New LSJL related studies

Here's a patent I found that has some insights in LSJL by Hiroki Yokota and Ping Zhang.

There have been a few LSJL related studies lately but unfortunately none have targeted bone lengthening like previous ones.  However, they can still offer insight into how LSJL works and the best way to perform LSJL.

The EIF2AK3 gene is associated with height.  Phosphorylation of EIF2alpha reduces protein synthesis.  So reduction of EIF2alpha phosphorylation is anabolic.

Loading- and Unloading-Driven Regulation of Phosphorylation of eIF2α

"Radiation, nutrient deprivation, hypoxia, and stress to the endoplasmic reticulum induce integrated stress responses (ISR), which activate phosphorylation of eukaryotic initiation factor 2 - subunit α (eIF2α). This activation of eIF2α phosphorylation decreases translational efficiency of a majority of proteins for preventing stress-driven apoptosis. We employed C57BL/6 wildtype and GCN2 knockout mice and applied ankle loading[LSJL] and hindlimb suspension. loading reduced the level of eIF2α phosphorylation regardless of the presence of GCN2 gene, while unloading elevated its phosphorylation."

So LSJL reduces EIF2alpha phosphorylation. EIF2alpha increases apoptosis.  It's possible that reducing apoptosis is beneficial to height increase.  GCN2 increases EIF2a-p.  14-week old mice were used.

"The loading was applied to the left ankle in the lateral-medial direction with 0.5 N force (peak-to-peak) at 5 Hz for 5 min. The right tibia was used as a sham loading control, where the right ankle was placed under the loading rod for 5 min in the same procedure used for the left ankle without applying a voltage signal to the loader."<-so this helps us know the importance of the pizeoelectric current on LSJL.  Note that bone formation generates a pizeoelectric current as well.

The pizeoelectric current and thus LSJL reduced EIF2alpha phosphorylation but not directly EIF2alpha levels.  The reduction in EIF2a-p was greater in GCN2-KO mice.

HRI, PERK, and PKR can also activate EIF2a-p.

"Compared to the control, the average amount of the reduction in the level of eIF2α-p was 29%".  The study also mentions a linkage between EIF2alpha and growth(see Fig. 4) so less EIF2alpha phosphorylation equals more growth.

EIF2alpha may relate to IGF2 which is why the LSJL scientists are focused on it.

Endoplasmic reticulum stress disrupts placental morphogenesis: implications for human intrauterine growth restriction.

"Eif2s1(tm1RjK) mice, in which Ser51 of eukaryotic initiation factor 2 subunit alpha (eIF2α) is mutated, display a 30w increase in basal translation. In Eif2s1(tm1RjK) placentas, we observed increased ER stress and anomalous accumulation of glycoproteins in the endocrine junctional zone (Jz), but not in labyrinthine zone where physiological exchange occurs. Placental and fetal weights were reduced by 15% (97mg to 82mg) and 20% (1009mg to 798mg) respectively. Mouse embryonic fibroblasts (MEFs) were derived from Eif2s1(tm1RjK) mutants. These MEFs exhibited ER stress, grew 50% slower and showed reduced Akt-mTOR signalling compared to wild-type cells. Conditioned medium (CM) derived from Eif2s1(tm1RjK) MEFs failed to maintain trophoblast stem cells in a progenitor state, but the effect could be rescued by exogenous application of FGF4 and heparin. ER stress promoted accumulation of pro-Igf2 with altered glycosylation in the CM without affecting cellular levels, indicating that the protein failed to be processed after release[so EIF2alpha helps process IGF2]. Igf2 is the major growth factor for placental development; indeed, activity in the Pdk1-Akt-mTOR pathways was decreased in Eif2s1(tm1RjK) placentas, indicating loss of Igf2 signalling. We observed premature differentiation of trophoblast progenitors at E9.5 in mutant placentas, consistent with the in vitro results and with the disproportionate development of the labyrinth and Jz seen in placentas at E18.5. Similar disproportion has been reported in the Igf2-null mouse. ER stress adversely affects placental development, and that modulation of post-translational processing, and hence bioactivity, of secreted growth factors contributes to this effect. Placental dysmorphogenesis potentially affects fetal growth through reduced exchange capacity."

"the change in placental structure and Akt signalling observed in the Eif2s1tm1RjK  mice is similar to the Igf2 null placenta, with the same disproportional reduction of the Jz and Lz, and smaller placenta size"<-Maybe EIF2alpha can mimic some of the effects of IGF2 which would make it even better for growth.

Moderate Joint Loading Reduces Degenerative Actions of Matrix Metalloproteinases in the Articular Cartilage of Mouse Ulnae

"A mouse elbow-loading model was employed. In the articular cartilage of an ulna, the mANA levels of a group of MMPs as well as their degenerative activities were determined. Elbow loading altered the expression and activities of MMPs depending on its loading intensity. Collectively, the data in this study indicate that 0.2 and 0.5 N joint loading significantly reduced the expression of multiple MMPs, that is, MMP-1, MMP-3{up in LSJL}, MMP-8, and MMP-13, and overall activities of collagenases or gelatinases in articular cartilage, while higher loads increased the expression and activity of MMP-1 and MMP-13. moderate loads at 1 N elevated the mANA level of CBP/p300-interacting transactivator with EO-rich tail 2 (CITED2){CITED2 is associated with the chondroinducer Sox9}, but higher loads at 4 N did not induce a detectable amount of CITED2 mANA. Since CITED2 is known to mediate the downregulation of MMP-1 and MMP-13, the result indicates that joint loading at moderate intensity reduces MMP activities through potential induction of CITED2."

This study is for articular cartilage but the same genetic up- and down- regulation is likely to apply in growth plate cartilage.

"Articular cartilage in a synovial joint is composed of chondrocytes embedded in an extracellular matrix(ECM), which is rich in type II collagen and proteoglyans."<-The same description could be applied to growth plate cartilage.

"we employed elbow loading with loading intensities ranging from 0.2 to 4 N(peak to peak). It is reported that loads at 0.5 N on the elbow were capable of stimulating bone formation throughout the ulna including the proximal and distal diaphyses"<-0.5N is the also the amount capable of inducing length growth.

"The tip of the loader had a contact surface of 3 mm in diameter, and the loading force at 2 Hz for 5 min was selected in the range of 0.2-4 N."<-For the length studies 0.5N at 5hz were used.

"In response to loads at 0.2, 0.5, and 2 N, the ulna metaphysis induced mean strains of 15, 39, and 93 microstrain"<-this is crazy low microstrain considering 1500 microstrain is estimated to be the threshold for bone adaptation.

"The mRNA levels of MMP-1, MMP-3, MMP-8, and MMP-13 were downregulated by loads of 0.2 and 0.5N, while the levels of MMP-1 mRNA and MMP-13 mRNA were upregulated by 2-N loads.  The
mRNA levels of TIMP-1{up in LSJL} and TIMP-2 were unchanged in response to 0.2 and 0.5 N, but they were elevated in response to loads at 2 N."<-Elevations in these compounds is not necessarily bad as it may allow for the formation of cartilage canals.

"CITED2 is a transcription regulator that is reported to mediate the suppression of MMP-driven tissue degradation in the articular cartilage"<-CITED2 was maximal at 1N and became increasingly reduced until it was minimal at 4N.

"In vitro MMP data pointed out that fluid shear at 1-10 dyn/cm2 reduced the expression and activities
of MMP-1 and MMP-13, while fluid shear at 20 dyn/cm2 increased them."<-This gives us information about the fluid shear induced by LSJL.  0.2 & 0.5N are induce 1-10dyncm2 fluid shear whereas 2N induces at least 20dyn/cm2.

"gentle joint rotation is reported to induce CITED2 expression and suppress degenerative actions of MMPs"<-So if overloading does increase MMP expression above the optimal level, moderate joint rotation which likely induces fluid shear between 1-10dyn/cm2 can be used to induce CITED2 expression and reduce MMPs.

This third study doesn't mention LSJL but it's by LSJL authors and deals with fluid flow.

RhoA-Mediated Signaling in Mechanotransduction of Osteoblasts

"Osteoblasts play a pivotal role in load-driven bone formation by activating Wnt signaling through a signal from osteocytes as a mechanosensor[the WNT pathway can induce osteogenesis over chondrogenesis so it may be neutral or not beneficial for height growth]. Using MC3T3-E1 osteoblast-like cells under 1 hr flow treatment at 10 dyn/cm(2)[so the equivalent of LSJL at 0.5N], we examined a hypothesis that RhoA signaling mediates the cellular responses to flow-induced shear stress. Flow treatment activated phosphoinositide 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) signaling as well as a circadian regulatory pathway. In response to flow treatment phosphorylation of Akt in PI3K signaling and phosphorylation of p38 and ERK1/2 in MAPK signaling were induced.  RhoA was activated by flow treatment, and an inhibitor to a Rho kinase significantly reduced flow-induced phosphorylation of p38, ERK1/2, and Akt as well as flow-driven elevation of the mRNA levels of osteopontin and cyclooxygenase-2. Collectively, the result demonstrates that in response to 1 hr flow treatment to MC3T3-E1 cells at 10 dyn/cm(2), RhoA plays a critical role in activating PI3K and MAPK signaling as well as modulating the circadian regulatory pathway."

So fluid shear has an effect on PI3K which is anabolic to all cells including stem cells and chondrocytes.

"1 hr flow treatment to [osteoblastic cells] activated a small GTPase, RhoA, and induced the phosphorylation of ERK1/2, p38, and Akt."<-Rho GTPase may play a role in chondrogenic differentiation.

"Rho GTPase signaling is known to be involved in cell proliferation through integrin and focal adhesion molecules"  Rho is involved in the transmission of forces from ECM to the cytoskeleton.  Rho was not detected at levels of shear strain from 2-5dyn/cm(2).  Actin cytoskeleton may be a bad thing to height growth as it reduces adaptation.  Since there was no Rho, that means there may be no LSJL induced cytoskeleton adaptation at 0.5N and below although we don't know how much force we are generating with LSJL.

"fluid flow on bone cells results in intracellular calcium mobilization"<-if this is true for stem cells it could induce chondrogenesis and it may be why frequency is so important to LSJL. Regular secretions of intracellular calcium mobilization may induce chondrogenesis and thus height.

In a pathway mentioned later in the study Rho signaling can stimulate PI3K activity.

So the latest LSJL studies don't have much to do with bone lengthening but it shows that LSJL can reduce EIF2alpha phosphorylation which can lead to cell growth.  That the effect of LSJL on various MMPs varies based on load. And that the level of load necessary to stimulate PI3K activity also stimulates Rho actin cytoskeleton assembly.  Therefore there must be a conditioning effect with LSJL.

What can other species teach us about height growth?

Other species have different genetic composition than us but we can mimic the effects that different genetics have on phenotype(our goal is a tall phenotype after all).  By analyzing species, that are taller than normal we can figure out why they are tall and see if we can mimic the conditions that made them tall?  For example, if they are taller due to high or low serum concentrations of certain compounds we can alter our own composition of those compounds.

We can also analyze the growth of species who are shorter than normal to see if we can reverse any processes that stunt growth.

The Ontogenetic Osteohistology of Tenontosaurus tilletti.

"Tenontosaurus tilletti is an ornithopod dinosaur. Here I describe the long bone histology of T. tilletti and discuss histological variation at the individual, ontogenetic and geographic levels. The ontogenetic pattern of bone histology in T. tilletti is similar to that of other dinosaurs, reflecting extremely rapid growth early in life, and sustained rapid growth through sub-adult ontogeny. But unlike other iguanodontians, this dinosaur shows an extended multi-year period of slow growth as skeletal maturity approached[so what we can learn from this dinosaur is how to extend the period of growth as skeletal maturity approaches]. Evidence of termination of growth(e.g., an external fundamental system) is observed in only the largest individuals[Interesting that termination of growth is only present in larger individuals perhaps the process of growth termination is height growth stimulating], although other histological signals in only slightly smaller specimens suggest a substantial slowing of growth later in life. Histological differences in the amount of remodeling and the number of lines of arrested growth varied among elements within individuals, but bone histology was conservative across sampled individuals of the species, despite known paleoenvironmental differences between the Antlers and Cloverly formations. The bone histology of T. tilletti indicates a much slower growth trajectory than observed for other iguanodontians (e.g., hadrosaurids), suggesting that those taxa reached much larger sizes than Tenontosaurus in a shorter time[so we can also use this dinosaur to learn how to increase growth rate]."

<-So these dinosaurs are shorter than normal and it's likely a result of growth rate slowing at senescence.  The largest dinosaurs were the ones who terminated growth the fastest.  This could possibly indicate that instead of trying to extend growth, we should be trying to make growth faster and then trying to form new growth plates with a method like LSJL.

Regardin Juvenile Tenontosaurus: "In all long bones of this size class, the cortex is thicker than the diameter of the medullary cavity and is comprised of woven primary bone tissues with large, longitudinally oriented simple vascular canals that do not open to the surface of the bone. The vascular canals are circular or subelliptical in cross-section and smaller in diameter compared to those of the perinate. These canals are almost always arranged circumferentially in the cortex, and often show strong radial patterns as well, especially in the humerus and ulna. Osteocytes are dense throughout the cortex. These are organized around the vascular canals, but in the interstices between them, the osteocytes are randomly arranged and randomly oriented. In all specimens examined, the medullary cavity was filled by calcite crystals, and in most cases a thin band of avascular lamellar bone lines the edge of the marrow cavity, delimiting the endosteal boundary of the cortex. This band of lamellae suggests a pause in the expansion of the medullary cavity at this stage of growth. No LAGs or secondary osteons occur in any of the bones of this size class."<-Note that these dinosaurs had a rapid growth in early life.  Some of these bone properties may have been responsible for that rapid growth.

Regarding SubAdult Tenontosaurus: "Subadults always retain a thick cortex relative to the size of the medullary cavity, but at this growth stage, cortical bone tissues are removed as the medullary cavity expands.

For all elements, the entire cortex is composed of well-vascularized woven bone. Except in the fibula, almost all of the visible cortical bone tissues are primary tissues vascularized by simple canals or (more commonly) primary osteons, but secondary osteons are visible endosteally in all subadult elements sampled. The amount and extent of secondary remodeling varies with the size (age) of the individual and by element. Younger individuals have secondary osteons only in the internalmost cortex, but these spread throughout the inner cortex and eventually into the mid-cortex in older animals. The tibia and femur show much less secondary remodeling compared to the humerus, ulna, and fibula.

All subadult elements show many longitudinally oriented canals throughout the cortex, and these are smaller in diameter compared to those of juveniles or perinates. More of these canals anastomose than in juveniles or perinates, and these anastomoses are longer (connect more longitudinal canals) in larger individuals[an anastomose is something that joins blood vessals, note that larger individuals had longer anastomoses which indicates that individuals who wish to grow taller should try to connect more longitudinal canals unless of course the longer anastomoses are the byproduct rather than the cause of height growth]. The anastomoses tend to be more circumferential in orientation in the tibia and femur, and more radial in the humerus and especially the ulna, but short connections occur in every direction in all elements. In larger subadults, true plexiform/laminar organization can be observed in the tibia, femur, and humerus. Interstitial osteocytes are dense throughout the subadult cortex and show no preferred arrangement relative to each other, nor a preferred orientation relative to the long axis of the bone.

All elements of subadult Tenontosaurus exhibited one or more LAGs[Lines of Arrested Growth or areas of broken bone deposition]. These often formed as “double LAGs”, i.e., two very closely-spaced LAGs that likely did not represent an entire year of growth between them. In many tibial sections, especially those from older individuals, the bone texture changed within each zone. In some elements, immediately following a LAG, a very thin band of parallel-fibered or weakly-woven bone tissue is present. This very quickly changes to woven bone, in which the collagen fibers were coarse and disorganized. The bone would become progressively less woven through the zone and leading up to the subsequent LAG. This suggests, for at least some elements, that the bone depositional rate decreased through the year, although lamellar-zonal bone (which is deposited much more slowly) was never observed in a subadult, and zonal width did not decrease dramatically in this size class."<-The sub adult region is likely when growth slowed down.

Regarding Adult Tenontosaurus: "[There were] signs of dramatically slowed growth in adult Tenontosaurus.  Extensive remodeling (i.e., several generations of secondary osteons) of the inner, mid- and even outer cortex was observed in all elements. Additionally, the zonal cycles of decreasing growth rates truncating in a LAG observed in the larger subadults is more pronounced in adults. Moving periosteally through the cortex, the woven-to-less-woven bone transition becomes a weakly-woven-to-parallel-fibered pattern, and ultimately lamellar bone tissue is deposited in the outermost cortex. These transitions are accompanied by trends in decreasing vascularity (in terms of number of canals, not their size), decreasing vascular complexity (number and kinds of anastomoses), decreasing numbers of osteocytes, and decreasing zonal width. Together, these signals indicate several years of sustained slow growth rates in adult Tenontosaurus before an ultimate truncation of growth."

"Throughout early ontogeny and into subadulthood, Tenontosaurus tilletti is characterized by bone tissues associated with fast growth. The cortex of all of the major long bones exhibits woven bone tissue, high levels of vascularization, complex patterns of vascular connectivity and organization, and high osteocyte density[so to grow taller we want to increase levels of woven bone tissue, more vascular connectivity and higher osteocyte density]. These histological characteristics suggest that T. tilletti maintained a rapid growth rate at least to the point of reproductive maturity. However, at some point in the subadult stage, Tenontosaurus transitioned to a slower growth regime. These slower growth rates are initially reflected in the woven texture of the cortex of subadults, but in adults, further decreases in osteocyte and vascular density, smaller zonal widths, and ultimately the presence of an EFS[External Fundamental System or the evidence of growth termination] suggest prolonged (several years) of slow growth rates before the termination of growth. These trends in bone histology strongly suggest asymptotic (“determinate”) skeletal growth for Tenontosaurus, consistent with what is observed in other dinosaurs"

"Beginning in subadulthood, Tenontosaurus changed its growth regime and began depositing parallel-fibered bone tissue in much narrower zones."

So what we can learn from these dinosaurs is that to grow taller we likely want to increase osteocyte density(osteocytes detect signals from interstital fluid flow which may be how it relates to height growth).  Osteocyte density is likely though a symptom rather than a cause of upcoming height increase.  More dense osteocytes indicate that the bone is underdeveloped relative to the number of osteocytes.

Woven bone formation is likely also a symptom rather than a cause of height growth.  Woven bone is a sign of new bone formation such as the case with gap repair.

That leaves vascularization which plays a role in distraction osteogenesis.  The fact that immature bone is so vascularly organized indicates that vascularization is not a symptom of bone that is ready to grow longer but rather a cause as you'd expect immature bone to be disorganized.  Adult dinosaur bone was characterized with less cartilage canals and less vascular complexity.

This indicates that proteins involved in vascularization like VEGF, MMP-9, and osteoclasts are important for height growth.  Mechanical stimulation for instance may increase levels of VEGF.  Of course VEGF is linked to Estrogen and Estrogen reduces height when levels are too high.  Thus it may not be a simple case of more of these compounds equals more height growth.  You may need a complex interplay of compounds to maximize vascularization to encourage height growth.

Given previous evidence of vascularization being linked to height growth, like hydrostatic pressure which can induce chondrogenesis being influenced by vascularization, this further exemplifies that vascular factors are huge influence upon height and anything that influences bone vascularization like VEGF, Estrogen, MMPs, etc. should be heavily analyzed for influence on height growth.


Temporal analysis of rat growth plates: cessation of growth with age despite presence of a physis.

"Despite the continued presence of growth plates in aged rats, longitudinal growth no longer occurs. We studied the growth plates of femurs and tibiae in Wistar rats aged 62-80 weeks and compared these with the corresponding growth plates from rats aged 2-16 weeks. During skeletal growth, the heights of the plates, especially that of the hypertrophic zone, reflected the rate of bone growth. During the period of decelerating growth, it was the loss of large hydrated chondrocytes that contributed most to the overall decrease in the heights of the growth plates. In the old rats we identified four categories of growth plate morphology that were not present in the growth plates of younger rats: (a). formation of a bone band parallel to the metaphyseal edge of the growth plate, which effectively sealed that edge; (b). extensive areas of acellularity, which were resistant to resorption and/or remodeling; (c). extensive remodeling and bone formation within cellular regions of the growth plate; and (d). direct bone formation by former growth plate chondrocytes. These processes, together with a loss of synchrony across the plate, would prevent further longitudinal expansion of the growth plate despite continued sporadic proliferation of chondrocytes."

"In rats, the rate of growth increases between 1 and 5 weeks, then declines until skeletal maturity, which is achieved by 11.5–13 weeks. Bones still continue to grow, albeit at a reduced rate, until ∼26 weeks of age, after which growth virtually ceases in rats"

"At 2 weeks, approximately half of the cartilaginous epiphysis (chondroepiphysis) had been replaced by the secondary ossification center. At this age, the height of the growth plate could not be taken as the distance between the primary spongiosa and the secondary ossification center (bony epiphysis), because approximately one third of that distance still represented the epiphyseal cartilage of the chondroepiphysis rather than growth plate cartilage."

"At 2 and 4 weeks, resorption took place at the epiphyseal and metaphyseal borders of the growth plates, whereas from 12 weeks onwards resorption was confined to the metaphyseal border. In old rats, TRAP activity was either completely absent or was present in the matrix at the reversal line which, in this case, marked the border between horizontal bone deposition and growth plate cartilage"

"features [of 82 week old rat growth plate versus 8 week old rat growth plate] are (1) horizontal deposition of bone matrix, (2) acellular areas, (3) within growth-plate remodeling, and (4) intralacunar bone formation."

"In the aged rats, spongiosa was absent in some regions, presumably as a consequence of resorption. In such areas, bone matrix was directly apposed to the cartilage, parallel to the growth plate, effectively sealing the growth plate with bone at the metaphyseal border."

"In the growth plates of young rats, the cartilage core, which forms the center of the spicules of primary and secondary spongiosa, is usually thin and is only detectable only at higher magnification. By contrast, some spicules of aged rats contained a wide core of cartilage that was evident even at low magnifications and often persisted well below the average height of the growth plate. Closer examination of these cartilage cores revealed that no cells were present. Such acellular regions could extend from the former reserve zone to the vascular front and beyond into the spongiosa, suggesting that the cartilage matrix was more resistant to resorption than adjacent cellular matrix. Acellular areas were also identified in the growth plates of 12-and 16-week-old animals, although less frequently. Adjacent to acellular regions, regions of high cellularity were frequently present"

"the osteogenic differentiation of chondrocytes in the old rats was not a gradual further differentiation of chondrocytes but was rather a transdifferentiation in which acquisition of the osteogenic phenotype coincided with loss of the chondrogenic phenotype."

All these changes seem consistent with genetic dysregulation rather than programmed senescence.


Aberrations of cell cycle and cell death in normal development of the chick embryo growth plate.

"The epiphyses of femurs from 7.5-15 day chicken embryos were studied by electron microscopy. Several forms of aberrant cell cycles were present: (1) in the perichondrium, polyploid metaphases, segmentating large (giant) cells, and mitotic catastrophe (midway between mitosis and apoptosis) were observed; (2) in the resting zone, premature chromosome condensation was found; (3) in the proliferative zone, approximately 5% of divisions were aberrant, representing most often mitosis restitution from metaphase and more seldom from the anaphase; (4) in all layers, 'dark chondrocytes' representing a premortal form of hypersecretory cells undergoing often a-mitotic nuclear segmentation were present. Many of the aberrations of cell cycle were combined with cell death. These deviations omitting or adapting the cell cycle check-points represent evidently the normal epigenetic mechanisms of development and repair. At the same time, by origin and appearances they seem very close to the loss of the growth control displayed by malignant tumours."

"All these aberrations were associated with tetraploidy and mostly with curtailments of the mitotic cycle. "

Changes in the expression of Fas, osteonectin and osteocalcin with age in the rabbit growth plate.

"The Fas receptor is a mediator of the apoptotic signal in some systems. We studied its expression in situ in growth plates of rabbits aged from five to 20 weeks. In addition, we investigated the immunolocalisation in the growth plates of the bone proteins, osteonectin and osteocalcin, and the changes in their expression with age. The Fas-positive chondrocytes were found mostly in the hypertrophic zone, as were the osteonectin-positive and osteocalcin-positive cells. The percentage of Fas-positive cells increased with age whereas little change was found in the number of osteonectin-positive and osteocalcin-positive chondrocytes. Many of the Fas-positive chondrocytes were also TUNEL-positive. This strongly suggests that apoptosis in the growth plate is mediated through the Fas system. Double immunostaining for osteocalcin and Fas showed that not all hypertrophic chondrocytes were of the same cell type. Some chondrocytes stained for osteocalcin only, others for Fas only, while some were positive for both"

Network based transcription factor analysis of regenerating axolotl limbs.

"We used the human orthologs of proteins previously identified by our research team as bait to identify the transcription factor (TF) pathways and networks that regulate blastema{a group of stem cells} formation in amputated axolotl{a water salamander} limbs. The five most connected factors, c-Myc, SP1, HNF4A{down in LSJL}, ESR1 and p53 regulate ~50% of the proteins in our data. Among these, c-Myc and SP1 regulate 36.2% of the proteins. c-Myc was the most highly connected TF (71 targets). Network analysis showed that TGF-β1 and fibronectin (FN) lead to the activation of these TFs. We found that other TFs known to be involved in epigenetic reprogramming, such as Klf4, Oct4, and Lin28{lin28b is up in LSJL} are also connected to c-Myc and SP1.

We found that the TFs, c-Myc and SP1 and their target genes could potentially play a central role in limb regeneration."

"Urodele amphibians (axolotls, salamanders and newts) regenerate amputated limbs perfectly throughout larval and adult life"

"Blastema cells originate by a reverse developmental process in which the tissue matrix near the amputation plane is degraded by proteases, releasing both mature cells that are reprogrammed to a mesenchymal stem cell-like state, and muscle stem cells (satellite cells). Within a few days after amputation, these cells accumulate under the apical epidermal cap (AEC), where they proliferate and are patterned into the missing limb parts."

" we found that TGF-β1 (transforming growth factor - beta 1) could potentially lead to the activation of SP1 and then to the expression of FN (fibronectin), which is produced by the blastema cells and the AEC. In turn, FN activates c-Myc via integrins and the Wnt pathway. Within these pathways we identified several TFs such as SMAD3 (mothers against decapentaplegic homolog 3), which may be involved in limb regeneration. In addition, Klf4 (kruppel-like factor 4), Oct4 (octamer-binding protein 4), and Lin28, TFs common to embryonic stem cells, induced pluripotent cells (iPSCs) and blastema cells, were also found to be connected to c-Myc and SP1."

Msx1 and Notch1 are also factors involved in limb regeneration.

"c-Myc activation from FN involves the cell adhesion proteins talin, FAK1 (focal adhesion kinase1), c-Src, Paxillin, ILK (integrin-linked protein kinase) and components of the canonical Wnt signaling pathway (GSK3-β-glycogen synthase kinase-3 beta, β-catenin, and Tcf/Lef (T-cell-specific transcription factor/lymphoid enhancer-binding factor 1)"

"molecular interactions of Wnt2b with Tbx5 that are responsible for limb identity and outgrowth"

Detailed comparison of LSJL genes and limb regeneration genes To Be Done.


Xenopus laevis as a novel model to study long bone critical-size defect repair by growth factor-mediated regeneration.

"We used the tarsus of an adult Xenopus laevis frog as an in vivo load-bearing model to study the regeneration of critical-size defects (CSD)[defects too large to be repaired naturally] in long bones. We found the CSD for this bone to be about 35% of the tarsus length. To promote regeneration, we implanted biocompatible 1,6 hexanediol diacrylate scaffolds soaked with bone morphogenetic proteins-4 and vascular endothelial growth factors. In contrast to studies that use scaffolds as templates for bone formation, we used scaffolds as a growth factor delivery vehicle to promote cartilage-to-bone regeneration. Defects in control frogs were filled with scaffolds lacking growth factors. The limbs were harvested at a series of time points ranging from 3 weeks to 6 months after implantation and evaluated using micro-computed tomography and histology. In frogs treated with growth factor-loaded scaffolds, we observed a cartilage-to-bone regeneration in the skeletal defect. Five out of eight defects were completely filled with cartilage by 6 weeks. Blood vessels had invaded the cartilage, and bone was beginning to form in ossifying centers. By 3 months, these processes were well advanced, and extensive ossification was observed in 6-month samples. In contrast, the defects in control frogs showed only formation of fibrous scar tissue."

"Instead of attempting to promote regeneration by direct bone deposition on a scaffold, we induced the intercalary regeneration of the missing bone by implanting a biocompatible scaffold loaded with growth factors specifically selected to induce cartilage to form in the defect, bridge the bone gap, and then convert this cartilage template to bone. We selected BMP-4 and VEGF as the growth factors to be used because BMP-4 is essential for the development of cartilage templates and is induced early in fracture repair, and VEGF induces blood vessel formation and is critical for the conversion of hypertophic cartilage to bone"

"The presence of the scaffold alone did not promote cartilage or bone formation in the tarsus gap."


Adipose-derived stem cells from the brown bear (Ursus arctos) spontaneously undergo chondrogenic and osteogenic differentiation in vitro.

"n the den, hibernating brown bears do not develop tissue atrophy or organ damage, despite almost no physical activity. Mesenchymal stem cells could play an important role in tissue repair and regeneration in brown bears. Our objective was to determine if adipose tissue-derived stem cells (ASCs) can be recovered from wild Scandinavian brown bears and characterize their differentiation potential. Following immobilization of wild brown bears 7-10 days after leaving the den in mid-April, adipose tissue biopsies were obtained. ASCs were recovered from 6 bears, and shown to be able to undergo adipogenesis and osteogenesis in monolayer cultures and chondrogenesis in pellet cultures. Remarkably, when grown in standard cell culture medium in monolayer cultures, ASCs from yearlings spontaneously formed bone-like nodules surrounded by cartilaginous deposits, suggesting differentiation into osteogenic and chondrogenic lineages. This ability appears to be lost gradually with age. This is the first study to demonstrate stem cell recovery and growth from brown bears, and it is the first report of ASCs spontaneously forming extracellular matrix characteristic of bone and cartilage in the absence of specific inducers."

"Remarkably, however, the cells from yearlings showed remarkable spontaneous cartilage and bone formation capacity. Interestingly, the spontaneous bone and cartilage formation appears to occur in a concurrent manner in and around the nodules, respectively, with mineralization characteristic of bone within the nodules and cartilage formation in the periphery."

"close cell-to-cell contact [is needed] for chondrogenesis to occur"

Do you need to feel fluid flow when performing LSJL?

One way I've hypothesized to identify whether you are performing LSJL effectively is whether you feel interstitial fluid flow in the diaphysis of the bone.  An increase in interstitial fluid flow in the diaphysis of the bone probably means that you are increasing hydrostatic pressure enough in the epiphysis of the bone to encourage chondrogenic differentiation of stem cells.  However, some like St.it. have grown without ever feeling this increase in interstitial fluid flow.  I have felt the increase in fluid flow whenever I have performed LSJL both with dumbells and with the clamp.  Maybe some have fewer nerve endings inside of their bones but a Vitamin D deficiency can result in bone marrow turning into fat(and thus less fluid flow).  One way to test this would be to see if someone could feel the fluid flow with something like LIPUS versus LSJL.

Interstital fluid is the fluid that is found in the interstitial spaces between cells.  Hydrostatic pressure is generated by the systolic force of the heart.

The water potential is created due to the ability of small solutes to pass through the walls of capillaries. This buildup of solutes induces osmosis. The water passes from a high concentration (of water) outside of the vessels to a low concentration inside of the vessels, in an attempt to reach an equilibrium. The osmotic pressure drives water back into the vessels. Because the blood in the capillaries is constantly flowing, equilibrium is never reached.
The balance between the two forces differs at different points on the capillaries. At the arterial end of a vessel, the hydrostatic pressure is greater than the osmotic pressure, so the net movement  favors water and other solutes being passed into the tissue fluid.  This could include growth factors that can induce chondrogenesis. Hydrostatic Pressure is what increases chondrogenesis.
At the venous end, the osmotic pressure is greater, so the net movement favors substances being passed back into the capillary. This difference is created by the direction of the flow of blood and the imbalance in solutes created by the net movement of water favoring the tissue fluid.

Let's look at Lateral Synovial Joint Loading induced fluid flow.

Knee loading dynamically alters intramedullary pressure in mouse femora.

"The number of daily loading cycles, bone strain, strain-induced interstitial fluid flow, molecular transport, and modulation of intramedullary pressure[we are looking to modulate the pressure in the epiphysis] have been considered as potential mediators in mechanotransduction of bone. Using a knee loading modality that enhances anabolic responses in mouse hindlimb, we addressed a question: Do oscillatory loads applied to the knee induce dynamic alteration of intramedullary pressure in the femoral medullary cavity? To answer this question, mechanical loads were applied to the knee with a custom-made piezoelectric loader and intramedullary pressure in the femoral medullary cavity was measured with a fiber optic pressure sensor{Maybe there could be some way to measure it like they do with blood pressure}. We observed that in response to sinusoidal forces of 0.5 Hz and 10 Hz, pressure amplitude increased up to 4-N loads and reached a plateau at 130 Pa. This amplitude significantly decreased with a loading frequency above 20 Hz."

So Lateral Synovial Joint Loading alters intramedullary pressure and if LSJL is inducing a pressure increase in the epiphysis you should feel the intramedullary pressure first as that is more dense than the epiphysis(thus it is easier for the pressure to increase there).   In the study, the peak frequency was around 0.5Hz which is not very much at all(which is good).  To do LSJL at 0.5 Hz would be to do the dumbell loading or clamping at 2 second intervals.  Their data indicates a huge speak at 2seconds so that is definitely the optimal frequency(Fig3D).  When you are clamping it may be best to turn the ratchet less than every two seconds.  Pizeoelectric current is the eletricity generate when an object is deformed(such as the bone as a result of pressure).  Peak pressure was observed at 80V(4N).  We'd have to get a strain gauge to measure peak force generated during LSJL.

So if you're not feeling fluid flow with LSJL then maybe you're loading too hard or too little.  Maybe you're deficient in Vitamin D.  Or maybe you don't have enough nerves in your bone to sense the fluid flow.  So try the simple changes to see if you can start feeling that fluid flow.  If that doesn't work then just feel content in that maybe your nerves aren't sensitive enough.

"Increasing loads to the knee elevated pressure alterations with the actuator voltage ranging from 10 V (0.5 N) to 80 V (4 N). The pressure elevation then reached a plateau and no significant increase was observed from 80 to 100 V"<-So past a certain point intramedullary/hydrostatic pressure doesn't increase.

130pascals is equivalent to 0.0013 Mega Pascals.  The stem cells seeded in type I collagen sponges that underwent chondrogenesis underwent 1 MPa of hydrostatic pressure.  Ordinary LSJL may not generate enough hydrostatic pressure.

Other interesting tidbits from the paper:

"best-fit linear regression analysis determined a calibration slope of 17.5 Pa/mV (pressure in the glass tube; r2 = 0.99) and 20.4 Pa/mV (pressure in the in vivo femur; r2 = 0.99), indicating that a voltage signal of 1 mV corresponded to pressure modulation of 17.5 Pa (glass tube) and 20.4 Pa (femur in vivo) "

"Prior to loading, the baseline intramedullary pressure was measured as 1290 ± 150 Pa (mean ± SD; equivalent to 9.5 ± 1.1 mm Hg)"

"Increasing loads to the knee elevated pressure alterations with the actuator voltage ranging from 10 V (0.5 N) to 80 V (4 N). The pressure elevation then reached a plateau and no significant increase was observed from 80 to 100 V. This two-phase trend was common with the loading frequency at both 0.5 Hz and 10 Hz. At 0.5 Hz, for instance, the pressure alteration (peak-to-peak) was observed as 0.34 ± 0.24 mV (mean ± SD) at 20 V, 2.8 ± 0.40 mV at 40 V, and 7.0 ± 0.72 mV at 80 V."

"The alteration of pressure signal (peak-to-peak) was estimated as 6.0 ± 1.0 mV with knee loading"<-So about 120 Pa pressure in the femur.

"[microparticles] motion along the length of the tube was modeled as αsin(2πft + θ0) − βt with α = 10.6 μm (amplitude of the oscillatory motion), β = 16.2 μm/s (linear translational speed) at f = 0.5 Hz, where “t” = time, and θ0 = phase angle"

"Although the observed pressure alteration with knee loading is 0.2 ~ 10% of the baseline intramedullary pressure, it is a dynamic change rather than static. Dynamic pressure oscillations in a tube have been shown to enhance solute dispersion even at a low-level fluctuation"

"The observed pressure amplitude (half of peak-to-peak) in the femoral bone cavity ranged from approximately 3 to 130 Pa depending on the loading conditions (0.5 to 4 N at 0.5 to 50 Hz). Note that 1 cm H2O is equivalent to 100 Pa, and therefore the observed maximum pressure alteration of 130 Pa corresponds to 1.3 cm H2O."

Of note is that they did not measure the pressure in the epiphysis but rather the diaphysis so the pressure in there may be much higher.

"First, increasing loads elevated the amplitude of modulation monotonously from 1 N to 4 N at the rate of ∼ 20 Pa per N, but no significant increase was observed above 4 N. Second, the loading effect was significantly reduced at a loading frequency above 20 Hz. The viscoelastic nature of tissues likely determines their ability to respond to loading and exhibits the lower response to higher frequencies. It is possible that at 4 N the bone structure reaches its elastic limit and further deformation is restrained. In the current in vivo studies, the cannula was filled with the saline solution and we occasionally observed that this saline solution was mixed with a small amount (< 0.05 ml) of fluid from the bone cavity. Therefore, the femur ex vivo might not faithfully represent the undisturbed condition for nominal knee loading. Nevertheless, our observations suggest intensity and frequency dependence of the pressure modulation and indicate an advantage of loading frequencies below 20 Hz to effectively alter intramedullary pressure."

Steven J. Warden is sort of an adjunct scientist to LSJL in addition to P. Zhang, Hiroki Yokota, and the late C.H. Turner.  Here's what he had to say about fluid flow and it's role in Lateral Synovial Joint Loading.

Breaking the rules for bone adaptation to mechanical loading 

"1) bone preferentially responds to dynamic rather than static stimuli, 2) only short durations of loading are necessary to initiate an adaptive response, and 3) bone cells accommodate to customary mechanical loading environments[this is likely due to an increase in resistance to load over time by the actin cytoskeleton]" 

"Bone experiences internal strain when mechanically loaded. strain refers to the change in length of a bone when load is applied. [Strain for] bone is often expressed in terms of microstrain (µε). As long bones are curved, they bend when axially loaded. This results in exposure of different tissue-level regions within the bone cross section to different levels of microstrain. Only those regions within the individual loaded bone that experience sufficient microstrain adapt{this may be why you don't grow taller with axial loading, all the strain is placed on the diaphysis and not on the epiphysis}. This has been demonstrated most evidently using the rodent ulna axial compression model, wherein tissue-level bone adaptation closely matches the tissue-level microstrain distribution"

"Applying low-level, compressive loading to the proximal tibial epiphysis of mice, they induced bone adaptation at a distant, nonloaded site (4-mm distal on the periosteal surface of the tibial diaphysis). That is, they found mechanical loading to stimulate bone formation at a site distant from the site of loading and distant from a site of significant microstrain."

Epiphyseal loading can induce adaptations in the diaphyseal region but not vice versa.  This is likely due to the travel of fluids from the epiphysis to the periosteal region.  The epiphysis is far more porous than the diaphysis so it is much easier for fluid to flow from the epiphysis to the diaphysis than vice versa.

"Bone is a porous tissue consisting of a fluid phase, a solid matrix, and cells. Mechanotransduction in the skeleton involves the movement of the fluid phase in relation to the solid matrix, which subsequently stimulates "detector" cells{osteocytes} and triggers a cascade of adaptive molecular events"

Our goal is to have these fluid phase to induce the cascade of adaptive molecular event of chondrogenesis in stem cells.  Here's a laterally loaded bone, you can see why it's so much easier for the fluid to flow from the epiphysis to the diaphysis than the other way around.


So you can see that if you are properly loading the epiphysis, you should be feeling fluid flow in the diaphysis as well.  Except if for some reason you aren't sensitive to the fluid flow in the diaphysis.

Here's a paper that states how much pressure is required to stimulate an osteogenic response, perhaps the threshold is similar for chondrogenic or stem cell response:

Skeletal adaptation to intramedullary pressure-induced interstitial fluid flow is enhanced in mice subjected to targeted osteocyte ablation.

Ablation refers to removal.

"Flow within the LCS[lacunar-canalicular system] was being generated at physiological levels{such as by compressive strains or jumping not LSJL induces lateral compressive strains}, and the inability for osteocyte ablation[ability of osteocytes to remove] to abrogate[cease] structural adaptation to pressure loading was not attributable to insufficient generation of lacunar-canalicular IFF."<-So osteocytes are unable to stop adaptation to pressure loading.  Indicating that pressure loading adaptions may come from another source like chondrocytes or stem cells.

"ImP[intramedullary pressure]-driven IFF is mediated by a non-osteocytic bone cell population"<-this hypothesis is very good for LSJL as it osteocytic bone population is not likely to generate height whereas other cell populations like stem cells differentiating into chondrocytes may.

"Osteocytes may mediate [the process of adaptative response to intramedullary pressure] in an antagonistic role by functioning as a cellular thermostat, halting bone formation initiated by mechanical loading once a sufficiently dense osteocytic network has been formed[so the greater the osteocytic network that has been formed the harder it is to get results from LSJL]. Recent studies demonstrating that deletion of osteoblastic and osteocytic gap junctions enhances load-induced bone formation"<-further evidence that load formation may be due to endochondral ossification[the type of bone growth that is height increasing] and not direct bone formation by osteocytes and osteoblasts.

"Dynamic pressurization of the intramedullary cavity results in significant flow into and out of the marrow cavity[which would include the epiphyseal bone marrow], potentially exposing surface-residing cells to enhanced flow in addition to osteocytes[which would include mesenchymal stem cells]"<-Thus, increasing intramedullary pressure does have the potential to stimulate mesenchymal stem cell chondrogenesis.

"A linear change in peak [intramedullary pressure] with pump flow rate"<-So the greater the pump flow rate the greater the intramedullary pressure within the bone and likely the greater the hydrostatic pressure within the epiphyseal bone marrow.

"No studies to date have demonstrated the capacity for bone cells to sense pressures less than 97.5 mmHg"<-however that does not mean that stem cells can't sense pressures less than 97.5mmHg.  This indicates that perhaps too high a pressure could inhibit stem cell stimulation as osteocytes have the ability to inhibit structural adaptation.

"[the ability of osteocytes to inhibit structural adaptation may be affected by] lower expression of the Dmp1 promoter in the mature (16 week-old mice in our studies)[LSJL has been performed on 16 week-old mice] versus immature skeleton (empty lacunae were quantified in 10 week-old mice in the studies of Tatsumi et al., though unloading studies were performed in 20 week-old mice), particularly given the role of Dmp1 in promoting mineralization and hydroxyapatite formation"<-so the ability of bone to stop IFF stimulated adaptation is higher in older individuals indicating that LSJL may actually be more effective in older individuals due to lower levels of Dmp1. Although the benefit of LSJL on bone length was greater in 8-week mice than 16-week old mice.  However, the pressure may have been lower than that required to induce osteocyte mediated bone structure adaptation inhibition in both 8-week and 16-week old mice thus making this a non-factor.  Note however, that the absolute increase in growth was greater for old versus young mice.

Perhaps maybe feeling IFF in the center of the bone is a negative indication for height growth as it indicates that the level of fluid flow is sufficient to induce osteocyte inhibition of bone adaptation.  However, it may be such that the osteocytes only inhibit adaptation at a local level and that locations that you don't feel fluid flow like the epiphysis may be able to adapt[stem cells may be able to differentiate into chondrocytes] as long as you don't feel fluid flow in that direct vicinity.

"Bone structural adaptation to intramedullary pressurization-driven IFF is similar or significantly enhanced in mice with targeted osteocyte ablation, particularly in trabecular bone, despite up to 50% of trabecular lacunae being uninhabited following ablation[osteocyte removal]. These exploratory data are consistent with the potential existence of non-osteocytic mechanosensory bone cells that sense ImP-driven IFF independently and potentially parallel to osteocytic sensation of poroelasticity-derived IFF within the LCS."<-So activity in the trabecular(the epiphysis is mostly trabecular bone) bone is affected by osteocyte activity regardless of whether the osteocytes are actually in the trabecular network.  So the potential for stem cells to differentiate into chondrocytes is potentially affected by osteocyte activity elsewhere in the bone.

Therefore, IFF fluid flow likely has an impact on the success of LSJL and perhaps too much of an osteocyte network may be detrimental to LSJL by lowering the threshold of hydrostatic pressure in which the osteocyte network inhibits the ability of non-bone cells to initiate an anabolic response(in this case stem cells differentiation into chondrocytes).  This indicates the possibility of a deconditioning period being beneficial to LSJL effectiveness by giving time for the osteocyte network to weaken.

According to this study LSJL induces hydrostatic pressure:

Biomechanics-driven chondrogenesis: from embryo to adult.

"Following tissue loading, hydrostatic pressure initially develops in the interstitial fluid[so LSJL should induce hydrostatic pressure in the epiphyseal bone marrow], which is followed by fluid flow-induced shear. However, in time scales greater than 10 micro seconds, the solid matrix begins to bear the applied load, resulting in deformation. Consequently, the cells residing in the matrix experience hydrostatic pressure, shear, compression, and, to a lesser extent, tension[this definitely occurs as we do LSJL for more than 10 microseconds!]. This mechanical stimulation produces a signaling cascade, resulting in increased gene expression, matrix protein production, and intracellular ion influx"

"precartilaginous condensation may be the result of mesenchymal progenitor cells exhibiting similar surface tensions rather than similar biomarkers."

"Chondrocyte progenitors secrete cartilage-specific matrix and decrease expression of cell-cell interaction proteins [post precartilaginous condensation]"

"HP does not result in deformation of incompressible media, so it is not expected to deform cells. Direct compression results in deformation of matrix and cells, which will also create fluid flow that is not observed with HP. "

"Under mechanical stimulation, mesenchymal stem cells migrate and chondrodifferentiate. Mechanical stimulation can be used to induce transdifferentiation[of say fibroblasts] into chondrocytes."

"Mechanical loading [10% strain, 1 Hz or HP between 3–10 MPa] of cultured mesenchymal stem cells can also promote chondrodifferentiation."

"Chondrogenesis involves the condensation of precartilaginous progenitor cells to form tightly packed cellular aggregates followed by differentiation into early chondrocytes"<-this is our goal with LSJL.

"Some groups postulate that mechanical forces contribute to de novo[a new] chondrogenesis from early stem cells"<-again this is our goal with LSJL.

"applying compressive loading in 3 dimensions enhances chondrogenesis of progenitor cells, generating up to 3-fold increases in matrix protein synthesis"

"hyperosmolarity up-regulates key cartilage genes, such as SOX9 and aggrecan"

"cellular deformation increases intracellular concentrations of Ca2+ and Na+ by enhancing Na+/H+ exchanger activity and stimulating stretch-activated ion channels. The influx of Ca2+ leads to the production of intermediate signaling molecules, such as inositol triphosphate and diacylglycerol, which activate kinase cascades that are crucial for cartilage homeostasis. Applying agents like histamine, which increase intracellular Ca2+ levels, has also been shown to modulate signaling intermediates like cyclic AMP"

"Spatiotemporal changes in progenitor cell adhesion molecule expression cause similar cells to transiently associate during chondrogenesis"

"2-photon laser microscopy and magnetic resonance imaging are used to reveal chondrocyte deformation at the single-cell level in response to muscle-induced mechanical loading, and at the tissue level during physiological loading"

"applying HP (5 MPa, 1 Hz) to murine embryonic fibroblasts results in 2-fold increases in collagen synthesis and GAG production. Similarly, HP (5 MPa, 1 Hz) increases chondrogenic gene expression in neonatal human dermal fibroblasts. Mechanical forces have also been postulated to induce chondrogenic gene and protein expression in smooth muscle cells following atherosclerotic calcification"

"Under mechanical stimulation, mesenchymal stem cells migrate and chondrodifferentiate"

Effect of fluid flow-induced shear stress on human mesenchymal stem cells: differential gene expression of IL1B and MAP3K8 in MAPK signaling.

"In response to different magnitudes and durations of fluid flow-induced shear stress, we observed significant differential gene expression for various genes in the MAPK signaling pathway. Independent of magnitude and duration, shear stress induced consistent and marked up-regulation of MAP kinase kinase kinase 8 (MAP3K8) and interleukin-1 beta (IL1B) [2-fold to >35-fold, and 4-fold to >50-fold, respectively]. We also observed consistent up-regulation of dual specificity phosphatase 5 and 6, growth arrest and DNA-damage-inducible alpha and beta, nuclear factor kappa-B subunit 1, Jun oncogene, fibroblast growth factor 1, and platelet-derived growth factor alpha. Our data support MAP3K8-induced activation of different MAPK signaling pathways in response to different profiles of shear stress, possibly as a consequence of shear-induced IL1B expression."

"IL-1 promotes a 10-fold increase in the induction of MAP3K8, a MAP kinase kinase kinase capable of acting on each of the ERK1/2, JNK and p38 signaling pathways."

" In response to 1, 5 and 10 dyn/cm2 shear stress we found pronounced up-regulation of interleukin-1 beta (IL1B) [24.2-, 15.5- and 11.6-fold, respectively] and MAP kinase kinase kinase 8 (MAP3K8, aka Cot/Tpl2; 6.6-, 8.6- and 13.5-fold, respectively"

"a roughly similar number of genes (450–700) were differentially expressed in response to each shear stress magnitude (1, 5 and 10 dyn/cm2) and duration (10 min, 1 and 24 h), although a 24 h duration resulted in over 1500 differentially expressed genes (2-h time point). In all cases, 55–60% of the genes were up-regulated at the 2 h time point, whilst after 24 h approximately 60% were down-regulated. "<-thus you likely don't want to load for over two hours.

"the most up-regulated gene was prostaglandin endoperoxide synthase 2 (PTGS2, aka COX2) [76-, 38- and 74-fold for 1, 5 and 10 dyn/cm2, respectively]"

Frequency-dependent enhancement of bone formation in murine tibiae and femora with knee loading.

"The left knee of C57/BL/6 [female 14 week old] mice was loaded with 0.5 N force at 5, 10, or 15 Hz for 3 min/day for 3 consecutive days"

"Compared with the sham-loading control, for instance, the cross-sectional cortical area was elevated maximally at 5 Hz in the tibia, whereas the most significant increase was observed at 15 Hz in the femur. Furthermore, mineralizing surface, mineral apposition rate, and bone formation rate were the highest at 5 Hz in the tibia (2.0-, 1.4-, and 2.7 fold, respectively) and 15 Hz in the femur (1.5-, 1.2-, and 1.8 fold, respectively). We observed that the tibia had a lower bone mineral density with more porous microstructures than the femur."<-this difference is interesting.  Maybe the higher BMD the more frequency needed to induce bone formation(and length increase).

"oscillatory alteration of intramedullary pressure in the femur was observed in response to sinusoidal loading with knee loading. Taken together, knee loading appears to affect motion of interstitial fluid as well as medullary fluid."

"Porosity is directly linked to the size of osteocyte population, which influences activities in bone remodeling. The relationship between osteocyte density and bone formation rate varies depending on skeletal site and developmental history. In human cancellous bone the inverse relationship between osteocyte density and bone formation rate was reported."

Height Increase with Lasers?

Chondrogenic mRNA expression in prechondrogenic cells after blue laser irradiation.

"For chondrogenic induction, ATDC5 cells were irradiated with a blue laser (405 nm, continuous wave) at 100 mW/cm(2) for 180 s following incubation in chondrogenic differentiation medium. Differentiation after laser irradiation was quantitatively evaluated by the measurement of total collagen contents and chondrogenesis-related mRNAs. The total amount of collagen and mRNA levels of aggrecan, collagen type II, SOX-9, and DEC-1 were increased relative to those of a non-laser irradiated group after 14 days of laser irradiation. On the other hand, Ap-2alpha mRNA, a negative transcription factor of chondrogenesis, was dramatically decreased after laser irradiation.  Intracellular reactive oxygen species (ROS) were generated after laser irradiation."

"Msx1 and 2, retinoic acid receptor, Zfp60, and Ap-2α have been found to suppress or delay chondrocyte differentiation"

"Light is thought to be absorbed by mitochondrial respiratory chain components, resulting in the increase of ROS, and adenosine triphosphate (ATP)/or cyclic AMP, and initiating a signaling cascade which promotes cellular proliferation and cytoprotection"

The effects of low-level laser therapy, 670 nm, on epiphyseal growth in rats.

" the aim of this study was to evaluate the use of LLLT, 670 nm, at three different doses on the epiphyseal growth of the right tibia of rats. Twenty-one Wistar rats, aged four weeks, were subjected to the application of LLLT, with dosage according to the group (G4: were submitted to the application of 4 J/cm(2); G8: were submitted to the application of 8 J/cm(2); G16: were submitted to the application of 16 J/cm(2)). After completion of protocol they were kept until they were 14 weeks of age and then submitted to a radiological examination (evaluation of limb length) and euthanised. The histological analysis of the growth plates (total thickness and hypertrophic and proliferative zones) was then performed. Comparisons were made with the untreated left tibia. No differences were observed in any of the reviews (radiological and histological), when comparing the right sides (treated) to the left (untreated). It was concluded that the treatment with LLLT within the parameters used caused changes neither in areas of the epiphyseal cartilage nor in the final length of limbs."

"Both the thermal, and nonthermal effects of ultrasound can produce changes in epiphyseal growth. Nonthermal effects occur through the alteration of membrane permeability to calcium influx, modulating the nuclear proliferation and also the RNA transduce. These effects are also found in low level laser therapy"

"The effect produced by LLLT irradiation is due to the absorption of energy by specific photoreceptors, such as porphyrin and cytochrome c oxidase. This generates increased production of oxygen, which stimulates the activity of mitochondria in ATP production, increases chemiosmosis, the production of DNA and the influx of calcium into the cytoplasm, which leads to mitosis and cell proliferation. These effects cause increased cell proliferation and migration, increased tissue oxygenation, and the modulation of cytokine levels, growth factors, and inflammatory mediators. As a result of these reactions, the laser produces anti-inflammatory and analgesic effects, promotes healing, the formation of blood vessels, and the stimulation of fibroblasts and bone cells"

"With respect to radiographic analysis, the medical report showed: soft tissue unchanged; bone texture preserved; joint surfaces smooth, and regular and symmetrical epiphyseal nucleus with normal dimensions"

Effect of GaAlAs laser irradiation on the epiphyseal cartilage of rats.

"To study the effect of an 830-nm gallium-aluminum-arsenic (GaAlAs) diode laser at two different energy densities (5 and 15 J/cm(2)) on the epiphyseal cartilage of rats by evaluating bone length and the number of chondrocytes and thickness of each zone of the epiphyseal cartilage.

A total of 30 male Wistar rats with 23 days of age and weighing 90 g on average were randomly divided into 3 groups: control group (CG, no stimulation), G5 group (energy density, 5 J/cm(2)), and G15 group (energy density, 15 J/cm(2)). Laser treatment sessions were administered every other day for a total of 10 sessions. The animals were killed 24 h after the last treatment session. Histological slides of the epiphyseal cartilage were stained with hematoxylin-eosin (HE), photographed with a Zeiss photomicroscope, and subjected to histometric and histological analyses. Statistical analysis was performed using one-way analysis of variance followed by Tukey's post hoc test. All statistical tests were performed at a significance level of 0.05.

Histological analysis and x-ray radiographs revealed an increase in thickness of the epiphyseal cartilage and in the number of chondrocytes in the G5 and G15 groups.

The 830-nm GaAlAs diode laser, within the parameters used in this study, induced changes in the thickness of the epiphyseal cartilage and increased the number of chondrocytes, but this was not sufficient to induce changes in bone length{likely as chondrocyte hypertrophy is the predominant increaser of bone length}."

"the chondrocyte layers that form the epiphyseal cartilage revealed that the resting cells, which are located on the boundary between the epiphyseal growth plate and osseous epiphysis, had a small number of chondrocytes distributed in the extracellular matrix. The general aspect of the cells suggested a small mitotic activity; also, cells were surrounded by reduced lacunar spaces, which is indicative of reduced cell activity."

"a case of a child with tissue damage in two distal phalangeal epiphyseal plates associated with the use of a CO2 laser for wart removal that resulted in deformity and shortening of the digits, requiring corrective osteotomy."


Low-level laser on femoral growth plate in rats.

"Thirty male Wistar rats aged 40 days were divided into two groups, G1 and G2. In G1 the area of the distal growth plate of the right femur was irradiated at one point using GaAlAs laser 830 nm wavelength, output power of 40 mW, at an energy density of 10 J/cm(2). The irradiation was performed daily for a maximum of 21 days. The same procedure was done in G2, but the probe was turned off. Five animals in each group were euthanized on days 7, 14 and 21 and submitted to histomorphometric analysis.

In both groups the growth plate was radiographically visible at all moments from both craniocaudal and mediolateral views. On the 21st day percentage of femoral longitudinal length was higher in G2 than G1 compared to basal value while hypertrophic zone chondrocyte numbers were higher in G1 than G2. Calcified cartilage zone was greater in G1 than in G2 at all evaluation moments. Angiogenesis was higher in G1 than in G2 at 14th and 21st days.

The low-level laser therapy negatively influenced the distal femoral growth plate."

"the rats were approximately 6 weeks of age on the first day of irradiation, a transitional period between a phase of accelerating growth (up to 5 weeks) and a time of decelerating growth (8-16 weeks), indicating that irradiation action occurs despite the longitudinal length rate of the bone."

"elevated angiogenesis may have contributed to early chondrocyte death and formation of calcified cartilage, contributing to reduction of the femoral longitudinal length."

"In another study, irradiation of the rat mandible growth center (Α=904 nm, 2000 Hz, pulse length 200 ns and output power 4 mW) on days 0, 2, 4, 6, 8 and 10, with the protocol repeated after a 50-day interval, produced higher growth than that observed in controls"

Increasing Height with Proepithelin?

The NF-kappaB pathway isn't typically associated with height growth. The only anabolic gene immediately associated with anabolic activity that's associated with NF-kappaB is PI3K. However, propethelin may stimulate growth plate chondrogenesis by means of the NFkappa-B pathway therefore exploring the NFkappa-B pathway may have other height increase applications.

Proepithelin Stimulates Growth Plate Chondrogenesis via Nuclear Factor- B-p65-dependent Mechanisms

"Proepithelin [is] an important regulator for cartilage formation and function. Proepithelin [mediates] induction of cell proliferation, differentiation, and apoptosis in the metatarsal growth plate. Proepithelin-mediated stimulation of metatarsal growth and growth plate chondrogenesis was neutralized by pyrrolidine dithiocarbamate, a known NF-κB inhibitor{propethelin stimulated growth plate chondrogenesis and therefore height growth, since propethelin's effects are mediated through NF-kappaB, NF-kappaB may be important for height growth as well}. In rat growth plate chondrocytes, proepithelin induced NF-κB-p65 nuclear translocation, and nuclear NF-κB-p65 initiated its target gene cyclin D1 to regulate chondrocyte functions. The inhibition of NF-κB-p65 expression and activity (by p65 short interfering RNA (siRNA) and pyrrolidine dithiocarbamate, respectively) in chondrocytes reversed the proepithelin-mediated induction of cell proliferation and differentiation and the proepithelin-mediated prevention of cell apoptosis. The inhibition of the phosphatidylinositol 3-kinase and Akt abolished the effects of proepithelin on NF-κB activation. Endogenously produced proepithelin by chondrocytes[so produced by the chondrocytes itself] is important for chondrocyte growth in serum-deprived conditions."

Stimulating proepithelin production by chondrocytes may be a way of increasing height.  Proepithelin may help without active growth plates as it stimulates cellular differentiation as well.

"Proepithelin is highly expressed in growth plate cartilage during embryonic and postnatal development. Its expression in musculoskeletal tissues appears to be restricted to chondrocytes, and it is concentrated in areas where ossification occurs, because it is localized exclusively in the lower proliferative and upper hypertrophic zones of the growth plate chondrocytes and is absent from osteocytes, osteoblasts, periosteum, and perichondrium. In vivo genetic knockdown studies of proepithelin showed a sharp reduction in skeletal length, bone volumes, and cortical bone thickness.  In patients with arthritis, both mRNA and protein levels of proepithelin were up-regulated, indicating that proepithelin may be critical for chondrogenesis"<-But does transgenic expression of proepithelin increase bone length further? As of now there are very few studies related to proepithelin so it is very under-explored in terms of increasing height.

"proepithelin-mediated activation of Akt is IRS-1-independent"

"Proepithelin, also known as granulin epithelin precursor 1, progranulin, PC cell-derived growth factor, or acrogranin, is the only known growth factor able to bypass the insulin-like growth factor receptor."<-IGFR is a possible negative feedback mechanism inhibiting height growth since proepithelin bypasses this negative feedback mechanism proepithelin has the ability to enhance height growth further than compounds like IGF-1 which cannot bypass the IGFR negative feedback mechanisms.

"proepithelin [may activate] AKT through an unidentified pathway"<-this might be how proepithelin exerts it's anabolic effects.

"Proepithelin is biologically active at an optimal effective dose of 240 nM"

"the NF- B subunit p65 facilitates growth plate chondrogenesis via the PI3K/Akt pathway"

"The addition of 10 M wortmannin (a PI3K inhibitor) or 10 M Akti1/2 (an Akt inhibitor) to the culture medium of proepithelin-treated chondrocytes significantly reversed the stimulatory effects of proepithelin on NF- B-p65 DNA-binding activity. In contrast, the addition of U0126 (a MAPK inhibitor), H-89 (a PKA inhibitor), or bisindolylmaleimide I (a PKC inhibitor) did not affect the proepithelin-mediated increase of NF kappaB-p65 activity. In the absence of proepithelin in the culture medium, none of these inhibitors had any effect on NF- B-p65 DNA-binding activity in chondrocytes"<-So proepithelin likely acts through NFkappaB-p65 through the PI3K/Akt pathway.

"cyclin D1 is known as a NF- kappaB induced gene that encodes molecules involved in cell proliferation"<-So cyclin D1 may be a mechanism in which NF-kappaB affects height growth.

"NF-kappa B was detectable in cyclin D1 promoter at 12 and 24 h after proepithelin stimulation."

"Proepithelin increased the height of the epiphyseal, hypertrophic, and proliferative zones of the growth plate

"endogenously produced proepithelin contributes to the ability of chondrocytes to grow in the absence of serum."

"Overexpression of proepithelin stimulates the proliferation of chondrocytes, and cartilage oligomeric matrix protein appears to be required for proepithelin-mediated chondrocyte proliferation in cartilage"<-this finding a way to induce overexpression of proepithelin may be a way to induce height growth.

"proepithelin regulates growth plate chondrogenesis and longitudinal bone growth by inducing the activity of NF-kappa B in growth plate chondrocytes"<-thus other activates of inducing NF-kappaB activity may be longitudinal growth stimulating as well.

"proepithelin may be an attractive medication with little toxicity to normal cells."<-So proepithelin may be a height increasing pill in the future.

So during development proepithelin stimulates longitudinal bone growth. Even afterwards it may help you grow taller by encouraging differentiation into chondrocytes. The scientists seems to believe that proepithelin should be developed into a pill. When this pill is developed, it's consumption may help you grow taller.

Gain Stature with Latency Associated Peptide?

An overgrowth disorder caused by TGF-Beta1 was only present when the gene coding for Latency Associated Peptide was modified and not TGF-Beta upregulation.  LSJL upregulates TGF-Beta (predicted but no change is observed in the gene expression data aside from a decrease in TGFbR1) but does it increase LAP too?

The single-molecule mechanics of the latent TGF-β1 complex.

"Together with its latency-associated peptide (LAP), TGF-β1 binds to the latent TGF-β1-binding protein-1 (LTBP-1), which is part of the extracellular matrix (ECM). Transmission of cell force via integrins is one major mechanism to activate latent TGF-β1 from ECM stores[LSJL can cause cell force]. Latent TGF-β1 mechanical activation is more efficient with higher cell forces and ECM stiffening.
We analyzed how forces exerted on the LAP lead to conformational changes in the latent complex that can ultimately result in TGF-β1 release. We demonstrate the unfolding of two LAP key domains for mechanical TGF-β1 activation: the α1 helix and the latency lasso, which together have been referred to as the "straitjacket" that keeps TGF-β1 associated with LAP. The simultaneous unfolding of both domains, leading to full opening of the straitjacket at a force of ~40 pN[a pico neuton which is 1 X10(-12) of a Newton so the amount of force in LSJL shouldn't be a problem], was achieved only when TGF-β1 was bound to the LTBP-1 in the ECM.
Our results directly demonstrate opening of the TGF-β1 straitjacket by application of mechanical force in the order of magnitude of what can be transmitted by single integrins. For this mechanism to be in place, binding of latent TGF-β1 to LTBP-1 is mandatory[does this occur in adult epiphyseal bone marrow]. Interfering with mechanical activation of latent TGF-β1 by reducing integrin affinity, cell contractility, and binding of latent TGF-β1 to the ECM provides new possibilities to therapeutically modulate TGF-β1 actions."

"Integrins activate latent TGF-β1 by at least two different mechanisms. One depends on proteases, which seem to be guided to the LLC[large latent complex which is composed of LAP and LTBP-1] by associating with integrins. The other is independent of proteolysis and involves transmission of cell traction forces to the LAP moiety of LLC"

"Whereas the overall LAP structure is predicted to be mechanically stable, two stretches in the molecule are prone to unfolding: the α1 helix and the latency lasso loop. Together both domains form a configuration that traps TGF-β1 in the SLC like a “straitjacket”"

The question is now does TGF-Beta form the complex with LAP and LTBP-1 in adult epiphyseal bone marrow?

According to this study TGF-Beta would be activated in LSJL(MMP-3 is the most significant gene upregulated by LSJL upregulated almost 5-fold):

The first stage of transforming growth factor beta1 activation is release of the large latent complex from the extracellular matrix of growth plate chondrocytes by matrix vesicle stromelysin-1 (MMP-3).

"Transforming growth factor beta-1 (TGF-beta1) is secreted in a biologically inactive form and stored in the extracellular matrix as a 290 kDa complex consisting of the mature TGF-beta1 homodimer (Mr 25 kDa), the latency-associated peptide (LAP; Mr 75 kDa), and the latent TGF-beta1 binding protein-1 (LTBP1; Mr 190 kDa). Latent TGF-beta1, composed of these three components, is known as the "large latent TGF-beta1 complex." In contrast, latent TGF-beta1 without LTBP1 is known as "small latent TGF-beta1." For all latent forms, dissociation of the TGF-beta1 homodimer from LAP is necessary for growth factor activation and acquisition of biological activity. Matrix vesicles produced by growth plate chondrocytes contain matrix metalloproteinases that can activate small latent TGF-beta1. The enzyme responsible for this is matrix metalloproteinase-3 (MMP-3), although matrix vesicles also contain MMP-2 and plasminogen activator. The present study tested the hypothesis that matrix vesicle enzymes are also involved in the release of the large latent TGF-beta1 complex stored in the extracellular matrix. Matrix vesicles were isolated from cultures of resting zone and growth zone chondrocytes and metalloproteinases present in the matrix vesicles extracted with guanidine-HCl. Chondrocyte extracellular matrices were prepared by lysing confluent cultures and removing the lysed cells. The matrices were incubated with matrix vesicle extracts and the release of total and active TGF-beta1 was determined. To determine if MMP-2 or MMP-3 was involved in the release, matrix vesicle extracts were preincubated with anti-MMP-2 antibody or anti-MMP-3 antibody to selectively deplete the enzyme activity. Matrices were also treated with rhMMP-2 or rhMMP-3. To determine the identity of the released protein(s), digests were separated on SDS-polyacrylamide gels and Western blotting analysis was performed using a specific antibody to LTBP1. Matrix vesicle extracts released both active and total (=latent + active) TGF-beta1 in a time-dependent manner, with peak release after 1 hour of incubation. The amount of total TGF-beta1 released was 10 times higher than the release of active TGF-beta1. The effect of the matrix vesicle extracts was dose-dependent; in addition, the amount and ratio of active to total TGF-b1 released was very similar, irrespective of the source of matrix or matrix vesicle extracts. Pre-incubation of matrix vesicle extracts with anti-MMP-3 antibody blocked the release of active and total TGF-beta1, whereas pre-incubation with pre-immune IgG or anti-MMP-2 antibody had no effect. The addition of rhMMP-3, but not rhMMP-2, caused a dose-dependent increase in the release of total, but not active, TGF-beta1. Both matrix vesicle extracts and rhMMP-3 released the large latent TGF-beta1 complex from the matrix. In addition to the expected 290, 230, and 190 kDa bands, samples run without reduction also contained proteins of molecular weights 110 and 50 kDa that reacted with the anti-LTBP1 antibody. When these same samples were electrophoresed after reduction, the high molecular weight immunoreactive bands disappeared and three bands of molecular weight 75, 32, and 25 kDa were observed. These results indicate that matrix vesicles contain enzymes, especially MMP-3, which are responsible for the release of TGF-beta1 from the matrix, most of which is in latent form. Further, the data suggest that release of the large complex occurs via cleavage at several novel sites in the 130 kDa LTBP1 molecule. Since matrix vesicle MMP-3 is also able to activate small latent TGF-beta1, these results suggest that the large latent TGF-beta1 complex protects against activation of the small latent TGF-beta1. release of the large latent TGF-bl complex from the matrix and activation of the latent growth factor are only two steps of what must be at least a three-step process[we need to make sure LSJL follows all three steps]."

Specificity of latent TGF-ß binding protein (LTBP) incorporation into matrix: role of fibrillins and fibronectin. states that LTBP-1 association depends on the fibronectin network.

Fibronectin is required for integrin alphavbeta6-mediated activation of latent TGF-beta complexes containing LTBP-1. identifies integrin alphavbeta6 as the key for activating latent TGF-Beta.  LSJL upregulates Integrin Alpha V which could lead to higher levels of integrin alphavebeta6.

Thus, intregin alphavbeta6, MMP-3, LAP, and LTBP-1 are all proteins to look for as possibly helping increase height by increasing active TGF-Beta levels.