Friday, April 6, 2012

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."

10 comments:

  1. Seeing how many people are not seeing results, I wonder if the loading time you suggested (I believe you said 1 min) is insufficient. In many of Dr. Yokota's research, he loads the mice knees at 3 minutes. Using basic logic, if he loads a mice at 3 minutes, shouldn't us humans be loading at a longer period. Hence, i've started loading for at least 5 minutes, and I'll probably do it longer to test it.

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  2. St.It is still within the age where growth could be occurring naturally. If he was 25 then it might lend more weight to the idea that you don't have to feel interstitial fluid flow.

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  3. also what would be the best possible nutrition pre and post lsjl ?

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  4. Im not expert in scientific field of chondrocytes and bones, but my logic tells me that directly seeking answers from the experts themselves would be a good idea.

    is there any way or do u have advise for me if say i wanted to get in contact with scientist? and ask them a simple question on what they suggest on growing taller?
    i figure it would be a wiser thing to do, after all. they can tell us straight up what they know, and i guess it would probably need a couple scientist to ask this question/

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    1. Ping Zhang is the scientist involved in LSJL most likely to be able to help with height increase as he has a grant in load driven bone lengthening. I have had no luck in direct contact with scientists.

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  5. http://www.ncbi.nlm.nih.gov/pubmed/22457138

    I don't know if all the puerarin talk is over, but this is a new study showing that puerarin promotes osteoblast bone formation.
    So, would puerarin be a good supplement for LSJL?

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  6. i heared about a site www.tallplace.com. This site ofered to increase height only legs up to 4 inch.

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  7. Tyler, You are an inspiration to me. Whether or not all this is true (evidence says yes, gut says no) the amount of work and research you put into this is simply amazing. Please keep the blog going and for the love of god, don't stop or give up hope for height growth. I check out your blog everyday and the sheer tenacity you display inspires me to no end!

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  8. In Fact, I'd have to say that one of my biggest fears is that I'll type in heightquest in my address bar and it won't be there.

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  9. btw, I've seen that the routine for LSJL has not been updated in quite a while. Is it still good? Moreover, I'd like it if you could point me towards your finger proof results. As I remember, It's been over a year now and I'd like to know if you've made any clearly visible progress.

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