Increase Height with Hyaline Cartilage

Scientists have shown that it is possible to recreate a growth plate within hyaline cartilage using a mesenchymal chondrocarsonoma. Since we know that growth plate fusion doesn't inhibit height growth, we can try to recreate a new cartilagenous growth plate within the epiphyseal fusion.

In vivo restoration of full-thickness cartilage defects by poly(lactide-co-glycolide) sponges filled with fibrin gel, bone marrow mesenchymal stem cells and DNA complexes. 

"A composite construct comprising of bone marrow mesenchymal stem cells (BMSCs), plasmid DNA encoding transforming growth factor-beta1 (pDNA-TGF-beta1), fibrin gel and poly (lactide-co-glycolide) (PLGA) sponge was designed and employed. To improve the gene transfection efficiency, a cationized chitosan derivative N,N,N-trimethyl chitosan chloride (TMC) was employed as the vector. The TMC/DNA complexes had a transfection efficiency of 9% to BMSCs and showed heterogeneous TGF-beta1 expression in a 10-day culture period in vitro. In vivo culture of the composite constructs was performed by implantation into full-thickness cartilage defects of New Zealand white rabbit joints, using the constructs absence of pDNA-TGF-beta1 or BMSCs as controls. Heterogeneous expression of TGF-beta1 in vivo was detected at 4 weeks, but its level was decreased in comparison with that of 2 weeks. After implantation for 12 weeks, the cartilage defects were successfully repaired by the composite constructs of the experimental group, and the neo-cartilage integrated well with its surrounding tissue and subchondral bone. Immunohistochemical and glycosaminoglycans (GAGs) staining confirmed the similar amount and distribution of collagen type II and GAGs in the regenerated cartilage as that of hyaline cartilage. The cartilage special genes expressed in the neo-tissue were closer to those of the normal cartilage. An overall score of 2.83 was obtained according to Wakitani's standard. By contrast, only part of the defects was repaired by the pDNA-TGF-beta1 absence constructs, and no cartilage repair but fibrous tissue was found for the BMSCs absence constructs. Therefore, combination of the PLGA sponge/fibrin gel scaffold with BMSCs and gene therapy is an effective method to restore cartilage defects." 

Well if you want to create a new growth plate this is the way to do it.  For height increase, it's preferable to use the existing hyaline cartilage but inserting new hyaline cartilage and sending bone marrow mesenchymal stem cells(with appropriate genetic expression) can have applications like fixing scoliosis. 

Recombinant human midkine stimulates proliferation of articular chondrocytes.

"Midkine, a heparin-binding growth factor, promotes population growth, survival and migration of several cell types.
Bromodeoxyuridine incorporation and MTT assays were performed to examine the proliferative effect of recombinant human midkine (rhMK) on primary articular chondrocytes. Activation of extracellular signal-regulated kinase (ERK) and phosphatidylinositol 3-kinase (PI3K) was analysed using western blot analysis. Systemic and local delivery of rhMK into mice and rats was preformed to investigate the proliferative effect of rhMK in vivo, respectively. Histological evaluation, including measurement of articular cartilage thickness, cell density, matrix staining and immunostaining of proliferating cell nuclear antigen was carried out. rhMK promoted proliferation of articular chondrocytes cultured in a monolayer, which was mediated by activation of ERK and PI3K. The proliferative role of rhMK was not coupled to dedifferentiation of culture-expanded cells. Consistent with its action in vitro, rhMK stimulated proliferation of articular chondrocytes in vivo when it was administered subcutaneously and intra-articularly in mice and rats, respectively. rhMK stimulates proliferation of primary articular chondrocytes in vitro and in vivo." 

If you have actively proliferating chondrocytes(either by your natural growth or via delivering stem cells to the hyaline cartilage growth plate) and inject the growth factor rhMK then you will grow taller.  More proliferating chondrocytes equals more chondrocytes dying and eventually being invaded by bone cells thus resulting in longer bones. 
Hyaline cartilage is the base for which long bones grow.  Stem cells differentiate into chondrocytes under orders from TGF-beta.  You inject hyaline cartilage into new areas and activate endochondral ossification then you get new growth possibilities.

Here's a patent involving using Midkine for hair growth.  Maybe there's a way to apply it to growth plate chondrocytes.

"MK is known to promote the migration of inflammatory cells such as macrophages and neutrophil, leading to inflammation."

"Pleiotrophin (PTN or HB-GAM) is a midkine family protein having approximately 50% identity at the amino acid sequence level to MK. Both MK and PTN comprise a high content of cysteine and basic residues. All the 10 cysteine residues are conserved in MK and PTN, and structurally, both can be divided into the N-domain and the C- domain. As a result of NMR analysis, it is known that these two molecules have very similar three-dimensional structures. Each domain consists of three β sheets, connected via a flexible linker region. K79, R81 , and K102, considered to be important to the binding of to chondroitin sulfate and heparin, are conserved between the two proteins[So pleiotrophin does bind to chondrogenic factors]. MK and PTN also share three-dimensional structures wherein these basic residues appear in the vicinity of the protein surface. Accordingly, PTN has been disclosed previously for the same medical indications as MK."

There doesn't appear to be an oral form of MK and PTN at this time.


Chondromodulin I and pleiotrophin gene expression in bovine cartilage and epiphysis.

"Pleiotrophin and chondromodulin-I are low molecular weight proteins that are abundant (20 microg/g tissue) in fetal cartilage and difficult to detect in adult cartilage. The results showed that the mRNAs for both proteins were abundant in fetal cartilage and while present in adult cartilage, were at 20-60-fold lower levels. Northern blotting revealed gradients of mRNA for both of these proteins in growth plate cartilage, with the highest levels in the resting zone, and the lowest in the hypertrophic zone. In contrast to pleiotrophin, chondromodulin-1 is down-regulated by retinoic acid with a pattern of expression similar to collagen type II and link protein, and may play a more specific role than pleiotrophin in modulating the chondrocyte phenotype."

"Ptn is upregulated by platelet-derived growth factor"

Growth Plates: They don't fuse

Note:  I am still a little sick.  I will start with pictures again this Thursday.  Virus is a confounding variable as the body may with-hold anabolic factors to stop the virus.  I'm still working out and performing LSJL but I'm waiting until I'm healthier to start measuring.  Also this page is inaccurate as the hyaline cartilage growth plate line does eventually fuse into bone but that does not inhibit height growth by new growth plate formation. 

Look at this page.  It's a pretty interesting explanation of all the ways that growth occurs but I want you to take note of one sentence:

6). The epiphysis ossify.

*Hyaline cartilage remains on the growth plate and the articulating surface.

The hyaline cartilage is not what's hypertrophying(differentiating) and then being ossified.  During primary ossification, stem cells turn into new chondrocytes(differentiate into chondrocytes) and then those chondrocytes are fused into bone.  The hyaline cartilage does not fuse into bone because there is already an extracellular matrix.  Stem cells within or transported to the hyaline cartilage by methods such as LSJL differentiate into chondrocytes.  Those chondrocytes hypertrophy and those chondrocytes are ossified into bone.  The chondrocytes within the hyaline cartilage do hypertrophy and eventually ossify.

Bone formed at the primary and secondary ossification centers push the articular(hyaline) cartilage away.  Some being at the end of the bone and others at the growth plate line.  The reason that growth doesn't occur indefinitely is a lack of stem cells in the hyaline cartilage growth plate line which is what LSJL tries to rectify.  The hyaline cartilage environment has genetic markers that shape the stem cells into hypertrophying and differentiating chondrocytes.  Methods like IGF-1 increase height growth by increasing stem cell proliferation thereby increasing growth.

Now, the reason that a growth plate line does not remain after fracture healing like in limb lengthening surgery is that in fracture healing endochondral ossification occurs as a result of periosteal progenitor cells which differentiate into chondrocytes and so on.  There's never any hyaline cartilage matrix so the entirety of the homeless chondrocytes are ossified.

So you see growth plates don't fuse.  New chondrocytes are created from stem cells and those chondrocytes are ossified.  Not the ones in the growth plate.

Mechanisms of Growth Plate Maturation and Epiphyseal Fusion

"Longitudinal growth occurs within the long bones at the growth plate. During childhood, the growth plate matures, its total width decreases and eventually it disappears at the end of puberty with complete replacement by bone along with cessation of longitudinal growth. The exact mechanism of epiphyseal fusion is still not completely understood and experimental studies are complicated by the fact that there is a species difference between humans and rabbits that do fuse their growth plates and rodents that do not. This mini review summarizes hypotheses and theories postulated in the literature regarding growth plate maturation and epiphyseal fusion. Growth factors, local regulators and hormones involved in growth plate maturation are described as well as four postulated hypotheses and theories regarding the final steps in epiphyseal fusion: apoptosis, autophagy, transdifferentiation and hypoxia. A better insight into the mechanisms of epiphyseal fusion may ultimately help to develop new strategies for the treatment of cartilage and growth disorders. "

Remember that growth plates have to do more than merely transform into bone otherwise they would never increase in length they have to deform the bone into a longer shape which is why I think apoptosis is the most logical final step as that could generate force deforming the bone.

"Senescence is a term for the structural and functional changes over time in the growth plate, such as a gradual decline in the overall growth plate height, proliferative zone height, hypertrophic zone height, size of hypertrophic chondrocytes and column density"<-senescence is what we try to prevent not fusion

"A new hypothesis is that proliferation is influenced by a multiorgan genetic program and that proliferation declines when this genetic program has reached a critical point"<-although this would not explain individuals of different proportions

"A most widely held hypothesis is that at the chondro-osseous junction site of the growth plate, terminally hypertrophic chondrocytes die by undergoing apoptosis leaving behind a scaffold of cartilage matrix for osteoblasts that invade and lay down bone[this apoptosis also releases water that generates force to induce bone deformation]. It is assumed that the same mechanism eventually also results in epiphyseal fusion. Studies in the rat showed that apoptosis-regulating proteins (the so-called caspases, which are cysteine proteases) are expressed in the growth plate and that there is an increased expression of proapoptotic factors with age. Typical morphological changes when cells undergo apoptosis include cell shrinkage with intact organelles and integrity of membranes, pyknotic nuclei by aggregation of chromatin, fragmented DNA, partitioning of the cytoplasm and nucleus into membrane-bound vesicles (apoptotic bodies) and absence of an inflammatory response. Interestingly, several recent studies failed to demonstrate a typical apoptotic appearance in the terminal hypertrophic chondrocytes and, therefore, these studies have questioned whether apoptosis is the final mechanism through which chondrocytes die in the terminal hypertrophic zone. Furthermore, we recently analyzed a unique piece of fusing human growth plate tissue during epiphyseal fusion and were not able to find signs of classical apoptosis"<-Note that if apoptosis is a critical role during endochondral ossification than it is logical not to find signs of apoptosis during fusion as this stage of growth may release factors to encourage further growth.  It may be at time of senescence apoptosis ceases and other forms of cell death occur.

"The oldest hypothesis is that at the chondro-osseous junction site of the growth plate terminal hypertrophic chondrocytes can transdifferentiate into osteoblasts. This theory is based on mostly organ and cell culture models, like for example chondrocytes in mice and murine metatarsal bone cultures that were able to transdifferentiate into osteoblasts producing bone matrix. Adams and Shapiro discussed that evidence in support of transdifferentiation is mostly circumstantial. It is based on microscopic examination of chondrocyte and osteoblast populations at the chondro-osseous junction and results from different studies are inconsistent. Although direct evidence is lacking, others speculate that transdifferentiation is present at the chondro-osseous junction because terminally differentiated cells are producing collagen type 1 together with extracellular matrix factors. In addition, to our knowledge human studies on transdifferentiation at the chondro-osseous junction in the growth plate have not been described."<-some chondrocytes may transdifferentiate into osteoblasts whereas others may undergo apoptosis.  Transdifferentiation is unlikely to induce bone deformation.

"In a unique human growth plate tissue specimen in the process of undergoing epiphyseal fusion, we observed a dense border of thick bone surrounding growth plate remnants at the site where normally the growth plate is located[just because apoptosis is not involved during fusion does not mean that it may not be involved in earlier stages of development]. In addition, signs of hypoxia and early necrosis were found. We postulated that the border of dense bone might function as a physical barrier for oxygen and nutrients to reach the fusing growth plate resulting in hypoxia and eventually cell death in a nonclassical apoptotic way through necrosis or a mixture of apoptosis and necrosis[if this is the case than osteoclasts can help continue endochondral ossification by degrading this dense bone]. In line with this new hypothesis, White et al recently demonstrated bridging bone in the center of a distal human tibial growth plate obtained from a 12.9-year-old girl, which might be an early sign of this shelling process. Signs of a hypoxia-related process were also reported by Stewart et al who observed an upregulated expression of hypoxia-inducible factor 2α mRNA during chick and murine chondrocyte differentiation in vitro. Hypoxia-inducible factor 2α knockout mice are small, which might indicate that this gene has an important role in the growth plate and subsequently in the regulation of longitudinal growth. Thus, epiphyseal fusion might be a hypoxia-related process leading eventually to cell death of growth plate chondrocytes."

It is likely that all four methods are involved with apoptosis being the most important but apoptosis is likely not present near fusion.

Growing Pains: The Need for Engineered Platforms to Study Growth Plate Biology

"Growth plates, or physis, are highly specialized cartilage tissues responsible for longitudinal bone growth in children and adolescents. Chondrocytes that reside in growth plates are organized into three distinct zones essential for proper function. Modeling key features of growth plates may provide an avenue to develop advanced tissue engineering strategies and perspectives for cartilage and bone regenerative medicine applications and a platform to study processes linked to disease progression. In this review, a brief introduction of the growth plates and their role in skeletal development is first provided. Injuries and diseases of the growth plates as well as physiological and pathological mechanisms associated with remodeling and disease progression are discussed. Growth plate biology, namely, its architecture and extracellular matrix organization, resident cell types, and growth factor signaling are then focused. Next, opportunities and challenges for developing 3D biomaterial models to study aspects of growth plate biology and disease in vitro are discussed. Finally, opportunities for increasingly sophisticated in vitro biomaterial models of the growth plate to study spatiotemporal aspects of growth plate remodeling, to investigate multicellular signaling underlying growth plate biology, and to develop platforms that address key roadblocks to in vivo musculoskeletal tissue engineering applications are described."

"the osteochondral interface is a transitional zone tissue that is composed of articular cartilage (90%), a calcified cartilage transitional tissue (5%), and subchondral bone (5%)."

"The calcified cartilage transitional zone is a permanent ≈20–250 µm thick layer that joins articular cartilage to subchondral bone in joints. This calcified cartilage is 10–100 times stiffer than articular cartilage but less stiff than subchondral bone when there is the same mineral content, and serves to reduce stress between mechanically dissimilar articular cartilage and bone. Another example of a transitional tissue is the enthesis, a permanent fibrocartilage tissue that joins connective tissue (e.g., tendons and ligaments) to bone to facilitate joint motion."

"The enthesis is a stratified fibrocartilaginous tissue ≈250–500 µm thick that contains gradients in cell phenotype, biochemical cues, mineral content, as well as matrix composition and alignment. Bone is 100 times stiffer than tendon and ligament, and the gradient within the enthesis protects it from failure by minimizing the strain concentrations between mechanically dissimilar tissues."

"Growth plates, or physis, are highly organized cartilage tissues ≈800–1000 µm thick that lie between the epiphysis and metaphysis at the ends of most long bones. However, unlike the osteochondral interface and tendon-bone or ligament-bone entheses which are permanent transitional tissues, growth plates are temporary cartilage tissues involved in development and growth that disappear at skeletal maturity in humans."

" growth plates are avascular and aneural—relying on the vascular systems of the adjacent bone tissue to maintain chondrocyte proliferation{manipulating the vascular system may be a way to grow atller for longer}—and are composed of chondrocytes embedded in an extracellular matrix"

"Cranial vault sutures differ from the growth plates of the long bones in that they do not facilitate bone growth via a cartilage intermediate (i.e., endochondral ossification). Rather, cranial vault sutures facilitate bone growth via a process of intramembranous ossification where bone growth occurs directly by mesenchymal stem cell differentiation in response to extrinsic growth signals caused by the expanding brain. Growth plates in the long bones facilitate bone growth via endochondral ossification and a cartilage intermediate, and possess intrinsic growth potential (i.e., no external signal is required)."

"Compositionally, bone is composed of inorganic calcium phosphate mineral (50–70%), organic matrix (i.e., collagen, 20–40%), water (5–10%), and lipids (<3%)."<-so calcium has the power to powerfully change bone.

" Long bones receive blood supply from arteries in the metaphysis and epiphysis, periosteal arteries, and the central nutrient artery. A dense capillary system within bone tissue is responsible for distributing oxygen and nutrients to bone resident cells: osteoblast, osteocytes, and osteoclasts. Osteoblasts are derived from mesenchymal stem cells, and they deposit collagen proteins that compose the bone extracellular matrix and are responsible for mineralization of these proteins. Osteoblasts terminally differentiate into osteocytes, bone cells that are embedded in the extracellular matrix and sense mechanical strain. Osteoclasts derive from mononuclear monocyte-macrophage precursor cells, and they are responsible for resorption of the mineralized bone matrix. Bone is constantly remodeled and maintained by reciprocal interactions mediated by osteoblasts and osteoclasts that are mediated through growth factor signaling. Notably, fibroblast growth factors (FGFs), transforming growth factor beta (TGFB), bone morphogenic proteins (BMPs), insulin-like growth factors (IGFs), and platelet-derived growth factor (PDGF) are all critical for bone homeostasis"

"Skeletal stem cells have been identified in the growth plates, and subpopulations of these cells can generate chondrocytes and osteocytes, but not adipocytes"

"he molecular mechanism of bony bridge formation, and their work suggests bony bridge formation begins immediately following injury. Namely, they identified pro-osteoblastic factors following growth plate injury including interleukin-6, bone morphogenic protein 2, and osteoprotegerin. The premature mineralization of the extracellular matrix and early osteogenic differentiation of mesenchymal and preosteoblast cells in the growth plate cartilage following a fracture leads to improper skeletal development"

"The resting zone cartilage is important for orienting the developing growth plates by producing a “growth plate-orienting factor” that may direct cells to align parallel to the long-axis of the bone. Additionally, vasculature originating in the epiphysis provides nutrients to chondrocytes in this zone."

"The middle layer of the growth plate is called the proliferative zone, and it primarily functions as a site of matrix production and cellular proliferation.  In the proliferative zone, chondrocytes appear flattened, begin to divide, and are arranged in columnar structures parallel to the long bones.  The rearrangement of chondrocytes into columns requires cell–cell adhesion and that chondrocytes elongate to take up space, and this is indicative of cell spreading event.  [In the proliferative zone there's no] cell separation, cell body contraction, or a leading edge which would have been an indication of cell migration.  Thus, they assert that chondrocyte rearrangement into columnar structures is more like cell spreading than cell migration.  The columnar architecture in the proliferative zone specifies the primary direction of growth in long bones. The long axis of the flattened chondrocytes is perpendicular to the long axis of the bone, and this columnar structure is maintained via cell–cell adhesion."

"n the hypertrophic zone, chondrocytes begin to separate from one another, become rounded, and expand in size. Chondrocytes in this zone enlarge to four times the size of chondrocytes in the proliferative zone, stop proliferating, and undergo apoptosis. Terminal differentiation of chondrocytes is associated with an increase in cell volume, intracellular calcium concentration, alkaline phosphatase enzyme activity, and synthesis and secretion of Collagen X.  Hypertrophic chondrocytes direct mineralization of the surrounding matrix and attract blood vessels and chondroclasts from the metaphysis. Blood vessels from the metaphysis invade the cartilage of the hypertrophic zone, form new vascular channels via angiogenesis, and provide access for other cell types involved in bone formation. Differentiating osteoblasts and osteoclasts invade the hypertrophic region via the perichondrium and remodel the cartilage tissue into bone tissue. This remodeling leads to new bone formation underneath the growth plate and thus bone elongation. There is some evidence indicating chondrocytes trans-differentiate into osteoblasts during vascular invasion of this zone. The hypertrophic zone is the weakest and is most likely to have a cleavage plane pass through leading to growth plate injury."

"compressive loading is detrimental [to the growth plate] when applied statically or dynamically; although, dynamic loading appears to be less detrimental than static loading [but loading is needed for proper growth plate development in general]"

"The primary extracellular matrix proteins in the growth plate are aggrecan, collagen, and hyaluronic acid"

"matrix vesicles containing mineral are deposited in the hypertrophic zone and serve as nodes for mineralization and subsequent bone formation. Aggrecan, a chondroitin sulfate proteoglycan, is the primary proteoglycan in growth plate cartilage and plays a structural role in the extracellular matrix. Aggrecan expression is upregulated during chondrocyte differentiation and its presence is important for chondrocyte-matrix and chondrocyte-chondrocyte interactions."

"Aggrecan plays an important role in growth factor regulation and signaling and is essential for chondrocyte organization, morphology, and survival during limb development."

"The glycosaminoglycans contained within aggrecan create a negatively charged molecule that binds growth factors required for chondrocyte maturation such as Indian hedgehog and this negative charge enables hydration of cartilage tissue."

"Aggrecan and hyaluronic interactions are stabilized by a link protein that interacts with both molecules, with hyaluronic acid necessary for chondrocyte-matrix stabilization. Free hyaluronic acid, synthesized by hypertrophic chondrocytes, is present in higher concentrations in the hypertrophic zone. Free hyaluronic acid is present between chondrocytes and their immediate extracellular matrix (i.e., sulfated proteoglycans, aggrecan), though the amount of free hyaluronic acid in the pericellular space increases as chondrocytes mature."

"CCN2 stimulates the proliferation and differentiation of growth plate chondrocytes.  CCN2 promotes endochondral ossification by acting on chondrocytes, osteoblasts, and osteoclasts."

"estrogen-resistant patients (i.e., patients with mutations in the estrogen receptor gene) grow into adulthood and lack proper growth plate fusion"

"estrogen can stimulate growth hormone and insulin-like growth hormone expression to further stimulate longitudinal bone growth."

"FGF receptor 3 (FGFR3) inhibits growth plate chondrocyte proliferation and limits the lengthening of the long bones"

"Growth plates are avascular and hypoxic, thus hypoxia-inducible factors are likely critical for survival, maintenance, and differentiation of growth plate chondrocytes. HIF1α is expressed in the hypoxic regions of the growth plates: the center portion of the proliferative zone and the upper portion of the hypertrophic zone. HIF1α enhances the expression of chondrocyte marker Sox9 and extracellular matrix components Collagen II and Aggrecan. HIF1α is also required for differentiation of hypoxic prechondrogenic cells. The primary hypothesis in cartilage biology is that hypoxia modulates differentiation and condensation of mesenchymal stem cells (MSCs) into chondrocytes by activating HIF1α. A downstream target of HIF1α is vascular endothelial growth factor, which is discussed in a later section. HIF1α is another excellent target for incorporation into specific zones where it is most relevant"

"Runt-related transcription factor 2 (RUNX2) drives proliferative chondrocytes to differentiate into hypertrophic chondrocytes.  RUNX2 is expressed in the proliferative and hypertrophic zones of the growth plate, and it is needed for osteoblast differentiation and chondrocyte maturation in osteogenesis. RUNX2 expression is regulated by PTHrP and induces Ihh signaling. RUNX2 is highly expressed in perichondral cells and in osteoblasts. RUNX2 also regulates chondrocyte proliferation via the upregulation of Anthrax toxin receptor 1 (ANTXR1).  Endochondral ossification proceeds normally in ANTXR1 negative mice, but they have shorter femur and tibia bones compared to wild-type mice."

Gli3

The hedgehog target Vlk genetically interacts with Gli3 to regulate chondrocyte differentiation during mouse long bone development.

"Endochondral bone development is orchestrated by the spatially and temporally coordinated differentiation of chondrocytes along the longitudinal axis of the cartilage anlage. Initially, the slowly proliferating, periarticular chondrocytes give rise to the pool of rapidly dividing columnar chondrocytes, whose expansion determines the length of the long bones. The Indian hedgehog (IHH) ligand regulates both the proliferation of columnar chondrocytes and their differentiation into post-mitotic hypertrophic chondrocytes in concert with GLI3, one of the main transcriptional effectors of HH signal transduction. In the absence of Hh signalling, the expression of Vlk (vertebrate lonesome kinase, also called Pkdcc) is increased{maybe if you got around this negative feedback mechanism and had both high Hedgehog levels and Vlk levels you could have additional height}. The shortening of limb long bones in Vlk-deficient mouse embryos is aggravated by additional inactivation of Gli3{Some Vlk and Gli3 is needed for optimal height growth}. Vlk and Gli3 synergize to control the temporal kinetics of chondrocyte differentiation during long bone development. Whereas differentiation of limb mesenchymal progenitors into chondrocytes and the initial formation of the cartilage anlagen of the limb skeleton are not altered, Vlk and Gli3 are required for the temporally coordinated differentiation of periarticular into columnar and ultimately hypertrophic chondrocytes in long bones. In limbs lacking both Vlk and Gli3, the appearance of columnar and hypertrophic chondrocytes is severely delayed and zones of morphologically distinct chondrocytes are not established until E16.5. At the molecular level, these morphological alterations are reflected by delayed activation and lowered expression of Ihh, Pth1r and Col10a1 in long bone rudiments of double mutant limbs.  VLK plays a role in the IHH/GLI3 interactions and Vlk and Gli3 cooperate to regulate long bone development by modulating the temporal kinetics of establishing columnar and hypertrophic chondrocyte domains."

"Inactivation of either Gli2 or Gli3 reduces the size of the ossification centres, a phenotype aggravated by concurrent inactivation of both genes, which results in severe shortening of limb skeletal bones"

"Vlk [is] expressed by columnar chondrocytes (CC) and the ossification centres (OC)"

Vlk appears to be important only in post natal development and not prenatal development.

Agrin

Agrin is highly expressed by chondrocytes and is required for normal growth.

"Agrin is a heparan sulfate proteoglycan that is best known for its crucial involvement in the organization and maintenance of postsynaptic structures at the neuromuscular junction.  Here we examined the early postnatal development of agrin-deficient mice in which perinatal death was prevented by transgenic expression of neural agrin in motor neurons. Such transgenic, agrin-deficient mice were born at Mendelian ratio but exhibited severe postnatal growth retardation. Growth plate morpholgy was markedly altered in these mice, with changes being most prominent in the hypertrophic zone. Compression of this zone was not caused by reduced viability of hypertrophic chondrocytes, as no differences in the apoptosis rates could be observed. Furthermore, deposition of the major cartilage matrix components collagen type II and aggrecan was slightly reduced in these mice. Consistent with a role for agrin in skeletal development, we show for the first time that agrin is highly expressed by chondrocytes and localizes to the growth plate in wild-type mice."

Agrin deficiency decreased .longitudinal bone growth and decreased chondrocyte proliferation.

"growth plate morphology is not significantly altered in Tg/agrn −/− mice until late in embryogenesis (E17) and that changes in the hypertrophic zone become more pronounced with age. "

Cholesterol can make you taller?

Cholesterol is subject to negative feedback mechanisms.  The more cholesterol you consume, the less your body produces.  Too much cholesterol also results in heart disease.  Is there a way to use cholesterol to make you taller? 

Control of chondrocyte gene expression by actin dynamics: a novel role of cholesterol/Ror-alpha signalling in endochondral bone growth. 

"The signalling pathways that regulate chondrocyte differentiation [include] the actin cytoskeleton and Rho GTPases.  Manipulation of actin dynamics in tibia organ cultures isolated from E15.5 mice results in pronounced enhancement of endochondral bone growth and specific changes in growth plate architecture. Global changes in gene expression were examined of primary chondrocytes isolated from embryonic tibia, treated with the compounds cytochalasin D, jasplakinolide (actin modifiers) and the ROCK inhibitor Y27632. Cytochalasin D elicited the most pronounced response and induced many features of hypertrophic chondrocyte differentiation. Bioinformatics analyses of microarray data and expression validation by real-time PCR and immunohistochemistry resulted in the identification of the nuclear receptor retinoid related orphan receptor-alpha (Ror-alpha) as a novel putative regulator of chondrocyte hypertrophy. Expression of Ror-alpha target genes, (Lpl, fatty acid binding protein 4 [Fabp4], Cd36 and kruppel-like factor 5 [Klf15]) were induced during chondrocyte hypertrophy and by cytochalasin D and are cholesterol dependent. Stimulation of Ror-alpha by cholesterol results in increased bone growth and enlarged, rounded cells, a phenotype similar to chondrocyte hypertrophy and to the changes induced by cytochalasin D, while inhibition of cholesterol synthesis by lovastatin inhibits cytochalasin D induced bone growth. Additionally, we show that in a mouse model of cartilage specific (Col2-Cre) Rac1, inactivation results in increased Hif-1alpha (a regulator of Rora gene expression) and Ror-alpha(+) cells within hypertrophic growth plates. We provide evidence that cholesterol signalling through increased Ror-alpha expression stimulates chondrocyte hypertrophy and partially mediates responses of cartilage to actin dynamics." 

Cholesterol activates Ror-alpha expression.  Ror-alpha stimulates chondrocyte hypertrophy.  Chondrocyte hypertrophy is one of the ways to stimulate height growth.   Note that Lovastatin may be better at stimulating chondrogenic differentiation while chosterol may be more helpful in chondrocyte hypertrophy.

"RhoA/ROCK signalling inhibits both early and late chondrocyte differentiation in vitro. Actin polymerization re-establishes chondrocyte gene expression in de-differentiated chondrocytes in vitro. Manipulation of the actin cytoskeleton promotes the differentiation of mesenchymal cells to the chondrocyte lineage in vitro."

"inhibition of ROCK signalling by Y27632 results in an expanded resting zone, whereas the proliferative and hypertrophic zones sizes remain unchanged"<-The resting zone is the zone most likely to induce an increase in adult height rather than just growth rate.

"Inhibition of actin polymerization by cytochalasin D resulted in a growth plate that was too unorganized to distinguish and measure any zones. Instead, the cellular morphology of cytochalasin D treated tibiae showed features of hypertrophic and resting chondrocytes; cells were larger but were also surrounded by an abundant matrix."<-Note cytochalasin D still resulted in an increase in longitudinal growth despite a disruption in growth plate zones.

CytD upregulates ATF3 by 9.3 which LSJL also upregulates.

"Hif-1α gene expression was found to be up-regulated in response to cytochalasin D treatment"

"Analysis of growth plate organization demonstrated that cholesterol treatment resulted in similar growth plate morphology as cytochalasin D. Chondrocytes were rounder and larger than control cells; however, cholesterol treatment did not alter growth plate architecture as severely as cytochalasin D treatment because we could still distinguish columnar stacks in the proliferative zone of cholesterol treated bones"

"Fabp4, Cd36, Lpl and Klf15 mRNA levels are all responsive to cholesterol signalling and their up-regulation in the microarray are likely due to the increase in Ror-α expression and activity"

"Cholesterol itself may also be regulating chondrocyte hypertrophy directly, through activation of Ror-α and downstream target genes (e.g. genes involved in lipid metabolism)."

Yeast hydrolysate induces longitudinal bone growth and growth hormone release in rats. 

"This study investigated the growth promoting effects of yeast extract (YH) fed to Sprague-Dawley male rats (3 weeks old) for 4 weeks. The negative (N)-control and positive (P)-control groups were given a daily oral administration of saline and foremilk (1 g/kg of BW), respectively, and the YH-1 and YH-2 groups were given daily administrations of YH (0.5 and 1 g/kg of BW, respectively). After 4 weeks, the YH-1, YH-2 and P-control groups showed significant differences in the body weight gain compared with the N-control group (p < 0.05). The YH-1 and YH-2 groups also had significantly different tibial bone growths (0.47 and 0.49 mm/day, respectively) and femur bone growths (0.52 and 0.53 mm/day, respectively) compared with the N-control group (0.37 mm/day of tibial growth and 0.42 mm/day of femur growth) (p < 0.05). The YH-1 and YH-2 groups had significantly different growth plate (proximal epiphysis) height increments (0.62 and 0.56 mm, respectively) compared with the N-control group (0.17 mm) (p < 0.05). Lastly, the YH-1 and YH-2 groups presented different growth hormone (GH) levels (1.77 and 2.10 ng/mL, respectively) than the N-control group (0.82 ng/mL) (p < 0.05). YH administration increased longitudinal bone growth and GH secretion in rats. Consequently, YH may offer an improved ability to treat GH deficiency-related disorders." 

Yeast Extract increases some forms of cholesterol.  What's interesting about the study is that the results were dose dependent.  The group that ate twice as much yeast had between 0.04-0.05mm of extra growth.  But remember growth rate does not always equal increased(or decreased) growth!  Yeast is available for sale(Marmite Yeast Extract 125gram - Pack of 2 Jars!) but I couldn't find hydrolized yeast extract and I'm not sure if that would make a difference. 

"Sung (2005) reported the effects of Nogjungtang(a traditional Korean deer decoction) on growth, feed efficiency and organ growth in Sprague-Dawley male rats (5 and 10 weeks). Yang et al. (2003) reported that GSM containing Eleutherococcus senticosus had a growth stimulating effect on longitudinal bone growth in vivo and in vitro. Finally, Park et al. (2007) reported that HM-10 (an herbal medicine mixture) showed potential growth promoting capacity in rats, including longitudinal bone growth and GH releasing properties."<-some compounds and studies that may have an impact on height.

Role of cholesterol in the regulation of growth plate chondrogenesis and longitudinal bone growth. 

"Inborn errors of cholesterol synthesis are associated with multiple systemic abnormalities, including skeletal malformations. The regulatory role of cholesterol during embryogenesis appears to be mediated by Shh, a signaling molecule in which activity depends on molecular events involving cholesterol. Based on this evidence, we hypothesized that cholesterol, by modifying the activity of Ihh (another of the Hedgehog family proteins) in the growth plate, regulates longitudinal bone growth. To test this hypothesis, we treated rats with AY 9944, an inhibitor of the final reaction of cholesterol synthesis. After 3 weeks, AY 9944 reduced the cumulative growth, tibial growth, and the tibial growth plate height of the rats. To determine whether cholesterol deficiency affects bone growth directly at the growth plate, we then cultured fetal rat metatarsal bones in the presence of AY 9944. After 4 days, AY 9944 suppressed metatarsal growth and growth plate chondrocyte proliferation and hypertrophy. The inhibitory effect on chondrocyte hypertrophy was confirmed by the AY 9944-mediated decreased expression of collagen X. Lastly, AY 9944 decreased the expression of Ihh in the metatarsal growth plate. We conclude that reduced cholesterol synthesis in the growth plate, possibly by altering the normal activity of Ihh, results in suppressed longitudinal bone growth and growth plate chondrogenesis." 

"metatarsal bone rudiments cultured in the presence of 10 μM lovastatin for 4 days grew significantly less than the controls"

"impaired cholesterol synthesis in the growth plate is responsible for suppressed longitudinal bone growth."

Cholesterol influences the expression of indian hedgehog and indian hedgehog controls the regulation of chondrocyte hypertrophy. 


P450 oxidoreductase expressed in rat chondrocytes modulates chondrogenesis via cholesterol- and Indian Hedgehog-dependent mechanisms.

"Cytochrome P450 oxidoreductase (POR) is the electron donor for microsomal cytochrome P450 enzymes and other non-P450 enzymes. Targeted deletion of POR expression in mice leads to a variety of embryonic defects, including bone abnormalities. In addition, POR mutations in humans are associated with impaired steroidogenesis and skeletal malformations. rat chondrocytes transfected with POR-specific short interfering RNAs exhibited decreased cell proliferation and differentiation and induced apoptosis. The reduced expression of POR in chondrocytes caused decreased intracellular cholesterol content. The addition of cholesterol in the culture medium prevented the POR small interfering RNA (siRNA)-mediated effects on chondrocyte proliferation, differentiation, and apoptosis. Because cholesterol is required for normal activity of the hedgehog proteins, we evaluated the effects of POR siRNAs on the expression of Indian hedgehog (Ihh), an important regulator of chondrogenesis. POR siRNA-transfected chondrocytes exhibited reduced Ihh expression, with such effect being neutralized by cholesterol. Lastly, recombinant human/mouse Ihh prevented the POR siRNA-mediated effects on chondrocyte proliferation, differentiation, and apoptosis. Our findings suggest that the bone malformations associated with defective POR activity are due to reduced cholesterol synthesis and, in turn, reduced Ihh expression in chondrocytes."

"the targeted inhibition of cholesterol synthesis in cultured metatarsal bones (through the activity of an inhibitor of 7-dehydrocholesterol reductase) results in suppressed longitudinal bone growth, chondrocyte proliferation, and chondrocyte hypertrophy"

"retinoic acid inhibits longitudinal growth of cultured rat metatarsal bones by suppressing chondrocyte proliferation, hypertrophy, and matrix synthesis "

Disp1 regulates growth of mammalian long bones through the control of Ihh distribution.

"Dispatched1 (Disp1){down in LSJL} is required for the release of cholesterol modified hedgehog (Hh) proteins from producing cells. We investigated the role of Disp1 in Indian hedgehog (Ihh) signaling in the developing bone bypassing the lethality of the Disp1(C829F) allele at early somite stages through the supply of non-cholesterol modified Sonic hedgehog (N-Shh). The long bones that develop in the absence of wild-type Disp1, while clearly shorter, have a juxtaposition of proliferating and non-proliferating hypertrophic chondrocytes that is markedly more normal in organization than those of ihh null mutants. Direct analysis of Ihh trafficking in the target field demonstrates that Ihh is distributed well beyond Ihh expressing cells though the range of movement and signaling action is more restricted than in wild-type long bones. Consequently, a PTHrP-Ihh feedback loop is established, but over a shorter distance, reflecting the reduced range of Ihh movement. These analyses of the Disp1(C829F) mutation demonstrate that Disp1 is not absolutely required for the paracrine signaling role of Ihh in the skeleton. However, Disp1 is critical for the full extent of signaling within the chondrocyte target field and consequently the establishment of a normal skeletal growth plate."

"TGFβ2 appears to be required for Sonic hedgehog (Shh), a surrogate for Ihh-mediated effects in vitro, but the growth of bones is not significantly affected in TGFβ2 mutants "

"Hh ligands undergo two lipid modifications; addition of a C-terminal cholesterol moiety in conjunction with cleavage of the precursor protein to its active signaling form, and a covalent attachment of an N-terminal palmitate. Each ligand signals through a single membrane receptor protein, patched1 (Ptch1), Hh-Ptch1 binding leads to the derepression of the seven-pass membrane protein smoothened (Smo), and pathway activation in the responsive cell. Interestingly, paracrine signaling by cholesterol modified hh ligands requires the action of a Ptch1 related membrane protein, Disp1, within Hh producing cells"

Regulation of growth plate chondrocytes by 1,25-dihydroxyvitamin D3 requires caveolae and caveolin-1.

"1,25-dihydroxyvitamin D(3) [1alpha,25(OH)(2)D(3)] regulates endochondral ossification in part through membrane-associated mechanisms, including protein kinase C (PKC) signaling activated by a membrane-associated 1alpha,25(OH)(2)D(3)-binding protein, ERp60. We tested the hypothesis that caveolae are required for 1alpha,25(OH)(2)D(3) action and play an important role in regulating chondrocyte biology and growth plate physiology.

Rat costochondral chondrocytes were examined for caveolae by transmission electron microscopy of cultured cells and of cells in situ. Western blots and confocal microscopy were used to detect caveolae proteins including caveolin-1 (Cav-1) and 1alpha,25(OH)(2)D(3) receptors. Caveolae cholesterol was depleted with beta-cyclodextrin (CD) and effects of 1alpha,25(OH)(2)D(3) on PKC, DNA synthesis, alkaline phosphatase, and proteoglycan production determined. Chondrocytes from Cav-1(-/-) and C57BL/6 wildtype mice were also treated with 1alpha,25(OH)(2)D(3). Epiphyses and costochondral junctions of 8-week-old male Cav-1(-/-) and wildtype mice (N = 8) were compared by histomorphometry and microCT. Data were analyzed by ANOVA and Bonferroni for posthoc comparisons.

Growth zone chondrocytes had caveolae and Cav-1, -2, and -3{up in LSJL}. Resting zone chondrocytes, which do not exhibit a rapid 1alpha,25(OH)(2)D(3)-dependent increase in PKC activity, also had these caveolins, but caveolae were larger and fewer in number. ERp60 but not VDR co-localized with Cav-1 in plasma membranes and in lipid rafts. CD-treatment blocked 1alpha,25(OH)(2)D(3) effects on all parameters tested. The Cav-1(-/-) cells did not respond to 1alpha,25(OH)(2)D(3), although 1alpha,25(OH)(2)D(3) increased PKC, alkaline phosphatase, and [(35)S]-sulfate incorporation in wildtype C57BL/6 cells. Histology and microCT showed that Cav-1(-/-) growth plates were longer and had more hypertrophic cells in each column. Growth plate changes were reflected in the metaphysis.

The membrane-mediated effects of 1alpha,25(OH)(2)D(3) require caveolae and Cav-1, and Cav-1 deficiency results in altered growth plate physiology."

"ERp60[receptor for Vitamin D] is required for protein kinase C α (PKCα) signaling in growth plate chondrocytes"

"the mechanism involves rapid activation of phospholipase A2 (PLA2) through PLA2-activating protein (PLAA). Arachidonic acid released by PLA2 can activate PKC directly. Prostaglandin produced through cyclooxygenase-1 binds its EP-1 receptor to activate PKC as well. In addition, lysophospholipid produced by PLA2 activates phospholipase C (PLC), resulting in formation of diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP3). DAG binds PKC and translocates it to the plasma membrane; IP3 initiates the release of Ca2+ from the endoplasmic reticulum, which can then serve as a co-factor for PKC activation."

"Specialized cholesterol-rich regions of the plasma membrane called lipid rafts/caveolae provide structural scaffolding for signal transduction and a principal component of caveolae that serves the scaffolding function, caveolin-1, was recently implicated in the function of membrane-associated estrogen receptors. The nuclear VDR has also been found in lipid rafts/caveolae, suggesting the hypothesis that these membrane microdomains play an important role in the mechanism of 1α,25(OH)2D3 action."

"ERp60 co-localized with lipid rafts"

How is your progress with Lateral Synovial Joint Loading?

I've seen Alex and Jory report results but I'm curious if anybody else has seen any results.  The first thing to do is if you are not getting results on Lateral Synovial Joint Loading is post a comment on the blog with the following information:

How long have you been performing Lateral Synovial Joint Loading and in what areas?
How much weight do you use and for how long?  Do you tap and if so how many times and with what load?
Are you doing intense cardiovascular exercise (to create an hypoxic environment which increases stem cell proliferation)?
Are you doing resistance training, if so what is the heaviest load(barbell or dumbell, not machines as load on machines is too relative) you use?  How frequently do you resistance train?
Do you do anything else that involves bone loading or anything that might stimulate the cartilage?

If you want to maximize gains then you want to bring up the weakest variables.  If you have an equation xyz=q and you want to maximize q(in our case height growth) and x+y+z=t(the amount of time you have) then you want x=y=z.

The point is that if you're not getting good gains with Lateral Synovial Joint Loading then you might want to focus on other factors to get those gains.  Lateral Synovial Joint Loading does put a stretching force on the cortical bone and that may explain very rapid gains in the beginning with diminishing returns(as the cortical bone may not be able to continue stretching without fresh stem cells coming into the system).  But, Lateral Synovial Joint Loading on rats did show an increase in activity in the growth plate so even if rapid beginner gains are attributable to cortical bone stretching further gains should be possible by getting stem cells to the hyaline cartilage growth plate line.

Anyways, the equation for height growth.  Stem Cell Proliferation(or progenitor cell profileration)+Stem Cell Differentiation+Chondrocyte Proliferation+Chondrocyte Differentiation=Height Growth via the Growth Plate.  If the stem cells are in the hyaline cartilage growth plate line then they will likely differentiate into chondrocytes due to their environment.

Lateral Synovial Joint Loading increases Interstitial Fluid Flow which helps stem cells in the red bone marrow get to the hyaline cartilage growth plate line.  It also places a compressive force on the epiphysis and a compressive force on the stem cells results in increased stem cell proliferation.  In addition, it causes microfracture in the trabecular bone which releases more stem cells.

The most basic thing you can do is increase the duration you perform LSJL for and you can increase the weight.  Optimal weights and duration have not yet been determined.  But between 30-70lbs is good and between 30seconds to 5minutes is good(though it's possible that more is better).

So what you can do to improve results here is to tap the epiphysis of the bone to create more microfractures and you can tap slightly below the hyaline cartilage growth plate line to cause shearing forces on the periosteum which releases progenitor cells which can also stimulate endochondral ossification.

The second thing you can do is intense cardiovascular training.  Intense cardio causes an oxygen debt which creates an hypoxic environment.  An hypoxic environment increases stem cell proliferation.  The womb is an hypoxic environment, remember?  You want the oxygen debt to be in the limbs that you want to grow so be sure the cardio involves the limbs you're trying to increase in length.

The third thing you can do is exercise in general and resistance training.  Exercise increases insulin sensitivity and insulin acts to increase height via the PI3K pathway.  Certain resistance exercises can also help increase short and irregular bone height.

X=Lateral Synovial Joint Loading.  Y=hypoxic environment.  Z=insulin sensitivity.  If you have LSJL in check manipulate those variables. 

Increasing Bone Growth with PI3K?

PI-3 Kinase is a subset of enzymes that are involved in cellular proliferation and differentiation.  Stem cells and chondrocytes are cells.  Increasing profileration of these cells could be responsible for increasing bone growth.

How does PI3K work to increase height?  How can we manipulate levels of this protein kinase? 

Insulin stimulates osteoblast proliferation and differentiation through ERK and PI3K in MG-63 cells. 

"To explore the mechanisms of action of insulin on osteoblast growth and differentiation, human osteoblastic cell line-MG-63 was used and stimulated by insulin in the presence or absence of ERK inhibitor PD98059, PI3-K inhibitor LY294002, or inhibitor PD98059 + LY294002. The results showed that insulin positively regulated the expression of its receptor. Insulin stimulated the proliferation of MG-63 cells in a time- and dose-dependent manner and blockade of both MAPK and PI3K pathways could inhibit the cell proliferation. In addition, ALP activity, the secretion of type I collagen, OC gene expression, and mineralized nodule formation were increased in the insulin treated group, whereas these indicators were decreased after treatment with blocking agents. However, treatment with PI3-K inhibitor LY294002 significantly reversed the down-regulation of Runx2 expression and treatment with ERK inhibitor PD98059 remarkably decreased up-regulation of Osx and IGF-1 expression after insulin treatment.  Insulin promoted osteoblast proliferation and differentiation through MAPK and PI3K pathway in MG-63 cells." 

This confirms that increasing insulin sensitivity can increase height at least in the torso where increasing osteoblast proliferation is what causes height increase there.  PI3K enzymes are needed to increase height but it is ultimately the level of insulin that regulates how much height is gained. 

Is PI3K involved in chondrocytes? 

Regulation of gene expression by PI3K in mouse growth plate chondrocytes. 

"Pharmacological inhibition of the PI3K signaling pathway results in reduced endochondral bone growth, and in particular, shortening of the hypertrophic zone in a tibia organ culture system. In this current study we aimed to investigate targets of the PI3K signaling pathway in hypertrophic chondrocytes. Through the intersection of two different microarray analyses methods (classical single gene analysis and GSEA) and two different chondrocyte differentiation systems (primary chondrocytes treated with a pharmacological inhibitor of PI3K and microdissected growth plates), we were able to identify a high number of genes grouped in GSEA functional categories regulated by the PI3K signaling pathway. Genes such as Phlda2 and F13a1 were down-regulated upon PI3K inhibition and showed increased expression in the hypertrophic zone compared to the proliferative/resting zone of the growth plate. In contrast, other genes including Nr4a1[up in LSJL] and Adamts5 were up-regulated upon PI3K inhibition and showed reduced expression in the hypertrophic zone. Regulation of these genes by PI3K signaling was confirmed by quantitative RT-PCR. We focused on F13a1 as an interesting target because of its known role in chondrocyte hypertrophy and osteoarthritis. Mouse E15.5 tibiae cultured with LY294002 (PI3K inhibitor) for 6 days showed decreased expression of factor XIIIa in the hypertrophic zone compared to control cultures. Discovering targets of signaling pathways in hypertrophic chondrocytes could lead to targeted therapy in osteoarthritis and a better understanding of the cartilage environment for tissue engineering." 

"inhibition of PI3K signaling results in reduced growth of tibiae"

#2 are genes upregulated by PI3K inhibition.  So genes downregulated by PI3K in growth plate chondrocytes.  #1 genes are genes upregulated in control but not #2 so genes upregulated by PI3K.

Akt is a downstream target of PI3K and LSJL increases p-Akt.

The role of Akt1 in terminal stages of endochondral bone formation: angiogenesis and ossification.

"The PI3K signaling pathway [has a role] in chondrocyte hypertrophy and bone growth. We aimed to investigate the role of Akt1, an important target of PI3K, in endochondral ossification. Akt1 KO mice showed reduced size compared to their littermates throughout life, but the largest difference in body size was observed around 1 week of age. Focusing on this specific developmental stage, we discovered delayed secondary ossification in the long bones of Akt1 KO mice. A delay in formation of a structure resembling a secondary ossification center was also seen in tibia organ cultures treated with the PI3K inhibitor LY294002. The expression of matrix metalloproteinase-14 (MMP-14) [Upregulated by LSJL], the main protease responsible for development of secondary ossification centers, was decreased in the epiphysis of Akt1 KO mice, possibly explaining the delay in secondary ossification centers seen in the Akt1 KO mice. Bone mineral density (BMD) and bone mineral content (BMC) measured in the proximal tibia of 1-year-old mice were decreased in Akt1 KO mice, suggesting that the original delay in ossification might affect bone quality in older animals."

"Akt1-deficient mice showed reduced body weight, dwarfism, and decreased bone mass (at 8 weeks of age). Akt2 mutants have a diabetes-like syndrome and mild growth deficiency"

"In Akt1 KO littermates we noticed vascular cartilage canals (VC) in the tibia and peripheral vascular invasion in the femur"

"[The] defect in ossification was due to a delay in secondary ossification formation in the Akt1 mutants compared to control littermates"

"reduced MMP-14 expression in our mutant mice contributes to the delay in secondary ossification"

So the PI3K enzyme is important for getting chondrocytes to hypertrophy.  Larger hypertrophy of chondrocytes should increase bone growth and that is what pushes the bones apart in the hyaline cartilage growth plate line.  

The proper function of the PI3K enzyme pathway is important for proper growth but ultimately it is other factors that ultimately manipulate growth like insulin.  There are PI3K inhibitors(for anti-cancer) but there are no PI3K stimulators on the market. 

To grow taller at times it seems as simple as looking at what they are trying to cure cancer and then do the opposite.  Not in this case however as the PI3K pathway that affects proliferation and differentiation seems to be either off or on.

Become Taller at your Primary Ossification Center?

Lateral Synovial Joint Loading increases height by initiating growth in the hyaline cartilage growth plate line at the secondary ossification centers.  Is there a method to become taller at the primary ossification center in the middle of the bone?  There's no hyaline cartilage remnant at the primary center of ossification like their is at the secondary centers of ossification.  The identity of stem cells is based on their environment and genetic expression of the surrounding cells.  There's no cartilage in the primary center of ossification(the middle of the bone).  But are there any possibilites at the primary ossification center to aid in growing taller?

[Expression of hypoxia inducible factor-1alpha in long bone development] 

"To observe dynamically the development of fetal long bone and detect the expression and distribution of HIF-1alpha,to investigate the expression pattern and possible effects of hypoxia inducible factor-1alpha (HIF-1alpha)[The best way to induce hypoxia is intense cardiovascular training like High Intensity Interval Training but it's only the high intensity parts that matter] in fetal long bone development of mouse. METHODS: E12.5, E13.5, E14.5, E15.5, E16.5 and E17.5 pregnant C57BL6 mice were sacrificed. After sacrifice, the embryos were delivered by caesarean section. The development of fetal long bone was dynamically observed by stereoscopic microscope, and the distributional expression of HIF-1alpha protein was detected by using method of immunohistochemistry. The expression of HIF-1alpha mRNA and osteoblast marker gene at various stage were also detected by using methods of reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: The cartilaginous long bone began to form and joints outline arised at E13.5, then the primary ossification center was observed at E14.5, showing opaque ossification under stereoscopic microscope,and then the osteogenesis expanded and extended to both sides. Immunohistochemistry demonstrated lots of HIF-1alpha protein positive chondrcytes in the center of primary ossification at E14.5, then they decreased dramatically. HIF-1alpha mRNA expressed at high level from E13.5 to E15.5, and then decreased to low level. CONCLUSION: Fetal long bone development pattern appeared to be endochondral osteogenisis process, existing hypoxia microenviroment may increase HIF-1alpha mRNA expression and thus initiate the cascade of endochondral osteogenisis." 

One of the keys to triggering growth in the primary ossification center is hypoxia(lack of air).  Hypoxia is best induced by intense cardio and not strangulation.  You want the hypoxia to be in your bone cells not in your brain cells. 

The transcriptional cofactor Lbh regulates angiogenesis and endochondral bone formation during fetal bone development. 

"Lbh is thought to act as a transcriptional cofactor and is highly conserved among species. Here we show that Lbh is expressed in chondrocytes, cells of the perichondrium, and the primary spongiosa in fetal growth plates of mice and chickens. Lbh overexpression in chick wings, using the RCAS-retroviral vector strategy, results in shortened skeletal elements and delayed hypertrophic chondrocyte maturation and bone formation. Additionally, osteoclast and endothelial cell invasion are delayed in the Lbh-overexpressing bones. Finally, we find a dramatic suppression of Runx2 and VEGF mRNAs in chondrocytes and osteoblasts that overexpress Lbh. Strikingly, this abnormal bone development in infected limbs can be rescued by concurrent overexpression of Runx2. These results suggest that during endochondral bone formation, Lbh may negatively regulate vascular invasion and formation of the early ossification center at least in part by interfering with Runx2 and/or VEGF expression." 

Runx2 and VEGF(two genes) are responsible for growth in the primary ossification center.  The primary spongiosa is the name for the initial trabecular bone network.  Heterotrophic Ossification may be something worth studying in the future as it is where new bone grows outside existing bone.  It would be interesting to see if Runx2 expression is responsible for this abnormal growth.  If bone can grow off of liver cells then bone can certainly grow back in the primary ossification center. 

Impaired bone development and increased mesenchymal progenitor cells in calvaria of RB1-/- mice. 

"We have previously shown that the retinoblastoma protein (pRb) can activate expression of Runx2-dependent, bone-specific genes in cultured cells. We now show that pRb also plays a role early in osteogenesis, and that in primary RB1(-/-) calvarial cells there is an increased osteoprogenitor pool. To understand pRb's function in vivo, we generated a conditional RB1-KO mouse in which pRb expression is efficiently extinguished in osteoblasts. These animals display an apparent developmental defect in bones, most strikingly in the calvaria. Cultured RB1(-/-) calvarial osteoblasts fail to cease proliferation upon reaching confluence or following differentiation. Re-plating assays of primary RB1(-/-) calvarial cells after differentiation showed a clear adipogenic ability with increased multipotency. RB1(-/-) osteoblasts display a severe reduction in levels of mRNAs expressed late in differentiation. In this study, we present strong evidence that pRb has multiple regulatory roles in osteogenesis. Furthermore, in the absence of RB1(-/-) there is a larger pool of multipotent cells compared with the WT counterpart. This increased pool of osteoprogenitor cells may be susceptible to additional transforming events leading to osteosarcoma, and is therefore key to understanding RB1 as a target in malignancy." 

Retinoblastoma protein can activate the genetic expression of Runx2 and possibility initiate bone growth at the primary center of ossification. 

Regulation of embryonic endochondral ossification by Smurf2. 

"Smurf2 is an E3 ubiquitin ligase that targets TGF-beta receptor activated Smad2 and Smad3 for the proteasome in primary articular chondrocytes, thus stimulating their hypertrophic differentiation. Comparatively, how Smurf2 functions in growth plate chondrocytes in a developing long bone is an open question. In this study, we measured the mRNA levels of endogenous Smurf2 and type X collagen in chick growth plate at different embryonic stages to monitor the correlation between the level of Smurf2 expression and chondrocyte maturational stage. We found that high levels of Smurf2 were associated with the differentiative and proliferative stages, while Smurf2 levels were thereafter decreased as the chondrocytes matured toward hypertrophy. In addition, we injected Smurf2-RCAS into chick wing buds at HH stage 20-23 and examined how the ectopic overexpression of Smurf2 in condensing chondrogenic mesenchyme affects the subsequent process of chondrocyte maturation and ossification during embryonic development. Histological analysis showed that overexpression of Smurf2 in a developing wing bud accelerated chondrocyte maturation and endochondral ossification, which may result from a decrease in TGF-beta signaling in the infected chondrocytes with Smurf2-RCAS." 

It seems like you want your Smurf2 levels in the primary ossification centers to be just right as you don't want chondrocytes to mature too quickly so the chondrocytes have more time to proliferate.  

There doesn't appear to be any special to induce growth in the primary ossification center.  However, there may be remnants of genes left over from earlier development.  New bones can grow from anywhere if the stem cells are there and the proper genetic expression is in place including within the primary center of ossification.

Height Boosting Progress with one week off of LSJL

Well I've been sick for the last week and so I haven't been performing LSJL as everything that's anabolic for your body cells is also anabolic for viral cells.  Here's last Weeks Pictures: Bone Length Increase | 3 Months In.  I didn't get any height increase so the height increase from LSJL must occur fairly rapidly after the stimulation.  When I return to LSJL I'll have to see if there's some kind of conditioning effect and if I get more results than normal after going back.  I tried going to the doctors office due to my virus as an excuse to get my height measured but they didn't have a device to measure height at least that I could visibly see.  Maybe it was broken or something.  Here's this weeks pictures.  I also took one from a sitting position to see if that way can see more visible gains.

Body Remodeling Review

Sky of EasyHeight, famously flew Antoine to New York to test his Body Remodeling theory.  The issue with Antoine is that he's never tried his techniques on himself.  Antoine has been performing his height increase method since 2007.  Antoine's system may use some kind of bone vibrator.   I couldn't find good information on good bone vibration machines but I assume it works like Ultrasound.

Antoine's traction device may cause spiral forces on the bone and may place a stretching force on the cortical bone in that manner.  Sky mentions that Antoine uses a 10lbs traction device, which although Sky may have mentioned might have been insufficient for longitudinal stretching with severe spiral forces may be sufficient.  Although it's hard to tell without actually seeing the device in action.

On the Body Remodeling site, Antoine mentions the evolution of specific bone cells being caused by his devices(stem cells?, chondrocytes?).  Still 3 years after 2007, Antoine only has one before and after picture(the first before and after picture is only an after picture at different magnifications).

Antoine's Body Remodeling has undergone a lot of criticism at the various height increase forums and I can only conclude that it is somewhat deserved.  Antoine needs to provide a lot more transparency into how his method works or he needs awesome before and after pictures.  If the method involves Ultrasound(bone viration machine) and Spiral Forces(his traction device) then perhaps he's keeping everything secret because he wants to overcharge versus what the equipment actually costs?  But, if he gave transparency then it would help height seekers and he could make the money with volume?

Height Increasement with Collagen?

The collagen in bone gives bone it's elasticity.  Collagen allows bone to temporarily lengthen(microstrain) and if microfractures occur while the bone is in this stretched state it stands to reason that the bone will maintain some of this state.  If we can increase the collagen content of bone, then we should be able to put more of a distraction force on the cortical bone.  Microfractures will maintain some of this distracted state.  This cortical bone diaphysis stretching is what is employed by Sky in his limb center.  And if it works(we're waiting on his before/after pictures), it's a viable alternative/supplement to lateral synovial joint loading.  How do we increase the collagen content in bone(for it's height increasement applications)?  Alternatively, is there anyway to enhance expression of the gene COL2A1 which controls the formation of hyaline cartilage(which is the resting zone of the growth plate)?

Collagen is formed by amino acids and peptides. 

Large-scale gene expression in bone marrow mesenchymal stem cells: a putative role for COL10A1 in osteoarthritis.

"To elucidate disease-specific molecular changes occurring in osteoarthritis (OA) by analysing the differential gene expression profiles of bone marrow mesenchymal stem cells (BM-MSCs) from patients with OA compared with those without OA. Expression profiles of BM-MSCs from eight paired patients with OA and patients with hip fracture without signs of OA were compared by DNA microarray expression analysis and significant differences were evaluated by computational Gene Set Enrichment Analysis. To validate the involvement of COL10A1 as part of the most downregulated gene set in OA, three tagging single nucleotide polymorphisms were genotyped in 191 patients with OA and 283 controls. COL10A1 expression was also assessed by quantitative RT-PCR in additional subjects. Expression levels in 9% (1967) of the overall transcripts were significantly different (p<0.05) between MSCs from patients with OA and controls (532 genes reached twofold differences: 240 were upregulated and 292 were downregulated). Cell development and differentiation were the functional categories accounting for most genes with altered expression. Interestingly, several genes related to the Wnt/-catenin pathway and collagen genes were downregulated in MSCs from patients with OA. The collagen gene set was clearly downregulated in OA. Furthermore, the expression of COL10A1 was significantly reduced in patients with OA. A genetic association between the COL10A1 rs11965969 polymorphism and OA was also found.  COL10A1 downregulation seems to have a role in the establishment of a defective and/or unstable subchondral cartilage matrix in OA disease. It is proposed that OA may be linked to the intrinsic defective regenerative potential of BM-MSCs resulting from its reduced expression of fate commitment-related genes."

Conclusion:  Collagen is increased by Mesenchymal Stem Cells.  To increase collagen content in bone or the hyaline cartilage growth plate line then one needs to increase expression of the genes that control mesenchymal stem cell lineage(such as COL2A1 for hyaline cartilage). 

The study states that it has supplement data detailing gene expression but I could not find it.

Applying an excessive mechanical stress alters the effect of subchondral osteoblasts on chondrocytes in a co-culture system. 

"Osteoarthritis (OA) sometimes occurs as a consequence of repeated microtrauma involved in parafunction, which may lead to microfracture in the subchondral bone. The aim of this in vitro study was to evaluate the effects of subchondral osteoblasts in loading with repeated excessive mechanical stress on the metabolism of overlying chondrocytes. A high-magnitude cyclic tensile stress of 15 kPa (30 cycles min(-1)) was applied to the cultured osteoblasts obtained from porcine mandibular condyles. The chondrocytes in alginate beads were then co-cultured with mechanically stressed or unstressed osteoblasts. Chondrocytes co-cultured with unstressed osteoblasts showed a phenotypic shift to hypertrophic chondrocytes, characterized by decreased expression of type II collagen{up}, aggrecan{up}, Sry-related HMG box (SOX-9){up}, and cartilage oligomeric matrix protein (COMP) genes and increased expression of type X collagen{up} and bone sialoprotein (BSP){Up} genes, suggesting that the co-culture may change the chondrocyte differentiation to some extent. These changes were more distinct in chondrocytes co-cultured with excessively mechanically stressed osteoblasts. After co-culture with stressed osteoblasts, the expressions of matrix metalloproteinase (MMP)1, MMP3{up} and MMP13 genes were also enhanced and the synthesis of DNA, proteoglycan and collagen were significantly decreased in chondrocytes. Alterations in cartilage metabolism can be induced by stressed osteoblasts, indicating a possible explanation for the onset and progression of OA." 

Subchondral osteoblasts expressed type I collagen at a 5:1 ratio to cartilage chondrocytes whereas cartilage chondrocytes expressed type II collagen at a 5:1 ratio to subchondral osteoblasts.

Mechanical stress upregulated TGF-B1 in mechanically stressed osteoblasts.

Mechanical stress studies are always interesting because they are something that we can perform on our own.  This study shows that chondrocytes co-cultured with osteoblasts are more likely to hypertrophy(one of the stages of growth).  Osteoblasts are prevalent in bone so there should be plenty of opportunities to co-culture near the hyaline cartilage.  In this case, loading is bad but it is very high magnitude loading.  This may have applications to lateral synovial joint loading as if you are not getting results it may be worth it to lighten the weight in case the load is "overstressing" the osteoblasts.  

So... 

Collagen is produced by Mesenchymal Stem Cells.  If we want to produce more collagen we need to enhance proliferation of MSCs but enhance the expression of genes that make MSCs undergo a collagenic lineage.  If you are not getting results with LSJL, try lowering the weights you use(10lbs is too light.  I think between 50-70lbs may be the sweet spot to maximize the positive benefits of mechanical loading whereas not overstressing say the osteoblasts). 

Type I collagen may help as well:

The predominant role of collagen in the nucleation, growth, structure and orientation of bone apatite.

"Type I collagen in vitro can initiate and orientate the growth of carbonated apatite mineral in the absence of any other vertebrate extracellular matrix molecules of calcifying tissues. Collagen matrix influences the structural characteristics on the atomic scale, and controls the size and the three-dimensional distribution of apatite at larger length scales."

"Circulating fluids continuously bathe the calcified connective tissue over both collagen inter- and intrafibrillar spaces"

"The 3D organization of collagen, similar to that described in human compact bone, has been reproduced in vitro by exploiting the capacity of collagen molecules to self-assemble spontaneously under high concentrations (at least 80 mg ml−1)"

"Collagen has a further impact on the hydration environment and local structure of phosphate in apatite"<-can we manipulate this to grow taller?

"Collagen can sequestrate large quantities of calcium, phosphate and carbonate ions, which precipitate spontaneously in apatite without additives. The concentration of calcium ions strongly influences the distribution of apatite crystals within the collagen matrix. Hence, the Ca-rich proteins in the ECF may play an important role as inhibitors of apatite precipitation whereas those in bone ECM may concentrate the ions locally in the gap regions described as the first site of nucleation for apatite crystals"

Other collagen types may help as well:

Minor cartilage collagens type IX and XI are expressed during embryonic stem cell-derived in vitro chondrogenesis.

"In vitro differentiation of murine embryonic stem (ES) cells via embryoid bodies (EBs) recapitulates the cellular differentiation steps of chondrogenesis. Differentiated chondrocytes lose their characteristic phenotype when they are kept in monolayer culture. The aim of this study was to further characterize the chondrogenic nodules derived by in vitro-differentiation of murine ES cells for the distribution of collagen types II, IX and XI{all three types of collagen are upregulated by LSJL} in comparison to in vitro dedifferentiating primary chondrocytes from murine embryonic ribs. Expression of cartilage collagens and other extracellular matrix proteins was analyzed. ES cell-derived chondrocyte differentiation starts with mesenchymal condensations synthesizing high amounts of fibronectin {Possible fibronectin related proteins like FNDC4 and FNDC6 are upregulated by LSJL}. Later, the matrix of the mature cartilage nodules consists of type II collagen, proteoglycans and the minor collagens type IX and XI {all of these proteins are upregulated by LSJL providing evidence that LSJL can form cartilage nodules that could potentially undergo endochondral ossification and make you grow taller}. The nodules show a three-dimensional structure with multiple layers of collagen type II-positive cells. At late differentiation stages these chondrocytes were located at lateral regions of the nodules. Similar to the distribution pattern of collagen type II positive cells, the cells staining positive for collagen type IX and XI were present in the surface regions, but not in the central areas of the chondrogenic nodules. During cultivation of the primary murine rib chondrocytes expression of chondrogenic marker genes such as collagen type II and aggrecan declined and many chondrocytes lost characteristic cartilage matrix proteins and converted to an elongated, fibroblastoid shape with prominent actin stress fibers."

"Early mesenchymal progenitor cells are characterized by the production of hyaluronan {hyaluronan synthase1 is upregulated 4.5-fold by LSJL}, fibronectin and collagen type I {upregulated by LSJL}. Condensations resulting from the aggregation of mesenchymal precursor cells can be visualized by their affinity to the lectin peanut binding agglutinin (PNA), binding to the cell surface of condensing cells"

"Collagen type IX was first described as a proteoglycan since it possesses a large glycosaminoglycan domain. The isoform mainly found in cartilage contains a large N-terminal globular domain (NC4) and is located on the surface of heterotypic type II/XI fibrils mediating cross-links either with collagen type ÍI fibrils or other collagen type IX molecules and interaction with glycosaminoglycans. Collagen type XI is a heterotrimeric protein located at the interior of collagen II/XI fibrils. A lack of the Col11a1 gene encoding the alpha1(XI) chain in mice results in autosomal recessive chondrodysplasia (cho) showing a shortened spine and thoracic skeleton with flared metaphyses"

"Collagen type XI has also been implied to have a function in the regulation of the diameter of collagen fibrils. Besides cartilage tissue, collagen type XI a1 chain mRNA expression was found in trabecular bone"<-so some COL11A1 expression can be explained by trabecular bone expression.

With the exception of Sox9 no proteins associated with the development of early mesenchymal condensations like PNA, N-cadherin, and Fibronectin were expressed over threshold in LSJL.

"Only some single cells expressed the extracellular matrix protein collagen type X characteristic for hypertrophic chondrocytes and peanut agglutinin, a marker for early mesenchymal condensations at three days after cell isolation. The same was true for N-cadherin"<-LSJL gene expression was taken at 49 hours which is below three days however unlike this study COL10A1 was upregulated above threshold.

"Collagen type IX seems to be preferentially expressed by cells isolated from “permanent” cartilage tissues, which do not undergo endochondral ossification"

Autologous Collagen-induced Chondrogenesis: Single-stage Arthroscopic Cartilage Repair Technique.

"Autologous collagen-induced chondrogenesis is a novel, single-staged arthroscopic cartilage repair technique using microdrilling and atelocollagen or fibrin gel application under carbon dioxide insufflation. Atelocollagen is a highly purified type I collagen obtained following the treatment of skin dermis with pepsin and telopeptide removal, making it nonimmunogenic. In this procedure, atelocollagen mixed with fibrinogen and thrombin in a 2-way syringe can maintain the shape of the articular surface approximately 5 minutes after application due to the reaction between the thrombin and fibrinogen. Carbon dioxide insufflation facilitates the application of the gel under dry conditions. Ten patients (mean age, 38 years) with symptomatic chondral defects in the knee who were treated arthroscopically with microdrilling and atelocollagen application were retrospectively analyzed. All defects were International Cartilage Repair Society grade III or IV and were 2 to 8 cm(2) in size intraoperatively. For the clinical assessment, Lysholm score was assessed preoperatively and at 2-year follow-up. All patients underwent morphological magnetic resonance imaging at 1.5-Tesla at 1-year follow-up. Mean Magnetic Resonance Imaging Observation of Cartilage Repair Tissue score at 1-year follow-up was 70.4±20.2 (range, 15-95). The Magnetic Resonance Imaging Observation of Cartilage Repair Tissue score for patellar lesions was similar to that of lesions in other locations: 73.3±11.7 vs 68.1±25.5, respectively. This technique had encouraging clinical results at 2-year follow-up. Morphological magnetic resonance imaging shows good cartilage defect filling, and the biochemical magnetic resonance imaging suggests hyaline-like repair tissue."

Can this technique be used to produce growth plate cartilage?

The issue is that it actually applies a gel so it might be complicated to do within the bone marrow and it's unclear whether there was any cartilage overgrowth within the knee joint.

Nanomechanics of collagen microfibrils.

"collagen-rich tissues are built with the collagen fibril as fundamental building block. These fibrils have a diameter in the range of 30 to 500 nm, a length up to the millimeter range and are assembled in complex hierarchical assemblies, whose structure depend on the particular tissue"

"In bone, the organic collagen protein matrix alone is not sufficient to provide the stiffness and resistance to compression required for this tissue (which has to carry considerable loads) and additional stiffening is provided by the inclusion of mineral hydroxyapatite crystals into collagen fibrils and particularly in the gap region"

"When subjected to mechanical load, collagen microfibrils feature two distinct deformation regimes. In the small-strain regime (<10%) the predicted Young’s modulus is ≈300 MPa, while in the large-strain regime (>10%) the microfibril shows a severely increased tangent stiffness, with a Young’s modulus of ≈1.2 GPa "