Experiment LIPUS routine

In my research, I've found that LIPUS does in fact generate hydrostatic pressure.  It also generates a rounded cell phenotype even in pre-osteoblastic cells and rounded cells are indicative of a chondrogenic phenotype.  The cells ended up returning to normal after the LIPUS(Ultrasound)-induced treatment stopped but with normal MSCs the cells may stay as differentiated chondrocytes.  The studies also indicated that heat helped encourage chondrogenesis.  Another reason to flex surrounding epiphyseal muscles during LSJL(which generates heat).  One study found that Ultrasound may affect the growth of the Mandibular condyle(the jaw).  That study found possible new cell differentiation up to applying the ultrasound for 10 minutes but only increases in growth rate after 10 minutes but before 20 minutes.

So I definitely think LIPUS is worthwhile to experiment on and I think we have some parameters to go on.

Here's the machine and here's the gel:ReliaMed Portable Ultrasound.  Parker Laboratories Aquasonic Ultrasound Gel .25 Liter Bottle.  You'll have to keep buying new gel as it runs out.

One of the studies found that the most effective was the highest one they used 407 mW per centimeters squared.  The max setting available on the machine is 4.6W or 4600 mW.  The effectiveness of this varies based on the size of your bone.

To perform Ultrasound you apply the gel to the epiphysis of the target bones.  And then move the pad around the epiphysis for around 10 to 20 minutes.  I haven't tried this yet.  I'll be buying an ultrasound soon though so I don't know any complications that can arise from this.

I would do either LIPUS or LSJL because to do both would take too much time.

All application times are 10 minutes.  Since you are doing both inner and outer some of the effects should come through when you're doing the other side of the leg.  You can do it while watching TV.

If you're bald do the top of the head as a control group as this bone is a little bit different.  If not try your heel bone or a tip of your finger.

Inner ankle
Outer ankle
Inner Tibia Epiphysis
Outer Tibia Epiphysis
Inner Femur epiphysis
Outer Femur Epiphysis

For the spine, you have to do one vertebrae at a time and both the tops and bottoms of each vertebrae so I don't think we should try it until we get everything else ironed out.

If you just want to help experiment.  Do one finger of your hand with LSJL and one with LIPUS.  Both for five minutes.

Grow Taller with Ultrasound

Previously, ultrasound was spoken about as a way to stimulate interstitial fluid flow in a manner similar to LSJL.  LIPUS also has the ability to stimulate signaling pathways(probably through IFF) and upregulate and down regulate genes.  What if sound was another mechanical signaling pathway like electromagnetic fields and the actin cytoskeleton(which can be modulated by cholesterol)?  Then Sound can be used for us high seekers to grow taller even when other methods have stalled.

The science of ultrasound therapy for fracture healing 

"Transforming growth factor-β (TGF-β) synthesis and adenylyl cyclase activation in osteoblasts are also stimulated by LIPUS." 

Both factors are anabolic. 

"Growth of the calcified portion of the metatarsal diaphysis in LIPUS-treated metatarsal rudiments was noted to triple during the week of treatment as compared to the control, untreated group (530 μm versus 180 μm). LIPUS directly affects osteoblasts and ossifying cartilage, resulting in more active ossification."  

LIPUS increases growth rate in osteoblasts.

"The osteosarcoma cell line, UMR-106, was subjected to 20-min doses of LIPUS. Expression of the immediate early-response genes c-fos and cyclooxygenase-2 (COX-2) were up-regulated by this treatment. Elevated mRNA levels for alkaline phosphatase and osteocalcin were also noted after LIPUS treatment." 

Both Alkaline Phosphatase and osteocalcin are anabolic to bones.

"Prostaglandin E2 (PGE2) production is regulated by COX-2. Both are thought to be vital for fracture healing, and can be affected by treatment with LIPUS. The effect of LIPUS on PGE2 regulation and on COX-2 mRNA expression was recently examined in the mouse osteoblastic cell line, MC3T3-E1. [There was] a three-fold increase in the amount of PGE2 produced by cells exposed to LIPUS for 60 min, as compared to control cells. Up-regulation of COX-2 was also noted after treatment of the cells with LIPUS; this up-regulation peaked at 60 min and subsided by 180 min.
PGE2 stimulates cellular responses by signaling through a cascade of enzymatic reactions. PGE2 binds to a guanine nucleotide-binding (G) protein-coupled receptor (GPCR), a class of heptahelical transmembrane receptors involved in cellular responses to external stimuli as diverse as photons, odorants, hormones (such as glucagon), and other extracellular mediators (such as epinephrine). The GPCRs, upon activation by ligand binding, activate diverse downstream pathways of intracellular signaling, ultimately leading to expression of certain genes associated with intracellular metabolism and with cell growth and proliferation.
If PGE2 production is related to fluid shear (thought to be created by LIPUS), then up-regulation of the downstream effectors of PGE2 should be similarly affected.  [There's an] effect of fluid shear stress on COX-2 gene expression in cultured MC3T3-E1 osteoblasts as well as in primarily cultured osteoblasts from mouse calvaria.  Fluid shear stress increased COX-2 gene expression within 30 min. This [is] under the direct control of extracellular signal-regulated kinase (ERK), one of the mitogen-activated protein (MAP) kinases known to be activated by growth factor receptors and by GPCRs, such as the PGE2 receptor."

So does LIPUS create a separate effect other than fluid shear strain(is it worth doing in addition to LSJL)? 

"At fracture sites, mesenchymal stem cells are differentiated into chondrocytes during the formation of the soft, cartilaginous callus. Mesenchymal stem cells, in primary culture, can be differentiated into chondrocytes by treatment with TGF-β. Aggrecan deposition, indicative of chondrocyte differentiation, was increased (8.7-fold versus 2.3 fold) if LIPUS was added to TGF-β, without increasing total protein content or cellular proliferation. Ultrasound increases chondrocyte differentiation of mesenchymal stem cells treated with TGF-β." 

Remember hydrostatic pressure increases chondrogenic differentiation as well. 

"Chondrocytes can be induced to proliferate and maintain chondrocytic lineage also via treatment with LIPUS. Chondrocytes from neonatal Wistar rats were isolated and cultured. Exposure to LIPUS induced and maintained expression of type II collagen and aggrecan, and blocked expression of type X collagen. It was noted to increase expression of TGF-β. Addition of TGF-β to the nontreated cells stimulated similar results, and blockade of TGF-β by addition of a neutralizing antibody abrogated the results seen after LIPUS stimulation. The mechanism of action of LIPUS likely involves TGF-β, in addition to other mechanisms, and that TGF-β is vital for chondrocyte responsiveness to LIPUS."  

LIPUS seems to have very similar effects to LSJL.  Loading of chondrocytes also increases type II collagen and aggrecan. Blocked expression of Type X Collagen is very good as Type X collagen is associated with fusion. 

"Production of VEGF messenger RNA and protein, NO, and hypoxia-inducible factor-1α (HIF-1α) were all stimulated by LIPUS. Inhibition of NO production reduced the VEGF response to LIPUS. The VEGF and HIF-1α responses to LIPUS are mediated by nitric oxide (NO) production. Integrin receptors, present in focal adhesions, are vital for this signaling cascade"  

Shear strain can also upregulate VEGF.  VEGF, NO, and HIF-1alpha are predicted to be upregulated by LSJL but such upregulation was not observed overthreshold thus maybe LIPUS as use as a supplement to LSJL.

Treatment of human mesenchymal stem cells with pulsed low intensity ultrasound enhances the chondrogenic phenotype in vitro.

"low intensity pulsed ultrasound (LIPUS) [was applied] on human bone marrow-derived mesenchymal stem cells undergoing chondrogenic differentiation. Aggregates of mesenchymal stem cells and mesenchymal stem cells seeded in three dimensional matrices were cultured in a defined chondrogenic medium and subjected to LIPUS for the first 7 days of culture. At 1, 7, 14 and 21 days, samples were harvested. Cell aggregates with daily treatment for 20 minutes showed no significant differences for proteoglycan and collagen content during chondrogenic differentiation. ultrasound application for 40 minutes daily resulted in a statistically significant increase of the proteoglycan and collagen content after 21 days in culture[so duration is important]. Aggregates treated for 20 minutes daily showed decreased expression of chondrogenic genes at all time points. 40 minutes of daily treatment of aggregates resulted in a significant increase of chondrogenic marker genes after an initial decrease at day 7 with time in culture. Ultrasound treated cell-scaffold constructs showed a significant increase of chondrogenic marker gene expression and extracellular matrix deposition. LIPUS can be used to enhance the chondrogenesis of mesenchymal stem cells in cell aggregates and cell-scaffold constructs."

"Pulsed low intensity ultrasound (30 mW/cm2) was applied for 0, 20 and 40 minutes per day, for the first 7 consecutive days of culture"  The specs used were "a carrier frequency of 1.5 MHz and with a 200 µs tone burst repeating at 1.0 kHz"<-That's crazy, that's 1,000 times per second!

"undifferentiated mesenchymal cells and differentiated chondrocytes differ significantly in terms of their responses to biomechanical forces"

"cyclic compressive loading can promote the chondrogenesis of rabbit bone marrow derived mesenchymal stem cells by inducing the synthesis of TGF-β1, which can stimulate the mesenchymal cells to differentiate into chondrocytes"

Low-Intensity Ultrasound (LIUS) as an Innovative Tool for Chondrogenesis of Mesenchymal Stem Cells (MSCs).

"LIUS not only induces chondrogenic differentiation of MSCs but also has diverse additional activities that enhance the viability of MSCs, increase possibly the integrity of the differentiated tissues and delays hypertrophic changes during differentiation. LIUS could be an innovative and versatile tool for chondrogenic differentiation of MSCs and for cartilage tissue engineering."

"preconditioning by LIUS for a short period (e.g., for one week) could program MSCs to differentiate well into chondrogenic lineages or to better maintain chondrogenic phenotypes during subsequent culture in the absence of additional LIUS stimulation."

"other groups used LIUS of pulsed wave at 30 mW/cm2, while we used continuous wave at 200 mW/cm2."

"LIUS signal appears to use the mechanotransduction pathway including integrins, stretch-activated ion channels, and interleukin-4 on the cell surface."

"LIUS could inhibit vascular invasion, as judged by immunohistochemical analysis for pallet/endothelial cell adhesion molecule-1 (PECAM-1) and could reduce the expression of angiogenic factors such as hypoxia inducible factor-1α (HIF-1α) and vascular endothelial growth factor (VEGF) in rabbit MSCs in vitro or in nude mice"

"LIUS induced chondrogenic differentiation of MSCs in the absence of added TGFβ both in vitro and in vivo"

"LIUS induced expression of tissue inhibitor of metalloproteinases-2 (TIMP-2) but showed no effect on the protein level of metalloproteinase-3 (MMP-3) or mRNA levels of MMP-13, and type I and X collagens. TIMP-2 functions as an inhibitor of MMPs and an anti-angiogenic factor that can inhibit vascular invasion following hypertrophy of chondrocytes during endochondral ossification. Type I and X collagens and MMP-13 are also involved positively in the hypertrophic changes of chondrocytes. LIUS might inhibit degradation of extracellular matrix (ECM) proteins and/or hypertrophic changes of differentiated MSCs, thereby enhancing the integrity of cartilage constructs."

"TGFβ is necessary and sufficient to induce chondrogenic differentiation of MSCs in vitro."<-LSJL induces TGF-Beta.

"LIUS [plays an] actively inducing role that directs MSCs to chondrogenic lineages without exogenous growth factors such as TGFβ. LIUS also had diverse additional functions during the chondrogenic differentiation of MSCs in vitro and in vivo. It enhanced (1) viability of MSCs by inhibiting apoptosis of cells, (2) regulated expression of genes involved in the integrity of the differentiated construct, and (3) delayed hypertrophic changes at the late stage of differentiation. "

So LIPUS can increase height but it doesn't seem to do so with a mechanism that's different than LSJL(both induced fluid flow/hydrostatic pressure).  So you should do either one or the other and LSJL is cheaper.  Electromagnetic fields operate differently because of the biophotons stored in intracellular DNA.


Integrin-mediated mechanotransduction pathway of low-intensity continuous ultrasound in human chondrocytes.

"US stimulation of chondrocytes induced phosphorylation of focal adhesion kinase (FAK), Src, p130 Crk-associated substrate (p130Cas), CrkII and extracellular-regulated kinase (Erk)[likely phosphorylated by LSJL]. Pre-incubation with inhibitors of integrin receptors, Src and MAPK/Erk kinase (MEK) reduced US-induced Erk phosphorylation levels, indicating integrins and Src are upstream of Erk in an US-mediated mechanotransduction pathway. US signals through integrin receptors to the MAPK/Erk pathway via a mechanotransduction pathway involving FAK, Src, p130Cas and CrkII."

"Human chondrocytes that were serum-deprived overnight were treated with 5.0 MHz US for 3 min"

"Focal adhesion complexes are formed by the interaction of the cytoplasmic tail of integrins with proteins such as paxillin, talin, vinculin, actin and FAK "

"FAK autophosphorylates at Y397, generating a high-affinity binding site for the Src homology 2 (SH2) domain of Src family kinases"

The effect of ultrasound stimulation on the gene and protein expression of chondrocytes seeded in chitosan scaffolds

"A low-intensity diffuse ultrasound (LIDUS) signal at 5.0 MHz (0.14 mW/cm2) was employed to stimulate bovine chondrocytes seeded in three-dimensional (3D) chitosan-based matrices. While the duration of application was constant at 51 s, US was applied once, twice, four times and eight times/day, and the impacts of US on the biosynthetic activity of chondrocytes and the expression of chondrocyte-specific genes were evaluated. When stimulated with continuous US for predetermined time intervals, chondrocytes had higher levels of type II collagen, aggrecan, L-Sox5 and Sox9{all but Sox5 was up in LSJL} mRNA expression when compared to controls; however, under the same conditions, the expression of MMP-3{up in LSJL} was downregulated. Both Sox5 and Sox9 genes coordinately responded to changes in US stimulation and generally mirrored the response of collagen type II transcript to changes in US stimulation. US stimulation increased the gene expression of cell-surface integrins α5 and β1. The expression of integrins α2 was downregulated by US treatment, suggesting that multiple integrin subunits may be involved in the regulation of chondrocytic function in response to US stimuli. The enhancement in the abundance of the mRNA transcripts upon US stimulation was observed to correlate with the protein expression of collagen type I, collagen type II, and integrins α5 and β1."

4 ultrasound applications per day increased cell count the most.

"indirect mechanical stimulation in the form of an acoustic signal [upregulates] the expression of integrins α5 and β1 and [downregulates] the expression of α2 at the mRNA level"

Low-intensity ultrasound stimulation enhances chondrogenic differentiation in alginate culture of mesenchymal stem cells.

"we investigated the effects of LIUS on the chondrogenesis of rabbit MSCs (rMSCs) in a 3-D alginate culture and on the maintenance of chondrogenic phenotypes after replating them on a monolayer culture. The LIUS treatment of rMSCs increased: (i) the matrix formation; (ii) the expression of chondrogenic markers such as collagen type II, aggrecan, and Sox-9; (iii) the expression of tissue inhibitor of metalloprotease-2 implicated in the integrity of cartilage matrix; and (iv) the capacity to maintain the chondrogenic phenotypes in a monolayer culture. Notably, LIUS effects were clearly shown even without TGF-beta treatment. LIUS treatment could be an efficient and cost-effective method to induce chondrogenic differentiation of MSCs in vitro for cartilage tissue engineering."

"MSCs were isolated from 2-week-old female New Zealand white rabbits"

"LIUS was treated for 10 min every 12 h for 1 or 2 weeks at a frequency of 1 MHz and an intensity of 200 mW/cm2 in a continuous-wave fashion. The intensity of 200 mW/cm2 was selected"

Note though that they were cultured in alginate beads which means the cells had adhesion capabilities.

Effect of therapeutic ultrasound on endochondral ossification.

"Ultrasound was tested on endochondral ossification of in vitro developing metatarsal long bone rudiments of 16- and 17-day-old fetal mice. Bone growth, calcification and resorption following exposure to several doses of pulse-wave (PW) or continuous-wave (CW) ultrasound were examined. PW was applied at intensities between 0.1 W cm-2 and 0.77 W cm-2 (Isatp) and CW intensities were 0.1 W cm-2 or 0.5 W cm-2 (Isata). After 1 week of culture, the metatarsal long bone rudiments were fixed and paraffin sections were prepared for histological evaluation and for measurement of the relative contribution of the various cartilage zones to the total bone length. In contrast to treatment with CW ultrasound, treatment of 16-day-old metatarsal long bone rudiments with PW ultrasound resulted after 4 days of culture in significantly increased longitudinal growth. Histology revealed a significant increased length of the proliferative zone, whereas the length of the hypertrophic cartilage zone was unaltered. This might indicate that proliferation of the cartilage cells is stimulated without influence on cell differentiation."

10% increase in growth rate was observed in 0.77 W cm-2 PW group.

"Doses of 0.1 or 0.5 W cm-2 continuous-wave (CW) ultrasound, applied for 1 or 5 min, had no significant effect on the longitudinal growth of the 16-day-old MT during culture, although the treated samples tended to show decreased growth"

"a dose of 0.77 W cm-2 causes an acoustic pressure amplitude of 276 kPa in the spatial peak in degassed water"

Mechanobiological conditioning of stem cells for cartilage tissue engineering.

"Mechanobiological conditioning (e.g. mechanical compression, hydrostatic pressure, osmotic, shear, ultrasound) [influences chondrogenic differentiation of dedifferentiated chondrocytes. Using an in vitro aggregate culture system enhanced chondrogenesis of mesenchymal progenitor cells, detected by an increased extracellular matrix deposition of collagen and aggrecan, could be shown under repeated cyclic hydrostatic pressure. An increase in chondrogenic differentiation of mesenchymal progenitor cells could be detected, when the cells were loaded in three-dimensional matrices and subjected to cyclic, compressive load or low-intensity pulsed ultrasound."

I do not have access to the full study.

Applications of Exogenous Mesenchymal Stem Cells and Low Intensity Pulsed Ultrasound Enhance Fracture Healing in Rat Model.

"[We] investigate the effects of combined treatment of exogenous mesenchymal stem cells (MSCs) and low intensity pulsed ultrasound (LIPUS) on fracture healing by comparing LIPUS-MSC, MSC and control (CTL) groups. Quantitative callus width/area demonstrated that the MSC-LIPUS group had the best healing, MSC group the second and CTL group the poorest with significant differences among each at different time points. MSC-LIPUS had the highest bone volume/tissue volume. [There was] a significantly faster remodeling in late phase in MSC-LIPUS and MSC groups. Combined treatment of MSCs and LIPUS was beneficial to fracture healing. Regenerative power and homing ability of MSCs were shown by promotion in fracture healing and locally found green fluorescent protein (GFP)-labeled MSCs at fracture calluses."

"three-month-old female Sprague-Dawley (SD) [were used]"

"a metal blade (weighted 500 g) dropping from a height of 35 cm, was used to create the fracture at the midshaft of the femur. The quality of fracture with fracture gap [was] smaller than 0.5 mm and displacement [was] less than 0.5 mm"

" Large amount of cartilaginous tissues bridged the osseous callus in the first week. At week 2, woven bone was observed in MSC-LIPUS and MSC groups with active endochondral ossification at the edge of cartilaginous tissues, while this phenomenon was shown in CTL group at week 3. The area of cartilaginous tissues tended to reduce in both MSC-LIPUS and MSC groups at week 3 and more woven bone replaced the cartilaginous tissues to bridge the fracture site in week 4, particularly in MSC-LIPUS group. There were, however, still large amounts of cartilaginous tissues in CTL group in week 4."

"LIPUS has been proven to increase blood flow around fracture site that might promote angiogenesis, MSCs migration and, hence, fracture healing."It's possible LIPUS could do this to normal bone too.

Combined Treatment of Alendronate and Low-Intensity Pulsed Ultrasound (LIPUS) Increases Bone Mineral Density at the Cancellous Bone Osteotomy Site in Aged Rats: A Preliminary Study.

"9-month-old Sprague-Dawley rats, with a unilateral proximal tibial osteotomy, were treated with alendronate (daily, 1 µg/kg) plus sham-LIPUS, saline plus LIPUS (20 min/day), alendronate plus LIPUS, or saline plus sham-LIPUS as a control for 4 weeks"

"The combined therapy significantly increased BMD at the osteotomy site at 4 weeks compared with the control, without affecting the contralateral, non-osteotomized tibia. Both alendronate and LIPUS alone also exerted a positive, albeit less, effect on BMD in the affected limb. Alendronate and LIPUS cooperate to enhance BMD during metaphyseal bone healing."

LIPUS and Alendronate were applied to both osteomy and non-osteomy bone.

"The ultrasound signals were transferred for 20 min per day with a spatial average-temporal average (SATA) intensity of 30 mW/cm2"

The region of interest was the epiphysis.  It is possible that LIPUS can increase other parameters besides BMD in non-osteomy bone.  BMD was the only parameter that was measured.

Low-intensity ultrasound increases FAK, ERK-1/2, and IRS-1 expression of intact rat bones in a noncumulative manner.

"daily 20-minute LIPUS [was] applied to intact tibias.

FAK, ERK-1/2, and IRS-1 expression and activation [was detected] from the stimulated intact tibias at 7, 14, and 21 days of daily 20-minute LIPUS.

LIPUS increased FAK expression (at 7 days), ERK-1/2 (at 14 days), and IRS-1 (at 7 days), but expression decreased 7 days later, indicating a noncumulative effect of LIPUS. As only FAK expression was detected at 21 days, these observations suggest LIPUS influences nuclear reactions that may be modulated by a major cellular mechanism preferentially inhibiting IRS-1 expression and not FAK expression. Increased ERK-1/2 expression at 14 days suggests the differing mechanisms for promoting ERK-1/2, FAK, and IRS-1 syntheses. IRS-1 expression behaved similarly to FAK expression; therefore, LIPUS may modulate growth factor pathways. LIPUS increased sustained FAK and ERK-1/2 activation, but not IRS-1, suggesting sustained ERK-1/2 activation is not the result of mechanically induced growth factor activation."

"[Male] WH rats, aged 12 weeks [were used]"

ERK 1/2 was phosphorylated at Tyr-204.  p-ERK 1/2 was the same in both control and LIPUS group at 21 days indicating a conditioning response.

"IGF-1 expression may be increased by LIPUS"

12 week old WH mice are still growing.

"Seven days after the augmentation of FAK (at 14 days) and ERK-1/2 (at 21 days) expression, their expression decreased, showing a noncumulative effect of LIPUS on intact bone and suggesting there is a regulatory mechanism in bone cells, similar to negative feedback, that is activated after the augmentation of FAK and ERK-1/2 expression. In healthy bone, this mechanism may have antineoplastic finalities, because FAK is persistently increased in neoplasia"

Mechanical means to improve bone strength: ultrasound and vibration.

"At  intensities of 5 to 300 W/cm2, [ultrasound] is used as a surgical tool to fragment calculi{mineral deposits}"

"LIPUS uses a significantly low intensity and does not yield major temperature changes (typically <1°C). However, even these minor temperature changes are hypothesized to stimulate enzymatic processes involving some sensitive enzymes, such as matrix metalloproteinase 1 and collagenase"

Low-intensity pulsed ultrasound (LIPUS) and cell-to-cell communication in bone marrow stromal cells.

"Single or daily-multiple LIPUS treatment at 1.5MHz, 30mW/cm(2), for 20min was applied to BMSC. BMSC form functional gap junctions and single LIPUS treatment significantly increased the intracellular dye transfer between BMSC. Activated phosphorylation of ERK1/2 and p38 by LIPUS stimulation was diminished when cells were treated with a gap junction inhibitor 18β-glycyrrhetinic acid (18β). We further demonstrated that 18β diminished the significant increase in alkaline phosphatase activity following LIPUS stimulation."

"Gap junctions mediate the intercellular exchange of regulatory ions and small molecules that allow metabolic cooperation between adjacent cells and control cell differentiation and growth. In bone, gap junctions have been suggested to be central to the transmission of biophysical stimuli"

LIPUS increased ALP by 30% and this was diminished by 18B.

The development and validation of a LIPUS system with preliminary observations of ultrasonic effects on human adult stem cells.

" a custom LIPUS system was designed and validated by exploring the effects of using different pulse repetition frequency (PRF) parameters on human adipose derived adult stem cells (hASCs) and bone marrow derived mesenchymal stem cells (hMSCs), two common stem cell sources for creating bone constructs in vitro. Changing the PRF was found to affect cellular response to LIPUS stimulation for both cell types. Proliferation of LIPUS-stimulated cells was found to decrease for hASCs by d 7 for all three groups compared with unstimulated control cells (P = 0.008, 0.011, 0.014 for 1 Hz, 100 Hz and 1 kHz PRF, respectively) and for hMSCs by d 14 (donor 1: P = 0.0005, 0.0002, 0.0003; donor 2: P = 0.0003, 0.0002, 0.0001; for PRFs of 1 Hz, 100 Hz, and 1 kHz, respectively). Additionally, LIPUS was shown to strongly accelerate osteogenic differentiation of hASCs based on amount of calcium accretion normalized by total DNA (P = 0.003, 0.001, 0.003, and 0.032 between control/100 Hz, control/1 kHz, 1 Hz/1 kHz, and 100 Hz/1 kHz pulse repetition frequencies, respectively)."

LIPUS increased calcium accretion per total DNA for hMSCs as well.

Application of low-intensity ultrasound to growing bone in rats.

"he longitudinal growth and bone density of the femur and tibia in young rats was measured after application of an ultrasound transducer emitting 1.5-MHz pulsed ultrasound (30 mW/cm2, SATA) for 20 min/day. After 28 days, no length difference was detected (< or = 2%) compared to the sham-treated leg or to unexposed controls. Also, no significant difference in bone mineral density (BMD) of the femur or tibia was found (< or = 6%). In a repeated experiment in which a periosteal trauma stimulus was created in the femoral diaphysis, the ultrasound also had no effect on growth or BMD."

"Four-week-old Sprague–Dawley male rats (approximately 90 g) [were used]"

Ultrasound was applied to a huge region which could be part of the problem and why there wasn't significant growth.

Ultrasound though seemed to increase femur length but decrease tibia length.

" in fracture experiments, disruption of the cortex may provide better access to activated endosteal and periosteal cells than in the intact limb."

Effects of low-intensity pulsed ultrasound on the differentiation of C2C12 cells.

"Pluripotent mesenchymal cell line C2C12 were cultured in differentiation medium with or without the addition of LIPUS stimulation. The ultrasound signal consisted of 1.5 MHz at an intensity of 70 mW/cm2{more than twice the 30mW/cm2 of LIPUS} for 20 min for all cultures. To verify the cell lineage after LIPUS stimulation, mRNA expression of cellular phenotype-specific markers characterizing osteoblasts (Runx2, Msx2, Dlx5, AJ18), chondroblasts (Sox9), myoblasts (MyoD), and adipocytes (C/EBP, PPARgamma) was studied s. The protein expression of Runx2 and activated phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) and p38 mitogen-activated protein kinase (p38 MAPK) were performed using Western blotting. The mRNA expression of Runx2, Msx2, Dlx5, AJ18, and Sox9 was increased markedly by the LIPUS stimulation, whereas the expression of MyoD, C/EBP, and PPARgamma was drastically decreased. In the Western blot analysis, LIPUS stimulation increased Runx2 protein expression and phosphorylation of ERK1/2 and p38 MAPK. LIPUS stimulation converts the differentiation pathway of C2C12 cells into the osteoblast and/or chondroblast lineage via activated phosphorylation of ERK1/2 and p38 MAPK{LSJL likely phosphorylates these two proteins as well}."

"C2C12 cells are a subclone of C2 myoblasts"

"Dlx5 expression correlates with osteoblast differentiation"

"The maximum expression of Dlx5 occurs in the final stages of osteoblast differentiation in vitro when the extracellular matrix mineralizes, suggesting that Dlx5 may be involved in the differentiation of the osteogenic cells. In contrast, Msx2 is predominantly expressed by proliferating osteoblasts and preosteoblasts, but decreases according to terminal osteoblast differentiation"

Modelling condensation and the initiation of chondrogenesis in chick wing bud mesenchymal cells levitated in an ultrasound trap.

"Chick wing bud mesenchymal cell micromass culture allows the study of a variety of developmental mechanisms, ranging from cell adhesion to pattern formation. However, many cells remain in contact with an artificial substratum, which can influence cytoskeletal organisation and differentiation. An ultrasound standing wave trap facilitates the rapid formation of 2-D monolayer cell aggregates with a defined zero time-point, independent from contact with a surface. Aggregates formed rapidly (within 2 min) and intercellular membrane spreading occurred at points of contact. This was associated with an increase in peripheral F-actin within 10 min of cell-cell contact and aggregates had obtained physical integrity by 30 min. The chondrogenic transcription factor Sox9 could be detected in cells in the ultrasound trap within 3 h (ultrasound exposure alone was not responsible for this effect). This approach facilitates the study of the initial cell-cell interactions that occur during condensation formation and demonstrates that a combination of cell shape and cytoskeletal organisation is required for the initiation and maintenance of a differentiated phenotype, which is lost when these phenomena are influenced by contact with an artificial substrate."

"[N-CAM and N-Cadherin while encouraging mesenchymal condensation, their] expression following condensation also inhibits chondrogenesis"  Cadherin 7 may also be key for mesenchymal condensation.

"loss of Sox9 occurs due to reorganisation of the F-actin cytoskeleton"

The pressure generated was 110 kPa.

"During condensation, cells come together and are compacted and take on a rounded morphology, show peripheral actin organisation, form adhesive complexes and gap junctions, then begin to express Sox9"

Low Intensity pulsed Ultrasound an alternative to LSJL

Low Intensity Pulsed Ultrasound has been shown to have profound benefits on the bone.  The theory behind Lateral Synovial Joint Loading is that it works by increasing interstitial fluid flow which in turn increases hydrostatic pressure.  Fluid flow transports stem cells to the hyaline cartilage growth plate line and hydrostatic pressure promotes chondrogenic differentiation of stem cells.  Joint Loading has been shown so far to increase ankle size and there have been sporadic but inconsistent reports of height increase(still self experimenting but need yet more experimenters).  The profound increase in ankle size shows that LSJL may be profoundly effective in increasing height in the spine.  The problem is finding an effective way to load the spine.  Enter LIPUS: Medical Products Online Professional Ultrasound Systems - AC Adapter 240/110 Volt International/Universal Adapter which can be purchased fairly cheaply(the goal is to induce shear strain and induce hydrostatic pressure exact degrees may not matter).

The machine listed above can go up to 150hz.  You can apply the ultrasound device to each of the vertabrae.  Like LSJL, LIPUS may enhance interstitial fluid flow.  So benefits of LSJL and LIPUS may intersect.

Pertussis toxin-sensitive Galphai protein and ERK-dependent pathways mediate ultrasound promotion of osteogenic transcription in human osteoblasts.

"Bone cells respond to mechanical stimulation via mechanoreceptors and convert biophysical stimulation into biochemical signals that alter gene expression and cellular adaptation. Pulsed acoustic energy treatment raises membrane potential and induces osteogenic activity. How membrane-bound osteoblast mechanoreceptors convert physical ultrasound (US) stimuli into osteogenic responses is not fully understood. We demonstrated that low-intensity pulsed US treatment (200-micros pulse, 1 kHz, 30 mW/cm2) elevated Cbfa1/Runx2 mRNA expression and progressively promoted osteocalcin mRNA expression in human osteoblasts. Pretreatment with pertussis toxin (PTX), but not with cholera toxin, suppressed US-augmented osteogenic transcription. This indicated that Gi proteins, but not Gs proteins, were involved in US promotion of osteogenic transcription. Further studies demonstrated US treatment could rapidly increase PTX-sensitive Galphai protein levels and subsequently enhanced phosphorylation of extracellular signal-regulated kinase (ERK). PTX pretreatment significantly reduced US promotion of ERK activation. Moreover, inhibition of ERK activity by PD98059 suppressed US augmentation of Cbfa1/Runx2 and osteocalcin mRNA expression. Membranous Galphai proteins and cytosolic ERK pathways acted as potent mechanosensitive signals in the response of osteoblasts to pulsed US stimulation[so interstitial fluid flow]."

The machine listed above only has a watt output of 8 and the amount tested in this study was 30(since our goal is to stimulate the spine and Runx2 is involved in osteoblasts which increase height in the spine we want to at least match the study).  I don't know where to find a higher powered ultrasound.

Identification of genes responsive to low-intensity pulsed ultrasound stimulations.

"This study was designed to compare the temporal changes of gene expression profile in osteoblastic cell lines (SaOS-2) treated with low-intensity pulsed ultrasound stimulation (LIPUS) using complementary DNA (cDNA) microarrays. SaOS-2 cells were treated with LIPUS for 20min. Thereafter, cells were harvested and RNA was extracted twice at 4 and 24h, respectively. Using cDNA microarrays, 7488 genes with changes in expression in SaOS-2 cells were identified for comparison. Microarray analysis revealed a total of 165 genes in SaOS-2 cells were regulated at 4 and 24h after LIPUS treatment. Except for 30 known LIPUS-regulated genes, our study demonstrated for the first time that over 100 genes were related to the underlying molecular mechanism of LIPUS and suggested that LIPUS might regulate a transient expression of numerous critical genes in osteoblastic cells."

Although this study relates to an osteoblastic cell line the same sort of genes may be upregulated in MSCs and chondrocytes.  And in the future we can use this study to compare to other studies to see which genes are upregulated to osteoblasts and which are upregulated due to other factors.

"(LIPUS, 200-μs burst of 1.5 MHz and 30.0 ± 5.0 mW/cm2) was able to stimulate osteogenesis"

" LIPUS induced transient expression of the immediate-early response gene c-fos and elevated gene expression for bone sialoprotein (BSP), insulin-like growth factor-1 (IGF-1), and osteocalcin (OC) using a mouse bone marrow stromal-derived cell line (ST2 cell), osteoblasts, and osteocytes"

"he LIPUS system used for this study generated 1.5 MHz ultrasound in a pulsed-wave mode (200-μs pulse burst-width with repetition frequency of 1 kHz at the spatial-averaged temporal-averaged intensity of 30 mW/cm2) "

Genes upregulated by LIPUS(noted if only upregulated at 4 or 24 hours) also by LSJL:
ITGBL1
IGFBP6(24hrs only)
GNAL(24hrs only){down}
LOX(24hrs only)

Downregulated:


Early gene response to low-intensity pulsed ultrasound in rat osteoblastic cells

"The aim of the current research was to quantify the changes in gene expression in rat bone marrow derived stromal cells (BMSC) to low intensity pulsed ultrasound (LIPUS) during early time points after the ultrasound application. LIPUS at 1.5 MHz, 30 mW/cm2 was applied to BMSC for a single 20 min treatment. Real-time PCR was carried out to quantify the expression of early response genes and bone differentiation marker genes 0.5, 1, 3, 6 and 12 h after the end of the LIPUS treatment. Compared with the controls, LIPUS treatment resulted in elevated transient expression of early response genes (c-jun{up}, c-myc, COX-2{up as PTGS2}, Egr-1{up}, TSC-22) as well as the bone differentiation marker genes, osteonectin and osteopontin{up}, at 3 h."

At 1 hour in response to LIPUS egr-1, c-jun, and PTGS2 were downregulated in contrast to LSJL where these genes were upregulated after the last loading.  These genes returned to baseline at about 6 hours so it's not likely that the upregulation in LSJL was due to residual effects.

Similar trends occurred with osteopontin and that was measured with RT-PCR whereas the responses to the other three genes were done with microarray for LSJL.