Gene Expression Patterns of Bone under normal mechanical load versus LSJL

Each day I go through studies to put into old posts and most of them are not that helpful but I found one that's full of one and insight's into the legendary microfracture theory sprouted by Sky and others.  Search for (*NEW*) to find it.  The study has a lot of great diagrams and the full study is available.  It's a rich informative study so it would be beneficial for other people to look into it.  Ultimately, you can see that the load required to induce a microfracture may be too much and if kicking with ankle weights induced the microfractures such as in this study you would notice them due to the massive appositional bone formation(thus if you did grow taller with such a method you'd have lots of bumps on your bones--chicks dig scars but do they dig microfractures?).  However, it may be possible for tapping with a sufficient load enough times may be enough to induce such a fracture.  I have no idea what that load is though.

CH Turner was one of the leading researchers behind Lateral Synovial Joint Loading before he passed away and he was the researcher who was most directly interested in bone(Hiroki Yokota for example was more interested in mechanotransduction).

CH Turner's studies are still coming out.  Here's one about the gene expression of bone on mechanical loading.  It will be interested to compare that to gene expression under LSJL.<-Read that study, it's important!

Gene expression patterns in bone following mechanical loading.

"The primary goal of this study was to determine the time sequence for gene expression in a bone subjected to mechanical loading during key periods of the bone-formation process, including expression of matrix-related genes, the appearance of active osteoblasts, and bone desensitization. A standard model for bone loading was employed in which the right forelimb was loaded axially for 3 minutes per day{so axial loading rather than our lateral loading, note that the epiphysis still gets loading under axial loading as there's no way to avoid it}, whereas the left forearm served as a nonloaded contralateral control. We evaluated loading-induced gene expression over a time course of 4 hours to 32 days after the first loading session. Six distinct time-dependent patterns of gene expression were identified over the time course and were categorized into three primary clusters: genes upregulated early in the time course, genes upregulated during matrix formation, and genes downregulated during matrix formation. Genes then were grouped based on function and/or signaling pathways. Many gene groups known to be important in loading-induced bone formation were identified within the clusters, including AP-1[The C-Fos and c-Jun complex]-related genes in the early-response cluster, matrix-related genes in the upregulated gene clusters, and Wnt/β-catenin signaling pathway inhibitors in the downregulated gene clusters. Chemokine-related genes, were upregulated early but downregulated later in the time course; solute carrier genes{these help with chondrocyte hypertrophy}, were both upregulated and downregulated; and muscle-related genes,  were primarily downregulated{This is interesting, as many anabolic pathways are shared between muscle and bone; this gives weight to the theory that muscle and bone compete for resources}."

The Peak Load used was 13N. "Compressive load was applied as an oscillating Haversine waveform for 360 cycles at a frequency of 2 Hz"  24 hours between loading.  Genes upregulated in supplementary material were taken from all time points from 4 hours to 32 days.  Mice were 20 weeks old.

"Bone responds in an anabolic manner to physiologic dynamic loading. For example, the midshaft humerus in the throwing arm of baseball pitchers and catchers showed enhanced bone mass, structure, estimated strength, and resistance to torsion compared with the nonthrowing control arm.{bone length was measured but the comparative length was not presented, something to ask Stuart J. Warden} In contrast, bone mineral density (BMD) in astronauts decreased 1.0% and 1.5% in the spine and hip, respectively, per month of spaceflight{This is not that much considering astronauts are reported to gain 3 inches in space, this shows you the potential of your intervertebral discs in terms of height gain}"

"Mechanical loading uses pathways currently being investigated for new drug development, such as low density lipoprotein receptor–related protein 5 (LRP5) and sclerostin"<-supplements to look into

"New osteoblasts appear on the bone surface 24 to 48 hours after initiating mechanical loading, and bone formation is observed within 96 hours of loading. Bone formation increases between 5 and 12 days after starting loading, but after 6 weeks of loading, bone formation returns to baseline levels."

This is for osteoblasts not chondrocytes which is what we are mainly looking for with LSJL but  it may take 12 days after starting LSJL to notice new osteoblast bone deposition(never mind that LSJL requires a chondrocyte phase beforehand).  A month is not enough time to measure results.

The shaft of the bone was used so stem cell genes should be detected.  So if any mesenchymal chondrogenesis occurred it should show up.

Looking at what was upregulated in axial Loading and Lateral Loading share many of the same pathways like TGF-Beta and WNT/B-Catenin in addition to many ECM related proteins.

Some genes involved in LSJL that were not involved in axial loading hyaluronan synthase(involved in hyaluronic acid).  No MMP3 in axial loading in contrast to lateral loading(MMP-3 is stimulatory to chondrocytes). 

And of course no induction of chondrogenic differentiation of stem cells in axial loading(that's more due to hydrostatic pressure though than genes).  Though it should be represented by genes which I think it is given the upregulating of ECM genes.  Chondrogenic differentiation produces ECM but ECM doesn't always indicate chondrogenic differentiation.

BMP-2 and TGF-Beta were produced by axial loading.  Both of which can induce chondrogenic differentiation.  Axial Loading + LIPUS may be enough to gain height.

Interleukin 1 receptor-like 1 was expressed by both axial and LSJL.  Stat3 was expressed which induces Lin28B expression.  It also downregulated FGF23 which may be involved in growth plate reactivation.

Upregulated Genes of Note(see supplementary material):
Acan(upregulated in LSJL)
ADAMTS1(up)
Adh7(up)
Angptl2(up)
Apcs(down)
Apln(up)
Arg1(up)
Bambi
Bgn(up)
BMP2(up)
c1qtnf5(up)
c3ar1(up)
Capn6(up)
Car8(up)
ccl2(up)
ccl7(up)
cd14(up)
cd276(up)
CCND1
Cdh15(down)
Cgref1(up)
Chgb(down)
Cit(down)
Cntn1(up)
Fn1
Follistatin
Cntn1(up in LSJL)
Col2A1 alpha1(up in LSJL)
Col3A1 alpha1(up in LSJL)
Col16A1(up)
crabp2(up)
creb3l1(up)
cspg4(up)
cthrc1(up)
cxcl1(up)
dbx1(down)
dlg4(up)
dnm1(up)
ENPP3(down)
fkbp10(up)
Gfpt2(up)
ggcx(up)
glrb(up)
GPR180(down in LSJL)
grin2d(up)
Hapln1(up in LSJL)
Hdlbp(down)
Hif1alpha
Hnf4a(down in LSJL)
HTRA1(up)
Id2(down in LSJL)
il1rl1(up)
IRS-1(down in LSJL)
Junb(also upregulated in LSJL)
Kcnn2(up)
Lepre1(up)
Leptin
lmna(up)
lox(up)
Lrat(up)
Mall(up)
Metrnl(down)
MMP2(up in LSJL)
MMP9
MMP14(up in LSJL)
NDRG4(up)
Neurod2(down)
Ninj1(down)
Nkx2.5
Nos3
Nr4a2(up)
Pacsin1(down)
Pcdhb2(up)
pcsk6(up)
pdgfc(up)
pdpn(up)
prrx1(up)
prss35(up)
PTHR1
PTGS2(up in LSJL)
PTN(up in LSJL)
RPL36al(down)
S100A4(up)
Scn1a(up)
Sct(down)
Serpina3n(up)
Serpine1(up)
Sept5(down)
Slc1a4(up)
Slc6a2(up)
Slc6a15(up)
Slco2a1(up)
Smad9(up)
Smpd3(down)
SOCS2(Anti-height gene)
SOCS3(upregulated in LSJL)
Sp7
Stat3
Syndecan 4(also upregulated in LSJL)
TGFbp1
TGFbp3
tnfrsf12a(up)
TIMP1(up in LSJL)
Vcan(up in LSJL)
VDR
Zfp36(upregulated in LSJL)

Axial loading upregulated a few chondrogenic genes like Acan and COL2A1 but nowhere near the amount of Collagens upregulated by LSJL which also upregulated Col9.  Also key, is that Sox9 is not upregulated in axial loading whereas it is in LSJL.

Downregulated genes of note:
Acacb(down)
Acsl6(down)
Anxa3(down in LSJL)
Arl6ip1(down)
Asb2(down)
Asph(up)
BMPR1B(up in LSJL)
Btla(up)
ccnb1(down in LSJL)
ccr1(up)
dpp4(down)
Egr1(up in LSJL)
Fgr(down)
Fnbp1(down)
GADD45A
Galc(down)
Gas6
Ghitm(down)
GHR
IGFBP6(up in LSJL)
Kynu(up)
Leptin Receptor
Mkrn1(down)
Mrps18b(up)
Myl1(up)
Nexn(down)
Ntn1(up)
Pcsk1(up)
Pdlim3(up)
Pkia(up)
Plag1
Ppp1r3c(up)
Prkaa2(down)
Prkg2(up in LSJL)
Pygl(down)
Rsad2(down as Pcaf)
Sdpr(down)
Sla(down)
Slc16a1(down)
Slc25a30(down)
Sost
Srpkg3(down)
TGFBR3
Tnnt3(down)
Trim55(down)
Tsc22d3(down)
Ucp2(down)
Vav1(down)
Vcam1(down in LSJL)

The differential expression of Egr and BMPR1B between Axial Loading and Lateral Loading could be key to LSJL's ability to induce chondrogenesis.

The gene expression data for LSJL was taken 1 hour after the last loading and in this study genes were taken 4 hours after the first loading.  So we can compare these early response genes to see how they compare to LSJL.

Upregulated:
Fosl1
Junb(up in LSJL)
Anxa2
S100A4(up)
S100A10
CCBP2
CCL2(up)
CCL7(up)
CXCL1(up)
CXCL13
IL1RL1(up)
IL1RL2
Osm
Osmr
Socs3(up)
Stat3
Tnfrsf12a(up)
Adamts1(up)
ECM1
Serpina3n(up)
Serpine1(up)
Tfpi2
CCND2
Clic1
Gpr1
KCNE4
Lep
Syndecan4 (up)

Regulatory mechanisms in bone following mechanical loading.

"The right forelimb [of rodents] was loaded axially for three minutes per day, while the left forearm served as a non-loaded, contralateral control. Animals were subjected to loading sessions every day, with 24 hours between sessions. Ulnas were sampled at 11 time points, from 4 hours to 32 days after beginning loading."

Mice were 20 weeks old.

"The peak load achieved during loading was 13 N"

Stat5B was upregulated 4 hours following loading. Stat5b is downregulated in LSJL

The expression of COL1 differs greatly with LSJL.  At 14 days there was almost no COL1 expression whereas at 14 days there was still extremely high COL1 levels with Axial Loading.

"The CREB-related transcription factors are important for bone formation, specifically ATF4, which is required for collagen synthesis by mature osteoblasts. The CREB motif was predicted to be positive at 2d, 4d, 6d, and 8d. The transcription factors that bind to the CREB motif include cAMP responsive element binding protein 1 (CREB1), cAMP responsive element modulator (CREM), activating transcription factor 1 (ATF1), ATF2, ATF3, ATF4, and ATF7. The CREB motif was present in the promoter of an important matrix gene, fibronectin 1 (Fn1), and in genes that promote collagen construction and cross-linking, including Lox, prolyl 4- hydroxylase beta polypeptide (P4hb), and procollagen C-endopeptidase enhancer (Pcolce)"

"at 32d, the system was less responsive to loading and had shifted from bone forming to baseline bone maintenance"<-maybe every 32 days take a break from LSJL?

Collagen 1 alpha 1 did not begin to rise until two days after loading.  The LSJL study took gene expression at 49 days after first loading.  In the axial loading Col1A was upregulated 3-fold seven days after loading whereas with LSJL it was upregulated only 2 fold.

Alternative Splicing in Bone Following Mechanical Loading

Alternative splicing means that gene expression was altered in mRNA.  The whole bone was ground.

"Compressive load was applied as an oscillating Haversine waveform for 360 cycles at a frequency of 2 Hz using a Bose ElectroForce 3200 Series electromechanical actuator"<-Peak Load was 13N.  Axial loading was used.

"Rats were subjected to loading sessions every day, with 24 hours between sessions."  Rats were 20 weeks old.  Gene expression data was taken up to 4 hours to 32 days.

The greatest alteration of gene expression occurred at 16 days or about 2 weeks.  Maybe this is when conditioning effect starts to inhibit gene expression?

According to this Study Sox9 mRNA was not altered at any time point.  Col2a1and Acan mRNA were altered.  Tgfbeta1 and Tgfbeta2 expression was altered.  The key stature genes HMGA2 and Lin28b were altered in LSJL but not here.

Altered genes of note:

Akt1

Akt2

BMPr1a

BMPr1b{up in LSJL}

BMP2{up in LSJL}

BMP4

CNP

CREB3l1

Esrra

Esrrb

Esr2{up in LSJL}

FGF2{up in LSJL}
FGF4

FGF21

FGFR1{up}

FGFR3

GH1{down in LSJL}

GHR

GHRHR

GPC3

HMGA1

ID2{down}

ID4

IGF1

IGF1R

IGF2R

IGF2bp1

NPR1

NPR2

NPR3

PLAG1

PRKG2{up}
RARA

Runx3

Shh

SHOX2

Smad1{down}

Smad2

Smad3

Smad4

Smad5

Smad7(inhibits BMP signaling, Smad6 which inhibits TGF-Beta signaling is not altered)

Smad9

Sox10

Sox11

Syn3

Twist1

Wnt4

Wnt5a

Here's the Partek GSEA Analysis to compare to LSJL for chondrogenic related genes, Bold means the p-value < 0.05, no fold changes were given and no fold cutoff is used:

Chondroblast Differentiation(6.77) 100%:
RARA
FGF4
FGF2
Cyr61

Chondrocyte Differentiation(5.16) 45%:
Col2a1
Creb3l2
MAPK14
Col11a2
TGFB1
Mef2d
FGFR1
OSR1
FGF9

Cartilage Condensation(8.32) 58.32%:
THRA
COL2A1
Tgfb2
Uncx
Ctgf
Bmpr1b
Acan

Chondrocyte Development(1.80) 33.33%<-this could be a key between LSJL and axial loading which has an enrichment score of 3.80.

Cartilage Development(12.70) 44.2%

Cartilge Development Involved in Endochondral Bone Morphogenesis(2.10) 42.8%

Growth Plate Cartilage Development(0.27) 14.29%

Endochondral Ossification(1.66) 29.41%

Systemic effects of ulna loading in male rats during functional adaptation.

"The aim of this study was to determine the effects of loading of a single bone on adaptation of other appendicular long bones and whether these responses were neuronally regulated. Young male Sprague-Dawley rats were used. The right ulna was loaded to induce a modeling response. In other rats, a second regimen was used to induce bone fatigue with a mixed modeling/remodeling response; a proportion of rats from each group received brachial plexus anesthesia to induce temporary neuronal blocking during bone loading. Sham groups were included. Left and right long bones (ulna, humerus, tibia, and femur) from each rat were examined histologically 10 days after loading. In fatigue- and sham-loaded animals, blood plasma concentrations of TNF-α, RANKL, OPG, and TRAP5b were determined. Loading the right ulna induced an increase in bone formation in distant long bones that were not loaded and that this effect was neuronally regulated{LSJL increased length in bones not loaded}. Distant effects were most evident in the rats that received loading without bone fatigue. In the fatigue-loaded animals, neuronal blocking induced a significant decrease in plasma TRAP5b at 10 days. Histologically, bone resorption was increased in both loaded and contralateral ulnas in fatigue-loaded rats and was not significantly blocked by brachial plexus anesthesia. In young, growing male rats we conclude that ulna loading induced increased bone formation in multiple bones. "

"The periosteum is the skeletal tissue with the greatest density of sensory nerve fibers, which are arranged in a dense netlike meshwork that is optimized for detection of mechanical distortion. Nerve branches or single neurons enter the bone cortex, often in association with the microvasculature, and connect individual bone cells to the central nervous system via unmyelinated sensory neurons."

"In the load and block + load groups, loading was performed for 1500 cycles at 4 Hz, with an initial peak strain of −3,750 µɛ (−18 N entered into materials testing machine, −16.8 N applied to ulna). In the fatigue and block + fatigue groups, cyclic loading was performed at 4 Hz. Loading was initiated at −16 N, and the load applied to the ulna was increased incrementally until fatigue was initiated. Loading then was terminated when 40% loss of stiffness was attained."

"TRAP5b is expressed on both immature and mature osteoclasts; plasma TRAP5b concentrations are proportional to osteoclast number."

(*NEW*)

Healing of non-displaced fractures produced by fatigue loading of the mouse ulna.

"Using adult (5 month) C57Bl/6 mice, we first determined that cyclic compression of the forelimb under load-control leads to increasing applied displacement and, eventually, complete fracture. We then subjected the right forelimbs of 80 mice to cyclic loading (2 Hz; peak force approximately 4N) and limited the displacement increase to 0.75 mm (60% of the average displacement increase at complete fracture). This fatigue protocol created a partial, non-displaced fracture through the medial cortex near the ulnar mid-shaft, and reduced ulnar strength and stiffness by >50%. Within 1 day, there was significant upregulation of genes related to hypoxia (Hif1a) and osteogenesis (Bmp2, Bsp) in loaded ulnae compared to non-loaded, contralateral controls. The gene expression response peaked in magnitude near day 7 (e.g., Osx upregulated 8-fold), and included upregulation of FGF-family genes (e.g., Fgfr3 up 6-fold). Histologically, a localized periosteal response was seen at the site of the fracture; by day 7 there was abundant periosteal woven bone surrounding a region of cartilage. From days 7 to 14, the woven bone became denser but did not increase in area. By day 14, the woven-bone response resulted in complete recovery of ulnar strength and stiffness, restoring mechanical properties to normal levels. In the future, the fatigue loading approach can be used create non-displaced bone fractures in transgenic and knockout mice to study the mechanisms by which the skeleton rapidly repairs damage."

Monotonic loading: "Both forelimbs of five mice were loaded by a displacement ramp (0.5 mm/sec) to complete, displaced fracture in order to determine monotonic mechanical properties. Mice were euthanized immediately after loading. Ultimate force (mean ± SD) was 4.32 ± 0.21 N, and stiffness was 3.98 ± 0.26 N/mm."

Fatigue loading:  "Both forelimbs of 14 mice were cyclically loaded at peak compressive forces (F) ranging from 2.1 to 3.5 N (50 to 80% of average ultimate force) until complete fracture. "

Force loading, to partial non-displaced fracture: "Right forelimbs of 80 mice were cyclically loaded at peak forces ranging from 3.75 to 4.10 N (70–75% of ultimate force) while displacement was monitored. Loading was terminated when peak displacement increased by 0.75 mm relative to the peak displacement at cycle 10."

The key here is to see if any chondrogenic genes were upregulated in partial, non -displaced fractures(so like a microcrack).  Col2a1 was highly upregulated at day 7.  BMP2[1.1-1.9 fold] and FGF2[1.1-1.8 fold] were moderately upregulated.  FGF2 was more highly upregulated in LSJL than here whereas BMP2 was more highly upregulated here than LSJL.  Hif1a the chondrogenically related transcription factor was more significantly upregulated peaking at 3.0 at day 3 and increasing before and decreasing after.

It should be noted that LSJL gene expression was done by microarray whereas this study was done with RT-PCR with the exception of BMP2 which was also done by PCR.

Here's what a bone microfracture-microcrack looks like:

M stands for marrow. CB stands for cortical bone.  WB stands for woven bone.

"Longitudinal sections of fatigue-loaded ulnae (H&E) show that the fracture occurred as a non-displaced, oblique crack through the medial cortex (arrows). On day 1 after loading, a clot is seen on both ends of the crack. On day 3, the periosteum is expanded and filled with cellular, fibrovascular tissue; nascent woven bone is seen sub-periosteally. On days 7 and 11 there is abundant woven bone on the medial periosteum. In approximately one-half of specimens the callus contained no cartilage (not shown), but in the others there was cartilage (*) in the center of the woven bone."

"Cyclic loading of the rat forelimb (~18 N peak force) to 85% of fracture displacement resulted in a non-displaced fracture localized to the medial cortex of the ulna and an associated loss of ulnar strength and stiffness of 55 and 80%, respectively. Because the fracture in the rat ulna was partial and non-displaced, and because the repair process involved negligible cartilage formation, we referred to this as a “stress fracture”, consistent with descriptions by others"<-now we've considered that that 0.5N in LSJL is equivalent to 100N on a 200lbs human which is already a challenge.  Imagine the challenge of generating 18N peak force.  It's possible that less force may be needed if the force is applied cyclically over a long period of time as long as that force causes residual damage in the bone.

"Cartilage was often observed at 7 and 11 day timepoints and appeared only on the medial surface, corresponding to the periosteal fracture location. By comparison, in studies of complete fracture in mice cartilage is seen on both sides of the bone as well as between the fractured ends"

The fracture occurred on the medial side and although the majority of the activity is on the medial side there is some enhanced activity on the lateral side giving weight to the possibility of gradually lengthening the bone through microfracture(as the unfractured side does seem to adapt).  Although the force required to induce a sufficient microfracture may be too large to be induced under normal physiological circumstances(and you would notice a bone adaptation as large as that depicted).  That does not preclude the possibility that rapid loading that is large enough to induce residual damage to bone is enough to induce such a microfracture as well.  Something like tapping.

Tibial loading increases osteogenic gene expression and cortical bone volume in mature and middle-aged mice.

"We examined this question in female BALB/c mice of different ages, ranging from young to middle-aged (2, 4, 7, 12 months). We first assessed markers of bone turnover in control (non-loaded) mice. Serum osteocalcin and CTX declined significantly from 2 to 4 months. There were similar age-related declines in tibial mRNA expression of osteoblast- and osteoclast-related genes, most notably in late osteoblast/matrix genes. For example, Col1a1 expression declined 90% from 2 to 7 months. We then assessed tibial responses to mechanical loading using age-specific forces to produce similar peak strains (-1300 µε endocortical; -2350 µε periosteal). Axial tibial compression was applied to the right leg for 60 cycles/day on alternate days for 1 or 6 weeks. qPCR after 1 week revealed no effect of loading in young (2-month) mice, but significant increases in osteoblast/matrix genes in older mice. For example, in 12-month old mice Col1a1 was increased 6-fold in loaded tibias vs. controls. In vivo microCT after 6 weeks revealed that loaded tibias in each age group had greater cortical bone volume (BV) than contralateral control tibias, due to relative periosteal expansion. The loading-induced increase in cortical BV was greatest in 4-month old mice (+13%). Non-loaded female BALB/c mice exhibit an age-related decline in measures related to bone formation. Yet when subjected to tibial compression, mice from 2-12 months have an increase in cortical bone volume. Older mice respond with an upregulation of osteoblast/matrix genes, which increase to levels comparable to young mice."

Unfortunately, no chondrogenic genes were studied.

Global gene expression analysis in the bones reveals involvement of several novel genes and pathways in mediating an anabolic response of mechanical loading in mice

"We applied mechanical loads[4-point bending] to the right tibias of the B6 mice at 9 N, 2 Hz for 36 cycles per day, with the left tibias used as unloaded controls"

"4 days of loading"

"Twenty-four hours after last stimulation"<-whereas LSJL was 1 hour after last stimulation.

"Ten-week-old C57BL/6J female mice"

Complete list of supplementary gene comparison to LSJL to be done.  Gene comparison of just genes on main paper and spot comparisons done below.
Genes upregulated in bone to four point bending also upregulated by LSJL:
Ptn
Ogn{down}
Itm2a
Lepre1
Col6a3
Col14a1
Col18a1
Matn2
Lox
Gas1
Timp1
Acta2
Ppfibp1{down}
Fer1l3
Spon2
Wnt2
Lmna
Sgk
Odz3
Anxa8
Chl1
Adamts4
Tcf12{down}
Col4a2
Junb
Tnc{down}
Bgn
Egfr
MMP2
BSP

Downregulated:
Mkrn1

Optimal Loads for Lateral Joint Loading

I finally got the full LSJL gene expression data and it shows that LSJL upregulates Sox9, COL2A1, Agc1, and MATN3 which is only expressed by cartilage all by at least 2 fold(See below).   Decreased expression of Id2 is also associated with chondrogenesis.  This provides support that LSJL can form new growth plates.  I'll have to finish my analysis to ascertain more insight.

Recently, David stated that he had been loading his right epiphysis by 120lbs(!) to try to correct a 1cm length discrepancy.  David used a hydraulic car jack and put two 45 lbs plates on his couch then he gradually used the jack to increase the pressure on his epiphysis.  Here's his ankle picture after several months:

You can see his right epiphysis is much bigger than his left.  David has reported no length change.  An increase in bone size is excellent news because it indicates some of or some combination of the below: stem cell differentiation into osteoblasts which deposit new bone; mechanical signaling triggering existing osteoblasts to lay down new bone(bone modeling); and shear strain on periosteum resulting in periosteal progenitor cells which differentiate into osteoblasts which deposit new bone.  Unfortunately, no height growth means no differentiation into chondroctyes.  Could excessive loads favor osteogenesis over chondrogenesis?  Or do you also need to load the articular cartilage to activate certain factors that favor chondrogenesis like Sox9 and TGF-Beta? 

Signalling cascades in mechanotransduction: cell-matrix interactions and mechanical loading. 

"Mechanical loading of articular cartilage stimulates the metabolism of resident chondrocytes and induces the synthesis of molecules to maintain the integrity of the cartilage[the articular cartilage connects to the hyaline cartilage of the growth plate so it can stimulate metabolism and synthesis of melcules there as well]. Mechanical signals modulate biochemical activity and changes in cell behavior through mechanotransduction. Compression of cartilage results in complex changes within the tissue including matrix and cell deformation, hydrostatic and osmotic pressure, fluid flow, altered matrix water content, ion concentration and fixed charge density[compression of cartilage changes hydrostatic pressure and one can presume growth plate hydrostatic pressure]. These changes are detected by mechanoreceptors on the cell surface, which include mechanosensitive ion channels and integrins that on activation initiate intracellular signalling cascades leading to tissue remodelling. Excessive mechanical loading also influences chondrocyte metabolism but unlike physiological stimulation leads to a quantitative imbalance between anabolic and catabolic activity resulting in depletion of matrix components[You shouldn't use too much load or risk depleting matrix components]. In this article we focus on the role of mechanical signalling in the maintenance of articular cartilage, and discuss how alterations in normal signalling can lead to pathology."

The bone begins as completely hyaline cartilage.  Any mechanical loading induced growth should lead to increased long bone growth as the articular cartilage is connected to the resting zone hyaline cartilage until later in development.

"Increased joint loading in athletes is associated with an increase in the area of the load-bearing surface rather than an increase in cartilage thickness"<-Joint Loading increases joint width but not height but can lateral loading of joints encourage joint height as it's on a different axis?

"cyclic tensile loading increased the mRNA level of MMP-1, MMP-3, MMP-9, IL-1β, TNF-α and TIMP-1 in cultured chondrocytes"<-tensile loading means stretch.  Aside from Interleukin Beta and TNF-Alpha these are the best genes for height growth to be expressed by chondrocytes.  So lateral joint loading directly on the cartilage may have height increase benefits as well.

"insulin-like growth factor-1 (IGF-1) and TGF-β increase chondrocyte cell surface expression of α3/α5 integrin subunits and stimulate adhesion of chondrocytes to fibronectin and type II collagen"<-IGF-1 and TGF-Beta encourage chondrocytes to adhere to the Type II collagen parts of the growth plate

"IL-4 not only mediates anabolic signalling by increasing aggrecan expression but can decrease catabolic events. In an animal model intra-articular injection of IL-4 decreased chondrocyte nitric oxide production and inhibited destruction of cartilage in instability-induced osteoarthritis. Pre-treatment with IL-4 (10 ng/mL) suppressed both MMP-13 and cathepsin B induction by mechanical stress, as well as cyclical tensile stress-induced IL-1β expression"<-IL-4 may be a promising height increase supplement for the future.

Gene expression profiles of dynamically compressed single chondrocytes and chondrons.

"A chondrocyte produces a hydrated pericellular matrix (PCM); together they form a chondron. Previous work has shown that the presence of the PCM influences the biological response of chondrocytes to loading. The objective of this study was to determine the gene expression profiles of enzymatically isolated single chondrocytes and chondrons in response to dynamic compression. Cartilage specific extracellular matrix components and transcription factors were examined. Following dynamic compression, chondrocytes and chondrons showed variations in gene expression profiles. Aggrecan[increases proteoglycan content], Type II collagen[Type II collagen is the basis for articular and hyaline cartilage, could possibly lead to height growth] and osteopontin[involved in bone modeling] gene expression were significantly increased in chondrons. Lubricin gene expression decreased[Lubricin is a joint lubricant so could be part of a negative feedback mechanism] in both chondrons and chondrocytes. Dynamic compression had no effect on SOX9 gene expression. Our results demonstrate a clear role for the PCM in interfacing the mechanical signalling in chondrocytes in response to dynamic compression. Further investigation of single chondrocytes and chondrons from different zones within articular cartilage may further our understanding of cartilage mechanobiology."

Dynamic loading of articular cartilage may enhance height growth by upregulating Type II Collagen expression. 

[Type II collagen fragment capacity to induce collagen cleavage and hypertrophy of articular chondrocyte] 

"The objective of this study was to determine whether a peptide of type II collagen which can induce collagenase activity can also induce chondrocyte differentiation (hypertrophy) in articular cartilage. At high but naturally occurring concentrations (10 microM and up) the collagen peptide CB12-II induced an increase in the expressions of MMP-13 (24h) and cleavage of type II collagen by collagenase in the mid zone (day 4) and also in the superficial zone (day 6). Furthermore the peptide induced an increase in proliferation on day 1 in the mid and deep zones extending to the superficial zone by day 4. There was also upregulation of COL10A1 expression at day 4 and of type X staining in the mid zone extending to the superficial zone by day 6. Apoptopic cell death was increased by day 4 in the lower deep zone and also in the superficial zone at day 7. The increase in apoptosis in the deep zone was also seen in controls. Our results show that the induction of collagenase activity by cryptic peptide sequence of type II collagen is accompanied by chondrocyte hypertrophy and associated cellular and matrix changes. This induction occurs in the mid and superficial zones of previously healthy articular cartilage. This response of the chondrocyte to a cryptic sequence of denaturated type II collagen may play a role in naturally occurring hypertrophy in endochondral ossification and in the development of cartilage pathology in osteoarthritis." 

So the Type II Collagen environment in the growth plate can trigger chondrocyte hypertrophy.  This could be why proliferative capacity is conserved in growth hormone deficiency as there is less surrounding Type II Collagen.  So overloading may result in the denaturing of Type II collagen which encourages ossification.
 
Genome-Wide Analyses of Gene Expression during Mouse Endochondral Ossification 

"Numerous molecular markers characterize the central stages of the chondrocyte life cycle. Chondrogenesis is typified by the expression of Sox transcription factors 5,6 and 9. Proliferating chondrocytes synthesize an ECM composed mainly of collagen II and aggrecan, among others, while the central ECM molecule expressed in hypertrophic cartilage is collagen X. Factors expressed at the chondro-osseous junction regulate chondrocyte apoptosis and mineralization of the cartilaginous ECM. Late hypertrophic chondrocytes express factors that promote angiogenesis, bone deposition and the secretion of bone-specific cell ECM[So chondrocyte hypertrophy encourages ossification without estrogen]. These factors include Vegf (vascular endothelial growth factor), Mmp13(matrix metalloproteinase 13), Mmp9 and Ibsp. Additional markers of the osteoblast and osteoclast phenotype, including core-binding factor alpha 1/runt-related transcription factor 2 (Cbfa1/Runx2), acid phosphatase 5, tartrate resistant (Acp5) and tumor necrosis factor (ligand) superfamily, member 11 (Tnfsf11; RANKL/receptor activator of NF-kappaB ligand) are upregulated in hypertrophic cartilage and cells in the zone of ossification."

"When chondrocytes terminally differentiate, they undergo apoptosis, leaving behind a calcified extracellular matrix (ECM) that is remodeled and degraded by invading blood vessels, osteoprogenitor cells and bone-resorbing cells."<-terminal differentiation proceeds fusion which involves remodeling left over Extracellular Matrix.  Ossification does not directly oppose cartilage growth.

"the expression of early stage chondrocyte markers such as Sox family members 5,6 and 9 (Sox5,6 and 9), Col2a1, growth differentiation factor 5 (Gdf5), Agc 1, Col11a1, Hapln1, Fgfr3 and Col9a2"<-These are the genes we are looking to be expressed by LSJL, not necessarily immediately but they should be expressed after LSJL is performed to indicate that LSJL was effective at inducing a new growth plate.



"several known growth plate markers [include] Sox9, Col2a1, Ihh and Cdkn1c" 

Here's the list of genes upregulated by LSJL:

Column A are upregulated genes whereas Column B are downregulated.  The genes involved are involved in the PI3K pathway and TGF-Beta pathway both of which are important to height growth.  Perhaps, loading the cartilage is important to activate these genes and pathways.  Note that the genes expressed shouldn't match the growth plate genes right away as it likely takes time for the growth plates to be formed.  However, you should expect to see growth plate genes eventually.

Here's additional genes modified by LSJL:

Under expressed proteins are blue and overexpressed are red.  There are no underexpressed proteins in the two pathways.  When one point shares more proteins for example BMP's some proteins can be underexpressed and some over.

Other genes of note:
" Genes highlighted in those pathways include inositol 1,4,5-triphosphate 3-kinase and phospholipase C (plc) in PI3K, collagen (col3α1, col4α1, col6α1), integrin β4, and thrombospondin 3 in ECM-receptor interaction, TGFβ receptor 1 and smad1in TGFβ signaling pathway, and wnt2, frizzled 2 and Wnt1-inducible protein 2 in Wnt signaling pathway."

Single genes that can be identified as being overexpressed: Integrin Alpha V(CD51) and Integrin Alpha 11.

Genes that were upregulated above 2 fold or below .5 fold compared to control samples(Bolded genes are pro-chondrogenic):

Barx2
CDC42bpa
Zfp9
Cdh10
FGF2-2.433
ECM2-2.027
Dmrt2
Zfp533-2.188(upregulated during cartilage formation)
Gli3-3.184(one form of Gli3 may inhibit chondrocyte proliferation)
Fzd3
Nov-3.563
THBS2
Aspn
Dkk3
Itgbl1
Scx
H19-2.174(H19 expression is liked to IGF-2)
Grem2
FGFR1
Cav3-2.001(Myostatin inhibitor)
MMP2
THBS4
WISP2
SOCS3-2.484
TUBB6
Sox9-3.148
IGFBP6-2.083
Pax1-2.166
Agc1-2.474
Jun
Zfp36
Dnm1-2.352
Gas1
HMGA2-2.137(increased expression can cause overgrowth)
Dhh
COL2A1-2.012
MATN4-2.166
Zfp410
IRS1-0.339
PLRG1
TRAPPC3
TLR7
NPC1
Zfp148
Zfp106
HMGB2-0.291(reduction in HMGB2 is associated with apoptosis, according to Expression patterns and function of chromatin protein HMGB2 during mesenchymal stem cell differentiation., HMGB2 inhibits chondrogenesis)
Zfp313
GPR108
Kif22
Zfp46
Rad17
Smad1-0.48
MAPK6
ADAMTS7-0.443
Zfp692
Zfp75
Zfp27
MAPK8
Zfp238
Id2-0.459(associated with early chondrogenic aggregates)
Gad1
GH-0.498
Lin9-0.471
STAT 5B-0.424(increases GH signaling)
Vcam 1-0.487(expressed by bone marrow stem cells, decrease in expression could indicate an increase in differentiation)
Accn1
MATN2-2.735

MATN3-2.485(Matrilin 3 is specific to cartilage matrix proteins)

Nkx3.2(also known as Bpax1)-0.49(this gene is pro-chondrogenic so it may be downregulated as part of a negative feedback mechanism)
Wnt2-3.089
Hey2-2.685
ADAMTS1-2.674
Hes1-2.405
Smad9-2.383
dusp14-2.32
Arg1-2.25
Sulf1-2.195
Vcan-2.144
Lin28B-2.143(Increases HMGA2 activity which in turn should increase height)
MMP14-2.055
HHIP-2.014
MMP7-0.491
CAMK2G-0.458
TGFBR1-0.411
Arg2-0.36
HTR2C-10.47(Serotonin Receptor)
PTGS2-7.63(synthesizes prostoglandin)
TNMD-6.185(Tendon Molecular marker)
IL6-3.051(activates STAT3)
BMPR1B- 2.166
COL9A1-2.455
COL11A1-2.029
HAPLN1-2.585(expressed in early chondrogenesis, associated with hyaluronic acid binding)
Dpt-3.144(increases in expression throughout chondrogenesis)
PRKG2-2.894(associated with cGMP)
Ptn-2.649(regulates cell cycle)
PDGFC-2.1
PKIA-2.013(inhibits cAMP)
CCNB1-0.437(cell cycle progression)
Sdc2-0.426(Syndecan 2, involved in pre-chondrogenic differentiation)
PDE6H-0.424(associated with cAMP)
HABP4-0.417(binds with hyaluronic acid)
CREB3L1-2.238(also called OASIS, may increase GH and IGF-1 levels)
COL10A1-2.012
HTRA1-2.322(interacts with BMP4, Identification of a novel HtrA1-susceptible cleavage site in human aggrecan: evidence for the involvement of HtrA1 in aggrecan proteolysis in vivo. states that HtrA1 is involved in Aggrecan breakdown)
HES5-2.562(In the data as BHLHB5)
GNAS-0.329(implicated in some forms of heterotropic ossification)

According to another LSJL study LSJL suppresses Sost and Dkk1.

Here's how these genes compare to MSCs normally undergoing endochondral ossification.

Gene Expression Profile during Chondrogenesis in Human Bone Marrow derived Mesenchymal Stem Cells using a cDNA Microarray.

"Chondrogenesis was induced by culturing human bone marrow (BM) derived MSCs in micromass pellets in the presence of defined medium for 3, 7, 14 or 21 days. Several genes regulated during chondrogenesis were then identified by reverse transcriptase-polymerase chain reaction (RT-PCR). Using an ABI microarray system, we determined the differential gene expression profiles of differentiated chondrocytes and BM-MSCs. Normalization of this data resulted in the identification of 1,486 differentially expressed genes. To verify gene expression profiles determined by microarray analysis, the expression levels of 10 genes with high fold changes were confirmed by RT-PCR. Gene expression patterns of 9 genes (Hrad6B, annexinA2, BMP-7, contactin-1, peroxiredoxin-1, heat shock transcription factor-2, synaptotagmin IV, serotonin receptor-7, Axl) in RT-PCR were similar to the microarray gene expression patterns."

Note that the LSJL gene expression study was done on rats whereas this was done on humans.  It's also possible that these genes were expressed at lower levels than were noted in the study.  Note there is definite signs of chondrogenesis like the genes related to proteoglycans.


These are different than the highly expressed genes in the LSJL study, AnnexinA2 facilitates actin cytoskeleton formation whereas in the LSJL study AnnexinA8 was present.  AnnexinA8 is an anticoagulant so it stops blood clotting likely to reduce hydrostatic pressure.

"The antigen which bound to SH2 antibody was identified as endoglin (CD105), a receptor for TGF-β3, which potentially plays a role in mediating the chondrogenic differentiation of MSCs and in their interactions with hematopoietic cells"<-endosialin is present instead in LSJL. Endosialin relates to calcium ion binding.

"Axl, synaptotagmin IV, Hrad6B, peroxiredoxin-1, BMP-7, heat shock transcription factor-2, annexin A2, contactin-1 and serotonin receptor-7 expressions were maintained in differentiating BM-MSCs until day 14."<-So maybe upregulating serotonin expression can upregulate chondrogenesis?

"BMP-7 is a strong chemotactic component in cartilage cells produced by mesenchymal stem cells, and it can promote cartilage cells to secrete specific extracellular matrix (proteoglycans and collagen type II). And BMP-7 can induce the differentiation of BM-MSCs into cartilage cells."

"Annexins bind to negatively charged phospholipids in a Ca2+-dependent manner."

"Contactin-1 is a cell surface adhesion molecule"

"IL-15 [which is expressed by MSCs] is a potent apoptosis inhibitor "

Hiroki Yokota wrote a paper that mentions gene expression in chondrogenesis:


Modelling and identification of transcription-factor binding motifs in human chondrogenesis.

Red are downregulated genes and green are upregulated genes.  Black is neutral.  We're looking for genes upregulated 1 day as that would be the time frame closest to that of the LSJL gene expression study.  LSJL genes were taken at 49 hours.  Column A is the observed profile.  ILR1(although in LSJL it's interleukin 1 receptor-like 1) and BMP2 is shared between chondrogenesis and LSJL.

The predicted regulatory model for Col2A1 "[predicts] that AP-1 and Smad would be the continuous stimulator, and Sox9 would be the inhibitor at day 1 and the stimulator at days 7 to 21".  c-Fos which LSJL upregulates is part of the AP-1 complex.

"AP-1 is reported to play the critical role in differentiation as a target of chondrogenetic growth factor such as bone morphogenetic protein-2 (BMP-2)"

"The model predicted that AP-1 would be the strong stimulatory factor during chondrogenesis. NFkB is known to control expression of BMP-2 and Sox-9 genes"

"The Sox-9 gene is one of the essential transcription factors in chondrogenesis by activating the enhancer element of a series of chondrogenetic marker genes such as Col2a1; Col9a2; Col11a2; and Aggrecan"

Can an increase in interstitial fluid flow in our bones make us taller?

Lateral loading the sides of the ends bones has multiple effects.  One of the effects may be to longitudinally stretch the Type I collagen fibers of our bones.  The second, is to induce chondrogenic differentiation of the mesenchymal stem cells contained within the epiphysis.   The third may involve the increase in interstitial fluid flow by laterally compressing our bones.  Interstitial fluid flow involves the travel of fluid through the lacunae of the cortical bone.  This may have potential height increasing effects by stretching the Type I collagen or by carrying mechanical growth signals over long distances.

MAP kinase and calcium signaling mediate fluid flow-induced human mesenchymal stem cell proliferation

"Mechanical signals are important regulators of skeletal homeostasis, and strain-induced oscillatory fluid flow is a potent mechanical stimulus. Although the mechanisms by which osteoblasts and osteocytes respond to fluid flow are being elucidated, little is known about the mechanisms by which bone marrow-derived mesenchymal stem cells respond to such stimuli. Here we show that the intracellular signaling cascades activated in human mesenchymal stem cells by fluid flow are similar to those activated in osteoblastic cells. Oscillatory fluid flow inducing shear stresses of 5, 10, and 20 dyn/cm2 triggered rapid, flow rate-dependent increases in intracellular calcium that pharmacological studies suggest are inositol trisphosphate mediated. The application of fluid flow also induced the phosphorylation of extracellular signal-regulated kinase-1 and -2 as well as the activation of the calcium-sensitive protein phosphatase calcineurin in mesenchymal stem cells. Activation of these signaling pathways combined to induce a robust increase in cellular proliferation. These data suggest that mechanically induced fluid flow regulates not only osteoblastic behavior but also that of mesenchymal precursors{it is possible that these precursors could make us taller}, implying that the observed osteogenic response to mechanical loading may be mediated by alterations in the cellular behavior of multiple members of the osteoblast lineage, perhaps by a common signaling pathway."

"like osteoblasts and osteocytes, MSCs are mechanoresponsive."<-with sufficient fluid flow we could conceivable stimulate the MSCs to become cells that make us taller.

"fluid flow would result in an increase in intracellular calcium concentration ([Ca2+]i) and the subsequent activation of downstream signaling proteins, such as ERK1/2 and calcineurin, which would in turn induce hMSC proliferation. is a vital and ubiquitous mediator in the processes by which extracellular signals are conveyed to the cell's interior and translated into a cellular response. Oscillations in [Ca2+]i regulate gene expression via numerous signaling cascades and have been shown to provide specificity among transcriptional activators.  The serine/threonine protein phosphatase calcineurin, for instance, responds to increases in [Ca2+]i and calmodulin binding by dephosphorylating and activating nuclear factor of activated T cells (NFAT) transcription factors.  Likewise, ERK1/2, members of the MAP kinase family, have been shown to be key regulators in the proliferation and differentiation of numerous cell types, including hMSCs and osteoblasts"

"During the static period, 1.4 ± 0.6% of cells exhibited a spontaneous increase in [Ca2+]i with an amplitude of 68.9 ± 9.1 nM. A significantly higher percentage of cells exhibited increases in [Ca2+]i when hMSCs were exposed to oscillatory fluid flow-inducing shear stresses of 5, 10, and 20 dyn/cm2 at 1 Hz (56 ± 2.4%, 87 ± 7.9%, and 89 ± 10.4%, respectively). Interestingly, the amplitudes of the observed [Ca2+]i increase in response to 5 and 10 dyn/cm2 (60.0 ± 10.2 and 96.8 ± 6.3 nM, respectively) were not statistically significantly different from spontaneous increases observed in static controls. In contrast, a significantly greater increase in [Ca2+]i was observed when cells were exposed to a shear stress of 20 dyn/cm2 (189 ± 12.4 nM)"<-it is possible that typical exercises do not generate sufficient fluid flow to induce the stimulus to make us and we need new exercises involving things like torsion to generate that stimulus.

"increased bone formation in response to mechanical loading is mediated by the response of bone cells to strain-induced fluid flow"<-since we see in this study other cells are stimulated too and these non bone cells could make you taller it's even conceivable that bone cells can make you taller.

Would increased interstitial fluid flow through in situ mechanical stimulation enhance bone remodeling? 

"Bone is a composite material made up of a collagen-hydroxyapatite matrix and a complex network of lacunae-canaliculi channels occupied by osteocyte and osteoblast processes, immersed in interstitial fluid. Changes in interstitial fluid flow velocity or pressure are the means by which an external load signal is communicated to the cell. Shear stress, induced by interstitial fluid flow, is a potent bone cell behavior regulator. One of the forms of altering interstitial fluid flow is through the mechanical deformation of skeletal tissue in response to applied loads[like via Lateral Synovial Joint Loading]. Other methods include increased intramedullary pressure, negative-pressure tissue regeneration, or external mechanical stimulation.  The efficacy of each method theoretically will depend on the mechanical efficiency of transmitting an external load and converting it into changes in interstitial fluid flow. [A] small mechanical percussion device could be placed directly in contact with the bone, thus inducing local interstitial fluid flow variations." 

"Within the confined geometry of the Haversian and lacunar–canalicular systems, interstitial fluid flow would impart shear stress upon cell membranes and processes. Fluid flow enhances cell proliferation and the expression of phenotypic markers of osteoblastic cells[it may enhance cellular proliferation of height increasing cells indirectly]. interstitial fluid flow induces the release of the paracrine factors[stem cells in the epiphysis may be able to benefit from this] necessary for the anabolic response of bone to mechanical loads.  Fluid flow was also shown to increase osteocytic prostaglandins[prostaglandins like PGE2 has mixed effects on height growth] and nitric oxide[Nitric Oxide pathway is involved in things like Guanyl Cyclase and CNP which can increase height growth]"

Increasing osteoblastic differentiation may increase height in some bones dependent on the location of the periosteum such as the flat bone of the skull and the calcaneus.

"The efficiency of inducing interstitial fluid flow can be improved if mechanical stimuli could be applied directly to the bone surface or within the intramedullary canal through a small mechanical, low-power, percussion device with programmable frequency and amplitude. This method of delivering mechanical stimuli would be more efficient, given that the stimulation would be applied directly to the bone, eliminating the shock-absorbing properties of muscles and joints, as occurs when stimulation is applied externally."<-mechanical stimuli on bone is more effective when applied directly to the bone due to the shock-absorbing properties of muscles and joints.  Hence, why individuals who lift extremely heavy weights like strong have not grown (much) taller.  Lateral Synovial Joint Loading is only blocked by the skin.

If sufficient fluid flow in the cortical bone requires direct mechanical loading to the bone it is likely that sufficient fluid build up(hydrostatic pressure) in the epiphysis requires direct mechanical loading as well.

Here's one study that shows how an increase in intramedullary pressure increase bone length(the mice were 8 weeks old):  

Femoral vein ligation increases bone mass in the hindlimb suspended rat. 

"Interstitial fluid flow (IFF) [is] due to intraosseous pressure changes. [We investigated] the role of IFF in bone in the absence of mechanical strain using an in vivo model, the hindlimb suspended rat.  Ligation of one femoral vein{veins take blood towards the heart while artieries take blood away from the heart so removing a vein would result in more blood in the limbs} was performed as a means to alter the IFF within the ipsilateral femur; the contralateral limb was sham-operated as control. Animals were suspended for a period of 19 days. Intramedullary pressure in the venous-ligated femurs increased relative to the sham-operated control femurs (27.8 mmHg vs. 16.4 mmHg), suggesting venous ligation increased IFF proportional to the pressure drop across the bone. Bone mineral content (BMC), when normalized to body weight, increased significantly in the venous-ligated femurs relative to control limbs (115.9 +/- 15.6% vs. 103.8 +/- 13.2%); similarly, gains in length (106.2 +/- 2.4% vs. 104.5 +/- 2.1%) and distal width (110.8 +/- 10.3% vs. 106.2 +/- 8.2%, p < 0.05) for the femurs with venous ligation were significantly greater relative to sham control. Furthermore, trabecular density was significantly higher in the femurs with venous ligation (351 +/- 12 g/cm3 vs. 329 +/- 11 g/cm3, p < 0.05). Daily administration of the cyclooxygenase inhibitor, indomethacin, via drinking water, suppressed the length increases observed for the venous ligated femur, suggesting a role for prostaglandins in IFF-mediated remodeling[COX2 was upregulated by LSJL as PTGS2]."  

"Bone contains a porous network of canaliculi that has been shown to facilitate substantial and rapid transcortical interstitial fluid flow (IFF). This fluid flow originates from leaky venous sinusoids in the intramedullary cavity and is driven radially outward through cortical bone by a transmural pressure gradient between the endosteal vasculature and the lymphatic drainage at the periosteal surface. Flow is steady in the absence of mechanical strain, but bending or compressive loads create pressure gradients that drive fluid from areas of compression to areas of tension[laterally loading the epiphysis generates a pressure gradient down the entire bone including through the growth plate]."

Ligation refers to tying off veins.


"A wire approximately twice the length of the tail was then folded in half and positioned on the tail such that wire ran along each side and the apex was at the dorsal tip of the tail. The wire was then secured in this position with athletic tape so that it was sandwiched between two layers. This wire was attached to a swivel hook from which the animal was suspended. With the hindlimbs fully outstretched, the feet were approximately 1 cm off the ground. Suspension lasted for 19 days."<-the mice were suspended.  Inversion essentially.

"Relative length and relative distal width of the venous-ligated limb was significantly greater in the HS rat compared with the sham limb"<-the hindlimb suspended mice grew taller.  Although, only the mice who had their veins tied off.


"Femoral vein occlusion has previously been shown to result in an increase in intramedullary pressure"<-So the mice were suspended to eliminate mechanical loading.  The height gain was due to the increase in intramedullary pressure and not another effect of mechanical loading.  Although an increase in intramedullary pressure would logically coincide with an increase in hydrostatic pressure in the epiphysis.

According to the study Growth of the Laboratory Mouse, mice start to slow down growth at day 42(6 weeks) but continue growing through day 98(14 weeks) so the height growth could be a modulation of hydrostatic pressure on the tail growth plates rather than new stem cell differentiation.  And it's unclear whether this is an increase in growth rate or absolute growth.  The rats in this study were female and 8 weeks old.


Maybe it's not intramedullary pressure that increased the bone length after all and it's the increase in prostaglandins in the interstitial fluid that cause an increase in length(it could still just be an increase in growth rate mind you).  But, inhibiting COX2(which is involved in PGE2 expression) only suppresed the length increase, it didn't totally eliminate it thus a change in intramedullary pressure has an effect independent of increased transport of PGE2 through the interstitial fluid.  Such as perhaps an induction of mesenchymal stem cells into chondrocytes.

Here's a study that explains the path of Bone fluid flow so we can analyze how much fluid flow goes to the epiphysis:

The pathway of bone fluid flow as defined by in vivo intramedullary pressure and streaming potential measurements.

"The pathway for intracortical fluid flow response to a step-load was identified in vivo using intramedullary pressure (ImP) and streaming potential (SP) measurements. An avian model was used for monitoring, simultaneously, ImP and SP under axial loading which generated peak strains of approximately 600 microstrain (microepsilon). ImP response to step-load decayed more quickly[thus you may have to load for a long time to keep Intramedullary Pressure high to induce chondrogenesis] than SP relaxation, in which multiple time constants were observed during the relaxations. While the initial relaxation of SP showed a decay on the order of 200 ms, ImP decayed on the order of approximately 100 ms[this is not very long at all so it's possible that LSJL provides must of it's growth stimulating benefits when it's being applied and there are low residual effects after]. After the initial decay (approximately 200 ms after loading), ImP quickly relaxed to base line, while SP continued to dominate relaxation. The decay of ImP is indicative of resistive fluid flow occurring primarily in the vasculature and other intraosseous channels such as lacunar-canalicular pores, and that SP represents the fluid flow in the smaller porosities, i.e., lacunar-canalicular system or even microspores[Since the epiphysis is mostly trabeculae it is not dense so it is likely to belong to the group that decays very quickly].  SP and ImP decays are determined by a hierarchical interdependent system of multiple porosities."

"Fuid can pass within the lacunar-canalicular porosity[this is defined as the space between the lacunae and the canaliculae, these two terms describe any open space in bone and the epiphysis has tons of them]"<-Thus fluid can pass through the epiphysis.

"Penetration of fluid in bone can be enhanced by dynamic mechanical loading"

"An increase in intramedullary pressure can influence bone’s fluid pathways through several coupling mechanisms. First, as the pressure in the medullary cavity increases towards the arterial blood pressure, fluid flow into the marrow cavity will be greatly inhibited and perhaps even stopped. However, the increase in fluid velocity out of the medullary canal will relieve much of the pressure buildup associated with dynamic loading[it's unknown however the pressure build that occurs in the bone epiphysis however fluid velocity out of the medullary canal should impact the epiphysis]. The outward flow does not provide complete compensation for this relief mechanism, and thus high marrow cavity pressures arise immediately after step loading. 

Second, considering that deformation of bone results in compression of the porous space within the cortical matrix, induced pore pressure is predicted at least one order of magnitude higher than load induced marrow cavity pressure.  This results in the kinematic loading induced fluid pressure buildup in cortical bone being much greater than that achieved within the marrow cavity, though net fluid motion may be far less. This implies the existence of a fluid-related coupling mechanism. When the ability of fluid to leave the intracortical pores is restricted because of the deformation of the pore space under loading, the marrow cavity may still provide a pathway for relief of the intracortical pressure because of the pressure difference between porous matrix and marrow cavity. Thus, while induced marrow cavity pressure can potentially reduce the fluid flow, the fluid relaxation pathways are still active, especially during unloading, in both venous and intracortical pores."

The epiphysis should be deformed as well but the pressure of the epiphysis should be much less than that of the cortical bone so fluid should flow into the epiphysis thus increasing hydrostatic pressure there.

Here's a study that involves dynamic pressure stimulating growth, it involves fractured bones but some of the principles may apply.

The influence of intermittent external dynamic pressure and tension forces on the healing of an epiphyseal fracture.

"the regenerative capacity of chondrocytes located in the growth plate of long bones revealed a potential for reparation[perhaps what they actually observed was an ability for MSCs to differentiate into chondrocytes?]. Newly formed bridging arteries crossing from the metaphysis to the epiphysis through the growth plate are thought to be responsible for the cell proliferation observed after Salter-Harris I and II lesions. We aimed to examine the influence of mechanical microstimulations on the growth or inhibition of the proliferation of the chondrocytes in the tibial growth plate.  Cell proliferation in the growth plate was not stimulated in the 1st week after distraction. The histological studies revealed an initial increase in proliferation of chondrocytes, especially between the 2nd and the 4th week."

"Chondrocytes exposed to relatively minimal intermittent tension and pressure react with an increased calcium and phosphate uptake"<-This means that if tension does not increase height but pressure does that increased calcium and phosphate uptake in chondrocytes is not the cause of the height increase.

"Fracture healing was completed histologically after 4 weeks. Immediately after induction of the fracture and stabilization of the fragment, no increase of the thickness of the specific cellular zones was seen, and no proliferation activity could be observed."<-so increase in pressure(can be induced by LSJL) had no height increasing impact until after the first week of fracture healing.  At that point the bone may not have been repaired enough to generate enough pressure to stimulate growth.  After the first week proliferative and hypertrophic zone were stimulated.

"Premature closure was frequently observed after epiphyseal distraction with a consequent loss of the bone length obtained."

"Arteries crossing from the metaphysis to the epiphysis through the growth plate are thought to be destroyed by the Salter-Harris lesions. These bridging capillaries must regrow after traumatic damage. They are important and responsible for cell proliferation, especially in the proliferative zone."

"Stimulation of the proliferative activity observed in our experiment i.e., induction of chondrogenesis in the proliferative and hypertrophic zones seems to be enhanced by the mechanical influence of the applied intermittent dynamic tension and pressure forces."

"Intermittent and alternating tension and pressure microstimulation of injuries of the growth plate and fractures of the growth region may be advantageous for the restoration of the injured growth plate."<-and in growth plates that are not injured and in people that do not have growth plates to form new ones.

Mechanically induced osteogenic differentiation--the role of RhoA, ROCKII and cytoskeletal dynamics.

"We examined whether oscillatory fluid flow, an exogenous mechanical signal within bone, regulates osteogenic, adipogenic or chondrogenic differentiation of C3H10T1/2 murine mesenchymal stem cells by measuring Runx2, PPARgamma and SOX9 gene expression, respectively. The small GTPase RhoA and isometric tension within the actin cytoskeleton are essential in flow-induced differentiation. oscillatory fluid flow induces the upregulation of Runx2, Sox9 and PPARgamma[fluid flow is definitely induced by LSJL and fluid flow upregulates Sox9].  the small GTPase RhoA and its effector protein ROCKII regulate fluid-flow-induced osteogenic differentiation. Activated RhoA and fluid flow have an additive effect on Runx2 expression. RhoA activation and actin tension are negative regulators of both adipogenic and chondrogenic differentiation. An intact, dynamic actin cytoskeleton under tension is necessary for flow-induced gene expression[this includes Sox9]."

There was no sign of RhoA activation in the LSJL gene expression study.  Nor Roc II.

"C3H10T1/2 mesenchymal progenitor cells [were exposed] to 1 hour of oscillatory fluid flow"

"Cytoskeletal mechanics and isometric tension within the actin cytoskeleton alters oscillatory fluid-flow-induced differentiation by the activation of RhoA, inhibition of ROCKII protein, inhibition of myosin II ATP hydrolysis, disruption of actin polymerization, and actin stabilization."

"ROCKII inhibition, myosin II inhibition, actin polymerization inhibition and actin stabilization [stopped] flow-induced SOX9 expression"

"LPA treatment[LPA activates RhoA] did not alter Sox9 basal expression levels and treated cells maintained their ability to upregulate SOX9 1.4±0.09-fold with oscillatory fluid flow, indicating that RhoA may not be a direct inhibitor of chondrogenic differentiation"

"inhibiting tension within the actin cytoskeleton promotes chondrogenic differentiation; however, an intact cytoskeleton is necessary for flow-induced alterations in Sox9 expression."

Gene expression of single human mesenchymal stem cell in response to fluid shear.

"An optical tweezer model has been employed to exert different levels of shear stress on a single non-adherent human bone marrow-derived mesenchymal stem cell to simulate physiological flow conditions. A single-cell quantitative polymerase chain reaction analysis showed that collagen type 1, alpha 2 (COL1A2), heat shock 70-kDa protein 1A (HSPA1A) and osteopontin (OPN){up in LSJL} are expressed to a detectable level in most of the cells. After exposure to varying levels of shear stress, there were significant variations in gene transcription levels across human mesenchymal stem cells derived from four individual donors. Significant trend towards upregulation of COL1A2 and OPN gene expression following shear was observed in some donors with corresponding variations in HSPA1A gene expression. Shear stress associated with vascular flow may have the potential to significantly direct non-adherent stem cell expression towards osteogenic phenotypic expression. these results are influenced by the selection process and donor variability."

Levels of baseline gene expression in cultured hMSC (CD_SEL)

18s COL1A2 COL2A1 ALP ACAN OPN HSPA CBFA1 SOX9 COX2

100% 92%     0% 4%   28% 46%  94%     19%  17%    25%

"The fluid velocity at 20, 40, 60 and 80 µm s−1 corresponds to a shear stress of 0.015, 0.030, 0.045 and 0.060 Pa, respectively."

So isolated shear stress on one stem cell is likely to induce osteogenic differentiation.  You need to achieve mesenchymal condensation to likely to induce chondrogenic gene expression.

Structure-function relationships in the stem cell's mechanical world B: emergent anisotropy of the cytoskeleton correlates to volume and shape changing stress exposure.

"The spatiotemporal organization of tubulin and actin elements of the cytoskeleton changes in response to volume and shape changing stresses [emulate] those during development, prior to the first beating of the heart or twitching of muscle. [We] quantify the change over baseline in spatiotemporal distribution of actin and tubulin in live C3H/10T1/2 model stem cells subjected to volume changing stresses induced by seeding at density as well as low magnitude, short duration, shape changing (shear) stresses induced by fluid flow (0.5 or 1.0 dyne/cm2 for 30/60/90 minutes). Upon exposure to fluid flow, both tubulin thickness (height) and concentration (fluorescence intensity) change significantly over baseline, as a function of proximity to neighboring cells (density) and the substrate (apical-basal height). Amplification of stress gradients (flow velocity) [occurs] with increasing distance to nearest neighbors and the substrate, i.e. with decreasing density and toward the apical side of the cell, tubulin adaptation appears to depend significantly on the magnitude of the stress to which the cell is exposed locally.  Adaptation of actin to the changing mechanical milieu is more global, exhibiting less significant differences attributable to nearest neighbors or boundaries than differences attributable to magnitude of the stress to which the cell is exposed globally (0.5 versus 1.0 dyne/cm2). changes in the actin cytoskeletal distribution correlate positively with one pre-mesenchymal condensation marker (Msx2) and negatively with early markers of chondrogenesis (ColIIaI alone, indicative of pre-hypertrophic chondrogenesis) and osteogenesis (Runx2). Changes in the tubulin cytoskeletal distribution correlate positively with a marker of pericondensation (Sox9 alone), negatively with chondrogenesis (ColIIaI) and positively with adipogenesis (Ppar-gamma 2). Exposure of MSCs to volume and shape changing stresses results in emergent anisotropy of cytoskeletal architecture (structure), which relate to emergent cell fate (function)."

"prescribed seeding conditions as well as seeding density can be used to subject multipotent stem cells (MSCs) to volume changing stresses and that changes in volume of the cell are associated with changes in shape, but not volume, of the cell nucleus."

"Tubulin resists compression and contributes to cell viscosity. In contrast, actin resists tension, contributing to cell stiffness and resistance to deformation"

"in contrast to the tubulin cytoskeleton which acts as a damper, resisting forces that compress the cell, the actin cytoskeleton acts like an elastic rope or spring to resist forces pulling on the cell"

Genes detected and what stage they are markers for: "pre- (Runx2 with Msx2) , peri-mesenchymal condensation (ColIa1, Sox9),  chondrogenesis (Sox9 and ColIIa1, and later AGC Aggrecan), osteogenesis (Runx2 without Msx2), adipogenesis (Ppar-γ2)"

"Cells seeded at LD (5,000 cells/cm2) [had] larger appearing cells and more defined actin microfilaments and microtubules than cells seeded at HD (35,000 cells/cm2) "

"The increase in apical[means at the apex or tip] and non-flow side tubulin in a cell is negatively correlated with ColIIaI, a marker of chondrogenesis. The non-flow side of tubulin is also positively correlated with Sox9, a marker of peri-mesenchymal condensation, and an earlier marker of chondrogenesis. The total amount of tubulin shows the same correlation with Sox9"<-So what you would want to do is increase the amount of the flow side tubulin to get both Col2a1 and Sox9.

"exposure to flow completely abrogates[abolishes] aggrecan expression"<-this is strange considering aggrecan was upregulated in LSJL.

"The increase in actin in the flow, non-flow, apical, and basal sides of the cell, is negatively correlated with ColIIaI, a marker of chondrogenesis. It is also negatively correlated with Runx2, a marker of pre-mesenchymal condensation as well as early chondrogenesis and osteogenesis. Furthermore, it is positively correlated with Msx2, a marker of pre-mesenchymal condensation"

Advances in assessment of bone porosity, permeability and interstitial fluid flow

"Bone tissue contains two types of fluid, blood and interstitial fluid. Through the arterial system blood arrives containing oxygen and nutrients; after passing through the bone capillaries the blood components then depart containing less oxygen and nutrients, and more carbon dioxide and other cellular waste products. Many substances, including amino acids, sugars, fatty acids, coenzymes, hormones, neurotransmitters, and inorganic compounds, are exchanged from the capillaries into the interstitial fluid in the vascular porosity in cortical bone. The vascular porosity (VP) is the space inside the Haversian and Volkmann canals that contains the soft tissue structures, blood vessels and nerves. The mineralized tissue matrix in bone contains lacunar pores and slender canalicular channels, forming a complex network that is called the lacunar–canalicular porosity (LCP). Lacunar pores are occupied by osteocytes, the most abundant cell type in bone, and the canaliculi contain the cell processes emanating from contiguous osteocytes, thus permitting communication between neighboring bone cells."

"Interstitial fluid flow is enhanced by the time-varying mechanical loads applied to bone, causing deformations that result in convection of interstitial fluid in and out of the LCP. The interstitial fluid in the LCP travels inside the canaliculi and lacunae, passing through the space between the osteocyte dendritic process and the canalicular wall, dragging the osteocyte's cell process and deflecting the tethering elements that attach the cell process to the canalicular wall. Focal adhesion complexes and integrins located at the cell process membrane are stretched out, which may lead to the genesis of molecules involved in signaling pathways (e.g., OPG, RANKL, NO, PGE2, ATP, sclerostin, DMP1, FGF23)"

"The largest pore size is associated with the vascular porosity (VP), which consists of the volume of all the tunnels in bone that contain blood vessels and includes all the osteonal canals (primary and secondary) as well as transverse (Volkmann) canals. The lacunar–canalicular porosity (LCP) contains the second largest pore size, and it is associated with the osteocyte lacunae and canaliculi channels. The space between the osteocyte and the lacunar–canalicular wall is filled by the osteocyte's glycocalyx and interstitial fluid. The smallest pore size in bone is found in the collagen-apatite porosity (CAP). The pores are spaced between the collagen and the crystallites of the mineral apatite. At this level, most of the water is bound to ionic crystals in bone. The typical lineal dimension associated with the vascular, lacunar–canalicular, and collagen-apatite porosities is 50 μm, 100 nm, and 1 nm, respectively"  The epiphysis contains blood vessels and possibly collagen-apatite so those are the areas of interestitial fluid flow we should be most concerned with for height growth.

Vascular porosity is age dependent and increases with age.  Vascular porosity is smaller in mice than humans.

"The pore size of the vascular porosity is sufficiently large to permit a rapid decay of a pressure pulse by diffusion, and thus should be a low-fluid-pressure domain. The fluid pressure magnitude in the vascular porosity domain is considered similar to the blood pressure in bone capillaries (40–60 mmHg, or correspondingly 5.3 kPa–8 kPa) because an interstitial fluid pressure in the vascular porosity significantly greater than 40–60 mmHg would collapse these blood vessels, and a prolonged increase in the interstitial fluid pressure significantly above the blood pressure would deprive the tissue of oxygen{maybe this deprivation of oxygen creates a pro-chondrogenic microenvironment?} and nutrients. The lacunar–canalicular system is a high-fluid-pressure domain because the pore size of the LCP is very small, leading to a slow decay of a pressure pulse. "

"Pressure magnitude gradients produced in bone with permeabilities on the order of 10−20 m2 are required to produce the fluid pressure necessary to convect fluid flow in the lacunar-canalicular system and allow fluid to flow against the trans-cortical pressure gradient. "

Influence of interstitial bone microcracks on strain-induced fluid flow.

"the presence of a microcrack in the interstitial osteonal tissue may drastically reduce the fluid velocity inside the neighbouring osteons. This fluid inactive zone inside osteons can cover up to 10% of their surface. Consequently, the fluid environment of bone mechano-sensitive cells is locally modified."<-Maybe microcracks are part of the conditioning response to LSJL.

"microdamage occurring inside the osteonal volume may generate a cell-transducing mechanism based on ruptured osteocyte processes"<-this mechanism may affect MSCs to differentiate into chondrocytes.

"the fluid velocity induced by cyclic loading is sufficient to stimulate cells [at] ||v|| > 5 × 10−8 m.s−1 where ||v|| designates the fluid velocity vector norm"

"osteocyte apoptosis may play a role in the signalling mechanisms by which bone is remodelled after microcrack formation"

"when a microcrack develops inside the interstitial matrix, the fluid velocities in the closest osteons fall below a threshold value, limiting the osteocyte solicitation and thus initiating the lining cells activation."

Migration of human mesenchymal stem cells under low shear stress mediated by mitogen-activated protein kinase signaling.

"hMSCs are able to detect and respond to shear stress due to blood and interstitial fluid flow through mechanotransduction pathways after transplantation{the idea with LSJL is to get the MSCs to respond by differentiating into chondrocytes}. We examined the effect of shear stress on hMSC migration and the role of mitogen-activated protein kinases (MAPKs) in their migration. Shear stress between 0.2 and 10 Pa, which was produced by the flow medium, was exerted on fluorescently labeled hMSCs. Cell migration was evaluated. hMSCs subjected to a shear stress of 0.2 Pa caused notably faster wound closure than statically cultured hMSCs, while migration in the 0.5- and 1-Pa shear stress group did not differ significantly from that in the control group. Shear stress >2 Pa markedly inhibited hMSC migration. hMSCs subjected to a shear stress of 0.2 Pa displayed an increase in extracellular signal-regulated kinases 1/2 (ERK1/2), c-Jun N-terminal kinases (JNK), and p38 MAPK activation for up to 60 min, while a shear stress of 2 Pa abrogated the activation. JNK and p38 MAPK inhibitors completely abolished the effect of shear stress on hMSC migration, while significant differences were observed between the ERK1/2 inhibitor-treated static control and shear stress groups.  Low shear stress effectively induces hMSC migration and that JNK and p38 MAPK play more prominent roles in shear stress-induced migration than ERK1/2."

It's possible also that an inhibition of migration is good as MSCs need to condense to differentiate into chondrocytes.

"laminar shear stress of 0.3 Pa significantly enhanced the migration of human umbilical vein endothelial cells (ECs) compared with a shear stress of 1.2 or 2 Pa"

"Treatment with the JNK inhibitor completely abolished the migratory ability of hMSCs, and shear stress did not compensate for the effect of inhibition. The same phenomenon was observed when p38 MAPK activation was inhibited"


Effect of fatigue loading and associated matrix microdamage on bone blood flow and interstitial fluid flow.

"We investigated the effect of a single period of cyclic fatigue on bone blood flow and interstitial fluid flow. The ulnae of 69 rats were subjected to cyclic fatigue unilaterally using an initial peak strain of -6000 muepsilon until 40% loss of stiffness developed. Groups of rats were euthanized immediately after loading, at 5 days, and at 14 days. The contralateral ulna served as a treatment control, and a baseline control group that was not loaded was also included. After euthanasia, localization of intravascular gold microspheres within the ulna and tissue distribution of procion red tracer were quantified. Microcracking, modeling, and remodeling (Cr.S.Dn, microm/mm(2), Ne.Wo.B.T.Ar, mm(2), and Rs.N/T.Ar, #/mm(2) respectively) were also quantified histologically. Cyclic fatigue loading induced hyperemia[increased blood flow] of the loaded ulna, which peaked at 5 days after loading. There was an associated overall decrease in procion tracer uptake in both the loaded and contralateral control ulnae. Tracer uptake was also decreased in the periosteal region, when compared with the endosteal region of the cortex. Pooling of tracer was seen in microdamaged bone typically adjacent to an intracortical stress fracture at all time points after fatigue loading; in adjacent bone tracer uptake was decreased. New bone formation was similar at 5 days and at 14 days, whereas formation of resorption spaces was increased at 14 days. A short period of cyclic fatigue induces bone hyperemia and associated decreased lacunocanalicular interstitial fluid flow, which persists over the time period in which osteoclasts are recruited to sites of microdamage for targeted remodeling. Matrix damage and development of stress fracture also interfere with normal centrifugal fluid flow through the cortex. Changes in interstitial fluid flow in the contralateral ulna suggest that functional adaptation to unilateral fatigue loading may include a more generalized neurovascular response."

"The number of cycles required to fatigue the ulna was 3076 ± 2584, and the peak load used to induce bone fatigue was − 23.8 ± 3.0 N"

"cyclic mechanical loading decreased osteocyte lacunar staining in both the fatigue-loaded and contralateral control ulna at all time points after loading, when compared with the baseline control group"

"an inverse or complex relationship may exist between bone blood flow and interstitial fluid flow in the lacunocanalicular network of bone over time. The existence of an inverse relationship between bone blood flow and interstitial fluid flow is also supported by studies showing that elevated intramedullary pressure and elevated interstitial fluid flow in bone are induced by reduction of venous blood outflow by femoral vein ligation"

"functional adaptation to unilateral cyclic fatigue loading includes a neurovascular response in the contralateral limb"<-Contralateral adaptation was also reported in LSJL.

This dabbled remodeling is much more specific than the massive hole present in the LSJL drilling study.  Intracortical damage is black arrows and bone remodeling is white arrows.  A is five days, B is 14 days.

The influence of load repetition in bone mechanotransduction using poroelastic finite-element models: the impact of permeability.

"there is a minimum time of rest between load cycles that is required to maximize fluid motion, which depends on the order of magnitude of the intrinsic permeability. We show that frequency and rest insertion may be optimized to deliver maximal mechanical stimulus as a function of permeability."<-couldn't get this full study yet.

Changes in interstitial fluid flow, mass transport and the bone cell response in microgravity and normogravity

" Fluids provide the most fundamental way to transport chemical and biochemical elements within our bodies and apply an essential mechano-stimulus to cells. Furthermore, the cell cytoplasm is not a simple liquid, and fluid transport phenomena together with viscoelastic deformation of the cytoskeleton play key roles in cell function. In microgravity, flow behavior changes drastically, and the impact on cells within the porous system of bone and [are influenced by] an expanding level of adiposity"

"up to one half of bone mass could be lost during a 3-year trip to Mars, resulting in mission-compromising low-energy bone fractures, complications from renal stones caused by skeleton-released calcium and an increased incidence of fragility fractures when returning to full or partial gravity"

"Gravity strongly affects fluid behavior by creating forces that drive and alter its motion. In the presence of gravity, fluid flow can also lead to altered phase interactions and processes that regulate gases. Controlling fluid flow in the absence of gravity creates both significant and novel challenges, where flow can be significantly complicated by temperature, capillary networks of different geometries, changes in fluid surface tension, droplets, and undesirable bubble formation. The near elimination of buoyancy, hydrostatic pressure and sedimentation cause adjustments to flow dynamics: liquids climb container walls, there is limited drainage of liquids, and liquids of different densities can stratify."

"In the body, electrostatic forces and energies (e.g., ion pairs, hydrogen bonds) are essential for the interaction of virtually all biological macromolecules. Due to the polar nature of water, the intercellular and intracellular interactions between water and hydrophilic and hydrophobic molecules, including polysaccharides, lipids, and proteins, are critical for healthy physiological processes."

"gravity’s impact on fluid flow is the creation of flows due to density differences (buoyancy-induced convection) as well as due to thermal convection. Gravitationally induced bulk convection is a type of natural convection caused by buoyancy variations that result from material properties other than temperature. With gravity, thermal convection occurs when heated fluids rise to the top along the gravity vector, which are then replaced by cooler fluids. Both bulk and thermal convection establish a fluid current in the body that is considered essential to driving mass transport and rapidly dissipating heat"

"Although slow, fluid flow nevertheless plays an important role in nutrient transport, soft tissue maintenance and remodeling, as well as the establishment and maintenance of the microenvironment, where limitations in the supply of vital nutrients lead either to tissue adaptation or necrosis."<-soft tissue maintenance and remodeling could increase height via creation of tissue that is capable of interstitial growth.

"The lacunae-canaliculi system (LCS) within bone tissue and its anatomical parameters vary according to bone type, location, age and health. The LCS is composed of larger lacunae (~10 µm) and smaller canaliculi (0.1–0.5 µm) inhabited by osteocytes, and this porous system facilitates the exchange of substances, with liquid flow providing nutrients, eliminating metabolic waste and generating fluid shear force to stimulate osteocyte viability and function."

"mechanically induced deformation of bone acts as a motive force for fluid displacement, generating fluid pressure gradients that drive interstitial fluid into the LCS and bony macrostructure. The forces generated act directly on bone cells, and this load-induced fluid flow is critical for mechanotransduction as well as enhancing convective solute transport within the macro- and microporosities."

"fluid shear stress can induce deformation of the bone cell membrane and alteration of membrane proteins, opening mechano-activated ion channels to allow the influx of cations, such as Ca2+, Na+, and K+, into the cell. Stretch-activated ion channels include the DEC/ENAC family of cation channels (named after Caenorhabditis elegans degenerins and mammalian Na+ channels), L-type (osteoblasts) and T-type (osteocytes) voltage-sensitive calcium channels, and annexin V voltage-gated calcium channels. Specifically, increases in osteoblast, osteocyte, and MSC membrane tension induce the opening of PIEZO1 channels. Mechanically activated nonselective Ca2+-permeable cation channels of the PIEZO family (PIEZO1 and PIEZO2) are recognized as the most important mediators of mechanotransduction and are crucial for bone formation. Mechanical stimulation enhances calcium flux into the cell, and the resulting calcium spikes mediate osteogenesis."<-I believe that it can stimulate other kinds of issue formation as well.

" Human bone marrow-derived stem cells respond to active mechanical stimulation, where 2%-8% uniaxial strain through tensile stretching resulted in osteogenic differentiation or subsequent bending resulted in both osteogenic and chondrogenic differentiation"<-Yes exactly chondrogenic differentiation!  This is what can impact height.

"in normogravity, deformation of the pores during loading induces motion in the fluid-like marrow, resulting in the generation of pressure and velocity gradients"

"a reduced bone volume resulted in an overall increase in bone deformation, leading to increased stimulation via microstrain to cells. Furthermore, an increased adipocyte content in the marrow resulted in lowering the microstrain levels to cells within the bone marrow, reportedly due to a shielding effect caused by the more compliant behavior of adipocytes."

Here's a review paper on fluid shear stress:

In Vitro Bone Cell Models: Impact of Fluid Shear Stress on Bone Formation

" Shear stress generated by interstitial fluid flow in the lacunar-canalicular network influences maintenance and healing of bone tissue. Fluid flow is primarily caused by compressive loading of bone as a result of physical activity{I believe that torsional loading is the best way to generate fluid flow}. Changes in loading, e.g., due to extended periods of bed rest or microgravity in space are associated with altered bone remodeling and formation in vivo. In vitro, it has been reported that bone cells respond to fluid shear stress by releasing osteogenic signaling factors, such as nitric oxide, and prostaglandins. This work focusses on the application of in vitro models to study the effects of fluid flow on bone cell signaling, collagen deposition, and matrix mineralization. Particular attention is given to in vitro set-ups, which allow long-term cell culture and the application of low fluid shear stress. In addition, this review explores what mechanisms influence the orientation of collagen fibers, which determine the anisotropic properties of bone. A better understanding of these mechanisms could facilitate the design of improved tissue-engineered bone implants or more effective bone disease models."

"Bone has the power to regenerate and repair constantly throughout the entire life."

"MSCs differentiate into osteoblasts under the appropriate stimuli, but they can also turn into cartilage, muscle, tendon, and fat cells"

"Osteocytes are terminally differentiated osteoblasts which became trapped within newly deposited bone matrix "

"Osteocytes, which are the most abundant cell type in bone (90–95% of total bone cells), are thought to respond to mechanical loading by releasing signal factors."

"mineralization is likely to be controlled by the small Ca2+-binding protein osteocalcin. Mechanotransduction is facilitated by glycoproteins, such as osteopontin and osteonectin, which can attach to integrins on cell surfaces. Osteopontin also enables the attachment of osteoclasts to bone surfaces. The small amount of lipids is crucial for cell signaling and ion flow"

How collagen fibers are formed:

"Collagen formation is initiated in the nucleus of collagen-producing cells, such as osteoblasts and also fibroblasts. In the nucleus, a particular segment of deoxyribonucleic acid (DNA) is transcribed into messenger ribonucleic acid (mRNA). After the mRNA has moved out of the nucleus into the cytoplasm, it is translated into polypeptide chains, known as pre-pro-collagen. Each chain is about 300 nm in length and 1.5 nm in diameter. They are characterized by a strict pattern consisting of multiple triplet sequences of Gly–Y–Z. Glycine residues (Gly) have to be present in every third position to allow proper folding of these chains later on. Although Y and Z can be any amino acid, they are commonly proline and hydroxyproline"

"Proline and lysine residues are then hydroxylated in the endoplasmatic reticulum (ER) which will aid cross-linking of peptide chains later. This enzymatic step requires ascorbic acid (vitamin C) as a cofactor. A lack of ascorbic acid would either result in the formation of looser collagen triple helices or prevent collagen synthesis altogether, resulting in diseases such as scurvy"

"The matrix-integrin-cytoskeleton pathway is thought to play an important role in bone mechanotransduction, since integrins directly connect bone cells with their ECM. Integrins are membrane-bound glycoproteins which allow rapid transmission of physical stimuli from the ECM via the cytoskeleton to the nucleus, where they could initiate changes in gene expression"

" The glycocalyx, which is a cellular coating rich in hyaluronic acid, might also contribute to bone cell mechanotransduction via force transmission to the cytoskeleton and integrins"

"Interstitial fluid (ISF) is a main component of body mass (up to 20%) and is distributed throughout the ECM. It provides cells with nutrients and waste removal and can also be found in cortical and cancellous bone where it fills the porosities within the tissue. The three levels of porosities in bone are: (1) the vascular porosity within the Volkmann canal and the Haversian canals (20 μm radius), (2) the lacunar-canalicular system (LCS), which are the channel structures within the mineralized bone tissue surrounding osteocytes and their processes (0.1 μm radius), and (3) tiny spaces between crystallites of the mineral hydroxapatite and collagen fibers (0.01 μm radius)"

"Mechanical loading results in bending of bones and matrix deformation. Compressive stress is generated on one side of the bone and tensile stress on the other. The resulting pressure gradient in the ISF is thought to drive the fluid from regions of compression to tension. ISF has to squeeze through the narrow channels, canaliculi, which connect osteocytes residing in small spaces, lacunae, within the mineralized bone matrix. Due to the small dimensions of the channels, high wall shear stress comparable to vascular wall shear stress is generated"

"Osteocytes subjected to FSS have been shown to release several physiological relevant messengers in vitro, including Ca2+, ATP, NO, and PGE2"

MOre on osteocytes since osteocytes are the cells directly most strongly impacted by fluid flow it is important to know whether there activities can make you taller:

Osteocyte Control of Osteoclastogenesis

"osteocytes can directly control the differentiation and activity of either osteoclasts or osteoblasts."

"Osteocyte apoptosis also increases in response to changes in biomechanical loading of the skeleton. Interestingly, both over-loading and unloading of the skeleton stimulate osteocyte apoptosis and both conditions result in increased bone resorption"

"apoptotic bodies produced by dying osteocyte-like cells in vitro are able to promote osteoclast formation in vitro and in vivo"