Thursday, July 29, 2010

Per1

A Negative Correlation Between Per1 and Sox6 Expression During Chondrogenic Differentiation in Pre-chondrocytic ATDC5 Cells.

"Pre-chondrocytes undergo cellular differentiation stages during chondrogenesis under the influence by different transcription factors such as sry-type high mobility group box-9 (Sox9) and runt-related transcription factor-2 (Runx2). We have shown upregulation by parathyroid hormone (PTH) of the clock gene Period-1 (Per1) through the cAMP/protein kinase A signaling pathway in pre-chondrocytic ATDC5 cells. Here, we investigated the role of Per1 in the suppression of chondrogenic differentiation by PTH. In ATDC5 cells exposed to 10 nM PTH, a drastic but transient increase in Per1 expression was seen only 1 h after addition together with a prolonged decrease in Sox6 levels. However, no significant changes were induced in Sox5 and Runx2 levels in cells exposed to PTH. In stable Per1 transfectants, a significant decrease in Sox6 levels was seen, with no significant changes in Sox5 and Sox9 levels, in addition to the inhibition of gene transactivation by Sox9 allies. Knockdown of Per1 by siRNA significantly increased the Sox6 and type II collagen levels in cells cultured for 24 - 60 h. Per1 plays a role in the suppressed chondrocytic differentiation by PTH through a mechanism relevant to negative regulation of transactivation of the Sox6 gene during chondrogenesis."

"2.2 kb of the 5′ flanking region of the mouse Sox6 gene contains one putative E-box (CACGTG), which is a target element recognized by the master clock molecules Bmal1/Clock heterodimer"

It seems that Sox9 levels were enhanced by Per1 but ColII levels were still way down.

"downregulation of Sox6 would lead to suppression of the gene transactivation mediated by the Sox9 trimeric complex upstream of the Col II gene whose translation is essential for chondrogenesis in ATDC5 cells."

Monday, July 26, 2010

Increasing Height with Hypnosis?

A lot of height seekers are trying hypnosis in an attempt to increase height.  In my last article about hypnosis, I stated that the hypnotherapist would have to be telekinetic to increase your height(or would have to unlock the inner telekinetic abilities inside of you).  Telekinesis within the body is referred to as biokinesis.  Hypnosis' ability to increase height is something that can be quantifiably measured somewhat.  Does it increase stem cell proliferation?  Does it inhibit myostatin?  Does it increase SOX9 or Beta-Catenin expression?  Does it increase insulin or IGF-1 sensitivity?

A lot of the studies on insulin and hypnosis are from the 60's and 70's so I couldn't find an abstract.  There were no relevant studies involving hypnosis stimulating the pituitary or parathyroid glands.  Height increase treatments operate basically in reverse of cancer treatments(in height increase you want to increase cellular proliferation+differentiation and in cancer you want to decrease it).  Most of the studies involving cancer and hypnosis involves the control of pain.  Here's something about the power of the mind... 

[Evidence about the power of intention] 

"Intention is defined as a directed thought to perform a determined action. Thoughts targeted to an end can affect inanimate objects and practically all living things from unicelular organisms to human beings. The emission of light particles (biophotons) seems to be the mechanism through which an intention produces its effects. All living organisms emit a constant current of photons as a mean to direct instantaneous nonlocal signals from one part of the body to another and to the outside world. Biophotons are stored in the intracelular DNA. When the organism is sick changes in biophotons emissions are produced.Direct intention manifests itself as an electric and magnetic energy producing an ordered flux of photons. Our intentions seem to operate as highly coherent frequencies capable of changing the molecular structure of matter. For the intention to be effective it is necessary to choose the appropriate time. In fact, living beings are mutually synchronized and to the earth and its constant changes of magnetic energy. It has been shown that the energy of thought can also alter the environment. Hypnosis, stigmata phenomena and the placebo effect can also be considered as types of intention, as instructions to the brain during a particular state of consciousness. Cases of spontaneous cures or of remote healing of extremely ill patients represent instances of an exceedingly great intention to control diseases menacing our lives. The intention to heal as well as the beliefs of the sick person on the efficacy of the healing influences promote his healing. In conclusion, studies on thought and consciousness are emerging as fundamental aspects and not as mere epiphenomena that are rapidly leading to a profound change in the paradigms of Biology and Medicine." 

So essentially a hypnotherapist(or yourself) would use these biophotons to change the molecular structure of your bone.  There have been studies done on biophotons. 

Biophotons as neural communication signals demonstrated by in situ biophoton autography. 

"Cell to cell communication by biophotons has been demonstrated in plants, bacteria, animal neutrophil granulocytes and kidney cells. Whether such signal communication exists in neural cells is unclear. By developing a new biophoton detection method, called in situ biophoton autography (IBA), we have investigated biophotonic activities in rat spinal nerve roots in vitro. We found that different spectral light stimulation (infrared, red, yellow, blue, green and white) at one end of the spinal sensory or motor nerve roots resulted in a significant increase in the biophotonic activity at the other end. Such effects could be significantly inhibited by procaine (a regional anaesthetic for neural conduction block) or classic metabolic inhibitors, suggesting that light stimulation can generate biophotons that conduct along the neural fibers, probably as neural communication signals. The mechanism of biophotonic conduction along neural fibers may be mediated by protein-protein biophotonic interactions. This study may provide a better understanding of the fundamental mechanisms of neural communication, the functions of the nervous system, such as vision, learning and memory, as well as the mechanisms of human neurological diseases." 

So the CNS can control biophotonic activity. 

Cancer growth and its inhibition in terms of coherence. 

"It is shown that a molecular origin for growth inhibition is rather unlikely because the cross-sectional area of inhibitory forces in a cell population cannot exceed more than about 10(-8) Dalton. A model of the time dependence of cell number N(t), where t is the time, is based on biophotons and explains without any contradiction to known experimental results growth regulation in terms of the factor a = 1/T, which stimulates the cell division rate dN/dt and the factor b = dT/dN(1/T(2)), which inhibits cell division. It accounts for the total cell division rate dN/dt = aN(t) - bN(2)(t). For adults, T is the coherence time of about 10(6) s, corresponding to the longest lifetime of cell organelles in men, while dT/dN = 10(-7) s corresponds to the resolution time of the cell population which is always the average time interval between two cell loss events. Our model follows a stringently holistic approach to describing a cell population as an entity, regulated by a fully coherent (biophoton) field." 

Biophotons have the ability to regulate cell populations.  So there's a method to measure biophotons called biophoton autography.  So in order to see if intent stimulus can increase height, one would need to measure how biophotons are emitted in response to different thoughts and how various biophoton emissions can affect cell populations.

Sunday, July 25, 2010

DHCR24

3β-Hydroxysterol-Delta24 reductase plays an important role in long bone growth by protecting chondrocytes from reactive oxygen species.

"Desmosterolosis is an autosomal recessive disease caused by mutations in the 3β-hydroxysterol-Delta24 reductase (DHCR24) gene, with severe developmental anomalies including short limbs. We utilized DHCR24 knockout (KO) mice to study the underlying bone pathology. Because the KO mice died within a few hours after birth, we cultured metatarsal bones from newborn mice. The growth of bones from KO mice was significantly retarded after 1 week of culture. Absence of proliferating chondrocytes in the growth plate and abnormal hypertrophy of prehypertrophic chondrocytes were observed in the bones from KO mice. Hypertrophic differentiation was evidenced by higher expression of Indian hedgehog, alkaline phosphatase, and matrix metalloproteinase 13. Since elevated levels of reactive oxygen species (ROS) during chondrogenesis are known to inhibit proliferation and to initiate chondrocyte hypertrophy in the growth plate, and since DHCR24 acts as a potent ROS scavenger, we hypothesized that the abnormal chondrocyte proliferation and differentiation in KO mice were due to decreased ROS scavenging activity. Treatment with an antioxidant, N-acetyl cysteine, could correct the abnormalities observed in the bones from KO mice. Treatment of bones from wild-type mice with U18666A, a chemical inhibitor of DHCR24, resulted in short broad bones with a disrupted proliferating zone. Treatment of ATDC cells with hydrogen peroxide (H(2)O(2)) induced hypertrophic changes as evidenced by the expression of the marker genes specific for hypertrophic chondrocyte differentiation. H(2)O(2)-induced hypertrophic change was prevented by adenoviral delivery of DHCR24. Induction of chondrocyte differentiation in ATDC cells by insulin was associated with increased ROS production that was markedly enhanced by treatment of ATDC5 cells with DHCR24 siRNA."

" From the 7th to 21st day the growth of the bones from KO mice was markedly slower with almost no further enhancement in bone length. However, a gradual increase of the length of the bones from WT mice was observed throughout the 21 days."

DHCR24 was expressed in all growth plate layers.

"The length of the metatarsal bones without U18666A (2.8 ± 0.1 mm) was significantly longer than that with U18666 (2.4 ± 0.2 mm)."

"Treatment with NAC, a potent antioxidant, reversed the abnormal findings observed in the bones cultured from KO mice."

"controlled ROS levels by DHCR24 are mandatory for long bone growth."

Friday, July 23, 2010

Actually, I've grown around 1/2" with LSJL

Okay, there were some measurement error problems but I find a good way to effectively measure height.  So I'm 5'9" up from 5'8 1/2" but not 5'9 1/4" like I thought.  Here's the previous pics:  Bone Length Increase.  Here's my height measurement:  I have a bump on my head(bone) so that's why it doesn't quite look like 69 inches but if you incorporate the hair which covers the bump then you get 69.

The tape measure is going up to 69 inches.

So you can see that it's the same place on the wall as with the other picture.  Here's tibia and fibula pics:
So now when I grow to 69 1/4" I'll be able to prove LSJL.

Wednesday, July 21, 2010

How to increase the height of your child

In my previous article about increasing kids height, I wrote more about increasing growth rate.  In this article, I'm going to write about how to increase your height at every stage of development.

How do we grow taller?  In long bones: stem cell proliferation + differentiation and chondrocyte proliferation + differentiation, and transport of new stem cells into the hyaline cartilage growth plate line.  In short bones:  Stem cell proliferation + differentiation and osteoblast proliferation + differentiation.  Their are also mesenchymal precursor cells that we can try to target as well.  It should be noted that most methods involving chondrocyte proliferation have been relatively unsuccessful due to a likely limited proliferative capacity of chondrocytes.  For long bones, you need to increase stem cell proliferation or get new stem cells.

How do we alter these factors?

In an embryo, you can alter genes.  You can also alter genes in adults as well(mutagenics).  You would want to alter genes that inhibit myostatin, enhance sensitivity to insulin and IGF-1, and enhance expression of the C-type natriuretic peptide.

With a baby, there's not much you can do.  A baby can't really exercise.  You can perform LSJL on baby.  Very light like with your thumb.  Adults grow from 10 to 20 times the size they were when they were a baby.  The impact of what you do for a baby could be multiplied by 10 or 20 times.  A baby is very soft and spongy with hyaline cartilage instead of bones.  If you alter genetic expression of the hyaline cartilage by very light lateral loading and increase fluid flow by that same loading how much of an impact do you think that could have later in life?  Mechanical loading has been shown to enhance cell proliferation.

For a kid, there's more you can do.  First, you want to make sure that estrogen levels are in the proper range to maximize cellular proliferation.  Testosterone inhibits Myostatin and myostatin inhibits cellular proliferation. So you can increase testosterone.  Of course, to do that you need advanced equipment to properly monitor levels of these factors.  If a doctor injects you with HGH or IGF-1 to stimulate cellular proliferation then you want to make sure that the body is responding and their effect is not diminished by negative feedback.  There is one cheap way however to increase a lot of anabolic factors.

Lithium.  Lithium has one outer electron and is highly reactive.  Lithium increases stem cell proliferation.

Exercise.  Exercise increases insulin sensitivity which increases cellular proliferation by the PI3K pathway.  Exercise increases sensitivity to IGF-1 and induces hypoxia(lack of oxygen) which is also anabolic.

There's also body fat percentage.  There's an optimal estrogen range to maximize cellular proliferation and too much or too little fat can affect that(fat cells produce estrogen).  Too much body fat inhibits your insulin sensitivity.  Body fat increases leptin which affects osteoblasts and chondrocytes so that is important as well.

You can also perform LSJL on kids but with more intensity.  Kids can also perform mechanical loading which again is highly anabolic to bone.  I know the common belief is no weight lifting before age 13 but it's much safer for a kid to weight train then it is to play a sport like football where you can't control what's going on.  Injuries can happen in any sport and weight lifting is safer due to the nature of it not involving the chaos of other people.  Compressive loading slows down growth rate but does not decrease final growth.  Weight lifting doesn't seem to increase growth on it's own but compressive loading increases vascularization to the growth plate and could enable easier delivery of new MSCs to the hyaline cartilage growth plate line via LSJL.  Loading of the muscles and the periosteum also alters genetic expression further encouraging anabolism.  And loading the bones could alter the PI3K pathway making it more sensitive to insulin and IGF-1.

So the younger you are the more options available for you to increase your height.  As you get older, you need to get all new stem cells into the hyaline cartilage by trabecular microfracture of the epiphysis.  But if you're younger, it's enough to enhance cellular proliferation of existing stem cells in the resting cell of the growth plate line and increase levels and enhance sensitivity through manipulation of various hormones or mechanical stimuli.

Sunday, July 18, 2010

Increase Height with Intramembranous Ossification?

Long bones naturally develop by endochondral ossification whereas short, flat, and irregular bones grow by intramembranous ossification.  Endochondral ossification involves hyaline cartilage and chondrocytes whereas intramembranous ossification does not.  Intramembranous ossification involves mesenchymal stem cells differentiating into osteoblasts which then lay down bone.

This form of ossification is even involved in healing bone fractures as there are stem cells within the trabeculae in the diaphysis of the long bones.  When enough stem cells come together and the appropriate genetic expression is in place the stem cells began to develop into osteoblasts.  The osteoblasts then produce trabecular bone, then the periosteum forms around the network of trabecular bone.  And cortical bone is formed between the trabecular bone and periosteum.

In intramembranous ossification there is no growth plate "fusion".  In some disorders, bone can form anywhere in the body.  Intramembranous ossification is truly organic bone growth.  Humans do not stay the same torso height once intramembranous ossification is complete.  The torso keeps growing due to hormonal changes causing the periosteum to produce new osteoblasts that lay down new cortical bone.  This is referred to as appositional growth.  The cortical bone increases the size of the bone resulting in an increase in height.  Further, intramembranous ossification within already formed bone should increase the number of trabeculae(bone density) as there is already a periosteum formed.  This increase in trabeculae can indirectly increase height by enhancing the periosteum thereby increasing the number of osteoprogenitor cells released as a result of shearing forces on the periosteum.

Appositional bone growth has been shown to be possible in runners.  Most likely due to the constant shearing forces on the periosteum altering genetic expression and releasing osteoprogenitor cells which lay down new cortical bone.  You get appositional bone growth in your spine, hip, pelvis, skull, or calcaneous and you'll grow taller.

Endochondral ossification-Grow taller by Lateral Synovial Joint Loading.  Cause microfractures in the trabecular bone of the epiphysis, increase interstitial fluid flow to send stem cells to hyaline cartilage growth plate line, the genetic expression of the hyaline cartilage growth plate line causes the stem cells to differentiate into chondrocytes

Intramemberanous Ossification-Grow taller by periosteal shear.  Cause shearing forces on the periosteum, like running does on the shin bone except for the spine, pelvic bone, skull bone, and calcaneus.  This shearing force releases osteoprogenitor cells that deposit new compact bone resulting in larger short, flat, and irregular bones resulting in a taller you.

I'm already performing tapping on my calcaneus(heel bone) and haven't really seen any results from it.  I'm going to start performing LSJL on it to see if LSJL can have the same benefits on an irregular bone.

Here's a study that states maybe there's really no such thing as intramembranous ossification and there's always a transitional chondrogenic phase:


Transient chondrogenic phase in the intramembranous pathway during normal skeletal development.


"Calvarial and facial bones form by intramembranous ossification, in which bone cells arise directly from mesenchyme without an intermediate cartilage anlage. However, a number of studies have reported the emergence of chondrocytes from in vitro calvarial cell or organ cultures and the expression of type II collagen, a cartilage-characteristic marker, in developing calvarial bones. Based on these findings we hypothesized that a covert chondrogenic phase may be an integral part of the normal intramembranous pathway. To test this hypothesis, we analyzed the temporal and spatial expression patterns of cartilage characteristic genes in normal membranous bones from chick embryos at various developmental stages (days 12, 15 and 19). Northern and RNAse protection analyses revealed that embryonic frontal bones expressed not only the type I collagen gene but also a subset of cartilage characteristic genes, types IIA and XI collagen and aggrecan, thus resembling a phenotype of prechondrogenic-condensing mesenchyme. The expression of cartilage-characteristic genes decreased with the progression of bone maturation. Immunohistochemical analyses of developing embryonic chick heads indicated that type II collagen and aggrecan were produced by alkaline phosphatase activity positive cells engaged in early stages of osteogenic differentiation, such as cells in preosteogenic-condensing mesenchyme, the cambium layer of periosteum, the advancing osteogenic front, and osteoid bone. Type IIB and X collagen messenger RNAs (mRNA), markers for mature chondrocytes, were also detected at low levels in calvarial bone but not until late embryonic stages (day 19), indicating that some calvarial cells may undergo overt chondrogenesis. On the basis of our findings, we propose that the normal intramembranous pathway in chicks includes a previously unrecognized transient chondrogenic phase similar to prechondrogenic mesenchyme, and that the cells in this phase retain chondrogenic potential that can be expressed in specific in vitro and in vivo microenvironments."

Of course just because there's mesenchymal condensation and that cartilagenous genes are expressed doesn't mean there's a transient cartilage phase.

Overgrowth disorders

Capillary-Venous Malformation in the Lower Limb.

"Regional capillary malformation of a lower extremity is associated with the overgrowth of bone or soft tissue in several disorders, most commonly Klippel-Trenaunay syndrome and Parkes Weber syndrome. We have observed a subset of patients with a capillary malformation of the leg, minor growth disturbance, and prominent veins. The objective of the current study is to describe a series of patients with regional capillary malformation of the lower extremity in association with phlebectasia. This is a retrospective series of 17 patients diagnosed with capillary-venous malformation of the lower extremity. We excluded patients with clinical or radiographic evidence of lymphatic or arteriovenous malformation.  In most patients the capillary malformation covered a large area without sharply demarcated borders. Four patients had one or more discrete, well-defined capillary stains involving less than 5% of the total surface area of the affected lower limb. Prominent veins were most common in the popliteal fossa and on the knee and dorsal foot. Approximately two-thirds of patients had a leg length discrepancy, with the affected leg being longer (n = 6) or shorter (n = 4); in many the affected leg was also slightly larger (n = 8) or smaller (n = 4) in girth. Radiographic imaging showed dilatation of superficial (n = 16), muscular (n = 9), and deep veins (n = 6). We characterize a subset of patients with regional capillary-venous malformation of the lower extremity with prominent veins and minor hypotrophy/hypertrophy that differs from Klippel-Trenaunay syndrome (capillary-lymphatic-venous malformation) but belongs at the minor end of the spectrum of vascular disorders with overgrowth."

In the patients aged 31, 40, and 33 there was no leg length discrepency.

Aire

Autoimmune regulator, Aire, is a novel regulator of chondrocyte differentiation.

" we aimed to identify a novel regulatory factor of chondrocyte differentiation using gene expression profiles of micromass-cultured chondrocytes at different differentiation stages. From the results of microarray analysis, the autoimmune regulator, Aire, was identified as a novel regulator. Aire stable knockdown cells, and primary cultured chondrocytes obtained from Aire-/- mice, showed reduced mRNA expression levels of chondrocyte-related genes. Over-expression of Aire induced the early stages of chondrocyte differentiation by facilitating expression of BMP2. A ChIP assay revealed that Aire was recruited on an Aire binding site (T box) in the Bmp2 promoter region in the early stages of chondrocyte differentiation and histone methylation was modified. Aire can facilitate early chondrocyte differentiation by expression of Bmp2 through altering the histone modification status of the promoter region of Bmp2. Taken together, Aire might play a role as an active regulator of chondrocyte differentiation, which leads to new insights into the regulatory mechanisms of chondrocyte differentiation."

Full-size image (84 K)
C lists other potential factors to look into for chondroinduction.  Aire was the only gene successfully validated in D.  Although Atoh8 and Erg could also be candidates.

"Aire−/− mice did not exhibit significant abnormal phenotypes in skeletal morphology. However, patients with mutations at the AIRE gene locus suffer from autoimmune polyendocrinopathy candidiasis ectodermal dystrophy (APECED) and some of them show reversible metaphyseal dysplasia (RMD) with notable progressive growth and abnormal endochondral ossification in adolescents. Cst10−/− mice developed and grew normally but showed abnormal phenotypes in formation of osteoarthritic osteophytes, age-related ectopic ossification and healing of bone fractures"

Saturday, July 17, 2010

Do aromatase inhibitors increase height in males?

Males and females can respond to sex hormones differently.  Estrogen may inhibit growth in females but augment growth in males. Estrogen is speculated to affect chondrocyte proliferative capacity.  Can the usage of aromatase inhibitors increase height in males?  Estrogen has been used to accelerate growth plate fusion(although cessation is more accurate a word as you stop growing as a result of exhaustion of mesenchymal stem cells from the hyaline cartilage growth plate line) in females but only by one or two inches.  And there's no real way to know how tall a female would've been if she had not been injected with excess estrogen.  What affect can aromatase inhibitors have on a naturally developing bone of a male?  Aromatase Inhibitors stop the body from converting Testosterone to Estrogen. 

Impaired body weight and tail length gain and altered bone quality after treatment with the aromatase inhibitor exemestane in male rats. 

"Estrogen deficiency induced by aromatase inhibitors may be a novel treatment modality for growth enhancement in short children, but may have adverse effects on bone, brain and reproduction.  26-day-old prepubertal rats received intramuscular injections with placebo or the aromatase inhibitor exemestane at a dose of 10, 30 or 100 mg/kg/week [E10, E30, E100(6)] for 6 weeks, completely covering the sexual maturation phase, or with 3 weeks E100 followed by 3 weeks placebo [E100(3)]. Growth parameters and histology of the testis, seminal vesicle and brain were analyzed. Bone architecture was studied with X-ray microtomography. Results: Exemestane dose-dependently decreased body weight and tail length gain, as well as liver and seminal vesicle weights, but did not affect nose-anus length gain, growth plate width or radial growth. E100(6) decreased trabecular thickness (epiphysis and metaphysis) and number (metaphysis). Normal IGF-I levels and brain, testis and seminal vesicle morphology were observed. E100(3) resulted in decreased tail length gain only. Exemestane treatment during sexual maturation did not augment linear growth in male rats, but caused impaired body weight and tail length gain and osteopenia." 

"However, estradiol levels in male rats are very low and are expected to be even lower during treatment with an aromatase inhibitor."<-So, Estrogen levels may be below equilibrium already and that's why height decreased.  An Estrogen inhibition regime seemed to slow down cellular proliferation which again seems to suggest being below equilibrium

Expression of vascular endothelial growth factor in the growth plate is stimulated by estradiol and increases during pubertal development. 

"Longitudinal bone growth is regulated in the growth plate. At the end of puberty, growth velocity diminishes and eventually ceases with the fusion of the growth plate through mechanisms that are not yet completely understood. Vascular endothelial growth factor (VEGF) has an important role in angiogenesis, but also in chondrocyte differentiation, chondrocyte survival, and the final stages of endochondral ossification. Estrogens have been shown to up-regulate VEGF expression in the uterus and bone of rats. In this study, we investigated the relation between estrogens and VEGF production in growth plate chondrocytes both in vivo and in vitro. The expression of VEGF protein was down-regulated upon ovariectomy and was restored upon estradiol (E(2)) supplementation in rat growth plates. In cultured rat chondrocyte cell line RCJ3.1C5.18, E(2) dose dependently stimulated 121 and 189 kDa isoforms of VEGF, but not the 164 kDa isoform. Finally, VEGF expression was observed at both protein and mRNA levels in human growth plate specimens. The protein level increased during pubertal development, supporting a link between estrogens and local VEGF production in the growth plate. We conclude that estrogens regulate VEGF expression in the epiphyseal growth plate, although the precise role of VEGF in estrogen-mediated growth plate fusion remains to be clarified." 

So Estrogen is linked to VEGF but does VEGF inhibit chondrocyte proliferation or anything that would cause growth plate cessation?  VEGF signaling is essential to proper endochondral ossification.  Inhibiting Estrogen while finding alternative methods to upregulate VEGF may be a way to grow taller.  TGF-Beta1 for instance can activate VEGF.

"VEGF-A has been shown to be expressed in the growth plate and also believed to be most important in the regulation of longitudinal bone growth"

"Vegfa conditional knockout mice driven by a Col2a1 promoter showed delayed invasion of blood vessels into the primary ossification center and delayed removal of terminal hypertrophic chondrocytes together with massive cell death in chondrocytes throughout the growth plate, demonstrating the importance of VEGFA in chondrocyte survival"<-lack of VEGF-A results in reduced height growth

"VEGF expression is up-regulated by estrogens"

"we confirmed that VEGF is expressed in the human pubertal growth plate and that the VEGF protein level increases with pubertal progression, supporting a link between estrogens and local VEGF production in the growth plate."

No link between VEGF and fusion was found in the study likely because fusion does not occur post cessation and since VEGF helps with chondrocyte survival it delays the point until cessation.  Since VEGF is essential, some mechanism to increase TGF-Beta1 levels may be needed to upregulate VEGF while taking aromatase inhibitors.

Estrogen deficiency leads to decrease in chondrocyte numbers in the rabbit growth plate. 

"In the pubertal growth plate, sex hormones play important roles in regulating the proliferation, differentiation, maturation, and programmed death of chondrocytes. Although many studies have been reported on the regulation of estrogen in long-bone growth, some of the mechanisms have remained unclear, including its role in cell kinetics in growth plate chondrocytes. The aim of this study was to clarify the effect of a deficiency of estrogen on growth plate chondrocytes. METHODS: We obtained growth plates of the femoral head from normal and ovariectomized Japanese white rabbits at 10, 15, 20, and 25 weeks of age. The effects of estrogen deficiency on the cell kinetics of growth plate chondrocytes were investigated immunohistochemically using antibodies for an apoptotic marker, caspase-3, and for proliferating cell nuclear antigen (PCNA). RESULTS: Both the length of the femur and the height of the growth plate in the ovariectomized rabbits tended to be larger than those in the normal rabbits. There were fewer chondrocytes in the ovariectomized rabbits than in the normal ones. Caspase-3-positive cells were detected mainly in the hypertrophic zone, whereas PCNA-positive cells were found in the proliferating to upper hypertrophic zones. The ovariectomized rabbits showed a higher caspase-3-positive rate at 20 weeks of age and a lower PCNA-positive ratio in all age groups than the normal rabbits. CONCLUSIONS: This study indicated that ovariectomy led to a decreased number of growth plate chondrocytes, which resulted from decreased cell-proliferating ability and probably acceleration of the number of chondrocytes undergoing apoptosis." 

Estrogen deficiency did not increase cell-proliferating capacity in females.  Oestrogen is the specific form of estrogen speculated to be responsible for growth plate senescence.  What's the conclusion? 

"Recent studies indicated that ER-α up-regulates cyclin D1, and PCNA then stimulates both cell progression
and prevents the apoptotic cascade, whereas ER-β down-regulates proliferation and has pro-apoptotic
properties"<-Both of these may be essential for growth has you need a reduction in proliferation and for apoptosis in the hypertrophic zone.


Normal bone growth requires optimal estrogen levels: negative effects of both high and low dose estrogen on the number of growth plate chondrocytes 

"Endochondral bone formation at epiphyseal growth plate consists of the synchronized processes of chondrogenesis and cartilage ossification. Estrogen, the major female sex hormone, plays an important role in this process, particularly during the pubertal growth spurt. However, its effects on the growth plate are not completely understood. The aims of this study were to clarify the effects of estrogen on the kinetics of chondrocytes in the growth plates of 10- to 25-week-old female rabbits by studying the effects of ovariectomy or high-dose administration of estrogen on the balance between cell proliferation and death. Forty-eight Japanese white rabbits were divided into three groups: sham operated, ovariectomized, or ovariectomized with subsequent weekly injection of high dose estrogen from 10 weeks. The chondrocyte kinetics was investigated by histomorphometry and immunohistochemistry, using antibodies for caspase-3, a marker of apoptosis, and for proliferating cell nuclear antigen. Both ovariectomized and estrogen-injected rabbits showed a declination of the chondrocyte number although the latter animals indicated a more dramatic effect. Estrogen-injected rabbits showed a decrease in the cell proliferating ability together with an increase in chondrocytes undergoing apoptosis while ovariectomy mainly reduced the cell proliferating ability. Given the known importance of estrogen for bone growth, one would expect that ovariectomy and high-dose administration of estrogen would have opposite effects. However, the present study indicated that both low and high concentration had a similar effect: a decrease in the chondrocyte number compared with control, suggesting that estrogen has to be maintained within a narrow range for optimal bone growth." 

So estrogen does affect cell proliferating ability but it enhances it however only to a certain point.  So you need estrogen at an equilibrium point for optimal bone growth.  This would be true even if you're trying to increase height post growth plate senescence either with LSJL or a fracture method.  You'd want your estrogen to be at that optimal level to maximize chondrocyte proliferative capacity. 


Marked increase of final height by long-term aromatase inhibition in a boy with idiopathic short stature.

"We present a 14.5-year-old boy with ISS and a height of 142.7 cm [standard deviation score (SDS) -2.79]. Based on the baseline bone age (BA) of 13.5-14 years, his predicted adult height (PAH) by Bayley/Pinneau was 154 cm (SDS -3.77)-158.2 (SDS -3.15). After a 5-year letrozole monotherapy, FH was 169 cm (SDS -1.57) showing a height difference between PAH and FH from 10.8 to 15 cm[So future height was actually higher than predicted height]. No permanent side effects of the medication have been observed. Both a transient occurrence and a spontaneous recovery of decreased bone mineral apparent density were seen, verified by dual-energy X-ray absorptiometry. Spinal magnetic resonance imaging revealed no vertebral abnormalities."

A novel mutation in the human aromatase gene: Insights on the relationship among serum estradiol, longitudinalgrowth and bone mineral density in an adult man under estrogen replacement treatment

"We report on a new case of human aromatase deficiency in a man of 26 years of age and present the results of five year follow-up during trandermal estradiol (tE2) substitution, focusing on bone growth and mineralization. The lack of patient's compliance to tE2 treatment [resulted] in low but detectable serum estradiol levels."

"Eunuchoid body proportions, unfused epiphyses, tall stature, osteopenia, increase fasting insulin, mild astenozoospermia and a history of right cryptorchidism were present. Baseline serum FSH was slightly above the normal range and estradiol was undetectable. Genetic analysis revealed a pattern of compound heterozygosity due to 23 bp deletion in exon IV and a point mutation in the first nucleotide of intron IX of the CYP19A1 gene, respectively. The closure of epiphyseal cartilage, the normalization of bone BMD and bone turnover markers, and the improvement of insulin levels were reached during tE2 only when serum estradiol raised above 73 pmol/L. Sperm parameters and overweight did not improve with substitutive therapy."

"Months of therapy  0                          24 months             60 months
Therapy               None             Estradiol gel 0.75 mg/day Estradiol gel 0.75 mg/day
Age (years)        26.8                    30.5                                33.6
Height (cm)        193                   193.6                              193.6
Weight (kg)        109                    117                                120
BMI (kg/m2)        29.3                    31.2                                 32
Arm span (cm)         212                    213                                213
Upper-to-the-lower segment ratio
                               0.89                    0.91                                0.91"

"The patient had a history of right cryptorchidism, which was surgically corrected when he was 3 year old. "<-he was missing a right testicle.


Pre-treatment he had almost twice as much testosterone as normal.  Estrogen levels were almost non-existant.  60 months of estrogen treatment halved his free testosterone levels.  PTH and AkP were halved by estrogen treatment.  Osteocalcin and fasting insulin levels went down by a third.

"GH response to a standard GHRH + Arginine test [before estrogen treatment] showed a severely impaired GH peak (2.8 μg/L; normal values > 9 μg/L), according to the cut-offs used in the general population together with a serum IGF-I in the lowest quartile of the normal range (20.7 nmol/L; n.v. 14.4–50.3)."

"[It's possible] that the long period of treatment was able itself to promote bone maturation notwithstanding the very low doses of estradiol due to the poor patient's compliance and finally that this is only one case report"

Thursday, July 15, 2010

Thrombospondin 2

 During development, TSP2 is upregulated in the periochondrium but downregulated in the more mature periosteum so TSP2 could play a partial role in the on/off switch for increasing height.

Thrombospondin-2 Secreted by Human Umbilical Cord Blood-Derived Mesenchymal Stem Cells Promotes Chondrogenic Differentiation.

"human umbilical cord blood-derived MSCs (hUCB-MSCs) promoted differentiation of chondroprogenitor cells by paracrine action. This paracrine effect of hUCB-MSCs on chondroprogenitor cells was increased by treatment with synovial fluid (SF) obtained from osteoarthritis (OA) patients, but was decreased by SF of fracture patients, compared to that of an untreated group. To identify paracrine factors underlying the chondrogenic effect of hUCB-MSCs, the secretomes of hUCB-MSCs stimulated by OA SF or fracture SF were analyzed using a biotin label-based antibody array. Among the proteins increased in response to these 2 kinds of SF, thrombospondin-2 (TSP-2) was specifically increased in only OA SF-treated hUCB-MSCs. In order to determine the role of TSP-2, exogenous TSP-2 was added to a micromass culture of chondroprogenitor cells. TSP-2 had chondrogenic effects on chondroprogenitor cells via PKCα, ERK, p38/MAPK, and Notch signaling pathways{Knockdown of the ERK pathway seemed to enhance the chondrogenic effects whereas knocking down the other pathways decreased the chondrogenic effect of TSP2.. Knock-down of TSP-2 expression on hUCB-MSCs using small interfering RNA (siRNA) abolished the chondrogenic effects of hUCB-MSCs on chondroprogenitor cells. In parallel with in vitro analysis, the cartilage regenerating effect of hUCB-MSCs and TSP-2 was also demonstrated using a rabbit full-thickness osteochondral-defect model. hUCB-MSCs can stimulate the differentiation of locally presented endogenous chondroprogenitor cells by TSP-2, which finally leads to cartilage regeneration."

Can TSP-2 encourage the chondrodifferentiation of MSCs and not just chondroprogenitor cells?

"Chondroprogenitor cells were isolated from the limb buds of 11.5 dpc ICR mouse embryos and
maintained as micromass cultures to induce chondrogenesis."

"TSP-2 released into the synovia promotes the differentiation of endogenous chondroprogenitor cells."

Wednesday, July 14, 2010

Why are males taller than females?

It is a secondary sex characteristic for females to be shorter than males.  Some have speculated that this may be due to estrogen which could define the proliferative capacity of chondrocytes.  What are some other possible causes for sex related height differences?

Ciliary neurotrophic factor inhibits bone formation and plays a sex-specific role in bone growth and remodeling. 

"Ciliary neurotrophic factor (CNTF) receptor (CNTFR) expression has been described in osteoblast-like cells, suggesting a role for CNTF in bone metabolism. When bound to CNTF, neuropoietin (NP), or cardiotrophin-like-cytokine (CLC), CNTFR forms a signaling complex with gp130 and the leukemia inhibitory factor receptor, which both play critical roles in bone cell biology. This study aimed to determine the role of CNTFR-signaling cytokines in bone. Immunohistochemistry detected CNTF in osteoblasts, osteocytes, osteoclasts, and proliferating chondrocytes. CNTFR mRNA was detected in primary calvarial osteoblasts and was upregulated during osteoblast differentiation. Treatment of osteoblasts with CNTF or CLC, but not NP, significantly inhibited mineralization and osterix mRNA levels. Twelve-week-old male CNTF ( -/- ) mice demonstrated reduced femoral length, cortical thickness, and periosteal circumference; but femoral trabecular bone mineral density (Tb.BMD) and tibial trabecular bone volume (BV/TV) were not significantly different from wild-type, indicating a unique role for CNTF in bone growth in male mice. In contrast, female CNTF ( -/- ) femora were of normal width, but femoral Tb.BMD, tibial BV/TV, trabecular number, and trabecular thickness were all increased. Female CNTF ( -/- ) tibiae also demonstrated high osteoblast number and mineral apposition rate compared to wild-type littermates, and this was intrinsic to the osteoblast lineage. CNTF is expressed locally in bone and plays a unique role in female mice as an inhibitor of trabecular bone formation and in male mice as a stimulus of cortical growth." 

So in males CNTF increases both limb height and torso height(cortical thickness+periosteal circumference).  In females, CNTF decreases the number of trabeculae.  So different responses to CNTF could explain height differences between males and females. 

"Femoral length was significantly lower in CNTF −/− mice of both sexes"

"Global deletion of CNTF resulted in a mild reduction in bone length, consistent with the short bone phenotypes of the gp130 knockout, the LIFR knockout, and the gp130ΔSTAT signaling mutant"

Sexual dimorphism in cortical bone size and strength but not density is determined by independent and time-specific actions of sex steroids and IGF-1: evidence from pubertal mouse models. 

"Although it is well established that males acquire more bone mass than females, the underlying mechanism and timing of this sex difference remain controversial. The aim of this study was to assess the relative contribution of sex steroid versus growth hormone-insulin-like growth factor 1 (GH-IGF-1) action to pubertal bone mass acquisition longitudinally in pubertal mice. Radial bone expansion peaked during early puberty (3 to 5 weeks of age) in male and female mice, with significantly more expansion in males than in females (+40%). Concomitantly, in 5 week old male versus female mice, periosteal and endocortical bone formation was higher (+70%) and lower (-47%), respectively, along with higher serum IGF-1 levels during early puberty in male mice. In female mice, ovariectomy increased radial bone expansion during early puberty as well as the endocortical perimeter. In male mice, orchidectomy reduced radial bone expansion only during late puberty (5 to 8 weeks of age), whereas combined androgen and estrogen deficiency modestly decreased radial bone expansion during early puberty, accompanied by lower IGF-1 levels. GHRKO mice with very low IGF-1 levels, on the other hand, showed limited radial bone expansion and no skeletal dimorphism. From these data we conclude that skeletal sexual dimorphism is established during early puberty and depends primarily on GH-IGF-1 action. In males, androgens and estrogens have stimulatory effects on bone size during late and early puberty, respectively. In females, estrogens limit bone size during early puberty. These longitudinal findings in mice provide strong evidence that skeletal dimorphism is determined by independent and time-specific effects of sex steroids and IGF-1." 

Remember as shown in the CNTF study, males and females could respond to chemicals differently.  Estrogen may inhibit bone growth in females but not in males.  

Use of aromatase inhibitors in children and adolescents: what's new? 

"Aromatase inhibitors have been reported to increase height prediction in boys with short stature, and in boys and girls with gonadotropin-independent precocious puberty. The following review discusses data published since 2008 regarding the safety and efficacy of aromatase inhibitors in pediatric patients. RECENT FINDINGS: Third-generation aromatase inhibitors in combination with antiandrogens appear effective in preventing bone age advancement and virilization in boys with familial male-limited precocious puberty (FMPP). Letrozole, but not anastrozole, decreased bleeding episodes and bone age advancement in girls with McCune-Albright syndrome (MAS), despite ovarian enlargement. Letrozole-treated boys with idiopathic short stature (ISS) had no loss of bone density but were noted to have more vertebral abnormalities than a placebo group. Two years of letrozole therapy did not increase predicted adult height in pre and peripubertal boys with ISS when re-assessed 4 years after the treatment period. SUMMARY: Aromatase inhibitors together with an antiandrogen appear to be a very promising treatment for FMPP. Further longer-term studies with letrozole are needed in MAS. The prevalence of vertebral deformities should be evaluated prospectively in patients treated with aromatase inhibitors. Adult height data are still lacking in pediatric patients treated with aromatase inhibitors. Two years of therapy in pre and peripubertal short boys does not appear to increase adult height. Hemogram, lipids, and bone density should be periodically assessed in treated patients. Further controlled studies are needed to demonstrate safety and efficacy of aromatase inhibitors in pediatric patients." 

So there's not enough evidence that aromatase inhibitors can increase final height in males. 

My theory:  Estrogen is responsible for decreasing height in women but not men.  Aromatase inhibitors in women will increase height but not men.  Estrogen therapy will decrease height in women but not men.  Men and women respond differently to estrogen.  

Skeletal Sexual Dimorphism: Relative Contribution of Sex Steroids, Growth Hormone - Insulin-Like Growth Factor-I (GH-IGF-I) and Mechanical Loading. 

"Structural gender differences in bone mass - characterized by wider but not thicker bones - are generally attributed to opposing sex steroid actions in men and women. Recent findings have redefined the traditional concept of sex hormones as the main regulators of skeletal sexual dimorphism. Growth hormone (GH) - insulin-like growth factor-I (IGF-I) action is likely to be the most important determinant of sex differences in bone mass. Estrogens limit periosteal bone expansion but stimulate endosteal bone apposition in females, whereas androgens stimulate radial bone expansion in males[radial = bone width]. Androgens not only act directly on bone through the androgen receptor (AR) but also activate estrogen receptor-alpha or -beta (ERalpha or ERbeta) following aromatization into estrogens. Both AR and ERalpha pathways are needed to optimize radial cortical bone expansion, whereas AR signaling alone is the dominant pathway for normal male trabecular bone development. Estrogen/ERalpha-mediated effects in males may - at least partly - depend on interaction with IGF-I. In addition, sex hormones and their receptors impact on the mechanical sensitivity of the growing skeleton. AR and ERalpha signaling may limit the osteogenic response to loading in males and females, respectively, while ERalpha may stimulate the response of bone to mechanical stimulation in the female skeleton. Overall, current evidence suggests that skeletal sexual dimorphism is not just the end result of differences in sex steroid secretion between sexes, but depends on gender differences in GH-IGF-I and mechanical sensitivity to loading as well." 

So you may not need an aromatase inhibitor but an aromatase receptor inhibitor. Sex related differences are due to how different pathways respond to stimulus.

The age of puberty determines sexual dimorphism in bone structure: a male/female co-twin control study.


"We hypothesized that sex differences in stature and bone structure are the result of sex differences in the duration but not the rate of prepubertal and pubertal growth.
We measured bone dimensions in 90 male/female co-twin pairs aged 7–18 yr using anthropometry and dual x-ray absorptiometry. Forty-two pairs had follow-up assessments. Within-pair differences were expressed as a percentage of the pair mean.
Thirty percent of the 1–1.5 SD sex difference in bone widths and midfemur bending strength observed in 11 postpubertal pairs was present in 43 prepubertal pairs. In prepubertal pairs, annual growth in leg length was about 1.5 times truncal growth, but neither rate differed by sex. During puberty, truncal growth in both sexes was higher than before puberty but did not differ by sex. The longer period of pre- and intrapubertal growth in males produced most of the sex difference in bone morphology observed in postpubertal twins.
Sex differences in bone morphology are the result of the later onset of puberty in males, not more rapid growth. Differences in bone widths are partly established before puberty."<-this means that age of puberty matters for height growth but not that other factors don't matter.

"Of the 90 male/female pairs, 66 were concordant for pubertal stage (43 pre-, 12 peri-, and 11 postpubertal), and 24 were discordant with females being more advanced in maturity than males (16 pre/peri, seven peri/post, and one pre/post). In the prepubertal twins, there were no sex differences in height, sitting height, leg length, weight, lean mass, fat mass, or hours of weight-bearing exercise (2.2 ± 0.3 vs. 1.7 ± 0.3 h/wk). However, bone widths were 4–7% (∼0.5 SD) greater in prepubertal males than females. This difference was one third of the 12–15% (∼1.5 SD) sex difference in the 11 mature twin pairs"

Thus perhaps constitutional delay can help you grow taller.


Sex-Specific Response of Rat Costochondral Cartilage Growth Plate Chondrocytes to 17β-Estradiol Involves Differential Regulation of Plasma Membrane Associated Estrogen Receptors.

"Both male and female rat growth plate chondrocytes express estrogen receptors (ERs); however 17β-estradiol (E(2)) induces membrane responses leading to activation of phospholipase A(2) (PLA(2)), phospholipase C (PLC), prostaglandin E(2) (PGE(2)) production, protein kinase C (PKC), and ultimately mitogen protein kinase (MAPK) only in female cells. Resting zone chondrocytes (RCs) showed 2-3 times more ERα in plasma membranes (PMs) from female cells than male cells. Tunicamycin blocked E(2)-dependent ER-translocation to the PM, indicating palmitoylation was required. E(2) induced complex formation between ER isoforms only in female RCs. To examine if the lack of response on PKC and PGE(2) in males is due to differences in signaling, we examined involvement of ERs and the role of PLC and PLA(2). Selective ERα (propylpyrazole triol, PPT) and ERβ (diarylproprionitrile, DPN) agonists activated PKC in female RCs only. The PLC inhibitor, U73122 blocked E(2)'s effect on PKC and the cytosolic PLA(2) inhibitor, AACOCF3 inhibited the effect on PGE(2) in female RCs, confirming involvement of PLC and PLA(2) in the mechanism. The PLC activator, m-3M3FβS activated PKC and PLAA peptide increased PGE(2) levels in male and female RCs, showing the signaling pathways are present."

"Although the binding affinity of E2 for ERs in growth plate chondrocytes was comparable in cells from both male and female rats, female cells had greater numbers of receptors than male cells"

"The nuclear fraction from both [male and female resting zone chondrocytes] exhibited all three ERα isoforms: ERα68, ERα46 and ERα36. Plasma membranes from female chondrocytes had all three isoforms, but male cells lacked ERα36. This was also the case for caveolae. ERβ59 was present in plasma membranes and nuclear fraction from female and male cells and was present in caveolae from female cells but not male cells "

Tuesday, July 13, 2010

Increasing Bone Length by increasing Chondrocyte Proliferative Capacity?

The phenomena of catch-up growth is said to be related to the fact that chondrocytes have a finite proliferative capacity. The chondrocytes simply stay inactive until there are appropriate circumstances for endochondral ossification to occur.  Stem cells have an unlimited proliferative capacity but if there are no stem cells in the hyaline cartilage growth plate line no further growth is possible.  It is the infinite ability of stem cells to proliferate that allows tall stature to occur in syndromes like Gigantism.  HGH increases IGF-1 which increases stem cell proliferation.  The reason that HGH on it's own doesn't usually increase height is that in Gigantism cellular homeostasis is altered(i.e. enlarged pituitary gland) whereas just simply injecting HGH will result in the bodies negative feedback mechanisms.

Is there anyway to increase the proliferative capacity of chondrocytes?  What mechanisms control it's proliferative capacity? 

Catch-up growth after glucocorticoid excess: a mechanism intrinsic to the growth plate. 

"In humans and other mammals, the release from growth-inhibiting conditions, such as glucocorticoid excess, leads to supranormal linear growth. The prevailing explanation for this catch-up growth involves a central nervous system mechanism that compares actual body size to an age-appropriate set-point and adjusts growth rate accordingly via a circulating factor. Although such a neuroendocrine "sizostat" was hypothesized more than 30 yr ago, its existence has never been confirmed experimentally. Here we show that suppression of growth within a single growth plate by locally administered glucocorticoid is followed by local catch-up growth that is restricted to the affected growth plate. Thus, the catch-up growth cannot be explained by neuroendocrine mechanism but, rather, must arise from a mechanism intrinsic to the growth plate. To explain this finding, we propose that the normal senescent decline in growth plate function depends not on age per se, but on the cumulative number of stem cell divisions, and that glucocorticoid administration, by suppressing stem cell proliferation, delays senescence, resulting in catch-up growth after the growth-inhibiting agent is removed" 

Now, in this study they propose that the mechanism is based on inhibiting stem cell proliferation.  If it did in fact affect stem cell proliferation then once could still increase bone length by increasing chondrocyte proliferation.  Now we know that myostatin inhibits stem cell proliferation.  Increasing testosterone inhibits myostatin.  Glutocorticoids may actually increase stem cell proliferation.  It makes no sense for catch up growth to be a restriction on stem cell proliferation as then the stem cells would still differentiate into chondrocytes and they would lose the number of stem cell proliferations during the time that the growth slows down.    It makes much more sense for it to be based on chondrocyte proliferative capacity.  

Depletion of resting zone chondrocytes during growth plate senescence. 

"With age, the growth plate undergoes senescent changes that cause linear bone growth to slow and finally cease. Based on previous indirect evidence, we hypothesized that this senescent decline occurs because growth plate stem-like cells, located in the resting zone, have a finite proliferative capacity that is gradually depleted. Consistent with this hypothesis, we found that the proliferation rate in rabbit resting zone chondrocytes (assessed by continuous 5-bromo-2'-deoxy-uridine labeling) decreases with age, as does the number of resting zone chondrocytes per area of growth plate. Glucocorticoid excess slows growth plate senescence. To explain this effect, we hypothesized that glucocorticoid inhibits resting zone chondrocyte proliferation, thus conserving their proliferative capacity. Consistent with this hypothesis, we found that dexamethasone treatment decreased the proliferation rate of rabbit resting zone chondrocytes and slowed the numerical depletion of these cells. Estrogen is known to accelerate growth plate senescence. However, we found that estradiol cypionate treatment slowed resting zone chondrocyte proliferation. Our findings support the hypotheses that growth plate senescence is caused by qualitative and quantitative depletion of stem-like cells in the resting zone and that growth-inhibiting conditions, such as glucocorticoid excess, slow senescence by slowing resting zone chondrocyte proliferation and slowing the numerical depletion of these cells, thereby conserving the proliferative capacity of the growth plate. We speculate that estrogen might accelerate senescence by a proliferation-independent mechanism, or by increasing the loss of proliferative capacity per cell cycle."

The basis for Estrogen inhibition is that Estrogen might decrease the proliferative capacity of chondrocytes per cell cycle.  This might explain the sex related height differences.  However, there are other possible explanations like testosterone inhibiting myostatin thus increasing stem cell proliferation or different genetic expression between males and females.  A lot of height seekers take aromatase inhibitors.  It's the basis for Height FX or Estrogenex Aromatase Inhibitor by Hi-Tech Pharmaceuticals - 90 Tablets.  More studies need to be done as to whether estrogen actually affects the proliferative capacity of chondrocytes.

"We have recently shown evidence that the resting zone chondrocytes serve as a pool of stem-like cells that generate columnar clones of proliferative zone chondrocytes[these resting zone stem like cells are basically stem cells but with a rounder shape which can be attained by hydrostatic pressure]. We have also found evidence suggesting that growth plate senescence is not a function of time per se but rather of the cumulative number of divisions that the chondrocytes have undergone[doesn't explain Gigantism though which seems to take place at a systematic level rather than at local growth plate levels]. Taken together, these previous findings suggest the following model: 1) stem-like cells in the resting zone have a finite proliferative capacity which is gradually exhausted[based on DNA-methylation and telomere length]; 2) as this proliferative capacity is exhausted, the proliferation rate of the proliferative zone chondrocytes (which are derived from the stem-like cells) decreases, causing longitudinal bone growth to slow with age and eventually cease."

"Although we attribute the decrease in proliferation with age to a decrease in the proliferative capacity, this decreased capacity is not necessarily cell autonomous and irreversible. The decrease in proliferative capacity could be context-dependent, and reversible[methylation status can be restored and telomeres can be lengthened, also you can get new stem cells to differentiate into chondrocytes in the epiphysis]. Previous studies suggest that the decrease is not due to hormonal or other systemic factors[But, again, what of Gigantism?], but the decrease could reflect a local change in concentrations of paracrine growth factors[possibly Ihh, Pth, etc.]."

"We speculate that estrogen might increase the loss of proliferative capacity that occurs with each cell cycle, or that estrogen might cause loss of proliferative capacity by a cell-cycle-independent mechanism[not that there seems to be a need for an equilibrium value for estrogen(not too high or too low)]. For example, if senescence is caused by epigenetic changes such as loss of DNA methylation with each cell cycle, then estrogen might act by decreasing expression of maintenance methylases, causing greater loss of methylation with each cell replication. The effects of estrogen on the resting zone chondrocytes could be mediated by the estrogen receptor- or -ß, both of which are expressed by resting zone chondrocytes in humans, rabbits, and rats. The combination of a decreased rate of growth (in most mammals) and an increased rate of senescence seems to be an effect specific to estrogen[again too little estrogen has been shown to have growth detrimental effects as well]. Other growth-inhibiting conditions, such as glucocorticoid excess and hypothyroidism, decelerate growth plate senescence. The mechanism responsible for this paradoxical effect of estrogen remains to be determined."

"The reserve region contains pairs of flattened cells[hydrostatic pressure may act to flatten stem cells making them look like reserve growth plate chondrocytes] that give the appearance of nascent proliferative columns. Finally, the concept is supported by functional studies; after surgical ablation, the reserve region can apparently be regenerated by epiphyseal chondrocytes[if you get new chondrocytes to the growth then you can grow taller], and the proliferative zone can apparently be generated by reserve chondrocytes"

Growth plate senescence is associated with loss of DNA methylation.

"The overall body size of vertebrates is primarily determined by longitudinal bone growth at the growth plate. With age, the growth plate undergoes programmed senescence, causing longitudinal bone growth to slow and eventually cease. Indirect evidence suggests that growth plate senescence occurs because stem-like cells in the growth plate resting zone have a finite proliferative capacity that is gradually exhausted. Similar limits on replication have been observed when many types of animal cells are placed in cell culture, an effect known as the Hayflick phenomenon. However, we found that the number of population doublings of rabbit resting zone chondrocytes in culture did not depend on the age of the animal from which the cells were harvested, suggesting that the mechanisms limiting replicative capacity of growth plate chondrocytes in vivo are distinct from those in vitro. We also observed that the level of DNA methylation in resting zone chondrocytes decreased with age in vivo. This loss of methylation appeared to occur specifically with the slow proliferation of resting zone chondrocytes in vivo and was not observed with the rapid proliferation of proliferative zone chondrocytes in vivo (i.e. the level of DNA methylation did not change from the resting zone to the hypertrophic zone), with proliferation of chondrocytes in vitro, or with growth of the liver in vivo. Thus, the overall level of DNA methylation decreases during growth plate senescence. This finding is consistent with the hypothesis that the mechanism limiting replication of growth plate chondrocytes in vivo involves loss of DNA methylation and, thus, loss of DNA methylation might be a fundamental biological mechanism that limits longitudinal bone growth in mammals, thereby determining the overall adult size of the organism."

So to increase chondrocyte proliferative capacity we want to increase levels of DNA methylation.  DNA methylation refers to the addition of the methyl group to DNA.  Of course, DNA methylation may be a necessary but not sufficient condition for chondrocyte replication so more pieces of the puzzle may be needed(telomere length is another example).

"growth plate senescence, appears to be a function, not of time per se, but rather of the number of replications that the growth plate chondrocytes have undergone"<-so just make more growth plate chondrocytes

"Some CG sequences in mammalian genomic DNA are methylated on the cytosine moiety. When DNA is replicated, the new strand is initially not methylated. However, DNA methyltransferase 1, a maintenance methylase, recognizes the hemimethylated CGs and adds the missing methyl groups"

"growth plate chondrocytes cultured at high density and exposed to a demethylating agent (5-azacytidine), undergo hypertrophic differentiation"<-so the signal to undergo chondrogenic differentiation doesn't involve hormonal control but rather due to cell concentration and methylation status

"Chondrocytes in primary culture were small, had a round or polygonal shape, and stained for alcian blue and alkaline phosphatase activity, but not for senescence related ß-galactosidase . In contrast, chondrocytes that were becoming senescent were larger, only stained faintly or not at all for alcian blue and alkaline phosphatase activity, but showed an increased senescence-related ß-galactosidase activity"<-Terminal differentiation depends less on alkaline phosphatase levels but rather more on B-galactosidase levels.

"For growth plate chondrocytes in vivo, three lines of evidence suggest that telomere shortening is not the primary cell-division counter causing replicative senescence. First, telomere length varies widely among different strains of mice with similar skeletal sizes. Indeed, some strains of mice have far greater telomere lengths than humans. Thus, telomere length does not correlate with skeletal size. Secondly, functional ablation of telomerase in mice leads to progressive shortening of telomere length in subsequent generations of the mice but has little effect on skeletal size. Thirdly, telomere length does not decrease significantly with growth plate senescence in mice"<-Telomere length may still affect the ability of mesenchymal stem cells to undergo chondrogenesis


Rescuing loading induced bone formation at senescence.

"We present an agent-based model of real-time Ca(2+)/NFAT signaling amongst bone cells that fully described periosteal bone formation induced by a wide variety of loading stimuli in young and aged animals. The model predicted age-related pathway alterations underlying the diminished bone formation at senescence, and hence identified critical deficits that were promising targets for therapy. Based upon model predictions, we implemented an in vivo intervention and show for the first time that supplementing mechanical stimuli with low-dose Cyclosporin A[ciclosporin(available by prescription)] can completely rescue[restart] loading induced bone formation in the senescent skeleton."

" NFAT activation dynamics (within minutes) is remarkably specific to Ca2+ amplitudes and frequencies known to influence distinct downstream cell functions, including proliferation, differentiation and apoptosis"<-LSJL downregulates NFAT5 and NFATC3.

"the model predicted age-related deficits in the ability of Ca2+ oscillations to dephosphorylate NFAT and in NFAT DNA binding capacity"

"Simulated restoration of age-related deficits in α and NFATnx (that are downstream of Ca2+ signaling) did not differentially influence Ca2+ oscillations in cells around the surface in response to the loading protocol"

"loading bone every 24 hrs vs 48 hrs does not differentially influence bone formation"

"supplementation with low-dose CsA would mitigate predicted deficits in loading (and Ca2+) induced dephosphorylation and nuclear translocation of NFAT (α) in part via CsA suppression of negative regulators such as p38 and/or GSK-3β"

"CsA supplementation could also effectively counteract predicted deficits in NFAT-DNA binding (NFATnx) by enhancing cooperative binding interactions between loading induced translocation of NFAT and CsA enhanced activation of transcription factor families such as AP-1"

Friday, July 9, 2010

I"ve started growing again by 1/4" with LSJL

I am now 5'9 3/4".  Here's the data from a while back:  Bone Length Increase | 3 Months 1 Week In.  It's been a while since I've grown with LSJL but I've been increasing my intensity with tapping with 10lbs dumbell, pressing down harder with 70lbs dumbell, and started using the table clamp.  I can even feel a larger separation in my knee.  Before I had to use my fingernail to feel the hyaline cartilage growth plate line in the epiphysis of my tibial bone but now I can feel it with my fingers.  If anyone can come up with an excuse for me to get a knee x-ray with my insurance covering it let me know.  Fibular measurement is 16 1/4" and Tibial measurement is 16 1/2".  Last time I took measurements:  Fibula was 16" and Tibia was 16".  Here's the pics:
It makes sense that eventually you have to increase the pressure you use as the trabecular bone in the epiphysis may model and become less apt to microfracture.  You can see that my eye level is slightly higher than the picture taken here.  And, I'm even standing further back in this picture than the other.  I'm going to have pictures of using the table clamp later today on the how to perform LSJL part of the page.  I think it might be more effective than dumbells, however, with a dumbell you can get rid of it immediately whereas with a table clamp you have to unscrew it to dump it.

I got my table clamp at Home Debot so I could test it out first.Bessey TK-6 Table Clamp.  It is available on amazon however.  My table clamp is 6 inches which is enough even to do my shoulder.

Thursday, July 8, 2010

Pitx1

Pitx1 knockout reduces height.

All-trans-retinoid acid (ATRA) may have inhibited chondrogenesis of primary hind limb bud mesenchymal cells by downregulating Pitx1 expression.

"Pitx1, which is expressed in the hindlimb bud mesenchyme, or its pathways may be etiologically responsible for the increased incidence of clubfoot. Here, we sought to investigate the mechanisms whereby Pitx1 regulated chondrogenesis of hindlimb bud mesenchymal cells in vitro. E12.5 embryonic rat hind limb bud mesenchymal cells were treated with ATRA at appropriate concentrations. Cell Counting Kit-8 (CCK-8) assay was performed to evaluate cell proliferation. Hematoxylin-safranin-O-fast-green staining assays were used to observe cartilage nodules, and Pitx1 expression was examined by immunofluorescent microscopy. Real-time quantitative PCR and immunoblotting assays were applied to determine the mRNA expressions of Pitx1, Sox9 and type II collagen (Col2al), respectively. ATRA inhibited the proliferation of hind limb bud cells dose-dependently. ATRA also induced a dose-dependent reduction in the number of cartilage nodules and the area of cartilage nodules compared with controls. The mRNA expression of Pitx1, Sox9 and Col2al were significantly downregulated by ATRA. Pitx1 was mainly expressed in the cartilage nodules and the levels of Pitx1, Sox9 and Col2al were also downregulated by ATRA dose-dependently. ATRA may decrease chondrogenesis of hind limb bud mesenchymal cells by inhibiting cartilage-specific molecules, such as Sox9 and Col2al, via downregulating Pitx1 expression."

" loss of Pitx1 expression in the developing mouse causes the hindlimb to assume the morphology and growth features of the corresponding bones in the forelimb"

"1 μM ATRA caused a 25% reduction at 24 h and a 25% reduction at 48 h in the growth of the mesenchymal cells compared to controls. Furthermore, 10 μM ATRA caused a 75% reduction at 24 h and a 75% reduction at 48 h in the growth of the mesenchymal cells compared to controls"

"Pitx1 was expressed in the cartilage nodules, mainly in the nucleus"

0.01 microM of ATRA did not have a chondroinhibitory effect.

"ATRA dose-dependently reduced the number of cartilage nodules and the area of cartilage nodules in rat hind limb bud"

IRE1alpha

 IRE1a relates to the UPR which is affected by Salubrinal.

IRE1α dissociates with BiP and inhibits ER stress-mediated apoptosis in cartilage development.

"Bone morphogenetic protein 2 is known to activate unfolded protein response signaling molecules, including XBP1S, BiP and IRE1α. Endoplasmic Reticulum stress is induced in chondrogenesis and activates IRE1α signal pathway, which is associated with ER stress-mediated apoptosis.  IRE1α interacts with BiP in unstressed cells and dissociates from BiP in the course of cartilage development. Induction of ER stress-responsive proteins (XBP1S, IRE1α, BiP) was also observed in differentiating cells. IRE1α inhibition ER stress- mediated apoptosis lies in the process of chondrocyte differentiation. Knockdown of IRE1α expression by way of the RNAi approach accelerates ER stress-mediated apoptosis in chondrocyte differentiation induced by BMP2, as revealed by enhanced expression of cleaved caspase3, CHOP and p-JNK1; and this IRE1α inhibition effect on ER stress-mediated apoptosis is required for BiP in chondrogenesis. Collectively, the ER stress sensors were activated during apoptosis in cartilage development, suggesting that selective activation of ER stress signaling was sufficient for induction of apoptosis. Activation of p-JNK1, caspase3 and CHOP were detected in developing chondrocytes and that specific ER stress signaling leads to naturally occurring apoptosis during cartilage development."

"The UPR includes three molecular branches (inositol requiring enzyme 1, IRE1; PKR-like ER resistant kinase, PERK and activating transcription factor 6, ATF6), which trigger both cell protective and cell death responses. If the cells are exposed to prolonged or strong ER stress, the cells are destroyed by apoptosis."

"IRE1 is involved in the switch between the pro-survival UPR and initiation of cell death pathways during ER Stress. Attenuation of IRE1 can switch the adaptive UPR to apoptosis, and persistent activation of IRE1 increases cell viability upon ER stress"<-So IRE1 may help ER stress have good anabolic effects rather than bad catabolic effects.

"sustained IRE1α RNase activation caused rapid decay of select microRNAs which repressed translation of caspase-2 mRNA, and thus sharply elevated protein levels of this initiator protease of the mitochondrial apoptotic pathway."

"BMP2 activates UPR transducers, such as PERK, OASIS, and ATF6. BMP2 stimulated ATF6 transcription by enhancing the direct binding of RUNX2 to the OSE2 (osteoblast-specific cis-acting element 2) motif of the ATF6 promoter region."

"BMP2 induces mild ER stress during chondrocyte differentiation and activates the IRE1α-XBP1 pathway, then XBP1S in turn enhances chondrocyte hypertrophy through functions as a cofactor of RUNX2. XBP1S controlled growth plate chondrocyte hypertrophy and differentiation. Besides, XBP1S can induce the growth and inhibits ER stress-mediated apoptosis via Erk1/2 signal activation and CHOP attenuation in ER stress"

" IRE1α [is in the] intracellular localization in the growth plate chondrocytes of developing cartilage "

"overexpression IRE1α can improve the BMP2-induced IRE1α"

Full-size image (40 K)
IRE1a can help you grow taller during development by preventing chondrocyte apoptosis.

Tuesday, July 6, 2010

Grow Taller Myths: Fact or Fiction

There are a lot of common myths about growing taller lets look at some of them.

1) The myth that Grow Taller 4 Idiots proliferated.  That it's possible to increase your disc height by stretching.  While stretching your intervertebral discs may activate genetic expression of certain growth factors, your discs can not stretch.  Your disc height is mainly determined by the fluid content of the nucleus pulposus.  Even if your discs are technically bigger(your lying down height increases but not your standing or sitting height) the nucleus fibrosus won't have the fluid content to support it.

2)HGH will increase your height.  Gigantism was found in cases of low serum level of growth hormone.  Gigantism is normally caused by an enlargement of the pituitary gland or something else that affects homeostasis.  The pituitary gland affects a lot more than HGH including negative feedback mechanisms.  Basically, you need a lot more than HGH. HGH has failed to increase height on it's own before.  Myostatin acts to inhibit cellular proliferation and differentiation.  That could be one reason why HGH on it's own does not increase height.

3)Inhibiting Estrogen will increase height.  Growth ceases due to a depletion of stem cells from the hyaline cartilage growth plate line.  It's not a race between ossification and chondrogenesis.  The growth plate conserves it's proliferative capacity in times of excess estrogen.  Estrogen affects growth rate and not final height.

4)Weight Lifting Stunts your Growth.  Compression only slows down growth rate, it doesn't affect final height.

5) The growth plates fuse.  Growth plates do not fuse. Hyaline cartilage remains on the growth plate line.  Stem cells in the hyaline cartilage differentiate into chondrocytes, those chondrocytes differentiate and die, and those chondrocytes ossify into bone.  The Hyaline cartilage it self does not.  The bones start out as a slab of hyaline cartilage and bone grows out of that.  New bone can grow out of the hyaline cartilage growth plate line.  The hyaline cartilage is visible on the x-ray.  It looks the same color of whiteness as the articular cartilage on the ends of the bones.  The proliferating and differentiating zone of the growth plate show up on the x-ray looking like a fracture as they don't have an extracellular matrix hence no whiteness.

Now, all theories are not totally wrong they are just much more complicated then they are made out to be.  Estrogen and HGH both play a role in growth.  But you won't grow taller simply by inhibiting Estrogen and stimulating HGH.  Inhibiting Myostatin maybe(which Testosterone inhibits by the way).  But basically a lot of theories are using an outdated model osteogensis versus chondrogenesis rather than the stem cell model.