as high as the free-swelling group containing AsA. Mechanical stimulation and strain appears to enhance the supply of nutrients and improve the synthesis of ECM via mechanotransduction pathways of chondrocytes. Mechanical stimulation is necessary for homogenous development of ECM in a cell-associated construct with a view to implantation of a large-sized articular cartilage defect."

"Even though A2P has no physiological activity, it can nevertheless reproduce the effect of VC activity after dephosphorylation by an alkaline phosphatase (ALP). Dephosphorylated A2P, or AsA, penetrates chondrocytes through a VC transporter, chiefly the sodium-dependent VC transporter 2 (SVCT2), and supports collagen synthesis as a cofactor in the rough endoplasmic reticulum. In free-swelling culture conditions, we only observed a small number of collagen molecules distributed in the construct. We also observed that the tangent moduli of both A2P dose groups were lower than the moduli of AsA(2.2). We must also consider the reaction rates of both dephosphorylation of A2P by ALP and the transport of AsA into the cytosol via SVCT2."

Effect of different ascorbate supplementations on in vitro cartilage formation in porcine high-density pellet cultures.

"Most strategies [for articular cartilage regeneration] require ascorbate supplementation to promote matrix formation by isolated chondrocytes. We evaluate ascorbate forms and concentrations on in vitro cartilage formation in porcine chondrocyte high-density pellet cultures. l-ascorbate[AsA], sodium l-ascorbate[NaAs], and l-ascorbate-2-phosphate[AsAP] were administered in 100 microM, 200 microM, and 400 microM in the culture medium over 16 days. Pellet thickness increased independently from the supplemented ascorbate form and concentration. Hydroxyproline content increased as well, but here, medium concentration of AsAP and low concentration of AsA showed a more pronounced effect. Proteoglycan and collagen formation could be proven in all supplemented cultures. Non-supplemented cultures, however, showed no stable matrix formation at all. Effects on the gene expression pattern of cartilage marker genes (type I and type II collagen, aggrecan, and cartilage oligomeric matrix protein (COMP)) were studied compared to non-supplemented control cultures. Expression level of cartilage marker genes was elevated in all cultures showing that dedifferentiation of chondrocytes could be prevented. All supplementations caused a similar effect except for low concentration of AsA, which resulted in an even higher expression level of all marker genes.  We could not detect a pronounced difference between ascorbate and its derivates as well as between the different concentrations."

"Ascorbate has a transcriptional effect on cartilage formation promoting type II collagen and prolyl-4-hydroxylase gene expression. At the post-transcriptional level, ascorbate is responsible for the reduction of iron to its ferrous state. This reaction is important for the hydroxylation of proline and lysine. Latter hydroxy amino acids are specific for type II collagens. Their non-covalent bonds support collagen triple helix and fibril formation. Collagen secretion from the cells is promoted by ascorbate-dependent hydroxylation within the endoplasmatic reticulum"

"Standard concentration of [Vitamin C is] about 280 μM"

"NaAs has the advantage of being non-acidic, while AsA is acidic in aqueous solutions such as culture medium. AsAP is not only non-acidic, but also characterized by a longer half-life in aqueous solutions compared to the other derivatives. It is internalized, dephosphorylated, and highly concentrated in the aqueous phase of the cells. AsA, NaAs and AsAP were administered in 100 μM, 200 μM, and 400 μM over 16 days. "

"The best results of 5 μg/mg were achieved by supplementation with 100 μM of AsA"

Friday, December 23, 2011

Twin Studies indicate the likelihood of environmental factors influencing height growth

Even if height is 100% genetic, genes are still manipulable through DNA Methylation status, telomere length, chromatin folding, and histone acetylation. Things like mechanical stimulation, hydrostatic pressure, and shear strain like that induced by Lateral Synovial Joint Loading are capable of upregulating and downregulating genes.  So too are chemicals capable of upregulating and downregulating genes.  Leptin for instance stimulates the PI3K pathway.

Twin studies can provide us insight on environmental factors that can effect height.  Even if height is determined solely by genetics, environmental factors can manipulate epigentic factors and genetic regulation.  If height is found to be 20% environmental in twin studies where twins engage in normal activities that means that supranormal activities like LSJL may affect height much more.  And remember bone is a substance and that means it shares the properties of all substances like being capable of being stretched.

An assessment of the individual and collective effects of variants on height using twins and a developmentally informative study design.

"In a sample of 3,187 twins and 3,294 of their parents, we sought to investigate association of both individual variants and a genotype-based heightscore involving 176 of the 180 common genetic variants with adult height identified recently by the GIANT consortium. First, longitudinal observations on height spanning pre-adolescence through adulthood in the twin sample allowed us to investigate the separate effects of the previously identified SNPs on pre-pubertal height and pubertal growth spurt. We show that the effect of SNPs identified by the GIANT consortium is primarily on prepubertal height[so genes mainly influence height prepuberty?  intersting]. Only one SNP, rs7759938 in LIN28B, approached a significant association with pubertal growth. Second, we show how using the twin data to control statistically for environmental variance can provide insight into the ultimate magnitude of SNP effects and consequently the genetic architecture of a phenotype. Specifically, we computed a genetic score by weighting SNPs according to their effects as assessed via meta-analysis. This weighted score accounted for 9.2% of the phenotypic variance in height, but 14.3% of the corresponding genetic variance."

"Twin and adoption studies suggest that height is highly heritable (∼80%)"<-20% however is a very large amount of unheritable variance.

Interestingly, according to table 1 there's a .25 cm increase in height males between age 20 and 29(however there are so many other possibilities for this increase rather than a .25cm growth in adult males between 20 and 29, note for instance how females show an average height reduction between 25 and 29).

"Shared environmental effects accounted for 9% (0%, 27%) and 11% (0%, 31%) of the variance in the intercept and slope, respectively"<-environment accounts for around 10% of height in normal cases and in extraordinary cases that could be much more like with extreme loading or by LSJL.

Heritability of adult body height: a comparative study of twin cohorts in eight countries.

"A major component of variation in body height is due to genetic differences, but environmental factors have a substantial contributory effect. In this study we aimed to analyse whether the genetic architecture of body height varies between affluent western societies. We analysed twin data from eight countries comprising 30,111 complete twin pairs by using the univariate genetic model of the Mx statistical package. Body height and zygosity were self-reported in seven populations[this could be a problem as people with higher self esteem could report their height as higher] and measured directly in one population[we still have this data though]. We found that there was substantial variation in mean body height between countries; body height was least in Italy (177 cm in men and 163 cm in women) and greatest in the Netherlands (184 cm and 171 cm, respectively). In men there was no corresponding variation in heritability of body height, heritability estimates ranging from 0.87 to 0.93 in populations under an additive genes/unique environment (AE) model. Among women the heritability estimates were generally lower than among men with greater variation between countries, ranging from 0.68 to 0.84 when an additive genes/shared environment/unique environment (ACE) model was used. In four populations where an AE model fit equally well or better, heritability ranged from 0.89 to 0.93. This difference between the sexes was mainly due to the effect of the shared environmental component of variance, which appears to be more important among women than among men in our study populations. Our results indicate that, in general, there are only minor differences in the genetic architecture of height between affluent Caucasian populations, especially among men."

"Twin correlations for MZ male pairs were uniformly high in all countries, ranging from 0.87 to 0.94 in male
and from 0.84 to 0.94 in female MZ pairs"<-Still however a significant part that is not correlated between twins.

"Heritability of body height is lower among women than among men and there is also greater geographic variation in the heritability estimates among women"<-The study also found little effect of sex-linked characteristics on height so it may be an estrogen production factor.  High levels of estrogen can cause apoptosis of growth plate chondrocytes and estrogen may be influenced by environmental factors.

This study found that genetic factors account for 90% when self-reporting bias is removed.  10% is still a large place for enivornmental factors however and gives the possibility for even more environmental influence when you have extraordinary stimulation like LSJL.

Bias, precision and heritability of self-reported and clinically measured height in Australian twins.

"Here we report a detailed quantitative genetic analysis of stature. We characterise the degree of measurement error by utilising a large sample of Australian twin pairs (857 MZ, 815 DZ) with both clinical and self-reported measures of height. Self-report height measurements are shown to be more variable than clinical measures. This has led to lowered estimates of heritability in many previous studies of stature. In our twin sample the heritability estimate for clinical height exceeded 90%. Repeated measures analysis shows that 2-3 times as many self-report measures are required to recover heritability estimates similar to those obtained from clinical measures. Bivariate genetic repeated measures analysis of self-report and clinical height measures showed an additive genetic correlation >0.98[well still 2% is environmental]. We show that the accuracy of self-report height is upwardly biased in older individuals and in individuals of short stature. By comparing clinical and self-report measures we also showed that there was a genetic component to females systematically reporting their height incorrectly; this phenomenon appeared to not be present in males. The results from the measurement error analysis were subsequently used to assess the effects of error on the power to detect linkage in a genome scan. Moderate reduction in error (through the use of accurate clinical or multiple self-report measures) increased the effective sample size by 22%; elimination of measurement error led to increases in effective sample size of 41%."

"This corresponds to a decline in height of 4.5 cm over a 40 year period"<-So posture correction and stimulating cartilage growth in the discs of the spine can restore height of at least 2 inches.

This study however found a higher percentage of environmental factors affected height.  It used self reported height but it measured the accuracy of self-reported height against height measurements.

Genetic and environmental influences on growth from late childhood to adulthood: a longitudinal study of two Finnish twin cohorts.

"Two cohorts of monozygotic and dizygotic (same sex and opposite sex) Finnish twin pairs were studied longitudinally using self-reported height at 11-12, 14, and 17 years and adult age (FinnTwin12) and at 16, 17, and 18 years and adult age (FinnTwin16). Univariate and multivariate variance component models for twin data were used.
From childhood to adulthood, genetic differences explained 72-81% of the variation of height in boys and 65-86% in girls. Environmentalfactors common to co-twins explained 5-23% of the variation of height, with the residual variation explained by environmental factors unique to each twin individual. Common environmental factors affecting height were highly correlated between the analyzed ages (0.72-0.99 and 0.91-1.00 for boys and girls, respectively). Genetic (0.58-0.99 and 0.70-0.99, respectively) and unique environmental factors (0.32-0.78 and 0.54-0.82, respectively)[this is what we're looking more for as unique environmental factors we can alter] affecting height at different ages were more weakly, but still substantially, correlated.
The genetic contribution to height is strong during adolescence. The high genetic correlations detected across the ages encourage further efforts to identify genes affecting growth. Common and unique environmental factors affecting height during adolescence are also important"

"Reliability of self-reported height, analyzed in sub-samples of both twin cohorts after completion of the last wave of questionnaires, was found highly correlated with measured height in FinnTwin12 (r = 0.99, N = 797) and FinnTwin16 (r = 0.99, N = 566) "

"Nutrition is universally the most important environmental factor affecting growth. For example, milk [likely due to lactoferrin and IGF-1] consumption has been found to be positively associated with height in children and adults"

"growth before puberty is the main determinant of adult stature"<-Thus why some believe you should delay puberty to grow taller.

Here's one study that shows that the environmental factor muscle loading may affect foot length.

Association between foot growth and musculoskeletal loading in children with Prader-Willi syndrome before and during growth hormone treatment.

"In 37 children with PWS, foot length (FL) before and after 6 years of growth hormone therapy (GHT) was retrospectively evaluated with parental and sibling's FL, height, and factors reflecting musculoskeletal loading, such as weight for height (WfH), lean body mass (LBM; dual energy X-ray absorptiometry, deuterium labeled water), physical activity (accellerometry), and walk age. Because of the typically biphasic evolution of body mass and the late walk age in PWS, 2 age groups were separated (group 1, >2.5 years; group 2, < or =2.5 years).
Children with PWS normalized height, but not FL after 6 years of GHT. Parental FL correlation with PWS's FL was lower than with sibling's FL. In group 1, FL positively correlated with WfH, LBM, and physical activity[after 2.5 years of age foot length correlated with physical activity, body weight, and muscle mass]. In group 2, FL negatively correlated with age at onset of independent ambulation. Foot catch-up growth with GHT was slower in group 2 compared with group 1.
In PWS, FL is positively associated with musculoskeletal loading. Small feet in children with PWS before and during long-term GHT may be more than just another dysmorphic feature, but may possibly reflect decreased musculoskeletal loading influencing foot growth and genetic and endocrine factors[decreased musculoskeletal loading may affect foot growth in all individuals and not just children with PWS]."

"In patients with GH deficiency or GH insensitivity syndrome, the size of hands and feet is reduced in proportion to the patient's body height, feet being relatively longer than hands, with both normalizing on GH therapy (GHT)"<-height is composed larger of long bone length whereas feet length is mainly determined by short and irregular bone size.  This may indicate that GH plays a larger role on short bone growth than long bone growth.

"GHT normalizing height and hand length, but not foot length (FL)"<-that feet would be influenced by different factors than hands is very interesting.

" In hemiplegic children, the inactive leg is shorter than the active one"

"Factors other than GH and the genetic background may have an impact on foot growth."

"the later the onset of musculoskeletal loading, the shorter the FL"<-this means that you may only need to pass a threshold of musculoskeletal loading to maximize foot length and that as long as you are passed that threshold your foot length growth is maximized.  The lack of catch-up growth is not good news for the possibility of environmental musculoskeletal stimulation being a significant way to increase foot length(as long as you are above threshold).

"in the older group of children only, there was a significantly positive correlation between FL and WfH, indicating that body mass may be a stimulus for foot growth"<-this data however is very positive for environmental musculoskeletal stimulation stimulating foot length with body weight correlating positively with foot length.

"in addition to height, parental FL variables of musculoskeletal loading were significant predictors of FL measures."<-So even in parents who did not have PWS, musculoskeletal loading was correlated with foot length[weight and exercise]

So environmental factors accounted for around at most 20% of height and less than 2% of height in some studies.  Musculoskeletal loading is one such environmental factor that was found to affect foot length.  Another corollary environmental factor to that is nutrition which affects body weight which is positively correlated to foot length(referred to as weight for height in the study).  Is it possible that foot length could be influenced by factors other than bone length?  There's arch size which may decrease with increasing loads(thus increasing foot length).  Fat mass and lean body mass can contribute to foot size(however, those things can contribute to hand size as well).

Less people load their hands than walk(less people provide mechanical stimulation to hands than feet).  Thus, if mechanical stimulation stimulates foot growth thus foot length should increase greater than hand length in normal individuals(as not as many people do push-ups as walk).  Thus, meaning PWS individuals are less far behind in hand length than foot length.  This too means that studying hand size with exercise would be an interesting study.  One twin does push-ups whereas another lives life normally.

Thursday, December 15, 2011

Being Taller due to PEMF?

Pulsed Electromagnetic Field Therapy is used to treat the healing of non-union fractures(fractures that don't heal).  Now studying non-union fractures is very important to us height seekers as fractures heal as a result of endochondral ossification(stem cells, to chondrocytes, to hypertrophic chondrocytes, to apoptotic chondrocytes, to being invaded by osteoblasts).  The primary goal of Lateral Synovial Joint Loading, is to induce bone marrow stem cells to differentiate into chondrocytes from the epiphyseal bone marrow.  Any technique with applications on non-union fractures(fractures where endochondral ossification does not occur) will have applications on Lateral Synovial Joint Loading.  Because it means that before the non-union endochondral ossification did not occur, however after the stimulation endochondral ossification did occur.  Therefore, the stimulation may be effective at inducing endochondral ossification.

Effects of Pulsed Electromagnetic Fields on Human Osteoblastlike Cells (MG-63): A Pilot Study. 

"Pulsed electromagnetic fields (PEMFs) are used [on] two groups of MG63 cells. One group was treated with PEMFs for 18 hours whereas the second was maintained in the same culture condition without PEMFs (control).
PEMFs induced the upregulation of important genes related to bone formation (HOXA10, AKT1), genes at the transductional level (CALM1, P2RX7), genes for cytoskeletal components (FN1, VCL), and collagenous (COL1A2)[COL1A2 is the gene coding fibrous cartilage which isn't as important for height growth as hyaline cartilage but could be useful nonetheless] and noncollagenous (SPARC) matrix components. PEMF induced downregulation of genes related to the degradation of extracellular matrix (MMP-11, DUSP4). {none of these genes altered in LSJL(even col1a2)  indicating that PEMF can be synergestic with LSJL}
PEMFs appear to induce cell proliferation and differentiation. PEMFs promote extracellular matrix production and mineralization while decreasing matrix degradation and absorption." 

PEMF's can induce cellular proliferation and differentiation.  PEMF's alters the expression of anabolic genes which could have positive benefits on height growth.  Being taller thanks to PEMF is a definitive possibility.   Even though this study showed the effects of PEMF on osteoblastic cells there may be spillover benefits on chondrogenic cells like the degradation of MMP-11.  Also DUSP4 inhibits cellular proliferation so that again can have spillover chondrocyte and stem cell benefits.

"PEMFs determine signal transduction by means of intracellular release of Ca2+ leading to an increase in cytosolic Ca2+ and an increase in activated cytoskeletal calmodulin. PEMFs induce a dose-dependent increase in bone and cartilage differentiation, and upregulation of mRNA expression of extracellular matrix molecules, proteoglycan, and Type II collagen"<-dose dependent means that the stronger the stimulus, the more that the positive height increase benefits will be magnified. The genes mentioned therein are definitely related to height growth and show that PEMF may have a direct benefit on height.

Genes upregulated by PEMF that are up- (or down-regulated by LSJL):

Genes downregulated by PEMF:

Effect of pulsed electromagnetic field on the proliferation and differentiation potential of human bone marrow mesenchymal stem cells. 

"PEMF stimulus was administered to BMMSCs[human bone marrow MSCs]  for 8 h per day during culture period. The PEMF applied consisted of 4.5 ms bursts repeating at 15 Hz, and each burst contained 20 pulses. Results showed that about 59% and 40% more viable BMMSC cells were obtained in the PEMF-exposed cultures at 24 h after plating for the seeding density of 1000 and 3000 cells/cm2, respectively. The growth rates of BMMSC during the exponential growth phase were not significantly affected, 20-60% higher cell densities were achieved during the exponentially expanding stage. Many newly divided cells appeared from 12 to 16 h after the PEMF treatment as revealed by the cell cycle analysis. PEMF exposure could enhance the BMMSC cell proliferation during the exponential phase and it possibly resulted from the shortening of the lag phase.  The PEMF-exposed BMMSC showed multi-lineage differentiation potential similar to the control group." 

"different characteristics of PEMF signals can reduce or enhance osteoclastogenesis of bone marrow cells"

"BMMSCcan repair a number of damaged tissues by the stimulation of mobilizing signals such as hypoxia, platelet-derived growth factor-AB (PDGF-AB) and insulin-like growth factor 1 (IGF-1)"

"voltage-gated delayed rectifier K+ current and Ca2+-activated K+ current channels were changed during progress from G1 to S phase, and functional expression of ion channels could regulate proliferation in undifferentiated rat mesenchymal stem cells. PEMF exposure changed the expression of ion channels and induced membrane hyperpolarization of BMMSCs and therefore resulted in the alteration of the cell cycle progression."

PEMFs can affect stem cell proliferation and this study shows that it can directly affect bone marrow stem cell proliferation and differentiation. In this study, only osteogenic differentiation was analyzed but PEMF helping with chondrogenic differentiation is likely.

Cytokine release from osteoblasts in response to different intensities of pulsed electromagnetic field stimulation. 

"We use an in-vitro osteoblast cell culture model to investigate the effects of low-frequency (7.5 Hz) pulsed electromagnetic field (PEMF) stimulation on osteoblast population, cytokines (prostaglandin E(2) (PGE(2))[PGE2 has effects on height growth], transforming growth factor beta1(TGFbeta1), and alkaline phosphatase (ALP) activity to find the optimal intensity of PEMF for osteoblast growth. PEMF can stimulate osteoblast growth, release of TGFbeta1, and, in addition, an increase of ALP activity. The synthesis and release of PGE(2) in the culture medium are reduced with increasing numbers of cells. Higher intensity does not necessarily mean increased osteoblast growth, and the most efficient intensity is about 2 mV/cm in this case." 

PEMF increased doubled osteoblast cell count.  PEMF increased PGE2 levels by 25%.  It also increased ALP levels by about 50%.

So PEMF has effects on transforming growth factor beta too?  This looks really promising for height growth.  The effect on cytokines may be a necessary side effect.  It's likely that for chondrogenic differentiation less than 2mV/cm will be used.   PEMF is really understudied in terms of inducing chondrogenic differentiation.  Right now, how much PEMF can help with growing taller is unknown.  TGF-Beta1 is what is responsible for initial chondrogenic differentiation which is key for height growth.

PEMF is for sale but I couldn't find a lot of options.  I know Amazon has a very strict refund policy that favors the consumer HealFast Therapy Equine PEMF Square Patch.  I can't verify how good this product is.  I also found Magnetic Therapy Set, Large.  It's cheap and it has pieces that are ideal of putting on the epiphysis of the bones.  You can see that they have one piece for the ankle and the other for the knee.

Here's a patent related to an electrical stimulation device.  The diagrams do show growth plate stimulation but they don't show if the device can get around chondrocyte finite proliferative capacity.  Two ways to get around it would be methods involving ECM secretion and inducing other stem cells not already a part of the growth plate to differentiate into chondrocytes. 

The patent mentions periosteal irritation, medullary plugging[likely refers to the medulla of the bone marrow so a hydrostatic pressure method], creation of an arteriovenous fistula[likely a hydrostatic pressure method], sympathetic denervation[removal of part of the sympathetic nervous system], heat, and foreign objects inserted into the epiphysis(like epiphyseal distraction) as previous attempts to grow taller. We'll have to do independent investigations on these things later.

"in vivo growth plate stimulation using signals of 2.5, 5.0, 10.0 and 20 volts peak-to-peak respectively"

" the group 1 animals (2.5 volts stimulation) experienced a 4.3% increase in length from 1.16 average ratio R/L to 1.21 average ratio R/L."

"For group 2 animals (5.0 volts stimulation) in Table II, a 9.2% increase in length was obtained, from 1.09 average ratio R/L to 1.19 average ratio R/L"<-so the benefit to length seems to be dose dependent.

"Table III and FIG. 4 illustrate that the group 3 animals (10.0 volts stimulation) experienced an 7.8% increase in bone length"<-the third group was not as much so results were biphasic.

"As shown in Table IV and FIG. 5, the group 4 animals (20.0 volts stimulation) experienced a 3.6% decrease in growth, from 1.16 average ratio R/L to 1.12 average ratio R/L. "<-further showing that results were biphasic

The study states that bimetallic strips were effective in inducing longitudinal growth[bimetallic strips convert temperature into mechanical stimuli]. A pulsed magnetic field of 1000mV was effective in inducing growth activity in a chick. An enhanced incorporation of 3H-thymidine is present in chondrocytes at 1166 V/cm^2 oscillating at 5 Hz.

The invention involves placing electrodes around the epiphysis and is AC[stands for alternating current which means that the current periodically changes direction likely to evade actin cytoskeleton adaptation] stimulation signal. Growth rate increase was demonstrated with the invention experimentation but it's uncertain whether that translates into higher adult height.

Method and device for treating osteoarthritis and cartilage disease, defects, and injuries in the human hip

"Anatomic, analytical, and planar circuit models are developed to determining the impedances, conductivities, and current flows in the human hip joint and its surrounding soft tissues and skin that are required to produce a 20 mV/cm electric field in the synovium and articular cartilage of the human hip. "

"The endogenous electrical currents, originally thought to be due to phenomena occurring at the surface of crystals in the bone, have been shown to be due primarily to movement of fluid containing electrolytes in channels of the bone containing organic constituents with fixed negative charges, generating what are called “streaming potentials.” Studies of electrical phenomena in cartilage have demonstrated a mechanical-electrical transduction mechanism that resembles those described in bone, appearing when cartilage is mechanically compressed, causing movement of fluid and electrolytes over the surface of fixed negative charges in the proteoglycans and collagen in the cartilage matrix. These streaming potentials apparently serve a purpose in cartilage similar to that in bone, and, along with mechanical strain, lead to signal transduction that is capable of stimulating chondrocyte synthesis of matrix components."

"it has been conjectured that direct currents do not penetrate cellular membranes and that control is achieved via extracellular matrix differentiation"

Here's a study about MSC's becoming proliferative due to electric currents and this may translate to it being easier to induce chondrogenic differentiation and therefore height growth:

Degenerate wave and capacitive coupling increase human MSC invasion and proliferation while reducing cytotoxicity in an in vitro wound healing model.

"We compared the effects of direct current (DC), capacitive coupling (CC), pulsed electromagnetic field (PEMF) and degenerate wave (DW) on cellular activities including cytotoxicity, proliferation, cell-kinetics and apoptosis by stimulating human-BMMSCs 3 hours a day, up to 5 days. In addition, migration and invasion were assessed using fluorescence microscopy and by quantifying gene and protein expression. We found that DW had the greatest proliferative and least apoptotic and cytotoxic effects compared to other waveforms. DC, DW and CC stimulations resulted in a higher number of cells in S phase and G(2)/M phase as shown by cell cycle analysis. CC and DW caused more cells to invade collagen and showed increased MMP-2 {upregulated by LSJL} and MT1-MMP {upregulated by LSJL as MMP14} expression[remember MT1-MMP is responsible for the formation of new cartilage canals]. DC increased cellular migration in a scratch-wound assay and all ES waveforms enhanced expression of migratory genes with DC having the greatest effect. All ES treated cells showed similar progenitor potential as determined by MSC differentiation assay[so all forms of electrical stimulation increase the likelihood of chondrocyte differentiation which is good for a method like LSJL that wants to induce chondrocyte differentiation]. ES can influence BMMSCs activities, especially DW and CC, which show greater invasion and higher cell proliferation compared to other types of ES. "

Degenerate wave mixing involves using all three electromagnetic waves.  So we wouldn't just want PEMF but the other two forms of waves as well.

"Recent studies have illustrated the invasive capacity of human MSCs requiring MMP-2 and MT1-MMP"<-So in order to form new cartilage canals and growth plates you likely need MMP-2 and MT1-MMP.

"DC treated cells also significantly over expressed several migratory genes including SDF-1/CXCR4, PDGFB-R and TGF-β1-R, IGF-1 and IGF-1R"<-So electrical stimulation increases the number of TGF-Beta and IGF-1 receptors meaning that the stem cells are more sensitive to anabolic height growth proteins like TGF-Beta and IGF-1.

Although the stem cells were obtained from patients undergoing hip replacement surgery so there is a chance that this electrical stimulation may not increase the proteins of healthy bones in the same way.
Here's a study on the chondroprotective effects of PEMF but that doesn't mean that PEMF doesn't stimulate chondrogenesis itself:

Chondroprotective effects of pulsed electromagnetic fields on human cartilage explants.

"pulsed electromagnetic fields (PEMFs) [were used on] human articular cartilage explants from patients with osteoarthritis (OA).  Explants cultured in the absence and presence of IL-1β were treated with PEMF (1.5  mT, 75  Hz) or IGF-I alone or in combination for 1 and 7 days. PG synthesis and release were determined. Explants derived from lateral and medial condyles scored OA grades I and III, respectively. In OA grade I explants, after 7 days exposure, PEMF and IGF-I significantly increased (35) S-sulfate incorporation 49% and 53%, respectively, compared to control, and counteracted the inhibitory effect of IL 1β[remember proteoglycan sulfation can increase height] (0.01 ng/ml). The combined exposure to PEMF and IGF-I was additive in all conditions. Similar results were obtained in OA grade III cartilage explants. PEMF and IGF-I augment cartilage explant anabolic activities, increase PG synthesis, and counteract the catabolic activity of IL-1β[maybe PEMF can help keep growth plates open longer] in OA grades I and III."

"Interleukin-1β (IL-1β), the main catabolic cytokine in OA, exerts its activity by stimulating the increase of matrix metalloproteinase gene expression and by suppressing the synthesis of type II collagen and proteoglycans (PGs)"<-This interleukin could have effects in the epiphyseal bone marrow or in the growth plate inhibiting chondrogenesis thus if PEMF inhibits thus it may help increase height.

"In vivo studies it has been reported that PEMFs stimulate PG synthesis in rats during endochondral ossification"

"PEMF can act in concert with IGF-I"<-So PEMF doesn't stimulate chondrogenesis in the same manner as IGF-1 but that doesn't mean that PEMF can work in concert with other forms of chondrogenic stimulation.

Effects of low frequency electromagnetic fields on the chondrogenic differentiation of human mesenchymal stem cells.

"HMSCs at cell passages five and six were differentiated in pellet cultures in vitro under the addition of human fibroblast growth factor 2 (FGF-2) and human transforming growth factor-β(3) (TGF-β(3) )[we should be looking for ways to upregulate these compounds in the epiphyseal bone marrow]. Cultures were exposed to homogeneous sinusoidal extremely low-frequency magnetic fields (5 mT) produced by a solenoid or were kept in a control system. After 3 weeks of culture, chondrogenesis was assessed. Under EMF, hMSCs showed a significant increase in collagen type II expression at passage 6. Aggrecan and SOX9 expression did not change significantly after EMF exposure[so PEMF can not induce chondrogenesis on it's own but it can aid it]. Collagen type X expression decreased under electromagnetic stimulation[Type X collagen is important for a growth plate phenotype so this may mean that PEMF is better at inducing articular cartilage versus growth plate cartilage]. Pellet cultures at passage 5 that had been treated with EMF provided a higher glycosaminoglycan (GAG)/DNA content than cultures that had not been exposed to EMF. Chondrogenic differentiation of hMSCs may be improved by EMF regarding collagen type II expression and GAG content of cultures. EMF might be a way to stimulate and maintain chondrogenesis of hMSCs."

"The differentiation of hMSCs into chondrogenic cells requires a high initial cell density, three-dimensional (3-D) culture conditions and the application of growth factors. "<-Bone marrow is already 3D. We can upregulate growth factor expression and LSJL may help getting the stem cells together via fluid flow so chondrogenesis can occur.

"The Earth's static magnetic field in the long axis of the solenoid was measured at 45 µT. Low frequency sinusoidal electromagnetic fields with a frequency of 15 Hz and a flux density of 5 mT using a current of 1.2 A root mean square (RMS) were applied three times a day (for 45 min every 8 h) during the entire differentiation period of 21 days. "<-This is unreasonable to apply to humans and some electromagnetic fields may be generated already due to bone deformation caused by LSJL. So we need to know if PEMF is additive to LSJL?

[Low frequence pulsed electromagnetic fields induce chondrocyte-like cells differentiation of rat bone marrow-derived mesenchymal stem cells in vitro].

"LFPEMFs can promote chondrogenic differentiation of rat bone marrow-derived mesenchymal stem cells (rBMSCs) in vitro. The rBMSCs were isolated by adherence method and the third-generation of the rBMSCs were randomly divided into LFPEMFs groups, chondrocyte-induced group and control group. LFPEMFs groups with complete medium were exposed to 50Hz, 1mT PEMFs for 30 min every day, lasting for 10, 15 and 20 d, respectively. Chondrocyte-induced group were treated with chondrogenic media, while control groups were only cultured with complete medium. The mRNA and protein expression level of Col-II and aggrecan were significantly higher in the LFPEMFs group or chondrocyte-induced group, compared to the control group."

Couldn't get full study.

Dose-dependent new bone formation by extracorporeal shock wave application on the intact femur of rabbits.

"Thirty Chinchilla bastard rabbits [one year old] were randomly assigned to 5 groups (EFD[energy flux densities] 0.0, 0.35, 0.5, 0.9 and 1.2 mJ/mm2) and treated with extracorporeal shock waves at the distal femoral region (1,500 pulses; 1 Hz frequency).  Animals were injected with oxytetracycline at days 5-9 after shock wave application and sacrificed on day 10.
Application of shock waves induced new bone formation beginning with 0.5 mJ/mm2 EFD and increasing with 0.9 mJ/mm2 and 1.2 mJ/mm2. The latter EFD resulted in new bone formation also on the dorsal cortical bone; cortical fractures and periosteal detachment also occurred."

"Shock waves also lead to increased biomechanical stability of tendons reattached to bone . At the same time, high-energy shock waves have been shown to cause tendon damage in a dose-dependent manner"

Here's some images from the study

"Extracorporeal shock-wave application to the distal femoral region of the rabbit resulted in macroscopically detectable hematomas in the focus zone of every animal in the group treated with EFD 1.2 mJ/mm 2 and slight hematomas were detected in the animals treated with 0.9 mJ/mm 2 . No hematomas were found in any other animal."

Extracorporeal shockwave treatment cause periosteal bone formation and trabecular bone fracture.

"Cortical fractures were filled with fibrous tissue"<-this is image d shown above.  Fibrous tissue should still be capable of interstitial growth.

"trabecular fractures showed chondrogenic and osteogenic callus tissue"<-this is image i as shown above

Electromagnetic fields enhance chondrogenesis of human adipose-derived stem cells in a chondrogenic microenvironment in vitro.

"We tested the hypothesis that electromagnetic field (EMF) stimulation enhances chondrogenesis in human adipose-derived stem cells (ADSCs) in a chondrogenic microenvironment. A 2D hyaluronoan (HA)-coated well (2D-HA) and a 3D pellet culture system (3D-pellet) were used as chondrogenic microenvironments. The ADSCs were cultured in 2D-HA or 3D-pellet, and then treated with clinical-use pulse electromagnetic field (PEMF) or the innovative single-pulse electromagnetic field (SPEMF) stimulation. The cytotoxicity, cell viability, and chondrogenic and osteogenic differentiations were analyzed after PEMF or SPEMF treatment. The modules of PEMF and SPEMF stimulations used in this study did not cause cytotoxicity or alter cell viability in ADSCs. Both PEMF and SPEMF enhanced the chondrogenic gene expression (SOX-9, collagen type II and aggrecan) of ADSCs cultured in 2D-HA and 3D-pellet. The expressions of bone matrix genes (osteocalcin and collagen type I) of ADSCs were not changed after SPEMF treatment in 2D-HA and 3D-pellet; however, they were enhanced by PEMF treatment. Both PEMF and SPEMF increased the cartilaginous matrix (sulfate glycosaminoglycan; sGAG) deposition of ADSCs. However, PEMF treatment also increased mineralization of ADSCs, but SPEMF treatment did not. Both PEMF and SPEMF enhanced chondrogenic differentiation of ADSCs cultured in a chondrogenic microenvironment. SPEMF treatment enhanced ADSC chondrogenesis, but not osteogenesis, when the cells were cultured in a chondrogenic microenvironment. However, PEMF enhanced both osteogenesis and chondrogenesis under the same conditions. Thus, the combination of a chondrogenic microenvironment with SPEMF stimulation can promote chondrogenic differentiation of ADSCs, and may be applicable to articular cartilage tissue engineering."

Method for non-invasive electrical stimulation of epiphyseal plate growth

"Epiphyseal growth plate stimulation in the bone of a living body is achieved by applying electrodes non-invasively to a body and supplying to said electrodes an AC signal in the range of about 2.5 to 15 volts peak-to-peak at a frequency of about 20-100 KHz."

"supplying to said electrodes an alternating current stimulation signal having a generally symmetrical sine waveform with a voltage amplitude within the range of about 5 to 10 volts peak-to-peak and a frequency of about 60 KHz for a sufficient period of time to effect an increase in the growth of said bone as compared with any bone growth that would occur naturally."

Growth was optimized at 5Vpp which can be seen in the attached pdf.

These next two studies are by the author of the patent:

Up-regulation of bone morphogenetic proteins in cultured murine bone cells with use of specific electric fields.

"BMP-2 through BMP-8, gremlin, and noggin were all normally expressed by MC3T3-E1[osteoblast precursor cells] cells, and could be significantly up-regulated by specific and selective capacitively coupled electric fields. However, mRNA expression for BMP-2, 4, 5, 6, and 7 was consistently up-regulated several times higher than that for BMP-3 and BMP-8, gremlin, and noggin under identical conditions. Concomitantly, BMP-2 protein production and alkaline phosphatase activity were both significantly increased in the same electrically stimulated cultures"

Up-regulation of chondrocyte matrix genes and products by electric fields.

"a 0.5-hour, 20 mV/cm, signal at 60 kHz up-regulated aggrecan gene expression approximately eightfold using a 50% duty cycle, whereas Type II collagen gene expression was up-regulated approximately fivefold using an 8.3% duty cycle. Using a compound signal (a 0.5-hour continuous period plus multiple 1-hour periods of 50% duty cycle for 7 days) both proteoglycan and collagen accumulation in vitro were increased approximately fivefold and twofold, respectively. Also, the most effective capacitively coupled electric signal was different for each of the two molecules studied (aggrecan, 50% duty cycle and 4-hour response time; Type II collagen, 8.3% duty cycle and 6-hour response time). Selective up-regulation of gene expression and matrix accumulation of cartilage structural macromolecules (such as aggrecan and Type II collagen) with specific capacitively coupled fields occurs in vitro."

Couldn't get full study.

Use of Magnetic Forces to Promote Stem Cell Aggregation During Differentiation, and Cartilage Tissue Modeling.

"Magnetic forces induce cell condensation necessary for stem cell differentiation into cartilage and elicit the formation of a tissue-like structure: Magnetically driven fusion of aggregates assembled by micromagnets results in the formation of a continuous tissue layer containing abundant cartilage matrix."

"Chondrogenesis was occasionally impaired by high internalized iron concentrations"

"associate magnetic nanoparticles with MSC in order to endow the cells with magnetic properties."

Patent for Eletrical stimulation device:

 Alternating Electric Current Directs, Enhances, and Accelerates Mesenchymal Stem Cell Differentiation Into Either Osteoblasts or Chondrocytes But Not Adipocytes  

"the alternating electric current is a current in the range from 10 μA to 40 μA [and a frequency of 10Hz]." A sinusodal waveform is used.

"the alternating electric current is for either a continuous or intermittent pattern and a duration of application of 6 hours per day for up to 28 consecutive days."

In several of the experiment modalities tested chondrogenic differentiation was not stimulated.

Electrical stimulation of masseter muscles maintains condylar cartilage in long-term organ culture

"When condylar cartilage is maintained under nonfunctional organ culture conditions, its phenotypic expression is altered to a premature form with less expression of the type II collagen characteristic of mature chondroblasts. The aim of this study was to examine whether, by electrical stimulation of the major masticatory muscle, the masseter muscle, chondrogenic expression could be maintained{interesting that the masseter was stimulated and not the lateral pterygoid muscle} under organ culture conditions in which the jaws with the craniomandibular joint were cultured in one block. Sixty BALB/c mice of both sexes were divided randomly into three groups of equal size. Two groups were decapitated at the age of 5 days. The cranial base and mandible were dissected out in one block, and the explant was placed on its cut surface on a culture dish. The masseter muscles of the explants in one group were stimulated with an electric pulsing device delivering an AC current of a frequency of 0.7 Hz and an amplitude of 5V with hourly active and silent periods. Five experimental and five control explants were fixed after culture periods of 1, 3, 7, and 14 days. The mice in the third group were used as in vivo controls. By electrical stimulation of the masseter muscle, the phenotypic characteristics of the condylar chondroblasts, such as the deposition of type II collagen and the thickness of the cartilage layers{thickness of the cartilage layers would contribute to height if it were in the joints that contribute to height but the big question is whether articular cartilage endochondral ossification is stimulated}, closely resembled the situation in vivo, while the controls in a non-functional environment gradually lost their characteristic form."

Couldn't get the full study

The Anabolic Effects of Electrical Stimulation on Endochondral Bone Formation

"Electrical stimulation (ES) is used to treat non-union fractures and enhance spinal fusion rates. Its effects on endochondral ossification have been studied both in vitro and in vivo with evidence that it is capable of both inhibitory or stimulatory effects. Literature on its mechanism, however, is limited. The purpose of this study is to determine what effects direct current (DC) and capacitive coupling (CC) have on endochondral ossification and examine their mechanisms.

Methods: Thirty-four femurs from 3-week old C57BLK6 mice were divided into 3 groups (DC, CC, and control) and maintained in culture. After 2 days, the femurs were transferred daily to a silicone chamber with phosphate buffered saline. The DC and CC group received 10 minutes of DC at 10V and CC at 10VPP/16Hz, respectively; the control group received no current. After 7 days in culture, all specimens were either placed in ethyl alcohol followed by 4% paraformaldehyde for micro-computed tomography (μCT) analysis and histomorphometry, or frozen immediately in liquid N2 for gene expression analysis using quantitative polymerase chain reaction (qPCR). Each ES specimen was compared to the control using parametric non-paired t-test with p<0.05 considered significant.

Results: Both DC and CC treatments exhibited significantly increased bone volume/tissue volume and bone area compared to control. The DC group additionally had significantly increased trabecular thickness with decreased bone surface/bone volume while the CC group had a significant increase in the trabecular number and decrease in the trabecular spacing. Histomorphometric analysis of von Kossa-stained specimens demonstrated a significant decrease in growth plate height in DC samples. There was also a significant increase in resting/proliferative zones and decrease in hypertrophic zones in both the DC and CC groups compared to the control. On Safranin O-stained sections, there was deeper staining and an increased area of red in the sub-epiphyseal spongiosa in both DC and CC specimens compared to control. Mason’s trichrome-stained sections showed a significant increase in the osteoid area/bone area in both DC and CC groups compared to the control. Analysis of qPCR data demonstrated that DC stimulation significantly increased expression of alkaline phosphatase (ALP) and type I collagen (Col1) while CC stimulation dramatically increased expression of ALP, Sox9, and type X collagen (Col10).

Conclusion: This study corroborates previous evidence that ES is capable of stimulating endochondral bone formation. The variances in results between DC and CC, however, suggest they work through distinct mechanisms. The increase in trabecular thickness and Col1 may indicate that DC functions through an osteogenic pathway while the increase in trabecular number, Sox9, and Col10 expression in CC stimulation may indicate it functions through a chondrogenic pathway. Significance: ES may prove to be an alternative method of growth plate modulation; however, further investigation is warranted."

Electrical stimulation drives chondrogenesis of mesenchymal stem cells in the absence of exogenous growth factors

"Electrical stimulation (ES) is known to guide the development and regeneration of many tissues. However, although preclinical and clinical studies have demonstrated superior effects of ES on cartilage repair, the effects of ES on chondrogenesis remain elusive. Since mesenchyme stem cells (MSCs) have high therapeutic potential for cartilage regeneration, we investigated the actions of ES during chondrogenesis of MSCs. Herein, we demonstrate for the first time that ES enhances expression levels of chondrogenic markers, such as type II collagen, aggrecan, and Sox9, and decreases type I collagen levels, thereby inducing differentiation of MSCs into hyaline chondrogenic cells without the addition of exogenous growth factors. ES also induced MSC condensation and subsequent chondrogenesis by driving Ca2+/ATP oscillations, which are known to be essential for prechondrogenic condensation. In subsequent experiments, the effects of ES on ATP oscillations and chondrogenesis were dependent on extracellular ATP signaling via P2X4 receptors, and ES induced significant increases in TGF-β1 and BMP2 expression. However, the inhibition of TGF-β signaling blocked ES-driven condensation, whereas the inhibition of BMP signaling did not, indicating that TGF-β signaling but not BMP signaling mediates ES-driven condensation. These findings may contribute to the development of electrotherapeutic strategies for cartilage repair using MSCs."

"intracellular ATP levels oscillate during chondrogenic differentiation and the ATP oscillations play critical roles in prechondrogenic condensation, and that extracellular ATP signaling mediates the ATP oscillations during chondrogenesis"

"ES induces differentiation of MSCs into not fibrocartilaginous tissues but hyaline cartilaginous tissues"

Monday, December 12, 2011

Big and Tall with FGF-2?

Effects of FGF-2 on human adipose tissue derived adult stem cells morphology and chondrogenesis enhancement in Transwell culture.

"We investigated the effects of FGF-2 on hADSC morphology and chondrogenesis in Transwell culture. hADSCs were obtained from patients undergoing elective surgery, and then cultured in expansion medium alone or in the presence of FGF-2 (10ng/ml).  Expression levels of SOX-9, collagen type II, and aggrecan were all significantly increased in hADSCs expanded in presence of FGF-2.  FGF-2 induced a slender morphology, whereas doubling time and trypsinization time decreased. FGF-2 induces hADSCs chondrogenesis."

"Adult MSCs [have] a large cytoplasmic volume when cultured without bFGF, while fetal and adult bone marrow-derived MSCs cultured with bFGF (5 ng/ml) have a smaller fibroblast-like morphology with a spindle-shaped cytoplasm"

"FGF-2 maintains the small cytoplasmic volume and spindle morphology of hADSCs over repeated passages."<-other means of maintaining small cytoplasmic volume and spindle morphology may be useful to increase height too.

This study seems like it could possibly involve an FGF-2 therapy for height growth:

Erythroid promoter confines FGF2 expression to the marrow after hematopoietic stem cell gene therapy and leads to enhanced endosteal bone formation.

"Transplantation of mouse Sca-1(+) hematopoietic stem/progenitor cells that are engineered to express a modified FGF2 leads to considerable endosteal/trabecular bone formation, but it also induces adverse effects like hypocalemia and osteomalacia. erythroid specific promoter, β-globin[this can be used for other compound treatments that we wish to confine to the bone marrow in attempts to increase height], leads to a 5-fold decrease in the ratio of serum FGF2 to the FGF2 expression in the marrow cavity when compared to the use of a ubiquitous promoter spleen focus-forming virus (SFFV). The confined FGF2 expression promotes considerable trabeculae bone formation in endosteum and does not yield anemia and osteomalacia. The avoidance of anemia in the mice that received Sca1(+) cells transduced with FGF2 driven by the β-globin promoter is likely due to attenuation of high-level serum FGF2-mediated stem cell mobilization observed in the SFFV-FGF2 animals. The prevention of osteomalacia is associated with substantially reduced serum Fgf23/hypophosphatemia, and less pronounced secondary hyperparathyroidism. Our improved stem cell gene therapy strategy represents one step closer to FGF2-based clinical therapy for systemic skeletal augmentation[and possibly height growth given FGF2's pro-chondrogenic capabilities]."

"due to a very short in vivo half-life of FGF2, daily injection of large quantities of FGF2 protein is necessary"

"the MLV-based HSC gene therapy yields FGF2 levels that are ∼100-fold higher than physiological concentrations"

"FGF2 expression led to a 2-fold increase in Bmpr1b, Bmp2 and Bmp4"<-This isn't that much at all.

"strong positive correlation between serum FGF2 and serum Fgf23"

Metabolic activities and chondrogenic differentiation of human mesenchymal stem cells following recombinant adeno-associated virus-mediated gene transfer and overexpression of fibroblast growth factor 2.

"Here, we examined the effects of recombinant adeno-associated virus (rAAV)-mediated overexpression of human fibroblast growth factor 2 (hFGF-2), a mitogenic factor also known to influence MSC differentiation, upon the proliferative and chondrogenic activities of human MSCs (hMSCs) in a three-dimensional environment that supports chondrogenesis in vitro. Prolonged, significant FGF-2 synthesis was noted in rAAV-hFGF-2-transduced monolayer and aggregate cultures of hMSCs, leading to enhanced, dose-dependent cell proliferation compared with control treatments (rAAV-lacZ transduction and absence of vector administration). Chondrogenic differentiation (proteoglycans, type-II collagen, and SOX9 expression) was successfully achieved in all types of aggregates, without significant difference between conditions. Most remarkably, application of rAAV-hFGF-2 reduced the expression of type-I and type-X collagen, possibly due to increased levels of matrix metalloproteinase-13, a key matrix-degrading enzyme. FGF-2 overexpression also decreased mineralization and the expression of osteogenic markers such as alkaline phosphatase, with diminished levels of RUNX-2, a transcription factor for osteoblast-related genes."

FGF-2 may have inhibitory effects at high levels due to FGFR1 and FGFR3.

Chondrogenic induction of human mesenchymal stem cells using combined growth factors for cartilage tissue engineering., states that FGFR-2 or FGF-6 with TGF-Beta2 are both able to induce chondrogenesis.

FGF-2 abolishes the chondrogenic effect of combined BMP-6 and TGF-beta in human adipose derived stem cells., states that FGF-2 can counteract the effects of BMP-6 and TGF-Beta in chondroinduction(and the mechanism is against BMP-6 not TGF as FGF-2 can promote chondrogenesis in the presence of tgf).  Thus, FGF-2 must be involved in some sort of feedback mechanism that in some circumstances can inhibit chondrogenesis.

"In ASC, it is reported that TGFβ receptor I (TGFβRI) is only expressed in the presence of BMP-6, which might be responsible for the reduced chondrogenic potential compared with BMSC. The chondrogenic potential of BMP-6 in alginate suspended ASC and a 100–350-fold upregulation for aggrecan mRNA and a 20–75-fold increase in collagen II mRNA expression after chondrogenic induction [was observed]. However, a concentration of 500 ng/mL BMP-6 was necessary to achieve the upregulation. In combination with TGF-β3, a concentration of only 10 ng/mL BMP-6 was sufficient to induce collagen type II expression on both the mRNA and protein level."

"BMP signaling acts through c-terminal phosphorylation of Smad1, Smad5 or Smad8. Activated Smad1 binds to the nuclear translocation factor Nup214 and initiates the transcription of chondrogenic genes. In the presence of FGF, MEK1 activates the MAPK pathway, which also phosphorylates Smad1. In this case, phosphorylation takes place at the linker region between MH1 and MH2 of Smad1, differing from the situation in normal BMP signaling. In contrast to c-terminally activated Smad1, linker-phosporylated Smad1 binds Smurf1, leading to polyubiquitination and finally to degradation of Smad1."<-So FGF-2 may degrade Smad1 resulting in an inability of BMP-6 to promote chondrogenesis although this doesn't explain why FGF-2 doesn't now promote chondrogenesis by it's own mechanism.

Fibroblast growth factor 2 enhances the kinetics of mesenchymal stem cell chondrogenesis.

"Treatment of mesenchymal stem cells (MSCs) with fibroblast growth factor 2 (FGF-2) {LSJL upregulates FGF2} during monolayer expansion leads to increased expression of cartilage-related molecules during subsequent pellet chondrogenesis. This may be due to faster differentiation and/or a durable change in phenotype. In order to evaluate changes over time, we assessed chondrogenesis of human MSCs at early and late time points during pellet culture. Marked enhancement of chondrogenesis was seen early compared to controls. However, the differences from controls in gene expression dramatically diminished over time. Depending on conditions, increases in glycosaminoglycan accumulation were maintained."

"FGF-2 may influence at least two aspects of MSC chondrogenesis. First, the growthfactor may speed the sequential, time-dependent pattern of gene expression that occurs during cartilaginous differentiation leading to earlier production of cartilage-associated molecules. Secondly, FGF-2 treatment may lead to an altered phenotypic state"

"Pellets from cells grown with 10 ng/mL of FGF-2 had significantly higher levels of Col II (92-fold), Col X (15-fold), ACAN (18-fold), and SOX9 (10-fold) gene expression compared to controls"

"FGF-2 may select for MSCs with inherent chondrogenic potential during monolayer culture"

"FGF-2 may generally enhance the chondrogenic potential of MSCs (priming mechanism) in part by increasing Sox9 protein levels. Our results support a priming mechanism"

Fibroblast growth factor control of cartilage homeostasis.

"FGF-2 selectively activates FGF receptor 1 (FGFR1){up in LSJL} to exert catabolic effects in human articular chondrocytes and IVD tissue via upregulation of matrix-degrading enzyme production, inhibition of extracellular matrix (ECM) accumulation and proteoglycan synthesis, and clustering of cells characteristic of arthritic states. FGF-18 most likely exerts anabolic effects in human articular chondrocytes by activating the FGFR3 pathway, inducing ECM formation and chondrogenic cell differentiation, and inhibiting cell proliferation. These changes result in dispersed chondrocytes or disc cells surrounded by abundant matrix. The role of FGF-8 has recently been identified as a catabolic mediator in rat and rabbit articular cartilage. The available evidence reveals the promise of FGF-2/FGFR1 antagonists, FGF-18/FGFR3 agonists, and FGF-8 antagonists (ie. anti-FGF-8 antibody) as potential therapies to promote cartilage regeneration."

"FGF-2 is produced endogenously in cartilage and has been proposed to be sequestered by
perlecan, a heparan sulfate proteoglycan (HSPG) localized in the extracellular matrix (ECM) of
articular cartilage. Upon cartilage injury, FGF-2 is released from its bound matrix and subsequently activates the ERK signaling pathway"<-Catabolic effects of FGF2 are mentioned which did not occur in LSJL.

"FGF-2 has been found to activate both FGFR1 and FGFR3, its catabolic activities were recently found to be specifically mediated by FGFR1"

"The binding of FGF-2 to FGFR1 leads to receptor phosphorylation, which in turn activates two critical signaling mediators, Ras and Protein kinase C delta. These molecules then integrate their signaling inputs into the Raf-MEK1/2-ERK1/2 cascade to regulate target gene expression"

"All three mitogen activated protein kinase (MAPK) subgroups (ERK, p38 and JNK){LSJL likely upregulates ERK and p38 but activation of JNK is unclear} converge on the transcription factor Elk-1, which transactivates MMP-13, ultimately promoting cartilage degradation. FGF-2 signaling also results in the activation of AP-1 and RUNX2, the latter of which may account for ADAMTS-5 induction"

In contrast to growth cartilage FGFR3 is anabolic in articular cartilage.

"Local delivery of adenovirus expressing fgf18 into the pinnae of nude mice induced the formation of auricular cartilage, type II collagen, proteoglycan accumulation, and chondrocyte proliferation"

"FGFR3 can both promote and inhibit chondrocyte proliferation depending on the stage
of development. FGF-18 signaling through FGFR3 may enhance chondrocyte proliferation in immature committed chondrocytes, even though it is well established that signaling through FGFR3 inhibits chondrocyte proliferation and differentiation in the mature proliferating chondrocyte zone of the growth plate"

"degradation of the ECM was promoted in the presence of FGF-8 and this degradation was enhanced when combined with interleukin-1 (IL-1). FGF-8 also induced the production of MMP-3 and prostaglandin E2 (PGE2) in rabbit articular chondrocytes"

Src and fibroblast growth factor 2 independently regulate signaling and gene expression induced by experimental injury to intact articular cartilage

"Protein tyrosine phosphorylation occurred within seconds of injury to the surface of intact articular cartilage, as did activation of MAPKs and IKK. Activation did not reoccur upon reinjury of cultured explants. The prominent tyrosine-phosphorylated proteins focal adhesion kinase, paxillin, and cortactin were identified as substrates of Src family kinases. The Src family kinase inhibitor PP2 blocked injury-induced tyrosine phosphorylation. It did not prevent activation of the MAPKs and IKK but differentially inhibited 8 of 10 inflammatory response genes that were induced by injury. In contrast, FGF signaling blockade with PD173074 reduced all MAPK and IKK activation by ∼50% and inhibited a different subset of genes but had no effect on Src-like signaling."

"Both FAK and paxillin are known to be tyrosine phosphorylated, and both were observed only in the lanes showing results for cartilage 10 minutes after dissection"

"Cartilage dissection caused phosphorylation of ATF-2 after 10 minutes, but PP2 caused no reproducible inhibition of this phosphorylation"

Gene expression comparison to LSJL to be done.

Fibroblast growth factor 2 (Fgf2) inhibits differentiation of mesenchymal stem cells by inducing Twist2 and Spry4, blocking extracellular regulated kinase activation, and altering Fgf receptor expression levels.

" fibroblast growth factor 2 (Fgf2) reversibly inhibited multilineage differentiation of primary mouse MSCs and now identify a unique compliment of signaling proteins that are dynamically regulated by this mitogen and whose expression levels are strongly correlated with inhibition of cell differentiation. Fgf2 selectively induced expression of Twist2 and Sprouty4 (Spry4) and repressed expression of soluble frizzled related receptor 2 (Sfrp2), runt-related transcription factor 2 (Runx2), and peroxisome proliferation activated receptor gamma (Pparg). In contrast, Wnt3a induced expression of Twist but not Twist2 or Spry4 and bone morphogenetic protein 2 (Bmp2) failed to alter expression of all three genes. Moreover, pretreatment of MSCs with Fgf2 delayed extracellular regulated kinase 1 (Erk1) and Erk2 phosphorylation and repressed bone-specific gene expression during an osteoinduction time course. Alternatively, pretreatment with Wnt3a had no effect, whereas Bmp2 pretreatment augmented Erk1/2 activation and bone-specific gene expression. Fgf2 also induced expression of Fgf receptor 1 (Fgfr1) and Fgfr4 and repressed Fgfr2 and Fgfr3 expression in MSCs, whereas Wnt3a and Bmp2 had the opposite effect. Twist and Spry4 [are] coexpressed in MSCs and Fgf2 treatment altered their subcellular distribution in a manner consistent with their mode of action. Inhibition of mouse MSC differentiation by Fgf2 is strongly correlated with upregulation of Twist2 and Spry4 and suppression of Erk1/2 activation."

"exogenous application of Wnt3a or ectopic expression of the Lpr5 receptor has been shown to stimulate MSC growth by inducing cyclin D (Ccnd1) and c-Myc expression. "

" Fgf2 upregulated expression of Twist2, Spry4, and Ccnd1 and depressed expression of Sfrp1 to a significant extent when compared with untreated cells. In contrast, Wnt3a stimulated expression of Twist (4.1-fold) and Ctnnb1 (1.4-folds) and downregulated expression of Sfrp1"

FGF-2 addition during expansion of human bone marrow-derived stromal cells alters MSC surface marker distribution and chondrogenic differentiation potential.

"Mesenchymal stromal cells were harvested from bone marrow of six patients and expanded in alpha-MEM or DMEM-LG. Starting in passage 2, 10 ng/ml FGF-2 was administered and non-supplemented media were used as controls. Growth indices were calculated from P0 to P4.  Standard chondrogenic, adipogenic and osteogenic differentiation protocols were applied.
Cell population growth indices were higher for those in FGF-2 supplemented media. Significant differences in surface marker distribution were observed for CD13, CD14, CD49, CD90, CD340 and STRO-1 depending on respective culture conditions. FGF-2 suppressed CD146 expression in both alpha-MEM and DMEM-LG. No differences in adipogenic and osteogenic differentiation potential could be observed, while FGF-2 significantly improved chondrogenic differentiation in DMEM-LG."

"acceleration of proliferation from the beginning of FGF-2 administration, and improved chondrogenic outcome for FGF-2 augmented groups."<-FGF2 may enhance MSC proliferation and therefore enhance mesenchymal condensation.

Grow Taller with Growth Hormone Binding Protein

In our study of Growth Hormone, we found that(in mice at least) that Growth Hormone and IGF-1 increased levels of GHR and GHBP.  And, that things that reduced height like dexamethasone lowered levels of GHBP and that things that increased height like thyroid hormone increased levels of GHBP.

Here's a study that shows which proteins active GHBP(and thus potentially which proteins we can manipulate):

The Extracellular Domain of the Growth Hormone Receptor Interacts with Coactivator Activator to Promote Cell Proliferation

"The presence of GH receptor (GHR) in the cell nucleus correlates with cell division[cell division is really good for growth plate chondrocytes and means more height growth], and targeting the GHR to the nucleus results in constitutive proliferation and transformation because of increased sensitivity to autocrine GH. Here we have sought additional mechanisms that might account for the enhanced proliferation seen with nuclear GHR, commencing with a yeast two-hybrid (Y2H) screen for interactors with the extracellular domain of the GHR [GH-binding protein (GHBP)]. We find that the GHBP is a transcriptional activator in mammalian cells, and this activity resides in the lower cytokine receptor module. Activity is dependent on S226, the conserved serine of the cytokine receptor consensus WSXWS box. By using parallel GHBP affinity columns and tandem mass spectrometry of tryptic digests of proteins bound to wild-type GHBP and S226A columns, we identified proteins that bind to the transcriptionally active GHBP. These include a nucleoporin and two transcriptional regulators, notably the coactivator activator (CoAA), which is also an RNA binding splicing protein. Binding of CoAA to the GHBP was confirmed by glutathione S-transferase pulldown and coimmunoprecipitation, and shown to be GH dependent in pro-B Ba/F3 cells. Importantly, stable expression of CoAA[the protein that binds to GHBP] in Ba/F3 cells resulted in an increased maximum proliferation in response to GH, but not IL-3[therefore if we stabilize the CoAA protein we will increase the cell response to GH]. Because CoAA overexpression has been identified in many cancers and its stable expression promotes cell proliferation and cell transformation in NIH-3T3 cells, we suggest CoAA contributes to the proliferative actions of nuclear GHR by the hormone-dependent recruitment of this powerful coactivator to the GHR."

So increase levels of CoAA to grow taller.

According to the study "The Coactivator activator CoAA regulates PEA3 group member transcriptional activity.": "Five CoAA interactors have been identified previously: the coactivator TRBP and the histone acetyltransferase CBP, the proto-oncogene coactivator SYT, the extracellular domain of the GH (growth hormone) receptor called GHBP (GH-binding protein), and the transcription factor RUNX2"<-So a likely way to manipulate CoAA is via RUNX2.  Diosgenin increases RUNX2 levels.  Parathyroid Hormone inhibits Zfp521 which allows for higher RUNX2.

Unfortunately it seems more likely that CoAA stimulates Runx2 than the other way around:

Co-activator activator (CoAA) prevents the transcriptional activity of Runt domain transcription factors.

"Runx factors bind DNA and co-factors to activate or repress genes crucial for bone formation [and] hematopoiesis. Co-activator activator (CoAA) is a nuclear protein that regulates gene expression, RNA splicing and is overexpressed in many human tumors. CoAA [is] a Runx2 binding protein. CoAA repressed Runx factor-dependent activation of reporter genes in a histone deacetylase-independent manner. CoAA also blocked Runx2-mediated repression of the Axin2 promoter, a novel Runx target gene. The carboxy-terminus of CoAA is essential for binding the Runt domains of Runx1 and Runx2. In electophoretic mobility shift assays, CoAA inhibited Runx2 interactions with DNA. These data indicate that CoAA is an inhibitor of Runx factors and can negate Runx factor regulation of gene expression. CoAA is expressed at high levels in human fetal osteoblasts and osteosarcoma cell lines. Suppression of CoAA expression by RNA interference reduced osteosarcoma cell viability in vitro, suggesting that it contributes to the proliferation and/or survival of osteoblast lineage cells."

So CoAA stimulates osteoblasts as well as chondrocytes.  Unfortunately, no way of increasing serum levels of the CoAA protein is known(which would stimulate GHBP).

Thursday, December 8, 2011

Are bone marrow cells capable of chondrogenesis?

The goal of LSJL is to induce chondrogenic differentiation of the mesenchymal stem cells in the epiphyseal bone marrow.  The chondrogenic potential of bone marrow cells is therefore of interest to us.  Are adult bone marrow cells capable of differentiating into chondrocytes?

Chondrogenesis of mesenchymal stem cells: role of tissue source and inducing factors.

"the cardio-vascular reparative effects attributed to MSCs appear to be mediated predominantly through the secretion of factors targeting cells at the site of repair"<-so what MSCs differentiate into depends on what factors are secreted at certain sites.  So at the sites where we want to grow taller we want our cells to secrete pro-chondrogenic factors.  LSJL encourages secretion of TGF-Beta which is pro-chondrogenic.

"MSC populations are heterogeneous cell populations whose composition depends on isolation methods and expansion conditions that differ largely among investigators. Nearly 50% of CFU-Fs from BM were tripotent MSCs while the remaining population of cells showed varied phenotypes"<-So the ability of MSCs to undergo chondrogenesis may vary by individual.

"SM[synovial membrane] MSCs [contain] two populations: 30% of cells were tripotent while the remainder displayed only osteo-chondral differentiation potential"<-so all synovial membrane cells have the ability to differentiate into chondrocytes.  However, the synovial membrane lines the articular cartilage and does not necessarily have access to epiphyseal bone marrow.

"embryonic stem (ES) cells can clearly be discriminated from MSCs by specific hypermethylation of numerous genes. The comparison of AT[adipose tissue] and BM MSCs revealed few differences. specific hypermethylation of numerous genes [occurs] in HSCs while the methylation patterns of MSCs from different sources were very similar"<-So DNA Methylation affects the differences between embryonic and adult stem cells much more than methylation differences between individuals.  We need to find out which specific methylations affect height.

" promoter hypomethylation is not predictive for the differentiation potential of cells, while hyper-methylation sets restrictions that define frames for differentiation potentials"<-so we may want to un hyper-methylate some genes.

"two cytosines in the COL10A1(Collagen Type X or terminal differentiation) promoter were consistently hypomethylated in MSCs in comparison with articular chondrocytes, correlating to the inducibility of COL10A1 expression and hypertrophy during in vitro chondrogenesis of MSCs "->so the hypomethylation of the COL10A1 gene in articular chondrocytes could be why articular chondrocytes do not ossify and undergo terminal differentiation.  For LSJL to work properly we need to ensure that the COL10A1 gene is hypomethylated.

"Histone modifications and histone-modifying molecules are regulated, while MSCs enter senescence in vitro and could be involved in the ensuing loss of differentiation potential. They are also actively involved in differentiation. Histone deacetylases, in particular HDAC4, may represent important regulators of chondrogenesis "<-So we need to worry about histones as well for inducing chondrogenesis.

"During embryogenesis the development of cartilage is initiated by a phase of condensation of mesenchymal precursor cells, and the cell-cell contact arising from condensation appears to be crucial for the onset of chondrogenesis. N-cadherin seems to be involved in cell-cell contact in pre-cartilage condensations, and functional N-cadherin was necessary for chondrogenesis of chick limb mesenchymal cells in vitro and in vivo. In human MSCs, N-cadherin is strongly up-regulated during the condensation phase during the first few days of chondrogenic induction in vitro . When MSCs are submitted to chondrogenic conditions in monolayer culture, they begin to condensate in response to the stimulus and form high-density three-dimensional cell aggregates. proper chondrogenic differentiation occurs also for MSCs embedded in gel-like biomaterials that keep cells apart from each other and thus limit direct cell-cell contact. This suggests that, although cell-cell contact facilitates chondrogenic induction of MSCs compared with monolayer culture, it does not represent an absolute requirement for in vitro chondrogenic differentiation of human MSCs in a three-dimensional structure."<-So cells may be able to differentiate into chondrocytes without contact with other MSCs.  This is good for LSJL as we only have to encourage chondrogenesis(and subsequent endochondral ossification) of one cell rather than several.

So evidence supporting chondrogenesis in epiphyseal bone marrow.  We need to learn more about methylation and histones that encourage height growth as we can manipulate those to grow taller.

The Therapeutic Effect of Bone Marrow Derived Stem Cell Implantation Following Epiphyseal Plate Injury is Abrogated by Chondrogenic Pre-Differentiation.

"Chondrogenesis was induced with TGF-Beta1 treatment in high-density monolayer cultures of BMSCs in vitro. The pre-differentiated or undifferentiated BMSCs were either seeded into agarose gels for continued in vitro culture, or injected into growth plate defects via an in situ gelling agarose. Pre-differentiated BMSCs had higher Sox-9, type II collagen, and aggrecan mRNA levels compared to undifferentiated cells after high-density monolayer culture. After transfer to agarose gels, pre-differentiated cells did not produce a cartilaginous matrix{so injecting chondrocytes into the bone may not help you grow taller}, even with continued TGF-Beta1 stimulation, whereas undifferentiated cells produced a cartilaginous matrix in this system. Three-dimensional images of the growth plate showed that delivery of either pre-differentiated or undifferentiated cells to defects resulted in a decrease in mineralized tether formation (fusion) in the growth plate tissue surrounding the defect to normal levels. Limb length discrepancy between injured and control limbs was corrected following treatment with undifferentiated but not pre-differentiated cells."

"delivery of an in situ gelling agarose to centralized growth plate defects in a rat animal model decreased limb length discrepancies, but did not fully restore function to the injured area"

"Growth factor stimulation is required to induce chondrogenesis of stem cells, although the specific treatment protocol depends on the tissue source from which the cells are derived"

"Bone marrow was flushed from diaphyses with alpha-MEM supplemented with 1% antibiotic/antimycotic" Our target is epiphyseal bone marrow but they should be similar to diaphyseal bone marrow stem cells.

"control samples continued to receive 1ng/ml FGF-2 and chondrogenic samples were switched to receiving 10ng/ml TGF-Beta1"

"BMSCs in high density monolayer showed very little proliferation after switching to chondrogenic media in both FGF-2 and TGF-Beta1 treated samples over the culture period"<-Maybe it's better to try to upregulate stem cell proliferation proteins when starting LSJL rather than inducing chondrogenesis?

"The limb length discrepancy was corrected in only the group treated with undifferentiated cells and the defect legs of this group were significantly longer then the empty defect limbs"<-overgrowth

Cultured mesenchymal stem cell transfers in the treatment of partial growth arrest.

"Mesenchymal stem cells were cultured from periosteum harvested from the tibias of New Zealand White (NZW) rabbits. An experimental model for growth arrest was created by excising the medial half of the proximal growth plate of the tibia of 6-week-old NZW rabbits. The cultured mesenchymal stem cells were embedded in agarose and transferred into the growth-plate defect after excision of the bony bridge in established growth arrest. Transfer of agarose alone and a periosteum flap without cells served as the control groups. In cases of transfer of mesenchymal stem cells, growth arrest with angular deformation and loss of length of the tibia was corrected. Transfer of agarose alone and a periosteum flap yielded poor results."

"periosteal derived MSCs delivered in agarose generated a new growth plate and completely corrected angular deformity"

Normal structure, function, and histology of the bone marrow.

Bone marrow accounts for 5% of body weight in humans.

Thus the natural bone marrow MSCs may actually be better than exogenous chondrocytes.

Biological properties of mesenchymal Stem Cells from different sources.

"Bone marrow MSCs (BM-MSCs) are a subpopulation of the stromal cells that line the endosteal surface of the marrow space. Cells in many respects identical to BMMSCs can be isolated from trabecular and compact bone and from non-hematopoietic bone marrow sites, such as the femoral head"

"MSCs have been first isolated from the bone marrow. They have been defined as nonhematopoietic, multipotential cells that support hematopoietic stem cells expansion in vitro and can differentiate into cells of various connective tissues. They are easy to harvest and they are hold in bone marrow in relatively high concentration"

Bone Marrow MSCs are rated as having very high chondrogenic differentiation capability second though to Umbillical Cord, Tendon, and Synovial Stem Cells.