Hyaluronan Synthase 1 to be tall?

LSJL upregulates hyaluronan synthase 1 by 4.1 fold.

Hyaluronan expressed by the hematopoietic microenvironment is required for bone marrow hematopoiesis.

"The contribution of hyaluronan (HA) to the regulatory network of the hematopoietic microenvironment was studied using knock-out mice of three hyaluronan synthase genes (Has1, Has2, and Has3). The number of hematopoietic progenitors was decreased in bone marrow and increased in extramedullary sites of Prx1-Cre;Has2(flox/flox);Has1(-/-);Has3(-/-) triple knock-out (tKO) mice as compared with wild type (WT) and Has1(-/-);Has3(-/-) double knock-out (dKO) mice. In line with this observation, decreased hematopoietic activity was observed in long term bone marrow cultures (LTBMC) from tKO mice, whereas the formation of the adherent layer and generation of hematopoietic cells in WT and dKO cultures was not different. 4-Methylumbelliferone (4MU) was used to pharmacologically inhibit the production of HA in LTBMC. Treatment with 4MU inhibited HA synthesis, decreased expression of HAS2 and HAS3, and eliminated hematopoiesis in LTBMC, and this effect was alleviated by the addition of exogenous HA. Exogenous HA also augmented the cell motility in LTBMC, which correlated with the HA-stimulated production of chemokines and growth factors. Conditioned media from HA-induced LTBMC enhanced the chemotaxis of hematopoietic stem/progenitor cells (HSPC) in response to SDF-1. Exposure of endothelial cells to 4MU decreased their ability to support HSPC rolling and adhesion. In addition, migration of transplanted HSPC into the marrow of 4MU-pretreated mice was lower than in untreated mice. HA depletion reduces the ability of the microenvironment to support HSPC, and HA [is] a necessary regulatory element in the structure of the hematopoietic microenvironment."

So the increase in HAS1 may be to increase hematopoiesis (formation of blood cells).

"LTBMC extend these results to show that HMW HA also increased production of several chemokines, including MIP-1α, MCP-1, CXCL16, SDF-1, and RANTES. In addition, HMW[High Molecular Weight] HA stimulated the production of growth factors including G-CSF, IGFBP-3, IL-12, LIX, IL-9, KC, and soluble VCAM-1"

Hyaluronan is required for cranial neural crest cells migration and craniofacial development.

"hyaluronan synthesized by Has1 and Has2 is necessary for the proper development of the visceral skeleton"

"While cell movement in vitro is promoted by hyaluronan, it is inhibited by degrading the hyaluronan itself or by blocking its binding to both of the hyaluronan receptors CD44 and RHAMM"

"CD44 could mediate the hyaluronan instructive effect that initiates signaling pathways and thus promotes NCC movements."

Protein Kinase-D

Inhibition of protein kinase-D promotes cartilage repair at injured growth plate in rats.

"Using a rat model, this study investigated potential roles of protein kinase-D (PKD) in regulating expression of osteogenic transcription factor osterix and the growth plate bony repair. 4 days post injury at the proximal tibial growth plate, rats received four once-daily injections of vehicle or 2.35mg/kg gö6976 (a PKD inhibitor), and growth plate tissues collected at day 10 were examined histologically and molecularly. In addition, effects of PKD inhibition on osteogenic and chondrogenic differentiation were examined in vitro using rat bone marrow mesenchymal stromal cells.
Compared to vehicle control, PKD inhibition caused a decrease in bone volume, an increase in % of mesenchymal tissue, and an increase in cartilaginous tissue[over twice as much] within the injury site{so maybe PKD inhibition can help form new growth plates?}. Consistently, gö6976 treatment tended to decrease expression of bone-related genes (osterix, osteocalcin) and increase levels of cartilage-related genes (Sox9, collagen-2a, collagen-10a1). In support, in vitro experiments showed that gö6976 presence in the primary rat marrow stromal cell culture resulted in a decrease of alkaline phosphatase(+) CFU-f colonies formed and an increase in collagen-2a expression in chondrogenic pellet culture.
 PKD is important for growth plate bony repair and its inhibition after growth plate injury may result in less bone formation and potentially more cartilage repair."


Growth plate height looks higher in go6976 treated group and the bone seems wider.



"mesenchymal tissue (Me), bony trabeculae (BT), cartilaginous tissue (Ca) and bone marrow (BM)"

Notice that there is no cartilage in the selected region in the vehicle.

Sox9 was upregulated 2 fold by Go6979 treatment which was less than LSJL where one isoform of Sox9 was upregulated 3 fold.

According to The potential use of protein kinase D inhibitors for prevention/treatment of epidermal tumors., Resveratol is Protein Kinase D inhibitor.

Resveratrol is available for sale: NutriGold Resveratrol GOLD, 500mg, 120 Vegetarian Capsules - trans-Resveratrol with Grape Seed and Red Wine Polyphenols (Featuring Patented ResVinol and ORAC-15M)

Height Gain and the Piezoelectric Current

The loading regime in Lengthening of Mouse Hindlimbs with Joint Loading involved a PiezoActuator.  Sky of Easyheight.com often spoke of a piezoelectric current.  Bone deformation itself generates a PiezoElectric current but it's nice to have a PiezoActuator because there's a strain gauge directly attached to it.  You can also get a more precise idea of the current generated by a PiezoActuator but nothing that can be achieved by the PiezoActuator cannot be achieved by loading.  What is the Piezoelectric current and how can it affect chondrocytes to help us gain height and grow taller?

Polymeric piezoelectric actuator substrate for osteoblast mechanical stimulation.

"Bone mass distribution and structure are dependent on mechanical stress and adaptive response at cellular and tissue levels. Mechanical stimulation of bone induces new bone formation in vivo and increases the metabolic activity and gene expression of osteoblasts in culture.Osteoblasts were grown on the surface of a piezoelectric material, both in static and dynamic conditions at low frequencies, and total protein, cell viability and nitric oxide measurement comparisons are presented."

Piezoelectricity can induce osteogenic differentiation.

"It has been suggested that forces capable of inducing cell deformation induce changes in membrane channels and on protein structure and that ultimately, cytoskeleton deformation exerts direct influence on cell nuclei"<-so if your cytoskeleton has adapted to the deformation you will not grow taller by mechanical means


"The advantages of using piezoelectric material for bone cells stimulation are: the control of mechanical ranges stimulation only requires the control of the amount of electrical energy applied; the quicker answer to electric stimulus allows working in physiological frequencies, as are the ones used 1 and 3 Hz, respectively."<-piezoelectricity is used over mechanical methods because it's easier to control the amount of electrical energy applied but electrical energy corresponds to a mechanical range

"The bone has piezoelectric properties,... mechanical stress applied to dried bone produces polarization and submission of bone to an electric field originates strain."<-mechanical stress such as lateral epiphyseal loading generates electric potential within the bone

Cartilage and conversely chondrocytes have electrical potential too.

Electrical behavior of cartilage during loading.

"When cartilage is deformed, it becomes electrically polarized. At least two mechanisms seem to underlie this phenomenon, namely, a short-duration, high-amplitude, piezoelectric-like response and a longer-duration, lower-amplitude response secondary to streaming potentials. The polarity of articular cartilage during loading could hypothetically facilitate joint lubrication."

Chondrocyte polarization may prevent chondrocyte apoptosis.  Although chondrocyte apoptosis is not involved in epiphyseal fusion(because fusion is a separate process than growth plate senescence), slowing down apoptosis may allow for a longer hypertrophic phase.

The antioxidant resveratrol protects against chondrocyte apoptosis via effects on mitochondrial polarization and ATP production.

"Chondrocytes and cartilage explants were isolated from OA patients undergoing knee replacement surgery. Effects of resveratrol in the presence or absence of interleukin-1beta (IL-1beta) stimulation were assessed by measurement of prostaglandin E(2) (PGE(2)) and leukotriene B(4) (LTB(4)) synthesis, cyclooxygenase (COX) activity, matrix metalloproteinase (MMP) expression, and proteoglycan production. To explore the mechanisms of action of resveratrol, its effects on mitochondrial function and apoptosis were examined by assessing mitochondrial membrane potential, ATP levels, cytochrome c release, and annexin V staining.
Resveratrol inhibited both spontaneous and IL-1beta-induced PGE(2) production by >20% (P < 0.05) and by 80% (P < 0.001), respectively; similarly, LTB(4) production was reduced by >50% (P < 0.05). The production of PGE(2) was inhibited via a 70-90% suppression of COX-2 expression and enzyme activity (P < 0.05). Resveratrol also promoted anabolic effects in OA explant cultures, by elevating proteoglycan synthesis and decreasing production of MMPs 1, 3, and 13[MMP-3 is good but MMP-13 is bad so Resveratrol on a whole may have a positive benefit on height growth]. Pretreatment of OA chondrocytes with resveratrol blocked mitochondrial membrane depolarization, loss of mitochondrial biomass, and IL-1beta-induced ATP depletion. Similarly, IL-1beta-mediated induction of the apoptotic markers cytochrome c and annexin V was also inhibited by resveratrol. Exogenous addition of PGE(2) abolished the protective effects of resveratrol on mitochondrial membrane integrity, ATP levels, expression of apoptotic markers, and DNA fragmentation.
Resveratrol protects against IL-1beta-induced catabolic effects and prevents chondrocyte apoptosis via its inhibition of mitochondrial membrane depolarization and ATP depletion. These beneficial effects of resveratrol are due, in part, to its capacity to inhibit COX-2-derived PGE(2) synthesis. Resveratrol may therefore protect against oxidant injury and apoptosis, which are main features of progressive OA."

Resveratrol is available for sale: Biotivia Resveratrol Bioforte 250mg , Full Spectrum Resveratrol Supplement, Capsules, 60-Count Bottle.  However, Apoptosis is a necessary part of endochondral ossification and completely preventing it may reduce height growth(however, increasing chondrocyte time before apoptosis may help height growth).  Apoptosis plays a role in growth plate function and complete elimination may reduce adult height. 


Resveratrol may stunt growth by preventing needed chondrocyte apoptosis but mechanical loading like LSJL may enhance membrane polarization and inhibit apoptosis until the proper time.


What is the optimal charge for height growth in chondrocytes?

The effects of fixed electrical charge on chondrocyte behavior.

 "In this study we have compared the effects of negative and positive fixed charges on chondrocyte behavior in vitro. Electrical charges have been incorporated into oligo(poly(ethylene glycol) fumarate) (OPF) using small charged monomers such as sodium methacrylate (SMA) and (2-(methacryloyloxy) ethyl)-trimethyl ammonium chloride (MAETAC) to produce negatively and positively charged hydrogels, respectively. The physical and electrical properties of the hydrogels were characterized by measuring and calculating the swelling ratio and zeta potential, respectively. Our results revealed that the properties of these OPF modified hydrogels varied according to the concentration of charged monomers. Zeta potential measurements demonstrated that the electrical properties of the OPF hydrogel surfaces changed on incorporation of SMA and MAETAC and that these changes in electrical properties were dose-dependent. Attenuated total reflectance Fourier transform infrared spectroscopy was used to determine the hydrogel surface composition. To assess the effects of surface properties on chondrocyte behavior primary chondrocytes isolated from rabbit ears were seeded as a monolayer on top of the hydrogels. We demonstrated that the cells remained viable over 7days and began to proliferate while seeded on top of the hydrogels. Collagen type II staining was positive in all samples, however, the staining intensity was higher on negatively charged hydrogels. Similarly, glycosaminoglycan production was significantly higher on negatively charged hydrogels compared with a neutral hydrogel. Reverse transcriptase polymerase chain reaction showed up-regulation of collagen type II and down-regulation of collagen type I on the negatively charged hydrogels[negatively charged chondrocytes are more likely to differentiate into chondrocytes and less likely to differentiate into bone]. These findings indicate that charge plays an important role in establishing an appropriate environment for chondrocytes and, hence, in the engineering of cartilage. Thus, further investigations into charged hydrogels for cartilage tissue engineering is merited."

A negative charge may be better for height growth.  This may be important for PEMF related methods.

Regulation of immature cartilage growth by IGF-I, TGF-beta1, BMP-7, and PDGF-AB: role of metabolic balance between fixed charge and collagen network.

"Cartilage growth may involve alterations in the balance between the swelling tendency of proteoglycans and the restraining function of the collagen network. Growth factors, including IGF-I, TGF-beta1, BMP-7, and PDGF-AB, regulate chondrocyte metabolism and, consequently, may regulate cartilage growth. Immature bovine articular cartilage explants from the superficial and middle zones were incubated for 13 days in basal medium or medium supplemented with serum, IGF-I, TGF-beta1, BMP-7, or PDGF-AB. Variations in tissue size, accumulation of proteoglycan and collagen, and tensile properties were assessed. The inclusion of serum, IGF-I, or BMP-7 resulted in expansive tissue growth[IGF-1 and BMP-7 can help result in height growth], stimulation of proteoglycan deposition but not of collagen, and a diminution of tensile integrity. The regulation of cartilage metabolism by TGF-beta1 resulted in tissue homeostasis[too much TGF-Beta1 may result in a lack of height growth by encouraging homeostasis], with maintenance of size, composition, and function. Incubation in basal medium or with PDGF-AB resulted in small volumetric and compositional changes, but a marked decrease in tensile integrity. These results demonstrate that the phenotype of cartilage growth, and the associated balance between proteoglycan content and integrity of the collagen network, is regulated differentially by certain growth factors."

IGF-1 and BMP-7 are good for height growth and these relate to charges.

"While it is possible that tissues can grow appositionally in the absence of remodeling, interstitial tissue growth must involve both growth and remodeling since accretion of a single tissue component will change the overall tissue structure and mechanical properties."<-LSJL involves interstitial growth.  Since remodeling is needed total inhibition of catabolic materials is not recommended like osteoclasts, etc.

"a higher tendency to grow is expected in cartilage of the growth plate where the proportion of GAG is higher and tensile strength and modulus are lower than those of articular cartilage"<-GAGs are like chondroitin, hyaluronic acid, and glucosamine.

"Indeed, a relatively high rate of axial growth is observed in the growth plate in vivo (up to ~400 μm/day), as well as in vitro where larger increases in length were observed in cartilages containing one or more osteogenic zones as compared to that of entirely cartilaginous explants"<-cartilage grows better than it's surrounded by bone(Type I collagen).

"The presence of serum, IGF-I or BMP-7 resulted in deposition of GAG that exceeded the deposition of collagen, creating swelling pressures that were excessive relative to the restraining ability of the collagen network. This metabolic imbalance facilitated a relatively loose and weak collagen network that allowed volumetric expansion"<-IGF-1 and BMP-7 results in GAG deposition creating swelling pressures(like hydrostatic pressure of LSJL).  This weak network allowed for volumetric expansion(Growing Taller).

"The proteoglycan constituent of the extracellular matrix provides the tissue with a fixed negative charge that increases the tissue’s propensity to swell and to resist compressive loading"<-so increasing proteoglycan content increases the negative charge and causes more swelling(and by extension more hydrostatic pressure)

So the negative charge helps cause tissue swelling.  GAGs such as hyaluronic acid help with this swelling and thereby help height growth.  Remember, HGH growth plates have a high ratio of ECM to cells.

Bone has piezoeletric potential and that potential can be unleashed by mechanical loading or by a piezoactuator.  Chondrocytes seem to prefer a negative charge and that can be acquired by GAG accumulation such as hyaluronic acid et al.  GAG production seems to be encourage further by negative charge making a good positive feedback loop.

Compounds that inhibit depolirization like Resvatrol may not be recommended as some depolarization is required to allow for apoptosis which is required for chondrocyte hypertrophy(so ossification can occur).  Otherwise, eventually the entire bone would turn into cartilage.

Grow Taller by Inhibiting Actin Polymerization

Inhibiting actin polymerization may be a way to force mesenchymal stem cells to commit to a chondrogenic lineage.  The actin cytoskeleton plays a huge role in mechanosensativity(or the sensativity of cells to load).  Compounds or other factors that all us to manipulate the actin cytoskeleon may help restore sensitivity to methods such as lateral synovial joint loading and also other loading regimes(like weight lifting).  There are compounds that inhibit actin polymerization.  Could they be a way to increase height growth?

Synthetic triterpenoids target the Arp2/3 complex and inhibit branched actin polymerization.

"Synthetic triterpenoids are anti-tumor agents that affect numerous cellular functions including apoptosis and growth inhibition. Here, we used mass spectrometric and protein array approaches and uncovered that triterpenoids associate with proteins of the actin cytoskeleton, including actin-related protein 3 (Arp3). Arp3, a subunit of the Arp2/3 complex, is involved in branched actin polymerization and the formation of lamellipodia. 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid (CDDO)-Im and CDDO-Me were observed to 1) inhibit the localization of Arp3 and actin at the leading edge of cells, 2) abrogate cell polarity, and 3) inhibit Arp2/3-dependent branched actin polymerization[CDDO and CDDO-Me can help you grow taller by inhibiting actin polymerization but it would probably need to be controlled as you need cell migration sometimes]. We confirmed our drug effects with siRNA targeting of Arp3 and observed a decrease in Rat2 cell migration. Taken together, our data suggest that synthetic triterpenoids target Arp3 and branched actin polymerization to inhibit cell migration."

Inhibiting cell migration may be one way to achieve mesenchymal stem cell condensation, one of the key steps for height growth.  However,  the application of CDDO or CDDO-Me would have to be precise as cell migration is needed to fight infections for instance.  You also need initial cell migration to get the stem cells all in one spot.

AlphaE-catenin regulates actin dynamics independently of cadherin-mediated cell-cell adhesion.

"alphaE-catenin binds the cell-cell adhesion complex of E-cadherin and beta-catenin (beta-cat) and regulates filamentous actin (F-actin) dynamics. In vitro, binding of alphaE-catenin to the E-cadherin-beta-cat complex lowers alphaE-catenin affinity for F-actin, and alphaE-catenin alone can bind F-actin and inhibit Arp2/3 complex-mediated actin polymerization. In cells, to test whether alphaE-catenin regulates actin dynamics independently of the cadherin complex, the cytosolic alphaE-catenin pool was sequestered to mitochondria without affecting overall levels of alphaE-catenin or the cadherin-catenin complex. Sequestering cytosolic alphaE-catenin to mitochondria alters lamellipodia architecture [lamellipodia are a part of the actin cytoskeleton on the outer portion of the skull, altering this architecture may also alter actin cytoskeleton sensitivity] and increases membrane dynamics[increase in membrane activity which would help with chondrogenic differentiation] and cell migration without affecting cell-cell adhesion. In contrast, sequestration of cytosolic alphaE-catenin to the plasma membrane reduces membrane dynamics. These results demonstrate that the cytosolic pool of alphaE-catenin regulates actin dynamics independently of cell-cell adhesion."

"During development, cells migrate to specific sites and then, upon contact with other cells, become stationary and differentiate into tissues"<-We want stem cells to migrate to one area, become stationary and differentiate into chondrocytes, so we want to only inhibit cell migration in one specific area(the area of the new growth plate)

"Membrane activity at the leading edge of cells, driven largely by Arp2/3 complex–mediated nucleation of branched filamentous actin (F-actin) networks, promotes cell movement and is involved in the initiation of intercellular contacts. After contact initiation in simple epithelial cells, the actin network associated with the plasma membrane is reorganized and eventually forms bundled filaments oriented parallel to the lateral contact between cells. These changes in actin organization coincide with dampening of membrane dynamics and cell migration and the establishment of strong cell–cell adhesion"<-just condensing the cells by itself by a mechanism such as Lateral Synovial Joint Loading induced hydrostatic pressure may be enough to inhibit cell migration

Actin polymerization is important but you need to inhibit the Arp2/3 complex at a specific point.  You'd want a localized inhibition where you'd want the new growth plate.  It's just too complex to be achieved right now.  Just keep doing LSJL like you're doing and cells should migrate when they're supposed to and adhese when they're supposed to.

Profilin1 regulates sternum development and endochondral bone formation.

"[The] actin cytoskeleton system is regulated by critical modulators including actin binding proteins. Among them, profilin1 (Pfn1) is a key player to control actin fiber structure and it is involved in a number of cellular activities such as migration. During the early phase of body development, skeletal stem cells and osteoblastic progenitor cells migrate to form initial rudiments for future skeletons. During this migration, these cells extend their process based on actin cytoskeletal rearrangement to locate themselves in an appropriate location within microenvironment.  Here, we examined the role of Pfn1 in skeletal development using a genetic ablation of Pfn1 in MPCs by using Prx1-Cre recombinase. We found that Pfn1 deficiency in MPCs caused complete cleft sternum. Notably, Pfn1 deficient mice exhibited absence of trabecular bone in the marrow space of appendicular long bone. This phenotype is location specific, as Pfn1 deficiency did not largely affect osteoblasts in cortical bone. Pfn1 deficiency also suppressed longitudinal growth of long bone[would overepxression of Pfn1 increase height?]."

Specific changes to the mechanism of cell locomotion induced by overexpression of beta-actin.

"Overexpression of beta-actin is known to alter cell morphology. Here we show that overexpressing beta-actin in myoblasts has striking effects on motility, increasing cell speed to almost double that of control cells. This occurs by increasing the areas of protrusion and retraction and is accompanied by raised levels of beta-actin in the newly protruded regions. These regions of the cell margin, however, show decreased levels of polymerised actin, indicating that protrusion can outpace the rate of actin polymerisation in these cells. Moreover, the expression of beta*-actin (a G244D mutant, which shows defective polymerisation in vitro) is equally effective at increasing speed and protrusion. Concomitant changes in actin binding proteins show no evidence of a consistent mechanism for increasing the rate of actin polymerisation in these actin overexpressing cells. The increase in motility is confined to poorly spread cells in both cases and the excess motility can be abolished by blocking myosin function with butanedione monoxime (BDM).  The additional motility shown by cells overexpressing beta-actin appears not to result from an increase in the rate of actin polymerisation but to depend on myosin function. This suggests that the additional protrusion arises from a different mechanism. We discuss the possibility that it is related to retraction-induced protrusion in fibroblasts. In this phenomenon, a wave of increased protrusion follows a sudden collapse in cell spreading."


Osteopontin Promotes Mesenchymal Stem Cell Migration and Lessens Cell Stiffness via Integrin β1, FAK, and ERK Pathways.

Osteopontin is typically a cell associated with osteoblasts but if it reduces in the dissolution of the actin cytoskeleton maybe it can enhance chondrogenesis.

"The expression of osteopontin (OPN){LSJL upregulates osteopontin} is elevated in response to injury and inflammation [and has a] role [in] rat bone marrow-derived mesenchymal stem cells (rMSCs)-directed migration. OPN activated focal adhesion kinase (FAK) and extracellular signal-regulated kinase (ERK) signaling pathways by the ligation of integrin β1 in rMSCs. Inhibitors of FAK and ERK pathways inhibited OPN-induced rMSCs migration, indicating the possible involvement of FAK and ERK activation in OPN-induced migration in rMSCs. OPN reduced cell stiffness in rMSCs via integrin β1, FAK, and ERK pathways, suggesting that the promotion of rMSCs migration might partially be contributing to the decrease in cell stiffness stimulated by OPN. To further examine the role of OPN on cell motility and stiffness, actin cytoskeleton of rMSCs was observed. The reduced well-defined F-actin filaments and the promoted formation of pseudopodia in rMSCs induced by OPN explained the reduction in cell stiffness and the increase in cell migration. OPN binding to integrin β1 promotes rMSCs migration through the activation of FAK and ERK pathways, which may be attributed to the change in cell stiffness caused by the reduction in the amount of organized actin cytoskeleton."

The effect of matrix stiffness on the differentiation of mesenchymal stem cells in response to TGF-β.

"Transforming growth factor β (TGF-β) can promote MSC differentiation into either smooth muscle cells (SMCs) or chondrogenic cells. Here we showed that the stiffness of cell adhesion substrates modulated these differential effects. MSCs on soft substrates had less spreading, fewer stress fibers and lower proliferation rate than MSCs on stiff substrates. MSCs on stiff substrates had higher expression of SMC markers α-actin and calponin-1; in contrast, MSCs on soft substrates had a higher expression of chondrogenic marker collagen-II and adipogenic marker lipoprotein lipase (LPL). TGF-β increased SMC marker expression on stiff substrates. However, TGF-β increased chondrogenic marker expression and suppressed adipogenic marker expression on soft substrates, while adipogenic medium and soft substrates induced adipogenic differentiation effectively. Rho GTPase was involved in the expression of all aforementioned lineage markers, but did not account for the differential effects of substrate stiffness. In addition, soft substrates did not significantly affect Rho activity, but inhibited Rho-induced stress fiber formation and α-actin assembly. MSCs on soft substrates had weaker cell adhesion, and that the suppression of cell adhesion strength mimicked the effects of soft substrates on the lineage marker expression. These results provide insights of how substrate stiffness differentially regulates stem cell differentiation, and have significant implications for the design of biomaterials with appropriate mechanical property for tissue regeneration."

"matrix stiffness did not significantly affect the activation of Smad2/3 in the absence or presence of TGF-β, suggesting that matrix stiffness regulated mechanotransduction in parallel to Smad2/3 signaling."

"RhoA activation significantly induced the expression of SMC markers on stiff substrates, and to a lesser extent on soft substrates. However, RhoA activation also significantly increased the expression of collagen-II and LPL on a soft substrate"

"RhoA activation drastically induced stress fibers and the assembly of α-actin into fibers in cells on stiff but not soft substrates, suggesting that soft substrates limit the function of Rho activity."

"focal adhesions of cells on soft substrates were fewer and smaller"

Insights into mesenchymal stem cell aging: involvement of antioxidant defense and actin cytoskeleton.

"MSCs [were] derived from young and old Sprague-Dawley rats. MSC concentration in bone marrow declines with age [and] their function is altered, especially their migratory capacity and susceptibility toward senescence. High-resolution two-dimensional electrophoresis of the MSC proteome, under conditions of in vitro self-renewal as well as osteogenic stimulation, identified several age-dependent proteins, including members of the calponin protein family as well as galectin-3.  Age-affected molecular functions are associated with cytoskeleton organization and antioxidant defense. [Aged MSCs have] lower actin turnover and diminished antioxidant power. Two main reasons for the compromised cellular function of aged MSCs: (a) declined responsiveness to biological and mechanical signals due to a less dynamic actin cytoskeleton and (b) increased oxidative stress exposure favoring macromolecular damage and senescence."

"[Hematopoietic stem cell] numbers do not necessarily decline with age, but for that cellular function is clearly compromised, for example with regard to mobilization, homing, and lineage choice. Cellular aging of HSCs has been attributed to various mechanisms that exhibit a partial cause and effect relationship to each other. For example, telomere shortening, as a cell-intrinsic trigger for replicative senescence, was shown to be associated with impaired HSC function due to reduced long-term repopulation capacities and increased genetic instability"

"Lifelong dietary restriction increases HSC frequencies and improved HSC function. The self-renewal capacity of HSCs depends on the control of oxidative stress, and additionally, progressive bone marrow failure is associated with elevated reactive oxygen species (ROS). Concordantly, treatment with antioxidative agents has prevented bone marrow failure and restored the reconstitutive capacity of HSCs deficient of Atm, whose gene product inhibits oxidative stress."

Vimentin and Transgelin two aging related actin cytoskeleton proteins are upregulated by LSJL.  The two proteins are also upregulated in aging.

"loss of galectin-3 has been associated with altered Indian hedgehog expression pattern at the growth plate, increased cell death of hypertrophic chondrocytes, and uncoupling of growth plate vascularization"

Decreasing F-actin is pro-chondrogenic.

Cyclic stretching promotes collagen synthesis and affects F-actin distribution in rat mesenchymal stem cells.

"Rat BMSC were harvested from adult rats and cultured to passage 4. Then the cells were seeded onto a silicone{silicone varies in stiffness} membrane loaded with an uniaxial cyclic stretching (10%, 1 Hz) during 3, 6, 12, 24 and 36 h. Stretching enhanced the synthesis of collagen types I and III in BMSC after 24 h stimulation. However, a decrease in fluorescence density of F-actin was observed after the stretching in a time dependent manner. The F-actin filaments seemed much thinner than those of static cells. Cyclic stretching [favors] the synthesis of collagen types I and III, but decreased the amount of F-actin in the BMSC."

Basketball effect on bone morphology

A cam deformity is an excess of bone along the upper surface of the femoral head.  It's unclear how this may affect height.

Growth Plate Alteration Precedes Cam-type Deformity in Elite Basketball Players.

"Vigorous sporting activity during the growth years is associated with an increased risk of having a cam-type deformity develop. The underlying cause of this osseous deformity is unclear. One may speculate whether this is caused by reactive bone apposition in the region of the anterosuperior head-neck junction or whether sports activity alters the shape of and growth in the growth plate. If the latter is true, then one would expect athletes to show an abnormal shape of the capital growth plate (specifically, the epiphyseal extension) before and/or after physeal closure.

We therefore raised three questions: (1) Do adolescent basketball players show abnormal epiphyseal extension? (2) Does the epiphyseal extension differ before and after physeal closure? (3) Is abnormal epiphyseal extension associated with high alpha angles?

We performed a case-control comparative analysis of young (age range, 9-22 years) male elite basketball athletes with age-matched nonathletes, substratified by whether they had open or closed physes. We measured epiphyseal extension on radial-sequence MRI cuts throughout the cranial hemisphere from 9 o'clock (posterior) to 3 o'clock (anterior). Epiphyseal extension was correlated to alpha angle measurements at the same points.

Epiphyseal extension was increased in all positions in the athletes compared with the control group. On average, athletes showed epiphyseal extension of 0.67 to 0.83 versus 0.53 to 0.71 in control subjects. In the control group epiphyseal extension was increased at all measurement points in hips after physeal closure compared with before physeal closure. In contrast, the subgroup of athletes with a closed growth plate only had increased epiphyseal extension at the 3 o'clock position compared with the athletes with a closed growth plate (0.64-0.70). We observed a correlation between an alpha angle greater than 55° and greater epiphyseal extension in the anterosuperior femoral head quadrant: the corresponding Spearman r values were 0.387 (all hips) and 0.285 (alpha angle > 55°) for the aggregate anterosuperior quadrant.

A cam-type abnormality in athletes is a consequence of an alteration of the growth plate rather than reactive bone formation. High-level sports activity during growth may be a new and distinct risk factor for a cam-type deformity."

"an increased incidence of the tilt deformity in adolescents [occurs] with compulsory sporting activity when compared with adolescents without compulsory sporting activity (24% versus 9%). "

"Increased extension of the physeal cartilage onto the metaphysis has been associated with abnormal growth of the femoral head in Perthes disease"

"(A) A radial-sequence MR image of the hip of a 20-year-old basketball player taken at the 2 o’clock position is shown. (B) The diameter (d) of the femoral head, through the center of the head-neck axis, and the distance from a line orthogonal to the diameter to the lateral-most extension of the epiphysis (e) is measured. The epiphyseal extension is defined as e/d. (C) The measurement of the alpha angle on this same MRI slice is shown."

"Athletes had greater epiphyseal extension than control subjects at all positions"

"When comparing subgroups, there was a marked increase in epiphyseal extension in athletes with open physes compared with control subjects. After physeal closure, epiphyseal extension remained greater in athletes compared with control subjects; however, the difference between the two groups was reduced"

"although we detected an altered physeal shape in athletes, the ultimate impact on morphologic features of the proximal femur and subsequent joint function remains unclear."

"One may speculate whether this pattern represents irregular growth velocity or early focal physeal closure. Early physeal arrest has been described predominantly in the upper extremities of overhead throwing athletes"

Editor's Spotlight/Take 5: Growth Plate Alteration Precedes Cam-type Deformity in Elite Basketball Players (DOI 10.1007/s11999-012-2740-6)

"An increased prevalence for cam-type morphologic features in adolescents is not unique for basketball players. It has been described for various kinds of sports including soccer, ice hockey, running, and other activities. High-impact activities of various kinds seem to affect the developing proximal femur. I would be very surprised if intensity level does not also play an important role. I believe it is likely a combination of impact and intensity that are responsible for the changes. The most important issue to me, however, is the time over which changes to the growth plate were detected. Alteration of the physeal extension occurred between the ages of 9 and 16 years, while the growth plate was still open."

What's the current status of Lateral Synovial Joint Loading research?

Since the publication of Lengthening of Mouse Hindlimbs with Joint Loading, there hasn't been papers directly related to LSJL likely due to CH Turner's death.  Are the scientists working on Lateral Joint Loading or do we have to rely on our own?

Here's what Stuart J. Warden's(wrote a paper related to LSJL and is CH Turner's mentee) working on:

NIH-NIAMS (R15 AR056858)
Long-term skeletal effects of exercise during growth
Role: Principal investigator (1.5 academic months)
$231,000

NIH-NIAMS (R01 AR052018)
The function of neurotransmitters in bone biology[should be interesting but not related to LSJL]
Role: Subcontract co-investigator (3 summer months) [PI-Bliziotes (Oregon Health and Science University)]
$1,480,725 (IU subcontract $608,000)

National Space Biomedical Research Institute (MA01604)
Extent, causes, and countermeasures of impaired fracture healing in hypogravity
Role: Subcontract principal investigator (1 academic month) [PI-Midura (Cleveland Clinic)]
$1,789,968 (IU subcontract $338,472)

Department of Defense
Secreted Wnt antagonists in disuse-induced osteoporosis
Role: Co-investigator (1 academic month) [PI-Robling (IU School of Medicine)]
$1,062,982

NIH-NICHD (R01 HD057126)
Supplemental vitamin D and functional outcomes in early adolescence[this should be very interesting given that the cyp27b1 enzyme related to Vitamin D[it converts Vitamin D to the active metabolite] has possible height shortening effects]
Role: Subcontract co-investigator (0.5 academic months) [PI-Lewis (University of Georgia)]
$1,076,127

Hiroki Yokota seems to still be working on it, in his research goals:  He states that his goals are:

"Analysis of molecular and cellular mechanisms in bone remodeling
Development of mechanical loading devices for strengthening bone[C-class clamps?]
Development of therapeutic agents for enhancing bone formation[chemical methods likely]"

Hiroki Yokota seems to be focused now on developing some sort of grow taller pill possibly:


"Hiroki Yokota, a professor of biomedical engineering at IUPUI developed a drug that stimulates bone growth. Normally a technology transfer might have occurred, but we were able to leverage a lot of resources through Indiana University (IU). Indiana Clinical and Translational Sciences Institute (CTSI) resources allowed us to hire a private contractor to create a new formulation that was better. Lilly scientists were invited to provide input on what they would do next. Kelley School of Business life sciences students helped develop a marketing plan that identified a potential market for broken bones in addition to osteoporosis. As a result, we hope to use this model to speed the process from the research bench to the bedside."<-this pill may upregulate TGF-Beta and may help people grow taller.  This is definitely part of his therapeutic agents.


Here's another article about it:  It's apparently a new compound.  Here's a paper discussing the drug:  Salibrunal activates genes involved in anabolic processes in bones <-these anabolic processes can play a role in helping you grow taller.  Salubrinal inhibits GADD34-PP1 phosphatase which can cause cell death.  The timeline for the drug estimates to be about 8 years.  Here's another article about Salubrinal.


"I was originally exploring mechanical stimulation, which is similar to exercise, to strengthen bones. But by studying these mechanisms, I came across a molecular pathway that became the beginning of this discovery."<-He discovered the pathways behind salubrinal while researching LSJL thus salubrinal may be related to the pathways involved in LSJL.


"Older populations are increasingly affected by weakened and broken bones as aging cells are no longer able to produce sufficient levels of collagen, the protein from which bones derive their strength. Salubrinal prevents this cellular decline by strengthening the body’s “protein-producing machinery,” which creates collagen and keeps bones strong."<-Type II collagen helps you grow taller so perhaps salubrinal could have height increase affects as well.


"the cells just enjoy the body’s rescue response without really experiencing any new negative pressure"<-So Salubrinal is anabolic.

Here's more about Salubrinal:


Salubrinal promotes healing of surgical wounds in rat femurs.


"Phosphorylation of eukaryotic initiation factor 2α (eIF2α), transiently activated by various cellular stresses, is known to alleviate stress-induced cellular damage. Here, we addressed a question: does elevation of eIF2α phosphorylation by salubrinal (a pharmacological inhibitor of eIF2α dephosphorylation) enhance healing of bone wounds? We hypothesized that salubrinal would accelerate a closure of surgically generated bone holes by modifying expression of stress-sensitive genes. To examine this hypothesis, we employed a rat wound model. Surgical wounds were generated on anterior and posterior femoral cortexes, and salubrinal was locally administered on the anterior side. The results showed that, compared to a contralateral control, the size of surgical wounds was reduced by 10.8 % (day 10) and 18.0 % (day 20) on the anterior side (both p < 0.001), and 4.1 % (day 10; p < 0.05) and 11.1 % (day 20; p < 0.001) on the posterior side. In addition, salubrinal locally elevated cortical thickness and increased BMD and BMC. Pharmacokinetic analysis revealed that subcutaneous injection of salubrinal transiently increased its concentration in plasma followed by a rapid decrease within 24 h, and its half-life in plasma was 1.2 h. Salubrinal altered the phosphorylation level of eIF2α as well as the mRNA levels of ATF3, ATF4, and CHOP, and suppressed cell death induced by stress to the endoplasmic reticulum. In summary, the results herein demonstrate that subcutaneous administration of salubrinal accelerates healing of surgically generated bone holes through the modulation of eIF2α phosphorylation."


<-we need to see if increasing mRNA levels of ATF3, ATF4, and CHOP will increase height.  This study suggested that joint loading reduced phosphorylation of EIF2alpha.  So Salubrinal does not operate in the same way as joint loading.  But joint loading increased mRNA levels of ATF3.  Individuals deficient in ATF4 show reduced height.  ATF3 is linked to chondrocyte development as suggested in the study.


"To alleviate cellular injury or initiate apoptotic cell death, cells induce an integrated stress response (ISR). During ISR, phosphorylation of the alpha subunit of eukaryotic initiation factor 2α (eIF2α) is activated, and this ISR-driven phosphorylation blocks an exchange process of eukaryotic translation initiation factor 2B from GDP-bound eIF2 to GTP-bound eIF2. Consequently, the global translation-initiation is suppressed except for a group of specific genes whose expression is presumably crucial for an adaptive response for survival. Thus, the modulation of a phosphorylated level of eIF2α potentially alters the fate of damaged tissues."<-so manipulating phosphorylation of EIF2alpha alters fate of damaged tissues so it may alter growth(there is always stress to cells even in healthy growth plates).  Note though that chondrocyte apoptosis may play a vital role in height growth.


"First, in response to 10 μM salubrinal, an increase in the phosphorylation level of eIF2α (eIF2α-p) was observed at 3 and 5 h. Second, this increase in eIF2α-p was dosage dependent for the administration of 10 and 50 μM salubrinal on days 1 and 2"

"The rationale for administration of salubrinal is that phosphorylation of eIF2α is cytoprotective during ISR, and salubrinal is used to protect cells from ISR and apoptosis"<-But again apoptosis may be important to growth plates.

"Using mice without bone wounds, that a salubrinal-treated femur had a higher BMD than a contralateral control femur and a vehicle control femur. These observations together with pharmacokinetic results indicated local effects of salubrinal, when applied subcutaneously. However, we also observed a gradual increase in body weight of the salubrinal-treated rats compared to vehicle controls, suggesting a possibility of a systemic side effect"<-could the increase in body weight be due to bone length?

The study: Reactive oxygen species and p38 MAPK regulate Bax translocation and calcium redistribution in salubrinal-induced apoptosis of EBV-transformed B cells states that salubrinal increased Ca2+ in B cells and Ca2+ uptake in mitochondria although this does not indicate that the same would occur in MSCs.  The general mechanism of action for salubrinal is described as "phosphorylated eIF2α-mediated survival has been known to lead to cap-dependent protein translation inhibition, activation of PI3K, induction of NF-kB, degradation of p53, and decreased load of nascent proteins in the ER, whereas phosphorylated eIF2α-mediated cell death has been known to decrease cyclin D1 translation and induce the ATF4-CHOP pathway"


He's in close proximity with Dr. Plopper who can help him with LSJL:

"Differentiation of human mesenchymal stem cells (hMSC) plated in/on defined ECM proteins: We are defining the effect of ECM contact on human mesenchymal stem cell differentiation. Our hypothesis is that contact with distinct ECM proteins stimulates specific signaling pathways that ultimately control the differentiation of these cells into bone-, cartilage-, or fat-producing cells."


This is key to us, as ECM proteins are one thing that are expressed during puberty but not post fusion(they can be induced though). Dr. Plopper even touches a course on ECM at Purdue.  Weeks 5-10 could help us grow taller, especially week 9 is about the ECM-cytoskeleton connection.

As could Dr. Kotha:


"Dr Kotha’s group studies the role of mechanical loading on damage and repair to bone at multiple length scales. They characterize how mineral and organic in bone support its deformation as load is applied. When this is combined with novel non-invasive ultrasound based technologies[Dr. Kotha could help us with LIPUS] being developed, the overall goal is to make devices that can be used to monitor the risk of bone failure at specific sites. They also evaluate how cells sense deformation of bone and what molecular pathways are activated in response to loading."


So maybe the reason why there haven't been LSJL studies lately is that Yokota, Kotha, and Plopper are working on something big.  If you want to go to the school of growing taller you should go to department of biomedical engineering at Rensselaer.


Here's what Ping Zhang is working on:

"Fabrication and application of unique loading devices."<-Again C-class clamp?

"Anatomical, physiological and mechanical characterization of bones and joints under mechanical loading[lateral joint loading].  Identification of cellular and molecular mechanisms involved in mechanotransduction of bone and joint cells."

Increasing Height with Acteoside and Harpagoside?

The herbalist Alkoclar has a new height increasing formula that's come out involving Acteoside and Harpagoside(Arth Aid - All Natural Relief for Arthritis - 100 Capsules).  Alkoclar was the one behind the Methyl Protodioscin and Desmethyl Icariin combo.  It's unlikely that any chemical method on it's own is likely to increase height as chemicals in general have not been found to be as effective at inducing chondrogenic differentiation with the exception of TGF Beta.  Osteoblasts are not as effective at increasing height likely due to the OPG/RANKL gradient not encouraging bone deposition on the longitudinal ends of bones.  Still herbal remedies like Acteoside and Harpagoside can act as supplements to a height increase regime.  They are also both anti-oxidants which are good for height growth although some free radicals are needed for certain chemical reactions.

How can Acteoside and Harpagoside(also known as Devil's Claw) help with increasing Height?


[Effect of a Harpagophytum procumbens DC extract on matrix metalloproteinases in human chondrocytes in vitro].

"interleukin 1beta (IL-1beta) and tumor necrosis factor alpha (TNF-alpha) [are inflammatory cytokines]. The increased release of cytokines leads to an enhanced production of matrix-degrading enzymes (MMPs). Direct antirheumatic effects of an extract of the secondary root of the African devil's claw (Harpagophytum procumbens DC) on the production of MMPs in IL-1beta-stimulated human chondrocytes were examined.  Extracts of Harpagophytum decreased significantly production of MMPs (MMP-1, MMP-3, MMP-9) in chondrocytes{remember though that an MMP-9 deficiency causes reduced height, MMP-3 also plays an important role in vascularizing the growth plate}. The IL-1beta-induced production of MMPs was significantly reduced by both a JM-extract (Jucurba) containing 210 mg dry extract and JF-extract (Jucurba forte) containing 480 mg dry extract.  The effect of JF-extract on the MMP-synthesis was more pronounced in untreated and cytokine-stimulated chondrocytes when compared with the effect of the JM-extract. The JF-extract showed a higher efficacy than the JM-extract."

Just look there's good and bad cholesterol there may be good and bad MMPs.  Devil's Claw seems to reduce the production of the good MMPs.

I couldn't get this full study.

Harpagoside does reduce COX-2 and COX-2 is bad for height growth(it's bad for height growth by way of MMP-13)  To grow taller we want to block MMP-13 expression and enhance MMP-3(LSJL upregulates MMP-3) and MMP-9 expression.

Harpagoside suppresses lipopolysaccharide-induced iNOS and COX-2 expression through inhibition of NF-kappa B activation.

"Preparations of Harpagophytum procumbens [is] known as devil's claw. Pharmacological evaluations have proven tthis herbal drug as an anti-inflammatory and analgesic agent. [We investigate] the mechanism of action of harpagoside, one of the major components of Harpagophytum procumbens, using human HepG2 hepatocarcinoma and RAW 264.7 macrophage cell lines. Harpagoside inhibited lipopolysaccharide-induced mRNA levels and protein expression of cyclooxygenase-2 and inducible nitric oxide in HepG2 cells. These inhibitions appeared to correlate with the suppression of NF-kappaB activation by harpagoside, as pre-treating cells with harpagoside blocked the translocation of NF-kappaB into the nuclear compartments and degradation of the inhibitory subunit IkappaB-alpha. Harpagoside dose-dependently inhibited LPS-stimulated NF-kappaB promoter activity[NF-kappaB can encourage chondrogenic differentiation however] in a gene reporter assay in RAW 264.7 cells, indicating that harpagoside interfered with the activation of gene transcription. Inhibition of the expression of cyclooxygenase-2 and inducible nitric oxide by harpagoside involves suppression of NF-kappaB activation."

But I don't think Harpagoside can help increase height as COX-2 and MMP-13 may actually benefit height increase.  Although LPS likely takes inflammatory molecules above equilibrium levels and COX-2 and MMP-13 are likely biphasic with an equilibrium level being most beneficial for height increase.

"Treatment of RAW 264.7 cells with LPS (100 ng/ml) for 24 h significantly increased the release of NO (37 ± 3 μM) by approximately 12-fold compared to that of the untreated cells (3 ± 2 μM)"<-So NO levels were likely well above equilibrium.

"translocation of IκB-α from the cytosol to the nucleus following LPS treatment was blocked by harpagoside."

Acteoside also inhibits MMP-9:

Acteoside inhibits PMA-induced matrix metalloproteinase-9 expression via CaMK/ERK- and JNK/NF-κB-dependent signaling.

"Acteoside [is] an active phenylethanoid glycoside found in bitter tea and many medicinal plants. [We] determine the effect of acteoside on tumor invasion and migration; the possible mechanisms involved in this inhibition were investigated in human fibrosarcoma HT-1080 cells. We employed invasion, migration and gelatin zymography assays to characterize the effect of acteoside on HT-1080 cells.  Acteoside suppresses phorbol-12-myristate-13-acetate (PMA)-enhanced matrix metalloproteinase-9 (MMP-9) expression at the protein, mRNA, and transcriptional levels through the suppression of NF-κB activation. Acteoside repressed the PMA-induced phosphorylation of ERK1/2 (ERK, extracellular regulated kinase) and JNK1/2. Acteoside decreased the PMA-induced influx of Ca(2+) and repressed PMA-induced calmodulin-dependent protein kinase (CaMK) phosphorylation. Treatment with BAPTA/AM, W7, or capsazepine markedly decreased PMA-induced MMP-9 secretion and cell migration, as well as ERK and JNK/NF-κB activation.  Acteoside inhibited PMA-induced invasion and migration of human fibrosarcoma cells via Ca(2+)-dependent CaMK/ERK and JNK/NF-κB-signaling pathways."

Acteoside also inhibited MMP-2.

"MMP-9 can be stimulated by inflammatory cytokines, epidermal growth factor, or phorbol-12-myristate-13-acetate (PMA)"

"calcium (Ca2+) regulates the expression and activation of MMPs, with particular involvement in the control of MMP-12 activity. Increased extracellular Ca2+ levels induce MMP-9 expression in human keratinocytes, inhibiting the influx of Ca2+ and decreasing the mRNA expression of MMP-2. Furthermore, the modulation of intracellular Ca2+ levels can alter the secretion of MMP-1 from migrating keratinocytes"

"TRP vanilloid-type 1 (TRPV1) is one of the several nonselective cationic channels, the activation of which induces Ca2+ influx; however, this influx can be inhibited by a specific antagonist, capsazepine (CPZ). Antagonists of cannabinoid receptors 1 and 2 either alone or in combination with CPZ significantly suppress both cannabidiol-induced tissue inhibitors of metalloproteinase (TIMP)-1 expression and the activation of MAPK p38 and extracellular regulated kinase 1/2 (ERK1/2)"

"TRPV1 can be phosphorylated by PKA, PKC, and Ca2+/calmodulin (CaM)-dependent kinase II (CaMKII)"

Acteoside may increase extracellular Ca2+ thereby inhibiting the influx of Ca2+ into the cell.  This is good if the levels of Ca2+ are too high but may inhibit anabolic effects if the Ca2+ are within physiological levels.

So at this time I don't believe either acteoside nor harpagoside has height increase potential as MMP-9 deficiency results in stunted growth.

Of course, if you inhibit Extracellular Matrix degradation in the cartilage you will get taller in your joints.  The height will go away if you stop taking it though.  In that case, you should just take Hyaluronic Acid which more clearly benefits the ECM.

Organic Development of chondrocytes

One of the thoughts as to why humans cannot grow post fusion is due to the belief that chondrocytes cannot grow organically.  There are lots of stem cells present in the bone marrow, as can be seen by the large amount of red dots present in the bone marrow such as in the link shown above.  If you look at these diagrams of epiphyseal distraction, you can see that everything else ossifies aside from the hyaline cartilage growth plate line(there is ossification within the hyaline cartilage as a result of microfracture; growth plate injuries often create a bony bridge in the fracture site; even in the very gradual epiphyseal distraction method there was still some ossification).

The goal of lateral synovial joint loading is to increase interstitial fluid flow by using methods such as the table clamp to send mesenchymal stem cells to the hyaline cartilage growth plate line where they will undergo a chondrogenic lineage.  If you look at the rat's under LSJL slide(right click and click on view image to see it in it's entirety) you can see stem cells being captured and taken into the hyaline cartilage growth plate line in slide B.  Can chondrogenesis be induced organically despite growth plate fusion?

Hydrostatic pressure enhances chondrogenic differentiation of human bone marrow stromal cells in osteochondrogenic medium. 

Interstitial fluid flow such as that created by using the table clamp or pressing on the epiphysis of the bone with the dumbell increases hydrostatic pressure. 

"This study demonstrated the chondrogenic effect of hydrostatic pressure on human bone marrow stromal cells[LSJL encourages chondrogenic differentiation of stromal cells, stem cells are a subset of stromal cells] (MSCs) cultured in a mixed medium containing osteogenic and chondrogenic factors. MSCs seeded in type I collagen sponges were exposed to 1 MPa of intermittent hydrostatic pressure at a frequency of 1 Hz for 4 h per day for 10 days[1 Hz means load for a duration of 1 second, 1 MPa is the type of pressure used in steam locomotives, standard atmospheric pressure is 0.1 MPa at sea level], or remained in identical culture conditions but without exposure to pressure. we compared the proteoglycan content of loaded and control cell/scaffold constructs with Alcian blue staining. We evaluate the change in mRNA expression of selected genes associated with chondrogenic and osteogenic differentiation (aggrecan, type I collagen, type II collagen, Runx2 (Cbfa-1), Sox9, and TGF-beta1)[Most of these genes are associated with chondrogenic differentiation with the exception of Runx2]. With the hydrostatic pressure loading regime, proteoglycan staining increased markedly. the mRNA expression of chondrogenic genes such as aggrecan, type II collagen, and Sox9 increased significantly. We also saw a significant increase in the mRNA expression of type I collagen, but no change in the expression of Runx2 or TGF-beta1 mRNA. This study demonstrated that hydrostatic pressure enhanced differentiation of MSCs in the presence of multipotent differentiation factors in vitro, and suggests the critical role that this loading regime may play during cartilage development and regeneration in vivo." 

"MSCs can differentiate along the chondrogenic lineage in three-dimensional culture conditions in the presence of soluble regulatory factors such as members of the TGF-β family. Other chondrogenic influences on the differentiation of mesenchymal cells are certain types of mechanical loading. Hydrostatic pressure and compressive loading[lateral joint loading results in compressive forces against the chondrocytes as well as well] increased cartilage matrix accumulation in cultured MSCs and in embryonic limb bud cells in vitro."

"Without the addition of TGF-β, the effects of mechanical stimuli are unclear; in some studies mechanical stimuli alone was sufficient to induce skeletal progenitor cells to adopt the cartilage phenotype"

"Loaded scaffolds were exposed to 1 MPa of pressure at a loading frequency of 1 Hz for 4 h per day, as this loading regime had previously been found to be effective in inducing chondrogenesis."

"The duration of the experiment was 10 days, as the results of a pilot study indicated that chondrogenic matrix accumulation could be visualized at this timepoint."

It's unclear whether the stem cells remained in an hypoxic environment.

"the hydrostatic pressure protocol increased the mRNA expression of both collagen I and II"

This mixture could lead to fibrocartilage rather than hyaline cartilage the authors speculate.

This was the medium used in the study: "1.0 mg/mL insulin, 0.55 mg/mL transferrin, 0.5 μg/mL sodium selenite, 50 mg/mL bovine serum albumin, 470 μg/mL linoleic acid), 10 nM dexamethasone, 50 μg/mL l-ascorbic acid, 1 mM sodium pyruvate, 40 μg/mL l-proline, 0.584 g/L l-glutamine, and 100 IU each penicillin and streptomycin."

Most of this aside for example the dexamethasone is available in the human body.

Previously, we found that periosteal loading increased expression of TGF-Beta whereas dynamic loading of chondrocytes increased expression of aggrecan and type II Collagen.  Dynamic loading of chondrocytes did not increase expression of Sox9 which is very important in endochondral ossification.  In the LSJL, studies Sox9 was not upregulated, however, the scientists only studied gene expression by bone and not by cartilage.  This study found that TGF Beta1 was not upregulated in contrast to LSJL which found that TGF Beta 1 was upregulated.  A possible explanation for this is that the collagen sponges contained no periosteum.   

Bone contains a lot of Type I collagen which was the medium used for the stem cells in the study.  So that part of the study is easy to recreate.  Hydrostatic pressure is pretty synonymous with interstitial fluid flow so that part of the study can be recreated by LSJL.  One thing you'll notice is that the stem cells were not in the hyaline cartilage growth plate line.  This means that chondrogenesis can occur organically even without an open growth plate.

Effects of hydrostatic pressure and transforming growth factor-beta 3 on adult human mesenchymal stem cell chondrogenesis in vitro.

"[What are] the effects of intermittent hydrostatic pressure (IHP) and transforming growth factor-beta 3 on chondrogenesis of adult human mesenchymal stem cells (hMSCs) in vitro? Chondrogenic gene expression was determined by quantifying mRNA signal levels for SOX9, a transcription factor critical for cartilage development and the cartilage matrix proteins, aggrecan and type II collagen. Extracellular matrix production was determined by weight and histology. IHP was applied to hMSCs in pellet culture at a level of 10 MPa[the amount of pressure used by a pressure washer] and a frequency of 1 Hz for 4 h per day for periods of 3, 7, and 14 days. hMSCs responded to addition of TGF-beta 3 (10 ng/mL) with a greater than 10-fold increase (p < 0.01) in mRNA levels for each, SOX9, type II collagen, and aggrecan during a 14-day culture period. Applying IHP in the presence of TGF-beta 3 further increased the mRNA levels for these proteins by 1.9-, 3.3-, and 1.6-fold, respectively, by day 14. Chondrogenic mRNA levels were increased with just exposure to IHP. Extracellular matrix deposition of type II collagen and aggrecan increased in the pellets as a function of treatment conditions and time of culture. This study demonstrated adjunctive effects of IHP on TGF-beta 3-induced chondrogenesis." 

"Normal hMSCs (positive for CD105, CD166, CD29, and CD44 and negative for CD14, CD34, and CD45) from normal human bone marrow"

"Hip joint contact pressures up to 18 MPa exist during some activities, such as rising from a chair[but note this pressure is not applied within the epiphysis of the bone]"

"The effect of IHP on SOX9 expression may involve changes in cytoskeletal organization, G-protein activation, and transcription factor translocation into the nucleus."

Here's an example of what a stem cell looks like with intermittent hydrostatic pressure applied:

The HE and Saf-O refers to a form of staining.  TGF-Beta3 +IHP is better but IHP is good enough to induce chondrogenic differentiation.  "IHP applied with and without addition of TGF-Beta3 also increased gene expression for all three chondrogenic proteins[Aggrecan, Sox9, Type II Collagen]."

I can't really think of a good way to measure pressure for LSJL as all the pressure gauges are for tires and water level.

Stem cells are sensitive to mechanotransduction which makes their mechanosensitivity important.  Hydrostatic Pressure induces dynamic compression as it's the flow of the fluid that makes the compression dynamic.


Dose- and time-dependent effects of cyclic hydrostatic pressure on transforming growth factor-beta3-induced chondrogenesis by adult human mesenchymal stem cells in vitro.

"MSCs were exposed to 0.1, 1, and 10 MPa of IHP at a frequency of 1 Hz for 4 h/day for 3, 7, and 14 days in the presence of transforming growth factor (TGF-beta3). Chondrogenesis was characterized by gene expression, macromolecule production, and extracellular matrix deposition. Exposure of hMSCs to 0.1 MPa of IHP increased SOX9 and aggrecan mRNA expression by 2.2- and 5.6-fold, respectively, whereas type II collagen mRNA expression responded maximally at 10 MPa. Production of sulfated glycosaminoglycan responded to IHP of 1 MPa and 10 MPa, whereas collagen levels increased only at 10 MPa. Morphologically, matrix condensation occurred with increased IHP, concomitant with collagen expression. Different levels of IHP differentially modulate hMSC chondrogenesis in the presence of TGF-beta3."

"hydrostatic pressure (50 MPa) [induces] heat shock proteins[HSP70]."  5MPa to cartilage increases TGFB3 levels.

"[Stem] Cells were positive for CD105, CD166, CD29, and CD44 and negative for CD14, CD34, and CD45."

Dynamic compression regulates the expression and synthesis of chondrocyte-specific matrix molecules in bone marrow stromal cells.

"[We] investigate the mechanotransduction of bovine bone marrow stromal cells (BMSCs) through the interactions between transforming growth factor beta1 (TGF-beta1), dexamethasone, and dynamic compressive loading. The addition of TGF-beta1 increased cell viability, extracellular matrix (ECM) gene expression, matrix synthesis, and sulfated glycosaminoglycan content over basal construct medium[Stimulating the other bone cells increases TGF-Beta1 which induces all these pro-chondrogenic effects]. The addition of dexamethasone further enhanced extracellular matrix gene expression and protein synthesis[dexamethasone is not needed]. There was little stimulation of ECM gene expression or matrix synthesis in any medium group by mechanical loading introduced on day 8. There was significant stimulation of ECM gene expression and matrix synthesis in chondrogenic media by dynamic loading introduced on day 16. The level of stimulation was also dependent on the medium supplements, with the samples treated with basal medium being the least responsive and the samples treated with TGF-beta1 and dexamethasone being the most responsive at day 16. Collagen I and collagen II gene expressions were more responsive to dynamic loading than aggrecan gene expression. Dynamic compression upregulated Smad2/3 phosphorylation in samples treated with basal and TGF-beta1 media. Interactions between mechanical stimuli and TGF-beta signaling may be an important mechanotransduction pathway for BMSCs, and they indicate that mechanosensitivity may vary during the process of chondrogenesis."

TGF-Beta1 affects mechanical sensitivity of bone marrow mesenchymal stem cells.

"TGF-β signals from the cell surface via a transmembrane serine/threonine kinase receptor complex. Upon ligand binding, the type II receptor subunit engages and transphosphorylates a type I receptor subunit (TβRI), which in turn phosphorylates the receptor-activated Smad proteins (R-Smads) Smad2 and Smad3[So we want TGF-Beta to be released from the osteoblast cell surface and then engage a type I receptor subunit in a mesenchymal stem cell]. A protein complex with Smad4 forms with the activated R-Smads and translocates into the nucleus, where the complex interacts with additional transcription factors, binding to the promoters of responsive genes and regulating their expression by cooperating with other activators or repressors. In addition to the Smad pathway, TGF-β  [activates] signaling pathways, including p38 mitogen-activated protein kinase (MAPK) and protein kinase C (PKC). TGF-β signaling [activates] the p38 MAPK pathway through activation of mitogen-activated protein kinase kinase 1 and subsequent ERK/ELK signaling. G-protein-dependent activation of PKC results from TGF-β stimulation of growth plate chondrocytes. TGF-β responsiveness may require the activation of the R-Smad2/4 complexes[lack of response to LSJL may be a failure of the R-Smad2/4 complex], as well as other signaling pathways"

"dexamethasone induced Sox9 upregulation in the pluripotent mesoblastic C1 line"

TGF-Beta1 plus Dynamic Compression(hydrostatic pressure) = chondrogenic differentiation = height growth

Evidence that hydrostatic pressure can induce differentiation in vitro:

The mechanical environment of bone marrow: a review.

"Bone marrow is a viscous tissue that resides in the confines of bones and houses pluripotent stem cells. Due to its confinement by bones, the marrow has a unique mechanical environment which has been shown to be affected from external factors, such as physiological activity and disuse. The mechanical environment of bone marrow can be defined by determining hydrostatic pressure, fluid flow induced shear stress, and viscosity. The hydrostatic pressure values of bone marrow reported in the literature vary in the range of 10.7-120 mmHg for mammals[This is the value we are trying to increase with LSJL], which is generally accepted to be around one fourth of the systemic blood pressure. Viscosity values of bone marrow have been reported to be between 37.5 and 400 cP for mammals, which is dependent on the marrow composition and temperature. Marrow's mechanical and compositional properties have been implicated to be changing during common bone diseases, aging or disuse. In vitro experiments have demonstrated that the resident mesenchymal stem and progenitor cells in adult marrow are responsive to hydrostatic pressure[Lateral Loading can affect mesenchymal stem cells], fluid shear or to local compositional factors such as medium viscosity. Therefore, the changes in the mechanical and compositional microenvironment of marrow may affect the fate of resident stem cells in vivo as well, which in turn may alter the homeostasis of bone."

"The close proximity of marrow within medullary cavities of bones subject the marrow to physiological loads as well"<-Lateral loading can affect the bone marrow.

"When the marrow hematopoietic activity increases, adipose tissue undergoes resorption to provide more space, or vice versa."<-If Adipose Tissue is inversly related to bone marrow activity then fat gain may be bad for height growth.

"Bone nutrient vessels enter the marrow cavity to make connections with marrow vessels. Small arteries of marrow also enter the bone, make a loop and return back to where they originated from"<-So it could be possible for chondrogenic stem cells to travel to where they are needed.

"The fixed femurs of rats in vivo [had loads applied] ranging from 0 to 12.25 kg for 1 min. Pressure values [rised] two-fold (12–14 mmHg increase) upon loading."12.25 kg is about 25 lbs so 25lbs increases hydrostatic pressure by about 12-144 mmHG, a very useful measurement for performing LSJL.  Note that larger animals like dogs tend to have larger hydrostatic pressure values than mice.

"Contraction of the quadriceps muscles with electrical stimulation [resulted] in a pressure increase of 60 mmHg"<-So if you contract your muscles at the same as performing LSJL you can get a larger pressure gain.

"In a tubular bone, the nutrient artery enters the marrow cavity, runs longitudinally in the center, then branches out toward the endosteum of the surrounding bone, leading to specialized vascular structures known as sinuses or sinusoids. Several of these sinuses may then combine to form collecting sinuses which lead to the central sinus or vein. This vein runs longitudinally next to the nutrient artery. Blood in marrow flows from the center toward the bone and then returns back to the center. This structural configuration yields high numbers of vessels and sinuses in the periphery (resulting in a slower flow rate of blood and higher surface area) where most of the exchange occurs. Therefore, hematopoiesis is maximal in the closer proximities to the bone surface leaving the central parts with relatively little hematopoietic activity. Due to this fact, it is possible to observe a transition region between red marrow and fatty marrow radially (red marrow being closer to the endosteal surfaces. Similarly, a longitudinal macroscopic distribution is observed as red marrow in the proximal half and fatty marrow in the distal half of the bones. Bone and marrow are connected by vasculature. Bone nutrient vessels enter the marrow cavity to make connections with marrow vessels. Small arteries of marrow also enter the bone, make a loop and return back to where they originated from.."

"There are numerous in vitro studies subjecting marrow-derived mesenchymal stem cells to high hydrostatic pressures (750–75,000 mmHg) in an effort to induce chondrogenic phenotype. Even though the chondrogenic differentiation of the progenitor cells does not take place in marrow cavity[meaning they eliminate the variable of blood loss via veins], those studies are related in indicating the sensitivity of the progenitors to the magnitude and the mode (cyclic, intermittent or static) of loading. In general, higher pressure (∼75,000 mmHg) over lower (∼750 mmHg) and intermittent loading over static have proved to be more effective in chondrogenesis"<-That's quite a lot of pressure.

Here's what the author(Ozan Akkus) has to say chondrogenic differentiation not taking place in the marrow cavity:

"It is more of an opinion and the statement of an observation. We believe that this may have something to do, in part, with the mechanical environment of the marrow, that it is not conducive to chondrogenesis. Otherwise, there may be other factors in play, such as suppression by neighboring hematopoietic cells. As we know, marrow is highly vascular and cartilage is avascular. So the vascularity is suppressive to chondrogenesis."

"Layout of bone marrow in a cross-sectional view of a tubular bone. Bone (B) is surrounding the bone marrow (BM). Central artery (CA) and central vein (CV) are running parallel to each other and longitudinally along the long bone (perpendicular to the plane of the page). The central artery and central vein branch toward the periphery to form arterioles (A) and sinusoids (S) which then combine and join with the central vein. Hematopoietic space (H) is interspersed by the sinuses. Developing red blood cells and granulocytic cells appear in the hematopoietic space. Megakaryocytes develop subjacent to the endothelium of marrow sinuses. It is possible to observe the radial distribution of marrow as the yellow marrow in the central regions and the red marrow in the periphery. (b) A toluidine-blue stained section taken transversely to the longer axis of a tubular bone. The micrograph displays the endosteal junction between bone and marrow (125×). The distribution of abundant number of red blood cells indicates that the bone marrow is hematopoietic"<-this is the cortical bone however and we are looking to induce new growth plates in the epiphysis.

Dynamic compression stimulates proteoglycan synthesis by mesenchymal stem cells in the absence of chondrogenic cytokines.

"Dynamic compression was applied to agarose hydrogels seeded with bone marrow-derived adult equine MSCs. In the absence of the chondrogenic cytokine transforming growth factor beta (TGFbeta), dynamic compression applied for 12 h per day led to significantly greater proteoglycan synthesis than in unloaded TGFbeta-free cultures, although at a rate that was approximately 20% to 35% of unloaded TGFbeta cultures. These data suggest that the emergence of aggrecan dominated a chondrogenic response to loading as increases in proteoglycan synthesis. Cross-sectional analyses were conducted to subjectively identify potential spatial distributions of heterogeneous differentiation. In loaded samples, cell viability and metachromatic staining was low near the porous compression platen interface but increased with depth[the increase in depth relates to hydrostatic pressure as the more depth the larger the HP], reaching levels in the lower portion of the hydrogel that resembled unloaded TGFbeta cultures. These results suggest that the combination of high hydrostatic pressure and low dynamic strain and fluid flow had a stronger effect on chondrogenesis than did low hydrostatic pressure coupled with high dynamic strain and fluid flow. Next, the 12-h per day loading protocol was applied in the presence of TGFbeta. Biosynthesis in loaded cultures was less than in unloaded TGFbeta samples[so perhaps you don't want to increase TGF-Beta levels when joint loading]. Taken together, these data suggest that the duration of loading necessary to stimulate mechanoinduction of MSCs may not be optimal for neo-tissue accumulation in the presence of chondrogenic cytokines[but maybe higher levels of TGF-Beta just mean loading has to be applied for a shorter period of time]."

"quantitative extracellular matrix (ECM) synthesis studies have demonstrate that MSCs are capable of synthesizing neo-tissue on the order of that reported for chondrocyte culture."<-The MSCs in the bone marrow are capable of forming new growth plates(which is a chondrocyte culture).

"Dynamic hydrostatic loading[hydrostatic loading is performed by submerging a compound deeper under water], on the order of what occurs during routine joint functioning, has been proven to upregulate chondrogenic gene expression and cartilage-like protein synthesis in the presence of TGFβ."

"Experiments used a sinusoidal dynamic compression protocol of 2.5% strain amplitude superimposed on a 7.5% static offset strain at a frequency of 0.3Hz in displacement control; these loading parameters are within the physiological range of moderate, low-amplitude strain when applied to intact cartilage explants. At the initiation of loading, these parameters created maximum stress response of approximately 2kPa[a Kilopascal is 1000 of a MegaPascal, usually Hydrostatic Pressure is measured in MPa so the pressure range was well below typical Hydrostatic pressure].
For loading in the absence of TGFβ, two dynamic compression duty cycles were explored. The first protocol was defined by 6-h cycles consisting of 45-min periods of compression followed by 5h and 15min of free-swelling culture[I don't think we'd be able to mimic this cycle]. Each 6-h cycle was applied four times, followed by 24h of free-swelling culture such that loading was applied on alternate days. In the second protocol, 45min of dynamic compression was followed by 45min of free-swelling culture throughout the loading period, resulting in 12h of loading per day. In the presence of TGFβ, MSC-seeded agarose samples were loaded using the second, 12-h/d dynamic compression protocol."

"Given the low levels of biosynthesis in adult equine MSC cultures in the absence of TGFβ[In alternate day loading], these effects of dynamic compression were considered negligible."  So during LSJL, the loading likely has to be performed at least daily.

"H-proline and S-sulfate incorporation in dynamic compression samples were 20% of those of TGFβ+ samples (p<0.05), whereas GAG accumulation in loaded cultures was 34% of that in TGFβ+ samples"<-this was twelve hours a day.

"Adjustment of the frequency of the applied loading from alternate day to daily loading, applied over 12h/d, resulted in significantly greater proteoglycan synthesis than in TGFβ− cultures. These data suggest a chondrogenic response to loading because the emergence of aggrecan production, the major proteoglycan in cartilage, probably dominates S-sulfate incorporation and GAG accumulation. Therefore, the duration of the application of loading appeared to be a critical factor in stimulating proteoglycan synthesis, as occurs during chondrogenesis."<-So sustained loading may work in the presence of low pressure
 
"In the three dynamic compression samples, the zones near the chamber base contained the highest density of viable cells, suggesting that chondrogenesis was preferentially stimulated in deep zones of maximal hydrostatic pressure."

"The decrease in ECM synthesis and accumulation demonstrated an inhibitory effect of loading in TGFβ."

"Previously, finite element analyses have predicted compression-induced hydrostatic pressures of less than 1kPa in agarose constructs compressed using near-physiological deformation levels. Although this value is significantly less than that measured in joints during normal activities, it is closer to the 0 to 3kPa reported for continuous passive motion."<-Normal motion involves 0 to 3 kPa.  So, if you walked for 12 hours a day at 3 kPascals you might be able to grow taller?

We're not going to be able to perform loading for the periods of time in this study but hopefully we're performing our stimulus at a pressure much higher than 2 kPa so we can do the loading perhaps 1/500th of the time for 1 MPa.