Does Bone Mineral Density affect Height?

Previously, it was though that bone density did not affect height and could only lower height by bone deformities.  However, this study suggests otherwise:

Usefulness of estimated height loss for detection of osteoporosis in women.

"Most of us are the tallest at around 30-35 years old because of the peak bone mass at that time"<-This suggests that higher bone density increases height.  If the authors of the study didn't believe that then they would have suggested that peak height is at age 25 or so.

"A lower bone mineral density had a greater influence on height loss in the femur than in the lumbar vertebrae"<-this is surprising as you'd expect it to have more of an impact in the vertebrae as weak vertebrae would result in poorer posture and thus lower height.  This leads credence to the theory that bone density has more impact on height than just bone strength influencing posture.  Bone Density may have a direct affect on the length of the bones.

The scientist who made these assertations is Yeoum SG.

Longitudinal change in height of men and women: implications for interpretation of the body mass index: the Baltimore Longitudinal Study of Aging.

"Among both men and women, cross-sectional height decreased with age"<-Including between 20 and 35.  However, the change in height between 20 and 40 is very small so it could be related to measurement or study error.  Other data in the study suggest an increasing slope in height between the ages of 17-30(Figure 2).

Increased physical activity is associated with enhanced development of peak bone mass in men: A five year longitudinal study.

"Peak bone mass is believed to be achieved before the end of the third decade in life, depending on 

bone site"<-So before age 39.

"Previous intervention studies in children, adolescents and young adults have reported that physical activity interventions result in increased bone mineral content (BMC), areal bone mineral density (BMD) and cortical bone size."<-So if exercise increases BMC and BMC increases height then exercise will increase height.

"For each hour of increased physical activity, aBMD of the lumbar spine and BMC of 

the total body increased with 0.005 g/cm-squared and 5.4 g, respectively, while cortical cross sectional 

area and total cross sectional area of the tibia increased with 0.36 mm-squared and 0.49 mm-squared,

respectively, between the baseline and follow-up visits"

In the study, the individuals who exercised 4 or more hours per week but hardly exercised at all went from an average of 182.4 height to 183 height.  With the mean age being about 18.9 at the start so natural growth cannot be excluded.  Those individuals who exercised before the study and stopped still increased average height from 181.3 to 181.8.  The individuals who exercised before the study and continued throughout the study had the exact same height growth of 181.3 to 181.8.

So perhaps the people who stopped the exercise maintained the benefits of 0.1cm in height.  The sedentary individuals who stayed sedentary went from 181.7 to 182.2.

Thus, exercise may add at least 0.1cm as individuals who added exercise grew 0.6cm over individuals in all other groups who only grew 0.5 cm.  Additional exercise may add additional height.  A confounding variable however is that the sedentary individuals who added exercise were taking 100mg more calcium then everyone else.  But Calcium relates to Bone Mineral Content so it still shows a trend of Bone Mineral Content being able to increase height.

The BMC increased by 5.4g for each hour of exercise between baseline and follow up visits.  The grew that grew the most exercised for 6.2 hours.  However, the group that started exercising for over four hours a week and ended exercising over four hours exercised for 7.9 hours and gained less height.

Here's another study that shows height loss in adults, unfortunately I could not find the full study for this:

Longitudinal shrinkage in lower legs: "negative growth" in healthy late-adolescent males.

"Forty-one healthy male subjects, aged 15.7 to 19.8 years participated in a study on lower leg length (LLL) growth, body height and weight increments. All subjects were measured and weighed at weekly intervals over a period of one year. Body height was determined by an anthropometer, body weight by conventional scales, and LLL was measured by a hand-held knemometer. The state of maturity of the proximal tibial epiphyses was determined by magnetic resonance imaging (MRI). Skeletal maturity was independently assessed by two radiologists. The growth plates were categorized as being open (category I), centrally but not completely fused (category II), or completely fused (category III).
Our findings demonstrate that in these adolescents, final tibial growth is characterized by longitudinal shrinkage. As shown in the MRI, when the proximal tibial growth plates have fused (i.e., at an age when lower leg growth is commonly believed to have completed), the lower leg of healthy adolescent males begins to shrink with a negative growth rate of -2.4 (SD 2.1) mm/year.
We assume that the final period of growth includes small reductions in bone length, possibly due to stabilization and rearrangements in the formerly growing cartilaginous tissues[so essentially bone modeling reduces height?]."

The height reduction with age is not likely related to something that varies like bone modeling but rather something like bone mineral content which decreases linearly with age.

There is not likely a linear relationship between BMC and height but rather a threshold value of BMC to maximize height.  Supramaximal values of BMC will not increase height past a further point.

Grow Taller with xenoestrogens?

Diethylstilbestrol is a synthetic xenoestrogen that has been reported to be available in the human diet indirectly through diary products. It has been known to cause neurological and sexuality issues. However, it's possible that these side affects could potentially be avoided by keeping the dosage low so as only to get the length gaining effects.


Developmental Exposure to Low Dose Xenoestrogens Alters Femur Length and Tensile Strength in Adult Mice.

"Developmental exposure to high doses of the synthetic xenoestrogen diethylstilbestrol (DES) has been reported to alter femur length and strength in adult mice. In this study we investigated the effects of developmental exposure to low doses of DES, bisphenol A (BPA)[bisphenol A is used in plastics like water bottles so you may already be getting low doses of bisphenol A], or ethinyl estradiol[this is present in oral contraceptive pills and it is also present in the urine of those who take oral contraceptive pills] (EE(2)) on bone geometry and torsional strength. C57BL/6 mice were exposed to 0.1 µg/kg/day DES, 10 µg/kg/day BPA, 0.01, 0.1, or 1.0 µg/kg/day EE(2) or vehicle from Gestation Day 11 to Postnatal Day 12 via a mini-osmotic pump in the dam[so they were exposed at a very young age for a very short period of time]. Developmental xenoestrogen exposure altered femoral geometry and strength assessed in adulthood by µCT and torsional strength analysis, respectively. Low dose EE(2), DES or BPA increased adult femur length. Exposure to the highest dose of EE(2) did not alter femur length, resulting in a non-monotonic dose response. Exposure to EE(2) and DES, but not BPA, decreased tensile strength. The combined effect of increased femur length and decreased tensile strength resulted in a trend toward decreased torsional ultimate strength and energy to failure. Taken together, these results suggest that exposure to developmental exposure to environmentally-relevant levels of xenoestrogens may negatively impact bone length and strength in adulthood."

All three of these compounds are present in common place sources like drinking water.  Thus, it may be possible to alter adult bone length by manipulating the consumption of potential sources of these compounds.  Estrogen operates best on height growth at an equilibrium quantity so we want to make sure that our estrogen is at just the right amount.

"Developmental exposure to 0.01 and 0.1 EE2 increased femur length 2.6% and 2.4% (t30 = -2.8, p = 0.008 and t25 = -2.17, p = 0.040, respectively)"

"Developmental DES exposure increased femur length 2.0% (t12 = -2.06, p = 0.031) in females and tended to increase femur length 1.3% (t9 = -1.03, p = 0.166) in males. Developmental BPA exposure, on the other hand, increased femur length 2.3% (t13 = -1.96, p = 0.036) in males and tended to increase femur length 1.0% in females (t14 = -0.84, p = 0.208) (Figure 2)."<-DES and BPA did not fail to increase femur length at the highest dose like EE2 so EE2 is the one you want to watch out for[EE2 is present in adult contraceptives]

However, "developmental exposure to 100 µg/kg DES, which is 1000 times greater than the dose used in the current study, resulted in shorter femurs that had increased total bone area and calcium content. These changes were the result of increased osteoblast activity as determined by an increased mineral apposition rate  and decreased resorption rate due to a decrease in the number of osteoclast cells"<-Extremely high levels of DES does reduce height growth.

Multigenerational exposure to ethinyl estradiol affects bone geometry, but not bone mineraldensity in rats.

"In F3 males, treatment with 10 ppb EE increased femur length compared with control, 2 ppb, or 50 ppb EE as a main effect."

"Sprague–Dawley rats for the parental (F0) generation were obtained from the NCTR breeding colony at weaning and housed two per cage until allocated to dose groups, after which time they were singly housed in polycarbonate cages with hardwood chip bedding. Ethinyl estradiol (EE, > 99% pure, Sigma, St. Louis, MO) was administered at doses of 0, 2, 10, and 50 ppb in Purina 5K96 chow (PMI Nutrition International, Richmond, IN), which is a modified NIH-31 diet that meets the nutritional specifications of NIH-31 but has had soy and alfalfa, major sources of phytoestrogens, removed and replaced by casein. These doses provided approximately 0, 0.1, 0.7, and 4 μg EE per kg body weight per day in males and 0, 0.2, 1, and 6 μg EE per kg body weight per day in females, and were selected such that the lower doses would approximate human oral contraceptive exposures, and the high dose would produce effects without overt toxicity"

"For the multigeneration reproductive study, males and females of the original parental generation (F0) were placed on a 5K96 diet at weaning and dosed feed was administered starting on postnatal day (PND) 42, approximately 1 month before breeding, and were maintained on dosed feed until termination at PND 140. For breeding, one male was cohabited with one female of the same treatment group for 14 days or until a vaginal plug was detected. Subsequent generations (F1–F4) were similarly bred. The F1 and F2 generations were exposed to the test compound administered in the diet continuously from conception through termination at PND 140. The F3 generation was removed from dose at weaning (PND 21)"<-So, estrogen increased height as long as it was below post natal day 21.  Perhaps, after day 21, the natural levels of estrogen increase so additional estrogen just puts it above equilibrium quantities of estrogen.

So what we can take from this is that when you're really young your levels of estrogen are too low to maximize height growth.  Then when development occurs estrogen levels become too high thus you likely want to lower these levels.

So to maximize height growth:

Baby/Toddler->Raise Estrogen to reach Equilibrium.

After->Lower Estrogen Levels.

The problem is finding the right levels.  Babies may not get the xenoestrogens they need to be tall from drinking water so other sources may be needed.

At some point, estrogen levels get too high for height growth so they have to be knocked down a little.

MMP13

F-Spondin which decreases height also increases MMP13.

Role of matrix metalloproteinase 13 in both endochondral and intramembranous ossification during skeletal regeneration.

"MMP13 is required for proper resorption of hypertrophic cartilage and for normal bone remodeling during non-stabilized fracture healing, which occurs via endochondral ossification. However, no difference in callus strength was detected in the absence of MMP13. Transplant of wild-type bone marrow, which reconstitutes cells only of the hematopoietic lineage, did not rescue the endochondral repair defect, indicating that impaired healing in Mmp13-/- mice is intrinsic to cartilage and bone. Mmp13-/- mice also exhibited altered bone remodeling during healing of stabilized fractures and cortical defects via intramembranous ossification. The bone phenotype occurs independently from the cartilage phenotype. MMP13 is involved in normal remodeling of bone and cartilage during adult skeletal repair, and that MMP13 may act directly in the initial stages of ECM degradation in these tissues prior to invasion of blood vessels and osteoclasts."

"MMP13 promotes both the resorption of hypertrophic cartilage from the growth plate and the remodeling of newly deposited trabecular bone during long bone development"

" At 3 days post-[non-stabilized]-fracture, we observed Mmp13 expression in regions of activated periosteum that also expressed Col1"

"At 6 days post-fracture, we observed Mmp13-expressing cells within the Col1 expression domain in Osteocalcin (Oc)-negative portions of the periosteum, indicating that Mmp13 is expressed during the early stages of healing by immature osteoblasts. The Mmp13 expression pattern differed from that of Mmp9, indicating that Mmp13 was not upregulated in newly recruited inflammatory cells and osteoclasts"

"At 10 days post-fracture, Mmp13 expression in the cartilage overlapped with Col10 and Vascular endothelial growth factor (Vegf) expression, indicating that Mmp13 is expressed by hypertrophic and late hypertrophic chondrocytes. At 14 days post-fracture, Mmp13 expression colocalized with Col1 and Oc expression, suggesting that by this time point Mmp13 is expressed in both immature and mature osteoblasts within the callus. Mmp13 expression was detected in osteoblasts throughout the remodeling phase of healing and was not associated with osteoclasts"

"the expression pattern of Mmp13 in both cartilage and bone tissues during fracture healing parallels that seen during development via endochondral ossification"

"there was a significantly greater volume of cartilage and of cartilage as a proportion of total callus volume in Mmp13−/− calluses at 7, 14 and 21 days post-fracture"

"At 28 days post-fracture, there was a significantly greater volume of spongy bone in Mmp13−/− calluses as compared to WT "<-But no difference in compact bone volume.

" Mmp9−/− mice, display aberrant cartilage formation at 10 days post-[stabilized]-fracture. Mmp13−/− stabilized fracture calluses did not display cartilage and were comparable to WT"

"[At 28 days], Mmp13−/− stabilized fracture calluses, were larger and contained more bone as compared to WT."

"the absence of MMP13 does not affect the overall amount of cartilage produced in the callus during non-stabilized fracture healing, but rather affects the removal of hypertrophic cartilage from the callus."<-So this is why MMP13 likely does not reduce but rather can increase height.

"MMP9 is strongly expressed by osteoclasts in the fracture callus, we observed that MMP13 was confined to chondrocytes and osteoblasts"

Knee loading reduces MMP13 activity in the mouse cartilage.

"Knee loading (0.5--3 N for 5 min) was applied to the right knee of surgically-induced osteoarthritis (OA) mice as well as normal (non-OA) mice, and MMP13 activity in the femoral cartilage was examined. The sham-loaded knee was used as a non-loading control. We also employed primary non-OA and OA human chondrocytes as well as C28/I2 chondrocyte cells, and examined MMP13 activity and molecular signaling in response to shear at 2--20 dyn/cm2.
Daily knee loading at 1 N for 2 weeks suppressed cartilage destruction in the knee of OA mice. Induction of OA elevated MMP13 activity and knee loading at 1 N suppressed this elevation. MMP13 activity was also increased in primary OA chondrocytes, and this increase was attenuated by applying shear at 10 dyn/cm2. Load-driven reduction in MMP13 was associated with a decrease in the phosphorylation level of p38 MAPK (p-p38) and NFkappaB (p-NFkappaB). Molecular imaging using a fluorescence resonance energy transfer (FRET) technique showed that Rac1 activity was reduced by shear at 10 dyn/cm2 and elevated by it at 20 dyn/cm2. Silencing Rac1 GTPase significantly reduced MMP13 expression and p-p38 but not p-NFkappaB. Transfection of a constitutively active Rac1 GTPase mutant increased MMP13 activity, while a dominant negative mutant decreased it.
Knee loading reduces MMP13 activity at least in part through Rac1-mediated p38 MAPK signaling."

LSJL was done in this study.

"Dynamic deformations of the epiphysis cause alterations in fluid pressure in the intramedullary cavity, driving oscillatory fluid flow and molecular transport in the lacunocanalicular network in the bone matrix and in the medullary cavity. Fluid flow may cause shear stress to osteocytes, leading to osteoblast differentiation and the initiation of bone formation"

"knee loading was applied at three levels of 0.5 N (mild), 1 N (moderate), and 3 N (strong), and fluid flow was given to induce shear intensity of 2 and 5 dyn/cm2 (mild), 10 dyn/cm2 (moderate), and 20 dyn/cm2 (strong)."


Loading+OA did not increase BMC or BMD anymore than control levels.

"MMP13 activity was decreased by 13% and 16% in response to loads at 0.5 N and 1 N, respectively. At the higher loads of 3 N, however, load-driven reduction of MMP13 was not observed"

"Loads at 0.5 and 1 N decreased the phosphorylated levels of p38 and NFκB in a load intensity-dependent manner. The reduction of their phosphorylation levels was larger at 0.5 N than at 1 N. Loads at 3 N, however, did not lower the levels of p-p38, and they increased the level of p-NFκB. "

"In C28/I2 chondrocyte cells, RNA interference with Rac1 siRNA significantly decreased the level of p-p38, but it did not alter the level of p-NFκB"

"Although oscillatory compressive stress at 0.2–0.5 atmospheric pressure (2–5 × 105 dyn/cm2) was also able to reduce the levels of MMP13 mRNA and p-p38 in C28/I2 chondrocyte cells, cells were ~105 times more sensitive to shear stress than compressive stress"

"Rac1 is necessary for development and maintenance of cartilage, and its chondrocyte-specific deletion results in severe dwarfism in mice. In response to fibronectin fragments, it is reported that Rac1 is required for the production of MMP13 in chondrocytes"

Becoming Taller with MicroRNA

In the future, there are likely to be a number of high tech mechanisms designed to induce height growth.  One of these mechanisms may include microRNA.

MicroRNAs regulate osteogenesis and chondrogenesis.

Being able to induce chondrogenesis is an important ability to induce height growth as you could then form new growth plates.  However, you may still need a demineralized bone surface.

"MicroRNAs (miRNAs) are a class of small molecules and non-coding single strand RNAs that regulate gene expression at the post-transcriptional level by binding to specific sequences within target genes[So we could get miRNAs into the body and then bind to certain genes to induce height growth]. Some miRNAs regulate the proliferation and differentiation of osteoblasts, osteoclasts and chondrocytes, eventually influencing metabolism and bone formation.  Here, we review the recent research progress on the regulation of miRNAs in bone biology, with a particular focus on the miRNA-mediated control mechanisms of bone and cartilage formation."

"[Endochondral ossification] involves the continuous growth and degradation of cartilage and the constant replacement of cartilage tissue by bone tissue."<-Cessation of growth involves the cessation of the growth of cartilage in the bone by getting new cartilage growth whether by LSJL or microRNA, you can get new height growth.

"As members of the Sox family of transcription factors, Sox9, L-Sox5, and Sox6 are necessary for chondrogenic differentiation, and they are expressed through the entire process of chondrogenesis, from mesenchymal condensation to the end of chondrocyte hypertrophy. In all of the cartilage-forming cells in the embryonic stage, Sox9 ensures cell survival and activates Col2a1 and other early cartilage marker genes"<-So we would look for miRNA that upregulates those genes or we would look for exercises/supplements that upregulate those genes.

"miRNA expression has both spatial and temporal specificity as well as tissue or cell specificity."<-thus if you use miRNA's to enhance height growth you have to be mindful of the potential effects on the rest of the body.

"Runx2 and Smads1/5 are necessary for osteogenic differentiation. miR-133 and miR-135 are down-regulated in the BMP-2-induced pluripotent mesenchymal cell line C2C12. Further, miR-133 is a negative regulator of Runx2. miR-133 directly binds to the predicted binding sequence in the 3′ UTR of Runx2 mRNA to inhibit the translation of Runx2 in C2C12 cells. Thus, miR-133 inhibits the osteogenic differentiation of C2C12 that had been induced by BMP-2. Meanwhile, miR-135 represses the osteogenic differentiation of C2C12 cells by targeting the Smad5 gene"<-So, miR-133 and miR-135 are potential targets to repress osteogenic differentiation so stem cells are more likely to differentiate into chondrocytes.

"With microarray technology and luciferase assays, our research group has confirmed that miR-145 acts directly on the 3′ UTR of target gene Sox9. Overexpression of miR-145 decreases the gene expression of MSCs such as Col2a1, Agc1, COMP, Col9a2, and Col11a1, and reduces the contents of GAGs. In contrast, the inhibition of miR-145 could significantly enhance the mRNA expression of the aforementioned genes and promote the contents of GAGs[so we should try to find supplements/exercises that inhibit miR-145]. Adipogenic differentiation marker genes, such as C/EBPδ and C/EBPβ, were not affected by miR-145. It is thus confirmed that Sox9 is the target gene of miR-145 and that miR-145 is a key negative regulator of chondrogenic differentiation, directly targeting Sox9 at an early stage of chondrogenic differentiation. A recent report confirmed the miR-145-mediated regulation of the Sox9 target gene in human articular chondrocytes. The increase in miR-145 could specifically reduce the expression of the cartilage extracellular matrix genes Col2a1 and aggrecan, as well as the tissue-specific miRNAs miR-675 and miR-140."

"cartilage-specific miR-675 could promote cartilage matrix type II collagen expression"<-increasing levels of miR-675 in already existing growth plates may help with height growth.

miR-145 repressions looks the most promising in inducing height growth but it's repression is also associated with several forms of cancer although that may be a symptom rather than the cause of cancer.

Anti-miR-145 may be a potential to lower levels of miR-145.

p53 represses c-Myc through induction of the tumor suppressor miR-145.

"The tumor suppressor p53 negatively regulates a number of genes, including the proto-oncogene c-Myc[Myc may increase height by increasing Lin28 so p53 is bad for height growth]]. One mechanism of the p53-mediated c-Myc repression may involve transcriptional regulation. We show that a putative tumor suppressor, miR-145, is expressed through the phosphoinositide-3 kinase (PI-3K)/Akt and p53 pathways. Importantly, p53 transcriptionally induces the expression of miR-145by interacting with a potential p53 response element (p53RE) in the miR-145 promoter[so inhibiting the p53 pathway may be a way to indirectly affect miR-145]. c-Myc is a direct target for miR-145. Although miR-145 silences the expression of c-Myc, anti-miR-145 enhances its expression. This specific silencing of c-Myc by miR-145 accounts at least in part for the miR-145-mediated inhibition of tumor cell growth both in vitro and in vivo. Finally, the blockade of miR-145 by anti-miR-145 is able to reverse the p53-mediated c-Myc repression. Together, these results define the role of miR-145 in the posttranscriptional regulation of c-Myc by p53 and suggest that, as a new member of the p53 regulatory network, miR-145 provides a direct link between p53 and c-Myc in this gene regulatory network."

pifithrin-α is a potential p53 inhibitor.  So what needs to happen is a supplemental form of Pifithrin-alpha needs to be made and it needs to make sure it doesn't cause cancer.  Likely though, p53 inhibition only accelerates cancer rather than acting as the direct cause.

MicroRNA-337 is associated with chondrogenesis through regulating TGFBR2 expression.

"The expression of six miRNAs in cartilage tissue during development was screened by real-time quantitative polymerase chain reaction (RT-qPCR). Rat models of bone matrix gelatin induced endochondral ossification, collagen-induced arthritis and pristane-induced arthritis were established to examine whether miR-337 is involved in chondrogenesis. Furthermore, the regulation of transforming growth factor-b type II receptor (TGFBR2) expression by miR-337 was determined with the luciferase reporter gene assay and Western blot. The expression of some specific genes relevant to cartilage tissue was tested by RT-qPCR after miR-337 mimic or inhibitor transfection.

MiR-337 expression was significantly down-regulated and almost disappeared in the maturation phases of endochondral ossification. The results of histology and RT-qPCR from three rat models showed that miR-337 is directly bound up with chondrogenesis[thus increasing levels of miR-337 may stimulate chondrogenesis]. Furthermore, the results from the luciferase reporter gene assay and Western blot indicated that miR-337 regulated TGFBR2 expression[Thus TGFBR2 may be key for chondrogenesis as well]. Our study also found that the enhancement of miR-337 may modulate the expression of cartilage-specific genes such as AGC1 in C-28/I2 chondrocytes.

We proved that miRNA-337 is associated with chondrogenesis through regulating TGFBR2 expression, and miRNA-337 can also influence cartilage-specific gene expression in chondrocytes. These findings may provide an important clue for further research in the arthritis pathogenesis and suggest a new remedy for arthritis treatment."

Of course, as always it's possible that miRNA-337 expression is caused by chondrogenesis rather than miRNA-337 causing chondrogenesis.

"AGC1 and ADAMTS4 transcripts in chondrocytes were significantly increased after treatment with miR-337 mimic, while MMP3 mRNA expression was markedly decreased and MMP13 did not obviously change"<-Thus lowering MMP3 levels may help increase height and raising AGC1 and ADAMTS4 levels may help individuals in terms of becoming taller.  According to the NCBI gene back back AGC1 is  a "mitochondrial amino acid transporter, acts both as a glutamate uniporter and as an aspartate-glutamate exchanger; involved in nitrogen metabolism and nitrogen compound biosynthesis".  According to WikiPathways ADAMTS4 is involved in the removal of cartilage matrix.  The full diagram of endochondral ossification on wikipathways can be viewed here.  ADAMTS4 is involved very late in the process of endochondral ossification.  AGC1 is also known as Aggrecan and is also involved late and leads into ADAMTS4 removal of cartilage matrix.

This indicates that removal of the cartilage matrix is very important for height growth which can be evidenced by removal of cartilage matrix being a prerequisite for invasion by osteoblasts.

According to the pathway ADAMTS4 and aggrecan are expressed very late in endochondral ossification however "miR-337 was very low and even hardly detected in the femoral head of adult rats, but highly expressed during the cartilage proliferation".  So miR-337 was evident much earlier.  So perhaps maybe it's MMP-3 expression reduction that was most height increase stimulating.  MMP-3 is not present in the pathway.

"miR-337 activity can promote the ability to anabolism of cartilaginous tissues."<-so miR-337 can help you become taller.

Unfortunately, there is not yet known a way to upregulate miR-337.

The microRNA expression profiles of mouse mesenchymal stem cell during chondrogenic differentiation.

"MiR-140 is tissue-specific expressed in cartilage during embryonic development and play important role in chondrocytes proliferation and differentiaion.It has been validated that histone deacetylase 4 (HDAC4) is its downstream target gene. miR-199 might effect its target gene SMAD1 to regulate chondrogenic differentiation of MSCs"

"miR-140*, miR-140, miR-30a, miR-132/212 and miR143/145 were found that the functions of their potential target genes were correlated with chondrogenic differentiation"

"two up-regulate miRNAs (miR-140*, miR-30a) were associated with decreased expression of their corresponding predicted target mRNA (ADAMTS5, Runx2)."

"two down-regulated miRNAs clusters (miR-132/212, miR-143/145) were associated with increased expression of their corresponding predicted target mRNA (Sox6, ACVR1B)."

Sox9 is associated with miR145.  Sox5 with miR 132/212.

"SMAD1 (miR-30a), SMAD2 (miR-132/212), SMAD3 and SMAD5 (miR-145)"

"suppressed activity of ERK1/2[activated by mir-132/212] resulted in significantly decreased secretion of type II collagen, suggesting ERK1/2 might positively regulate chondrogenesis"


Expression of microRNAs during chondrogenesis of human adipose-derived stem cells.

"Twelve miRNAs were found to be differentially expressed pre- and post- chondrogenic induction by over a two-fold change, including 8 up-regulated miRNAs (miR-193b, miR-199a-3p/hsa-miR-199b-3p, miR-455-3p, miR-210, miR-381, miR-92a, miR-320c, and miR-136), and 4 down-regulated miRNAs (miR-490-5p, miR-4287, miR-BART8*, and miR-US25-1*)"<-different miRNAs than are typically associated with chondrogenic differentiation.

"When mouse mesenchymal stem cells (MSCs) were compared with mature mouse chondrocytes, miR-29a and miR-29b were revealed to directly target 3’ UTR of Col2a1 encoding type II collagen, and their activity was under the regulation of Sox9. miR-199a(*), a bone morphogenic protein 2-responsive microRNA, significantly inhibited early chondrogenesis, as revealed by the reduced expression of early marker genes for chondrogenesis such as cartilage oligomeric matrix protein (COMP), type II collagen, and Sox9, whereas anti-miR-199a(*) increased the expression of these chondrogenic marker genes via direct targeting to smad1 in C3H10T1/2 stem cells"<-so anti-miR-199a could help you grow taller. miR-199a targets Smad1.

"miR-124a was strongly upregulated during chondrogenesis while the expression of miR-96 was substantially suppressed"

"miR-455-3p regulated TGFβ signaling, suppressing the Smad2/3 pathway during chondrognesis"

"[a] predicted candidate target gene, NFKBIA (IKBA), binds NF-κB and inhibits its function"

"the activation of NF-κB was associated with inhibition of chondrogenesis of MSCs by both IL-1β and TNFα in a dose-dependent manner"

"A regulatory circuit comprised of NF-κB, Lin28 {upregulated by LSJL}, Let-7 miRNA, STAT3 and IL-6{upregulated by LSJL}, and a minicircuitry of miR-124, IL6R, STAT3, miR-24, miR-629 and HNF4α {downregulated by LSJL}, was involved in cell transformation"

The role of microRNA-23b in the differentiation of MSC into chondrocyte by targeting protein kinase A signaling.

"both H-89 and microRNA-23b induced differentiation into chondrocyte of hMSCs through down-regulation of protein kinase A (PKA) signaling. The small molecule, H-89, was identified by PCA analysis as a potential mediator of chondrogenic differentiation. H-89 induced the expression of the chondrocyte marker, aggrecan, as well as miR-23b. We searched that miR-23b regulates protein level of PKA. When miR-23b was transfected into hMSCs, chondrogenic differentiation was induced. We confirmed the target of miR-23b using a reporter gene assay. Furthermore, not only H-89 or miR-23b-treated cells, but also cell co-treated with H-89 and miR-23b differentiated into chondrocytes. Our results indicate that H-89 induces the expression of endogenous miR-23b, thereby inducing chondrogenic differentiation by negatively inhibition of PKA signaling."

"miR-145 attenuates differentiation of MSCs into chondrocytes via targeting on the 3′-UTR of Sox9"

LSJL decreases PKIG which inhibits PKA.  However it increases PKIA which also inhibits PKA, so maybe PKA inhibition does play a role in LSJL chondroinduction.

miR-181a promotes osteoblastic differentiation through repression of TGF-β signaling molecules.

"miR-181a [is] highly upregulated during BMP induced osteoblastic differentiation of C2C12 and MC3T3 cells. Overexpression of miR-181a led to upregulation of key markers of osteoblastic differentiation as well as enhanced ALP levels and Alizarin red staining, indicating the importance of this miRNA for osteoblastic differentiation. miR-181 isoforms (181a, 181b, 181c) are expressed during different stages of mouse calvarial and tibial development, implying their role in both endochondral and intramembranous ossification. miR-181a promotes osteoblastic differentiation via repression of TGF-β signaling molecules {So miR-181a is likely anti-chondrogenic therefore miR-181a inhibition is likely pro-chondrogenic} by targeting the negative regulator of osteoblastic differentiation Tgfbi (Tgf-beta induced) and TβR-I/Alk5 (TGF-β type I receptor) {down}. Rgs4 and Gata6 are direct targets of miR-181a."

"miR-181 is upregulated during rat articular cartilage development"

Genes downregulated by miR-181:
Dclk1{up}
Grem2{up}

More gene expression analysis to be done.

A microRNA signature associated with chondrogenic lineage commitment

"human USSC[unrestricted somatic stem cells] isolation and expansion were carried out"

"downregulation of mir-376a expression and upregulation of mir-630, mir-624 and mir-1268 expression during chondrogenesis. There were 20-fold to 30-fold greater amounts of mir-630 and mir-624 in 21-day chondrocytes compared to undifferentiated USSCs."

"The expression of mir-1268 was 4-fold greater on day 21 than on day 1."

"most of the downregulated miRNAs such as mir-376a, mir-376c, mir-377, mir-337–5p, mir-376a, mir-495, mir-1185, mir-379, mir-127–3p and mir-654–3p are located on chromosome 14. On the other hand, chromosome 19 encodes many of the upregulated miRNAs such as mir-99b, mir-637, mir-181d, mir-515–5p and mir-638."

Anti-let7f increased transcription   AS did pro mir-6230 and mir-624.  Anti mir-221 repressed Sox9.

Mir's good for height growth:

mir-624 genes "SMAD2, WNT5A, PCDH19, CTNNB1"
mir-630 genes "TOB2, GJC1, PDGFRA, TGFBR2"
mir-1268 genes "SOX12, CAMK2G{down in LSJL}"
A more detailed analysis of MiRNA's and target gene comparison to be done.

Height Increasing by Inhibiting Fra-1?

LSJL may have an effect on Fra-1.  No change in expression over 2 fold or under 0.5 fold was observed but a change was expected.

Fra-1 governs cell migration via modulation of CD44 expression in human mesotheliomas

CD44 encourages chondrogenesis so inhibition is bad.

"Silencing of Fra-1, a component of the dimeric transcription factor, activator protein-1 (AP-1), inhibits mRNA expression of c-met and cd44 in rat mesothelioma cells and is causally linked to maintenance of the transformed phenotype. In a panel of human MM cells, Fra-1 mRNA expression in MM is complex and regulated by extracellular signal-regulated kinase (ERK1, ERK2), Src, and phosphatidyl-inositol-3-kinase (PI3K) pathways in a tumor-specific fashion. Cell lines with PI3K-dependent Fra-1 expression were SV40 positive and expressed the lowest basal Fra-1 levels. Levels of Fra-1 expression correlated with amounts of CD44 expression that were greater in simian virus 40 negative (SV40-) MM cells. Using dominant negative (dn), short hairpin (sh) and small interference (si) RNA constructs, we next demonstrate that expression of CD44, the principal hyaluronic receptor in MMs, correlates with Fra-expression in both simian virus 40 positive (SV40+) and SV40- MMs. Both Fra-1 and CD44 expression are linked to cell migration in SV40- MM cells. Lastly, in contrast to normal lung tissue, Fra-1 was expressed in 33 of 34 human MMs, and that all CD44+ tumors were SV40-. Fra-1 is associated with cell migration in human MMs and that Fra-1 modulation of CD44 may govern migration of selected MMs."

"CD44 is a type I transmembrane glycoprotein (85–200 kDa) and functions as the major cellular adhesion molecule for hyaluronic acid (HA), a component of the extracellular matrix (ECM)."

The below study confirms the inhibitory role of Fra-1 on chondrogenesis.

Fra-1/AP-1 impairs inflammatory responses and chondrogenesis in fracture healing.

"Transgenic overexpression of Fra-1, a component of the transcription factor activator protein-1 (AP-1), in various tissues progressively and globally enhances bone formation. We created a transverse fracture of the mouse tibial diaphysis and examined fracture healing radiologically, histologically, and immunologically. Strikingly, fracture union was delayed even though the bone formation rate in callus was higher in Fra-1 transgenic (Tg) mice. In these mice, chondrogenesis around the fracture site was impaired, resulting in accumulation of fibrous tissue, which interferes with the formation of a bony bridge across the callus. Immediately after fracture, induction of the inflammatory mediators TNF-alpha, interleukin (IL)-6, and Cox-2{LSJL downregulates Cox-2 and TNF-alpha and upregulates IL-6} was significantly suppressed in Fra-1 Tg mice followed, by the reduced expression of Sox-9 and BMP-2. Because serum prostaglandin E(2) (PGE(2)) levels were dramatically low in these mice, we administered PGE(2) to the fracture site using a slow-release carrier. The accumulation of fibrous tissue in Fra-1 Tg mice was significantly reduced by PGE(2) administration, and chondrogenesis near the fracture site was partially restored. The Fra-1-containing transcription factor AP-1 inhibits fracture-induced endochondral ossification and bony bridge formation presumably through suppression of inflammation-induced chondrogenesis."

So LSJL  increased Fra-1 expression based on the effects on TNF-alpha and COX-2.

"Fos family proteins, such as c-Fos, Fra-1, Fra-2, and FosB, heterodimerize with Jun proteins to form the dimeric transcription factor activator protein-1 (AP-1)"<-LSJL upregulates c-Fos.

Green tea increases Fra-1 levels according to Green tea polyphenol stimulates a Ras, MEKK1, MEK3, and p38 cascade to increase activator protein 1 factor-dependent involucrin gene expression in normal human keratinocytes.

Estrogen induces Fra-1 according to this study, Estrogen-induced expression of Fos-related antigen 1 (FRA-1) regulates uterine stromal differentiation and remodeling.

According to this study C-Fos can also inhibit chondrogenic differentiation

Inhibition of chondrocyte differentiation in vitro by constitutive and inducible overexpression of the c-fos proto-oncogene.

"ATDC5 chondrogenic cells, undergo a well-defined sequence of differentiation from chondroprogenitors to fully differentiated hypertrophic chondrocytes. We constitutively overexpressed exogenous c-fos in ATDC5 cells. Several stable clones expressing high levels of exogenous c-fos were isolated and those also expressing the cartilage marker type II collagen showed a marked decrease in cartilage nodule formation.  Two clones, DT7.1 and DT12.4 were capable of nodule formation in the absence of c-fos. However, upon induction of exogenous c-fos, differentiation was markedly reduced in DT7.1 cells and was virtually abolished in clone DT12.4. Pulse experiments indicated that induction of c-fos only at early stages of proliferation/differentiation inhibited nodule formation, and limiting dilution studies suggested that overexpression of c-fos decreased the frequency of chondroprogenitor cells within the clonal population. Rates of proliferation and apoptosis were unaffected by c-fos overexpression under standard conditions, suggesting that these processes do not contribute to the observed inhibition of differentiation. Gene expression analyses demonstrated that the expression of the cartilage markers type II collagen and PTH/PTHrP receptor were down-regulated in the presence of exogenous c-Fos and correlated well with the differentiation status. Induction of c-fos resulted in the concomitant increase in the expression of fra-1 and c-jun, further highlighting the importance of AP-1 transcription factors in chondrocyte differentiation."

This is only ATDC5 cells, c-fos may have a different effect in HBMMSC's.

So decreasing c-Fos levels will also decrease Fra-1 levels and both these decreases will enhance chondrogenesis.

MS-275, a benzamide histone deacetylase inhibitor, prevents osteoclastogenesis by down-regulating c-Fos expression and suppresses bone loss in mice. states that MS-275 may be a potential c-Fos inhibitor.
BMP treatment of C3H10T1/2 mesenchymal stem cells induces both chondrogenesis and osteogenesis.

"Both BMP-7 and BMP-2 induced C3H10T1/2 cells [murine mesenchymal stem cell line] to undergo a sequential pattern of chondrogenic followed by osteogenic differentiation that was dependent on both the concentration and the continuous presence of BMP in the growth media. Differentiation was determined by the expression of chondrogenesis and osteogenesis associated matrix genes. Experiments using BMP-7 demonstrated that withdrawal of BMP from the growth media led to a complete loss of skeletal cell differentiation accompanied by adipogenic differentiation of these cells. Continuous treatment with BMP-7 increased the expression of Sox9{up in LSJL}, Msx 2, and c-fos{up in LSJL} during the periods of chondrogenic differentiation after which point their expression decreased. Dlx 5 expression was induced by BMP-7 treatment and remained elevated throughout the time-course of skeletal cell differentiation. Runx2/Cbfa1 was not detected by ribonuclease protection assay (RPA) and did not appear to be induced by BMP-7. The sequential nature of differentiation of chondrocytic and osteoblastic cells and the necessity for continuous BMP treatment to maintain skeletal cell differentiation suggests that the maintenance of selective differentiation of the two skeletal cell lineages might be dependent on BMP-7-regulated expression of other morphogenetic factors. Wnt 5b, Wnt 11, BMP-4, growth and differentiation factor-1 (GDF-1), Sonic hedgehog (Shh), and Indian hedgehog (Ihh) were endogenously expressed by C3H10T1/2 cells. Wnt 11, BMP-4, and GDF-1 expression were inhibited by BMP-7 treatment in a dose-dependent manner while Wnt 5b and Shh were selectively induced by BMP-7 during the period of chondrogenic differentiation. Ihh expression also showed induction by BMP-7 treatment, however, the period of maximal expression was during the later time-points, corresponding to osteogenic differentiation. BMP-7 activity could be further enhanced twofold by growing the cells in a more nutrient-rich media. The murine mesenchymal stem cell line C3H10T1/2 was induced to follow an endochondral sequence of chondrogenic and osteogenic differentiation dependent on both dose and continual presence of BMP-7 and enhanced by a nutrient-rich media. The induction of osteogenesis is dependent on the secondary regulation of factors that control osteogenesis through an autocrine mechanism."

"Depending on the concentration used, 5-azacytidine stimulated C3H10T1/2 to differentiate into myotubes, adipocytes, or chondroblasts"  5-azacytidine interferes with DNA methylation which can silence gene expression.

"In the high-dose BMP-7 treated cells, type II collagen expression increased dramatically and reached peak expression by 4 days after the initiation of BMP-7 treatment and was maintained at this high level until day 8, after which it sharply declined. The low-dose BMP-7 treated cells maintained their type II collagen expression longer than the untreated cells but never obtained the same maximal level of expression seen in the 250 ng/ml dose group. "

"C-fos exhibited almost no expression in the control cultures but showed a very strong peak of expression in the BMP-7 treated groups during the period of maximal chondrogenic expression. Fra 2 expression was initially high in all groups of cultured cells with a slight increase in expression over time in all groups. Fra 1 was also expressed initially by all groups but decreased to near absent levels by day 2"

Shear- and compression-induced chondrocyte transcription requires MAPK activation in cartilage explants.

"Mitogen-activated protein kinase (MAPK) pathways are activated by static and dynamic compression of cartilage, which simultaneously induce intratissue fluid flow, pressure gradients, cell, and matrix deformation. We applied dynamic shear to bovine cartilage explants. We measured ERK1/2 and p38 activation at multiple time points over 24 h. Distinct activation time courses were observed for different MAPKs: a sustained 50% increase for ERK1/2 and a delayed increase in p38 of 180%. Cartilage explants were preincubated with inhibitors of ERK1/2 and p38 activation before application of 1-24 h of three distinct mechanical stimuli relevant to in vivo loading (50% static compression, 3% dynamic compression at 0.1 Hz, or 3% dynamic shear at 0.1 Hz). mRNA levels of selected genes involved in matrix homeostasis were measured. Most genes examined required ERK1/2 and p38 activation to be regulated by these loading regimens, including matrix proteins aggrecan and type II collagen, matrix metalloproteinases MMP13, and ADAMTS5, and transcription factors downstream of the MAPK pathway, c-Fos, and c-Jun. Thus, we demonstrated that the MAPK pathway is a central conduit for transducing mechanical forces into biological responses in cartilage."

"In cartilage explants, static compression can induce the phosphorylation of extracellular signal-regulated kinases (ERK1/2) and p38, and dynamic compression can induce ERK activation. In isolated chondrocytes, fluid shear activates ERK1/2"

"The increase in the phosphorylated ERK1/2 level reached a maximum value at 3% shear strain and slightly decreased at 4.5% shear strain; therefore time course experiments were performed at the 3% shear strain, 0.1 Hz condition. Interestingly, there was a dramatic increase in the phosphorylated ERK1/2 level even at 0% compression over the free swelling condition"

"The mechano-induced up-regulation of transcription factors Sox9, c-Fos, and c-Jun was partially suppressed by U0126[ERK inhibitor] under all loading conditions"

"p38 signaling contributes toward transforming growth factor-β-stimulated proteoglycan synthesis and chondrocyte proliferation (36, 37), and ERK1/2 is activated by insulin-like growth factor-1, which strongly promotes anabolic chondrocyte behavior"

Fos/AP-1 proteins in bone and the immune system.

"Induction of receptor activator of nuclear factor (NF)-kappaB ligand (RANKL) signals by activated T cells and subsequent activation of the key transcription factors Fos/activator protein-1 (AP-1), NF-kappaB, and NF for activation of T cells c1 (NFATc1) are in the center of the signaling networks leading to osteoclast-mediated bone loss. Conversely, nature has employed the interferon system to antagonize excessive osteoclast differentiation, although this counteracting activity appears to be overruled under pathological conditions."

"Mesenchyme-derived chondrocytes and osteoblasts are able to rebuild the resorbed bone by producing the extracellular matrix that eventually gives rise to the known mineralized skeletal elements and also to joints and tendons"

"the Fos proteins (Fos, FosB, Fra-1, and Fra-2) can only heterodimerize with members of the Jun family, the Jun proteins (Jun, JunB, and JunD) can both homodimerize and heterodimerize with Fos members to form transcriptionally active complexes"

"Chimeric mice obtained from Fos-overexpressing embryonic stem cells develop chondrogenic tumors, implying a function of Fos in chondrogenesis. Surprisingly, Fos overexpression in an in vitro model of chondrogenesis inhibited the differentiation of chondrocytes. Ectopic Fos expression from a ubiquitous promoter in transgenic mice has no noticeable effects on cell differentiation in most organs, but it results in the specific transformation of osteoblasts leading to osteosarcoma formation"

"NFATc1 was originally described as a Fos target gene in osteoclastogenesis, but recent evidence has shown that it is also regulated by NF-κB."

Enhanced expression of TGF-beta and c-fos mRNAs in the growth plates of developing human long bones.

"The highest levels of TGF-beta mRNA [are] associated with the growth plates. This mRNA was localized predominantly in the osteoblasts and osteoclasts of the developing bone, in periosteal fibroblasts and in individual bone marrow cells. TGF-beta may have a role in stimulation of type I collagen production and bone formation. Only a low level of TGF-beta mRNA was detected in cartilage where type II collagen mRNA is abundant. the highest levels of c-fos mRNA were detected in epiphyseal cartilage. [The] two cell types with high levels of c-fos expression [were] the chondrocytes bordering the joint space and the osteoclasts of developing bone."

"Deregulated expression of c-fos under the metallothionein promoter in transgenic mice was found to specifically interfere with the development of long bones"

"TGF-B [may] not [be] associated with the chondrocytes of the growth plate"<-this study is from 1988 though.

"osteoclasts also contain high levels of c-fos transcripts"
Distribution and expression of mRNAs for the proto-oncogenes c-fos and c-jun in bone cells in vivo.

"Femurs of 4-week-old rats were examined. C-fos and c-jun exhibited similar distribution in growth plate and bone tissue. Expression of c-fos and c-jun mRNAs in growth plate was observed in the proliferative zone and partly in the upper layer of the hypertrophic zone. In spongy bone, high expression of c-fos and c-jun mRNAs was observed in the osteoblast cytoplasm. There was little expression in bone lining cells. In the bony trabeculae, slight expression of c-fos and c-jun was observed in the premature osteocytes situated close to the bone surface, but no expression was detected in osteocytes that possessed relatively large lacunae in the center of the trabeculae. C-fos and c-jun were also slightly expressed in osteoclasts. c-fos and c-jun are involved in regulating chondrocyte proliferation as immediate early genes, and may also be involved in the gene expression of bone matrix proteins as transcription factor (AP-1) in vivo. Strong expression [of c-Fos and c-Jun]  in osteoblasts but hardly any expression at all in bone lining cells [suggests] that these genes are involved in oteoblast activation."

We need to find a selective c-Fos inhibitor that doesn't inhibit the NF-kappaB pathway also.  However there have been studies that report a stimulatory effect of c-Fos on chondrogenesis.

annexin a8 & LSJL

LSJL upregulates Annexin VIII(Annexin a8) 3.1 fold.

Annexin VIII is differentially expressed by chondrocytes in the mammalian growth plate during endochondral ossification and in osteoarthritic cartilage.

"Fetal bovine growth plate chondrocytes as opposed to epiphyseal chondrocytes [genetic expression was compared]. Annexin VIII [is] expressed by growth plate chondrocytes and not by epiphyseal chondrocytes. Immunohistochemistry of the fetal bovine growth plate identified a gradient of increasing annexin VIII protein from the proliferative to the hypertrophic zone. annexin VIII [is] largely [localized] to the chondrocyte cell membrane.
We examined the distribution of annexin VIII in normal and osteoarthritic (OA) articular cartilage. In OA cartilage, the protein was located in a subset of mid- to deep zone chondrocytes and in the matrix surrounding these cells; no annexin VIII was detected in normal articular cartilage. annexin VIII is a marker for chondrocyte differentiation during normal endochondral ossification."

"The annexin repeats form a planar cyclic structure with a central pore region. The pore is lined with hydrophilic residues and has been found to act as a calcium channel in vitro and in vivo."

"Annexin VIII is expressed by the differentiating chondrocytes of the growth plate but not by the phenotypically stable epiphyseal chondrocytes [suggesting] that it has a specific differentiation-associated role within the growth plate"

Bone matrix regulates osteoclast differentiation and annexin A8 gene expression.

"We cultured murine bone marrow-derived osteoclasts on either cell culture plastic or devitalized mouse calvariae. Annexin A8 (AnxA8) mRNA was markedly up-regulated by bone. AnxA8 protein was present at high levels in osteoclasts present in human tissues recovered from sites of pathological bone loss. The presence of bone mineral was required for up-regulation of AnxA8 mRNA since osteoclasts plated on decalcified bone express AnxA8 at low levels as did osteoclasts plated on native or denatured type I collagen. AnxA8-regulated cytoskeletal reorganization in osteoclasts generated on a mineralized matrix.  Anxa8 [is] a gene strongly induced late in osteoclast differentiation and a protein that regulates formation of the cell's characteristic actin ring."

"AnxA8 has been shown to bind F-actin and phospholipids in a calcium-dependent manner, consistent with the idea that AnxA8 may regulate cytoskeletal reorganization, a process central to osteoclastic resorption"

"Mononuclear cells, stromal cells, and fibroblasts did not stain positively for AnxA8 expression"

"AnxA8 can disrupt the actin cytoskeleton and/or suppress lysosomal trafficking, leading to failure of osteoclast spreading and function"

"AnxA8 knockdown inhibits formation of actin rings"

"Osteoclasts on native or denatured type I collagen fail to express AnxA8."<-However AnnexinA8 was expressed on osteoclasts on bone so osteoclasts could have been the source of AnxA8 with LSJL.

Osteoclasts are responsive to fluid flow Fluid flow-induced calcium response in early or late differentiated osteoclasts., and the change in calcium response is consistent with alterations in AnxA8 expression.

Effect of fluid flow-induced shear stress on osteoclast formation induced by osteocyte.

"MLO-Y4 osteocyte-like cells were exposed to fluid flow-induced shear stress(12dyn/cm(2))for 0, 1, 2, 4, 6, 12, and 24 hours. Osteocyte exposed to shear stress at different time points were used in co-culture system for 9 days. On the 9(th) day the amount of positively stained osteoclasts were counted and compared. The expressions of osteoprotegerin (OPG) and receptor activator of nuclear factor-kappa (RANKL) were detected.

Compared with bone cells without stimulation with fluid flow-induced shear stress, the amount of osteocytes significantly decreased at all time points after the application of fluid flow-induced shear stress. The OPG expression at mRNA levels was significantly up-regulated in the first 12 hours, the RANKL mRNA expression was significantly down-regulated in the first 4 hours, and the RANKL/OPG ratio significantly decreased within 12 hours. However, all these indicators showed no significant difference at 24 hours when compared with the pre-stimulation level."

LSJL gene expression was done at 49 hours but one hour after the last stimulation so osteoclast activity could have influenced annexin a8 levels.

Tie2

Tie2 is upregulated during distraction osteogenesis.

Identification of progenitor cells that contribute to heterotopic skeletogenesis.

"Individuals who have fibrodysplasia ossificans progressiva develop an ectopic skeleton because of genetic dysregulation of bone morphogenetic protein (BMP) signaling in the presence of inflammatory triggers.
We used Cre/loxP lineage tracing methods in the mouse to identify cell lineages that contribute to all stages of heterotopic ossification. Specific cell populations were permanently labeled by crossing lineage-specific Cre mice with the Cre-dependent reporter mice R26R and R26R-EYFP. Two mouse models were used to induce heterotopic ossification: (1) intramuscular injection of BMP2/Matrigel and (2) cardiotoxin-induced skeletal muscle injury in transgenic mice that misexpress BMP4 at the neuromuscular junction. The contribution of labeled cells to fibroproliferative lesions, cartilage, and bone was evaluated histologically by light and fluorescence microscopy. The cell types evaluated as possible progenitors included skeletal muscle stem cells (MyoD-Cre), endothelium and endothelial precursors (Tie2-Cre), and vascular smooth muscle (Smooth Muscle Myosin Heavy Chain-Cre [SMMHC-Cre]).
Vascular smooth muscle cells did not contribute to any stage of heterotopic ossification in either mouse model. Despite the osteogenic response of cultured skeletal myoblasts to BMPs, skeletal muscle precursors in vivo contributed minimally to heterotopic ossification, and this contribution was not increased by cardiotoxin injection, which induces muscle regeneration and mobilizes muscle stem cells. cells that expressed the vascular endothelial marker Tie2/Tek at some time in their developmental history contributed robustly to the fibroproliferative, chondrogenic, and osteogenic stages of the evolving heterotopic endochondral anlagen. endothelial markers were expressed by cells at all stages of heterotopic ossification. muscle injury and associated inflammation were sufficient to trigger fibrodysplasia ossificans progressiva-like heterotopic ossification in a setting of chronically stimulated BMP activity.
Tie2-expressing progenitor cells, which are endothelial precursors, respond to an inflammatory trigger, differentiate through an endochondral pathway, contribute to every stage of the heterotopic endochondral anlagen, and form heterotopic bone in response to overactive BMP signaling in animal models of fibrodysplasia ossificans progressiva. Thus, the ectopic skeleton is not only supplied by a rich vasculature, but appears to be constructed in part by cells of vascular origin. dysregulation of the BMP signaling pathway and an inflammatory microenvironment are both required for the formation of fibrodysplasia ossificans progressiva-like lesions."

"a recurrent heterozygous missense mutation in the BMP type-I receptor, activin receptor IA/activin-like kinase-2 (ACVR1/ALK2) [can cause heterotopic ossification]"

"a mutation in ACVR1 results in dysregulation of BMP signaling"

"Tie2, a receptor tyrosine kinase for angiopoietins, plays a critical role in the development of the embryonic vasculature and is ubiquitously expressed in early endothelial precursors during development and postnatal tissue repair"<-LSJL upregulates ANGPTL1(which interacts with Tie2) and ANGPTL2(which is related to inflammation which this study claims is needed for heterotopic ossification).

"While hematopoietic stem cells express Tie2, recent bone marrow transplantation studies have shown that cells of the hematopoietic system do not contribute to fibroproliferative, chondrogenic, or osteogenic stages of BMP-induced heterotopic ossification"

" Intramuscular injection of BMP2 or BMP4 protein is also sufficient to elicit ectopic skeletogenesis"

"the preosseous skeletal anlagen is derived from cells of nonhematopoietic origin"

"the labeled cells in heterotopic lesions of Tie2-Cre;R26R mice arise from the endothelium of the local vasculature, in response to injury and BMP signaling."<-We need to study the endothelium and local vasculature of adult bone marrow.

"BMP receptors are highly expressed on endothelial cells in vivo, and the BMP-Smad pathway potently activates the endothelium"

"BMPs have the ability to redirect the differentiation of connective tissue progenitor cells to orchestrate an endothelial-to-mesenchymal transition in these cells, often through inflammatory cell intermediates"

"isexpression of constitutively active ACVR1/ALK2, a BMP type-I receptor and the gene mutated in fibrodysplasia ossificans progressiva, is sufficient to stimulate an endothelial-to-mesenchymal transformation in endothelial cells of the heart"

"BMP4, as well as hypoxia and inflammatory cytokines—conditions and factors that are present in the earliest preosseous lesions of heterotopic ossification—upregulate Tie2 in endothelial cells, which contributes to the angiogenic response"

Tie2 ligands angiopoietin-1 and angiopoietin-2 are coexpressed with vascular endothelial cell growth factor in growing human bone.

"we investigated the expression of the angiopoietins (Ang-1 and Ang-2) in human neonatal ribs. Ang-1 and Ang-2 exhibited similar patterns of staining in the growing rib. In the cartilage, expression of Ang-1 and Ang-2 increased with chondrocyte maturation. Ang-1, Ang-2, and VEGF were not detected in the resting zone except adjacent to vascular canals, and maximum expression was detected at the cartilage bone interface {Ang1, Ang2, and VEGF may play critical roles in cartilage canal formation}. In the cartilage, Ang-2 was more highly expressed than Ang-1 or VEGF, with staining observed in the proliferating, hypertrophic, and mineralized zones. In the bone, Ang-1, Ang-2, and VEGF were detected in modeling and remodeling sites. Ang-1 was detected in the majority of osteoblasts, osteoclasts, and in some marrow space cells. Ang-2 was expressed at variable levels by osteoblasts and osteoclasts in modeling and remodeling bone. VEGF was detected in cells at bone surfaces and in the marrow spaces. Strong staining for VEGF was observed in osteoblasts and osteoclasts in modeling and remodeling bone. In the perichondrium, Ang-1, Ang-2, and VEGF were most highly expressed adjacent to the hypertrophic zone and at sites of bone collar formation. In the periosteum, Ang-1, Ang-2, and VEGF expression colocalized with alkaline phosphatase expression. The distribution of Ang-1, Ang-2, and VEGF indicate these factors may play key roles in the regulation of angiogenesis at sites of endochondral ossification, intramembranous ossification, and bone turnover in the growing human skeleton."

"transgenic overexpression of Ang-2 resulted in failure of angiogenesis and normal vascular development due to disruption of endothelial-pericyte interactions"

"vascular invasion of the cartilage from the perichondrium was only observed in the resting zone, with focal expression of Ang-1, Ang-2, and VEGF in chondrocytes adjacent to the invading vascular canals."

"In the bone, all three factors were detected at sites of bone modeling and remodeling"<-So maybe we don't need to do anything special to induce these factors and just LSJL will be enough.

COMP-Ang1 promotes chondrogenic and osteogenic differentiation of multipotent mesenchymal stem cells through the Ang1/Tie2 signaling pathway.

"cartilage oligomeric matrix protein angiopoietin1 (COMP-Ang1), an Ang1 variant which is more potent than native Ang1 in phosphorylating Tie2 receptor was developed. The Ang1/Tie2 signaling system not only plays a pivotal role in vessel growth, remodeling, and maturation, but also protective and recruit effect on MSCs. Thus, the aim of the present study was to investigate the differentiate effect of Ang1/Tie2 signaling on MSCs in the presence of chondrogenic, osteogenic and adipogenic induction medium, and to determine the possible mechanisms.  MSCs cultured in each induction medium with COMP-Ang1 revealed strongly chondrogenic and osteogenic morphological change (3.5- and 2-fold, respectively){COMP-Ang1 is more chondrogenic than osteogenic} as well as up-regulate each gene, except for adipogenic differentiation. Phosphorylation of Tie2 expression lead to phosphorylation of p38 and AKT and then accelerating each differentiation of MSCs to chondrocytes and osteoblasts."

Comp-Ang1 increased cell proliferation maximally at 300 nanograms.  It also increased cell viability.  It also increased phosphorylation of Tie2 and this was downstream of the p38 and Akt pathway.

Epigenetics and Height

The main components to epigenetics are DNA Methylation, Chromatin Folding,  telomere length, and histone acetylation.  Even if height was 100% genetic that gives us a great degree to manipulate genetics especially since genes can be upregulated and downregulated.  Here's a study that shows that paternal activity influences height growth:

Parental body size and early weight and height growth velocities in their offspring

"Whereas weight or height at a given age are the results of the cumulative growth experience, growth velocities allows the study of factors affecting growth at given ages.
235 parent-child trios belonging to 162 families [were] examined in 1999.
Weight and height growth velocities from birth to seven years were estimated from a modelling of individual growth curve and correlated with parent's body size in 1999.
Ponderal index and length at birth were significantly associated with maternal but not paternal BMI and height[babies spend time in the mothers womb but not the mans]. In the first six months, height growth velocity was significantly associated with maternal stature (at three months: 0.12+/-0.05 and 0.02+/-0.05 cm/month for a 10 cm difference in maternal and paternal height respectively) and weight growth velocity with paternal BMI (at three months: 5.7+/-2.8 and 1.9+/-2.3g/month for a difference of 1 kg/m(2) in paternal and maternal BMI respectively). Between two and five years, height growth velocity was more significantly associated with paternal height whereas weight growth velocity was more closely associated with maternal BMI.
Early childhood growth is characterised by alternate periods associated specifically with maternal or paternal BMI and height. This novel finding should trigger the search for specific genetic, epigenetic or environmentally shared factors from the mothers and fathers."

Maybe a parents height upregulates and downregulates genes that affect growth rate.  It's logical that the larger you are the faster you have to produce new bone and cartilage cells, maybe that rate is expressed in epigenetic mechanisms that can be passed down to the offspring.

"In a study published in 1954, weight at birth and in early infancy were more correlated with maternal than paternal stature, but this difference disappeared before adulthood"<-so it's likely an epigenetic change that influences genes that affect more growth velocity rather than final height so likely genes that affect the rate of cellular proliferation.

"The mother's height was more strongly associated with the offspring's height growth velocity in the first six months of life than father's height.  At one year, the height growth velocity was not associated with parental stature[maybe at this point the epigenetic mechanisms that affected stature growth are now adjusted for]. From two to five years, height growth was strongly associated with the father's height, whereas significant associations with the mother's height reappeared at four years and its effect remained much smaller than those with father's height"<-If height growth is affected by sex linked characteristics then it makes sense for fathers height to play a large impact on height growth.  If father is X(short)Y then he will carry those genes in height.  Whereas, Mother can be X(tall)X(short) and if short is recessive she will still be tall but still have potential to pass on X(short).  Mothers have the ability to carry sex-linked genes recessively and not show them in phenotype whereas males do not have that ability.

"over-expression of paternal alleles in the IGF2 gene in Beckwith–Wiedemann syndrome which results in a marked acceleration of bone growth in infancy and early childhood "<-an example given of epigenetics.  But children with Beckwith-Wiedemann syndrome usually grow to a height predicted by their parents height.  IGF-2 is strongly associated with growth velocity and IGF-2 expression decreases with age.

Secular trends in growth of African Pygmies and Bantu.

"The evolution in height of West Pygmies and Bantu farmers from 1911 to 2006 was evaluated using data from the literature as well as data gathered by our research team during an expedition to Cameroon in 2006.
During the last century, no secular trend in west Pygmies is apparent, as height changed from 151 cm to 155 cm in males and from 143 cm to 146 cm in females. A small though significant (p=0.026), increment (about 2 cm) was observed only in female subjects during the last ten years. By contrast, Bantu heights show a significant change from 1943 to 2006 for both males (from 159 cm to 172 cm; p=0.025) and females (from 148 cm to 160 cm; p=0.029).
Over the last century, the Bantu population exhibited a significant secular trend for height, whereas West Pygmies did not increase their linear growth. The lack of secular trend in Pygmies possibly suggests that their stature reflects adaptation to the forest lifestyle. We may hypothesize that not only environmental but epigenetic factors have also contributed to their growth potential."

"Most studies have concluded that the Pygmy growth pattern is normal up to the time of puberty and that their short stature is primarily due to insufficient growth acceleration at puberty."

"the BMI values of males and females in Bantu were significantly higher compared to those in Pygmies"<-Leptin which is produced by bodyfat stimulates longitudinal growth.

"it is quite possible that their short stature is epigenetically determined to facilitate adaptation to life in the dense tropical forest and endurance against starvation. Furthermore, their small body size could favour their life in the hot, humid climate of a tropical forest by minimizing the body’s heat production during exercise. With regard to possible pathophysiological mechanisms, it must be mentioned that [some have] reported normal GH secretion but low IGF-I levels. We also recently reported that reduced IGF-I and GHBP levels in Pygmies are associated with a marked decrease of the GHR gene expression which is not associated with variants in the sequence of the GHR gene. This supports the hypothesis of a genetically determined short stature in Pygmies"<-To test this take a kid born from Shaq and put him in the same forest that Pygmies grow up in and see how tall he grows. The excessive heat may downregulate pro-height growth genes or cause DNA damage and that may be the epigenetic cause for pygmy height.

Short stature may be more prone to epigenetic variations than tall stature.

Common variants show predicted polygenic effects on height in the tails of the distribution, except in extremely short individuals.

"Common genetic variants have been shown to explain a fraction of the inherited variation for many common diseases and quantitative traits, including height, a classic polygenic trait. The extent to which common variation determines the phenotype of highly heritable traits such as height is uncertain, as is the extent to which common variation is relevant to individuals with more extreme phenotypes. To address these questions, we studied 1,214 individuals from the top and bottom extremes of the height distribution (tallest and shortest ∼1.5%), drawn from ∼78,000 individuals from the HUNT and FINRISK cohorts. We found that common variants still influence height at the extremes of the distribution: common variants (49/141) were nominally associated with height in the expected direction more often than is expected by chance (p<5×10(-28)), and the odds ratios in the extreme samples were consistent with the effects estimated previously in population-based data. To examine more closely whether the common variants have the expected effects, we calculated a weighted allele score (WAS), which is a weighted prediction of height for each individual based on the previously estimated effect sizes of the common variants in the overall population. The average WAS is consistent with expectation in the tall individuals, but was not as extreme as expected in the shortest individuals (p<0.006), indicating that some of the short stature is explained by factors other than common genetic variation. The discrepancy was more pronounced (p<10(-6)) in the most extreme individuals (height<0.25 percentile). The results at the extreme short tails are consistent with a large number of models incorporating either rare genetic non-additive or rare non-genetic factors that decrease height. We conclude that common genetic variants are associated with height at the extremes as well as across the population, but that additional factors become more prominent at the shorter extreme. "

We'd hope to have found that there are some elements of tall stature that are not explained by genetic variation.  This leads to the suspicion that there was no existing environmental method that could epigenetically induce height growth.  However,  LSJL was not known at the time of the study and there are very few individuals who would go to extremes for height growth like hanging or the rack.  Several teenagers though weight train, run, and do sports so that rules out those methods at inducing epigenetic height growth.  Again though, if the stimulus required was very extreme than it's not likely to be significant statistically.

Here's a study that suggests epigenetic involvement in height growth specifically maternal exposure to Ultraviolet B:

Insights into the programming of bone development from the Avon Longitudinal Study of Parents and Children

"We examined associations between proxy measures of in utero nutrition and total body bone mineral content (BMC), bone area (BA), and bone mineral density (BMD) assessed at age 9.9 y in the Avon Longitudinal Study of Parents and Children (ALSPAC). There were positive relations between birth weight and BMC, BA, and BMD. These associations were explained by the co-association of birth weight with body size in later childhood. In height- and weight-adjusted analyses, an inverse association was observed between birth weight and BMD at age 9.9 y, which suggests that birth weight had a negative influence on bone mass after relations with bone and body size were taken into account. In analyses of associations between bone mass at age 9 y and background ultraviolet B exposure during the third trimester of pregnancy (a proxy measure for maternal vitamin D status), maternal ultraviolet B exposure was positively related to BMC, BA, and BMD. After adjustment for height, these associations were only partially attenuated, which suggests that maternal ultraviolet B exposure affected skeletal size and mass independently of longitudinal growth, possibly by the increase of periosteal expansion[so maternal ultraviolet B exposure increased longitudinal growth but it also increased skeletal mass independently of longitudinal growth as well]. There was a positive relation between maternal folate intake and BMD of the spine subregion independent of body size. Although a co-association with folate intake in childhood could explain this relation, the maternal methylenetetrahydrofolate reductase (MTHFR) genotype affected spine BMD independently of the child MTHFR genotype, which suggests that maternal folate status has an independent effect on bone development of offspring. Together, these results confirm that there is a relation between bone development in childhood and several proxy measures for nutritional status in utero."


Most of the light that gets through the atmosphere is UVA light.  UVB light is between 315 and 280 nm which is rare.  This lamp(Cole-Parmer 6-watt UV Lamp with 365nm and 302nm Wavelength Light Tubes, 115VAC), however, has a 302nm light tube.

"We also observed a positive association in ALSPAC between background ultraviolet B amounts and height at age 9 y, in keeping with the known season of birth effects on adult height."<-Although we don't know if maternal exposure to Ultravoilet B results in increased adult height.

"For example, an association was seen between maternal dietary intake of folate and BMD and aBMC at the spine subregion, which may point to a role of epigenetic changes in the programming of skeletal development because folate status was likely to influence the availability of methyl donors for methylation during gestation, which is a key mechanism in epigenetic gene silencing."<-So folate intake by the mother does likely affected by epigenetics as evidenced by the effects of BMD.  However, the lack of effect on height may indicate that methylation status has little effect on height.  Although, the effect on height is unclear as it is not directly mentioned whether Folate consumption impacts height at age 9 in the study(it's definitely mentioned that Ultraviolet B affects height however and it's speculated by the scientists that this effect is related to Vitamin D).

Magnesium is another compound that when taken by the mother influences the childs height.  The study mentions too that Magnesium and Folate maternal consumption has only a minor impact on skeletal development.

Impact of physical activity and doping on epigenetic gene regulation.

"The abuse of pharmaceuticals which improve athletic performance may alter the expression of specific genes involved in muscle and bone metabolism by epigenetic mechanisms, such as DNA methylation and histone modifications. Moreover, excessive and relentless training to increase the muscle mass, may also have an influence on the health of the athletes. This stress releases neurotransmitters and growth factors, and may affect the expression of endogenous genes by DNA methylation, too."

Couldn't get full study.

Epigenetic anomalies in childhood growth disorders.

"Fetal growth restriction is associated with morbidity among small for gestational age (SGA) neonates as well as in children and adults who were former SGA. Imprinted genes (whose expression is restricted to a single parental allele) have a critical role in controlling mammalian fetal growth. The human chromosome 11p15 encompasses two imprinted domains regulated by their own differentially methylated imprinted control region (ICR1 at the H19/IGF2 domain, and ICR2 at the KCNQ1/CDKN1C domain). Loss of imprinting at these two domains is implicated in two clinically opposite growth disorders. Indeed, our group has identified a loss of DNA methylation (LOM) at ICR1 in over 50% of patients with Russell-Silver syndrome (RSS) characterized by intrauterine and postnatal growth retardation with spared cranial growth, dysmorphic features, frequent body asymmetry and severe feeding difficulties. By contrast, gain of methylation at ICR1 is found in 10% of patients with Beckwith-Wiedemann syndrome (BWS), an overgrowth syndrome with an enhanced childhood tumor risk. We have now identified over 130 RSS patients with 11p15 LOM. This 11p15 epimutation is a frequent and specific cause of RSS as it has not been identified in non syndromic SGA patients. These new findings in the pathophysiology of RSS allow long-term follow-up studies to be performed based on molecular diagnosis. This will help to define appropriate clinical guidelines regarding growth, rapid bone age advance during puberty and feeding difficulties. Remarkably, we have also recently found that ∼10% of RSS patients and ∼25% of BWS patients showed multilocus LOM at imprinted regions other than ICR1 or ICR2 11p15, respectively."

Couldn't get this full study unfortunately.

Epigenetic regulation of osteogenic and chondrogenic differentiation of mesenchymal stem cells in culture.

TGFB1 and TGFB3 can promote chondrogenesis.


"In the cell laboratory, cartilage differentiation of MSCs can be performed in a pellet culture system. Approximately 2 × 105 cells (passages 2-3) must be condensed in to a pellet by centrifugation at 300 g for 4 minutes, followed by incubation in an atmosphere of 37˚C and 5% CO2 in a 0.5 ml chondrogenic medium. The chondrogenic medium should be composed of 10 ng/ml TGF-β3, 500 ng/ml BMP-6, 100 nM dexamethasone, 50 µg/ml ascorbic 2-phosphate, 50 µg/ml ITS and 1.25 mg/ml bovine serum albumin. Addition of Lithium Chloride and a small molecule refereed to as SB216763 can enhance glycoseaminoglycal deposition in the human marrow-derived MSC chondrogenic culture"


"DNA methylation levels of CpG-rich promoters of chondrocyte-specific genes were mostly maintained at low levels"


"Sox9 associates with CREB-binding protein (CBP)/p300 via its carboxyl termini activation domain and functions as an activator for cartilage tissue-specific gene expression during chondrocyte differentiation"

"p300 potentiated Sox9-dependent transcription through hyperacetylation of histones. P300/ CBP acts as a coactivator to cartilage homeoprotein- 1 (Cart1) through acetylation of the conserved lysine residue adjacent to the homeodomain"

"Histone deacetylation by HDAC1 has been reported to have a critical inhibitory role in cartilage noncollagenous matrix deposition during cartilage differentiation. Cartilage oligomeric matrix protein (COMP) is a noncollagenous matrix protein in cartilage. In a study using Sox-9-null mice, the COMP gene was inhibited by a transcription repressor,the negative regulatory element (NRE)-binding protein by recruiting HDAC1 to the COMP promoter.  Rat chondrosarcoma cells and BMP-2-treated C3H10T1/2 progenitor cells, it was observed that the leukemia/ lymphoma-related factor, a POZ domain-containing transcriptional repressor, interacted with HDAC1 and inhibited COMP gene expression and chondrogenesis"

"HDAC4 regulates chondrocyte hypertrophy and endochondral bone formation by inhibiting the activity of Runx2 which is a transcription factor necessary for chondrocyte hypertrophy.  HDAC4-null mice display premature ossification of developing bone; and conversely, over expression of HDAC4 in proliferating chondrocytes in vivo inhibits chondrocyte hypertrophy and differentiation"

"HDAC promotes collagen II expression by suppressing the transcription of Wnt-5a"

MSCs versus chondrocyte gene expression

Differential Protein Expression between Chondrogenic Differentiated MSCs, Undifferentiated MSCs and Adult Chondroctyes Derived from Oryctolagus cuniculus in vitro.

"This preliminary study aims to determine the differentially expressed proteins from chondrogenic differentiated multipotent stromal cells (cMSCs) in comparison to undifferentiated multipotent stromal cells (MSCs) and adult chondrocytes (ACs). Methods: ACs and bone marrow-derived MSCs were harvested from New Zealand White rabbits (n = 3). ACs and cMSCs were embedded in alginate and were cultured using a defined chondrogenic medium containing transforming growth factor-beta 3 (TGF-β3). Chondrogenic expression was determined using type-II collagen, Safranin-O staining and glycosaminoglycan analyses. Two-dimensional gel electrophoresis (2-DE) was used to isolate proteins from MSCs, cMSCs and ACs before being identified using liquid chromatography-mass spectrometry (LC-MS). The differentially expressed proteins were then analyzed using image analysis software. Results: Both cMSCs and ACs were positively stained with type-II collagen and safranin-O. The expression of glycosaminoglycan in cMSCs was comparable to AC at which the highest level was observed at day-21 (p>0.05). Six protein spots were found to be most differentially expressed between MSCs, cMSCs and ACs. The protein spots cofilin-1 (CFL1) and glycealdehyde-3-phosphate dehydrogenase (GAPD) from cMSCs had expression levels similar to that of ACs whereas the others (ie. MYL6B, ALDOA, TAGLN2, EF1-alpha), did not match the expression level of ACs. Conclusion: Despite having similar phenotypic expressions to ACs, cMSCs expressed proteins which were not typically expected."

"TAGLN2 was significantly down-regulated in cMSC. Absence of TAGLN2 has shown to increase the activity of Rho GTPase that phosphorylates MYL6B and induces formation of stress fibres"

"Activity of GAPD in cMSC and ACs has increased three folds as compared to MSCs."

"ALDOA expression was lower in cMSCs than both the MSCs and ACs. ALDOA is a glycolytic enzyme for energy generation in chondrocytes, particularly marked at cell maturity. Reduction of energy modulated by ALDOA may limit the ability of ACs to restore GAG matrices. Recently, both GAPD and ALDOA have been found to interact with S100A1 and S100B proteins that suppress hypertrophic differentiation and mineralization of chondrogenic cells"

"[EIF-1A] was significantly down-regulated in cMSC"

"Phosphorylated EF1-alpha by type-I TGFβ receptor kinase at Serine 300 exerted a direct inhibitory on protein synthesis, therefore reducing cell proliferation"

"MYL6B, ALDOA, TAGLN2 and EF1-ALPHA were not as highly modulated during MSC chondrogenesis as compared to ACs."

Growing Taller with a Bioengineered Cartilage Pellet

Growth plate transplants have shown signs of being successful.  A growth plate transplant is really nothing more than a layer of chondrocytes within bone which is what we're trying to induce with hydrostatic pressure LSJL.  A pellet usually refers to something of a very small size so likely utilizing bioengineered cartilage pellets would be much easier than limb lengthening surgery.

Restoration of longitudinal growth by bioengineered cartilage pellet in physeal injury is not affected by low intensity pulsed ultrasound

"We investigated the application of a Bioengineered Cartilage Pellet (BCP) in repairing a rabbit physeal fracture model, and the possible effects of Low Intensity Pulsed Ultrasound (LIPUS) treatment. Rabbits with physeal fracture created were assigned to the NC group (no BCP, no LIPUS), GC group (BCP, no LIPUS), and GT group (BCP and LIPUS). Femoral lengths and cartilage area were assessed at 4, 8, and 16 weeks post-defect. After transplantation, the BCP showed continuous growth in the host and demonstrated resemblance to a natural growth plate. The GC group showed 34.1, 32.1, and 41.1% advantage in lengthening over the NC group and the GT group showed 51.1, 41.6, and 26.9% improved lengthening than the NC group, at 4 (p = 0.203), 8 (p = 0.543) and 16 weeks (p = 0.049), respectively. Cartilage area was shown to be significantly higher in GC and GT group compared to NC group (p < 0.05). No significant difference was found between GC and GT group. Femoral longitudinal growth was shown to be improved by the BCP, however no additional enhancement effect was shown to be provided by LIPUS[maybe they targeted LIPUS on the wrong cells or the LIPUS stimulation wasn't enough?]. "

"mesenchymal stem cells (MSC) [can be used] together with a composite scaffold to regenerate cartilage [and it's possible to] use the cultured autologous chondrocytes together with a demineralized bone matrix to prevent bone bridging and restoring growth in damaged growth plate in rabbit tibia. "<-We've seen the importance of a demineralized bone matrix before.  And it's possible that a demineralized bone matrix can increase height without the presence of external stem cells or chondrocytes.

"[LIPUS has been] shown to increase chondrocyte proliferation, proteoglycan synthesis, and aggrecan gene expression."<-Which is why LIPUS may help increase height.

"LIPUS treatment started 1 day after introduction of the physeal defect and each rabbit in the GT group received 20 min of treatment, 5 days per week until time-points were reached. Coupling gel was applied to the ultrasound transducer with air bubbles carefully removed as they would block transmission of the ultrasound waves. The transducer was then placed directly on the medial side of the defective knee. The LIPUS devices delivered 200-μs bursts of 1.5 MHz ultrasound signals repeated at 1.0 kHz with intensity of 30 mW m−2."<-So the LIPUS device was applied laterally to the knee which is how we would apply LIPUS to induce stem cell differentiation into chondrocytes.  Maybe the fracture inhibited LIPUS effectiveness as the fracture may reduce hydrostatic pressure.  A study involving drilling done by the LSJL scientists showed that holes can reduce hydrostatic pressure.

"Our findings suggest skeptics that LIPUS may accelerate endochondral ossification in physeal fractures, which will cause premature physeal plate closure that is not always desirable in physeal injuries."<-So LIPUS may be bad for height growth during development?

"The lack of efficacy of LIPUS in stimulating the BCP in its transplantation site could be due to variations in ultrasound transmission to intact and fractured bone via an entry point. The bony ends of the physeal defect might have deflected off some of the ultrasound waves. This may be related to others' observation in the enhancement of fracture healing by LIPUS, but no positive stimulatory effect on bone mineral density in intact bone."<-So it's possible that LIPUS might not go deep enough into the bone to increase hydrostatic pressure and induce chondrogenesis.

Here's the study that describes how pellet culture was performed unfortunately I don't have access to it:

Bioengineering and Characterization of Physeal Transplant with Physeal Reconstruction Potential

"In this study, we attempted to treat proximal tibial physeal damage in rabbits with transplanted bioengineered physeal tissue. Resting chondrocytes from the reserve zone of costal cartilages of 6-week-old rabbits were pellet cultured in centrifuge tubes. The pellets were characterized histologically and biochemically with reference to the normal physis. The bioengineered tissue was then transplanted into partially damaged proximal tibial physis. Histological changes and proteoglycan metabolism of the transplants were monitored until 7 weeks posttransplantation. Our results showed that chondrocytes cultured by three-dimensional pellet exhibited cell division and the derived cells arranged in short columns similar to normal physis. They synthesized and deposited cartilaginous matrix and differentiated into hypertrophic chondrocytes marked by increases in cell size and alkaline phosphatase activity. The transplant incorporated well in host tissue with no sign of rejection for up to 7 weeks posttransplantation. A further 3-fold increase in thickness of the transplant within the host was observed. Endochondral ossification was demonstrated at 7 weeks posttransplantation."

Here's another study however that explained the method to form chondrocyte pellets:

Hyaline cartilage engineered by chondrocytes in pellet culture: histological, immunohistochemical and ultrastructural analysis in comparison with cartilage explants

"Fertilized White Leghorn chicken eggs were incubated at 37 °C for 16 days. The distal part of the sternum was removed from chick embryos, predigested in 0.2% collagenase for 30 min, and further digested in fresh collagenase solution for 3 h[likely to get red of the extracellular matrix]. Chondrocytes were suspended in supplemented Dulbecco's modified Eagle medium  at a concentration of 107 mL−1. Four × 106 chondrocytes in 0.4 mL of cell suspension were transferred into each 0.75-mL tube . Chondrocyte pellets were formed by centrifugation at 500 g for 10 min[so they use centrifugal force to get all the chondrocytes at the end of the test tube]. The culture medium was DMEM plus 10% fetal bovine serum, 50 µg mL−1 ascorbate, 2 mm glutamine and 0.2% penicillin/streptomycin. The pellets were cultured at 37 °C under a gas mixture of 95% air/5% CO2. The pellets were transferred into Petri dishes at day 3 and continued to grow for 2 weeks."

In the bone marrow there are no chondrocytes and thus no cartilagenous matrix to digest.  The medium is mostly things that already exist in the bone marrow.  What we can do is use centrifugal force to get more mesenchymal stem cells in one location.  The development of cartilage has been shown to be initiated by a condensation of mesenchymal precursor cells.  Maybe Kojima's rotation device can help you grow taller...  Kojima's green light could demineralize a bone surface and then the rotation could get a bunch of stem cells onto that bone surface.

So maybe after LSJL you need to use a bicycle to get stem cells onto a bone surface which has been demineralized by hydrostatic pressure.