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.