Wednesday, September 22, 2010

Grow Taller with Tensile Strain

Tensile strain is where an object such as a bone is being stretched.  This is the mechanism being applied by the limbcenter technique(bone stretching).  Now if you pull a pencil apart you're not getting very much tensile strain but if you try to bend the pencil you get much more tensile strain with much less force.  If you bend the pencil equally in both directions you will get equality of tensile strain.  Note that tensile strain is also important in creating gap fractures(a gap fracture is a microfracture induced in an osteon while the osteon is being stretched; new bone is then secreted from an osteocyte within the osteon allowing it to heal in a longer state).  What specific adaptations are induced by tensile strain and how can we use those to grow taller?

Tensile strain and magnetic particle force application do not induce MAP3K8 and IL-1B differential gene expression in a similar manner to fluid shear stress in human mesenchymal stem cells.

"We reported a potentially important role for mitogen-activated protein kinase kinase kinase 8 (MAP3K8) and interleukin-1beta (IL-1B) in MAPK signalling in MSCs exposed to fluid shear stress[fluid shear stress is induced by LSJL]. In this follow-up study, we examined the expression of these genes in MSCs exposed to other types of mechanical force: uniaxial tensile strain (3% cell elongation) and forces generated through the exposure of magnetic particle-labelled MSCs to an oscillating magnetic field (maximum field strength 90 mT). Exposure to both types of mechanical force for 1 h did not significantly alter the gene expression of MAP3K8 or IL-1B over the 24 h period subsequent to force exposure. These data demonstrate that uniaxial tensile strain and magnetic particle-based forces do not induce MAP3K8-related MAPK signalling in the same manner as does fluid flow-induced shear stress. This illustrates divergence in the process of mechanotransduction in mechanically stimulated MSCs"

"[There's a] consistent, significant and marked upregulation (2–50-fold) of the mitogen-activated protein kinase kinase kinase 8 (MAP3K8) and interleukin-1β (IL-1B) genes [in MSCs] in response to fluid shear stress"

MAPK regulates cellular proliferation and differentiation.  So LSJL activation of this is very important.  Now, it's important to note that the scientists only elongated the cells and not the entire bone.  Elongating the entire bone may also increase MAPK growth factors.

How much strain does it take to cause gap fractures?

Microcracking damage and the fracture process in relation to strain rate in human cortical bone tensile failure.[Osteons are the bone units of cortical bone]

"Traumatic failures in-vivo are more likely to be orders of magnitude faster than the quasistatic tests usually employed in-vitro. We have reported recently [The effect of strain rate on the mechanical properties of human cortical bone.] results from tests on specimens of human femoral cortical bone loaded in tension at strain rates (epsilon ) ranging from low (0.08s(-1)) to high (18s(-1)). Across this strain rate range the modulus of elasticity generally increased[modulus of elasticity is the tendency of bone to deform in response to strain; since it increased as strain increased bone became more deformed/lengthened], stress at yield[the yield stress is the point where the bone doesn't return to normal i.e. the bone stays lengthened] and failure and strain at failure decreased for rates higher than 1s(-1), while strain at yield was invariant for most strain rates and only decreased at rates higher than 10s(-1). The results showed that strain rate has a stronger effect on post-yield deformation than on initiation of macroscopic yielding[macroscopic means visible change in length; strain rate is the speed in which the strain is applied; so applying a strain faster was important then more length in the bone]. In general, specimens loaded at high strain rates were brittle, while those loaded at low strain rates were much tougher. Here, a post-test examination of the microcracking damage reveals that microcracking was inversely related to the strain rate. Specimens loaded at low strain rates showed considerable post-yield strain and also much more microcracking[microcracks occurred more when the strain was applied slowly; an increase in bone length may not be due to microcracks]. Partial correlation and regression analysis suggested that the development of post-yield strain was a function of the amount of microcracking incurred (the cause), rather than being a direct result of the strain rate (the excitation). Presumably low strain rates allow time for microcracking to develop, which increases the compliance of the specimen, making them tougher. This behaviour confirms a more general rule that the degree to which bone is brittle or tough depends on the amount of microcracking damage it is able to sustain. More importantly, the key to bone toughness is its ability to avoid a ductile-to-brittle transition for as long as possible during the deformation. The key to bone's brittleness, on the other hand, is the strain and damage localisation early on in the process, which leads to low post-yield strains and low-energy absorption to failure."

So, the faster the bone was increased in length the more likely the bone was to maintain the length post removal.  However, a slower change in length gave bone more time to microcrack(and thus for new bone to form in those gaps) thereby making the bone sturdier in the end.

Tensile strain may also have a positive effect on growth plate chondrocytes.

The effect of mechanical loading on the metabolism of growth plate chondrocytes.

"This study was aimed at evaluating the effect of tensile loadings with various frequencies on the proliferation of growth plate chondrocytes and extracellular matrix synthesis. The chondrocytes obtained from rib growth plate cartilage of 4-week-old male Wistar strain rats were cultured by day 4 and day 11 and used as proliferating and matrix-forming chondrocytes, respectively. Intermittent tensile stresses with different frequencies were applied to each stage chondrocyte. DNA syntheses were examined by measuring the incorporation of [(3)H]thymidine into the cells. Furthermore, the rates of collagen and proteoglycan syntheses were determined by measuring the incorporation of [2,3-(3)H]proline and [(35)S]sulfate into the cells, respectively. At the proliferating stage, intermittent tensions with the frequencies of 30 cycles/min and 150 cycles/min significantly (p < 0.05) upregulated the syntheses of DNA, which indicates the promotion of chondrocyte proliferation. At the matrix-forming stage, collagen, and proteoglycan syntheses also enhanced with increase of the loading frequency. In particular, the intermittent tension with the frequencies of 30 cycles/min and 150 cycles/min increased significantly (p < 0.05 or p < 0.01) both the collagen and proteoglycan syntheses. These results suggest that the proliferation and differentiation of growth plate chondrocytes are regulated by the mechanical loading and that the chondrocyte metabolism enhanced with increase of loading frequency."

A little like muscle growth, size of strain influences proliferation and differentiation(hypertrophy) while frequency of strain increases collagen and proteoglycan synthesis(like muscle nutrition).

Tensile damage and its effects on cortical bone.

Is length increase one of the effects?


"Plexiform bovine bone samples are repeatedly loaded in tension along their longitudinal axis. In order to induce damage in the bone tissue, bone samples are loaded past their yield point. Half of the bone samples from the damaged group were stored in saline to allow for viscoelastic recovery while the others were decalcified. Tensile tests were conducted on these samples to characterize the effects of damage on the mechanical behavior of the organic matrix (decalcified samples) as well as on bone tissue (stored in saline). The ultimate strain of the damaged decalcified bone is 29% higher compared to that of non-damaged decalcified (control) bone. The ultimate stresses as well as the elastic moduli are similar in both decalcified groups. This phenomenon is also observed in other collagenous tissue (tendon and ligament). This may suggest that damage in bone is caused by shear failure of the organic matrix; transverse separation of the collagen molecules or microfibrils from each other. In contrast, there is a trend towards lowered ultimate strains in damaged bone, which is soaked in saline, with respect to control bone samples (not damaged). The damaged bone tissue exhibits a bi-linear behavior in contrast to the mechanical behavior of non-damaged bone. The initial elastic modulus (below 55 MPa) and ultimate strength of damaged bone are similar to that in non-damaged bone."

"Damage in the form of cracks can start and develop by tensile failure of collagen molecules/fibrils and/or mineral, by shear failure between collagen molecules/fibrils, and/or by debonding of the organic matrix from the mineral platelets"<-Do these forms of damage lengthen bone?


"Under excess load, deformation of collagen fibers involves stretching, slippage of laterally adjoining elements and separation (in collagen molecule and/or in collagen fibril levels) and ultimately defibrillation of the fibrils from the overlap regions under shear force transmission"<-Note the usage of the word stretching, the collagen molecules stretch in until there is a slippage of adjoining elements until they separate.

"This behavior seems to be a characteristic of type I collagen as experiments on self assembled type I collagen fibrils indicate that a decrease in the diameter of the collagen microfibrils (thinner microfibrils) increases the toe region (low-strain elastic modulus) while an increase in the length of the collagen microfibrils increases the elastic modulus at higher strains as well as the ultimate strength"<-increase in the length of the collagen microbils likely leads to a height increase

"microcracks (<10 μm) were initiated at 0.4% tensile strain in equine bone and there was a considerable growth in microcrack density when tensile strain reached 0.8%"<-0.4% tensile strain are needed to cause microcracks and thereby height increase.  Calculating 0.4% tensile strain is unknown at this time.

Tensile strain(bone stretching) can enable you to grow taller by stretching collagen microfibrils until the point of breaking.

The importance of the elastic and plastic components of strain in tensile and compressive fatigue of human cortical bone in relation to orthopaedic biomechanics.

"The longevity, success, or failure of an orthopaedic implant is dependent on its osseointegration especially within the initial six months of the initial surgery. The development of strains plays a crucial role in both bone modelling and remodelling. For remodelling, in particular, strains of substantial values are required to activate the osteoblastic and osteoclastic activity for the osseointegration of the implant. Bone, however, is subject to "damage" when strain levels exceed a certain threshold level. Damage is manifested in the form of microcracks; it is linked to increased elastic strain amplitudes and is accompanied by the development of "plastic" (irrecoverable, residual) strains[what we would want is a residual strain such that increases the length of the bone]. Such strains increase the likelihood for the implant to subside or loosen. The present study examines the rates (per cycle) by which these two components of strain (elastic and "plastic") develop during fatigue cycling in two loading modes, tension and compression. The results of this study show that these strain rates depend on the applied stress in both loading modes. It also shows that elastic and plastic strain rates can be linked to each other through simple power law relationships so that one can calculate or predict the latter from the former and vice versa. We anticipate that such basic bone biomechanics data would be of great benefit to both clinicians and bioengineers working in the field of FEA modelling applications and orthopaedic implant surgery."

The difference between stress and strain is that strain actually changes the shape of the object in this case the bone.  To grow taller, strain must occur that causes microcracks not just stress.

Obviously tensile strain is very powerful.  If gradual tensile strain is applied to a bone, microcracks should appear in the osteons and the bone will grow longer one osteon at a time.  The question is:  How do we apply tensile strain to the bone?  If we do the medieval rack, the other types of tissues of the body are likely to fail far sooner than the bone.  And how do we do the vertebrae? 

Lateral Joint Loading does apply a distraction force on the bone(it applys a downward compressive force on the epiphysis and a stretching force along the entire shaft) but is it enough?  Sky's limbcenter method may show promise but he is being cryptic and not forthcoming with the effectiveness of the treatments.  Then there is the problem of loading the arms and upper leg(femur).

Here's an article about changes in genetic expression due to tensile strain:

Effects of Wnt3A and mechanical load on cartilage chondrocyte homeostasis.

"Chondrocytes were pre-stimulated with recombinant Wnt3A for 24 hours prior to the application of tensile strain (7.5%, 1 Hz) for 30 minutes. Activation of Wnt signalling, via β-catenin nuclear translocation and downstream effects including the transcriptional activation of c-jun, c-fos and Lef1, markers of chondrocyte phenotype (type II collagen (col2a1), aggrecan (acan), SOX9) and catabolic genes (MMP3, MMP13, ADAMTS-4, ADAMTS-5) were assessed.
Physiological tensile strain induced col2a1, acan and SOX9 transcription. Load-induced acan and SOX9 expression were repressed in the presence of Wnt3A[Wnt3A encourages cell spreading and cellular condensation is an important preliminary part of chondrogenesis]. Load induced partial β-catenin nuclear translocation[stabilized beta-catenin may inhibit stem cells from undergoing a chondrogenic lineage]; there was an additive effect of load and Wnt3A on β-catenin distribution, with both extensive localisation in the nucleus and cytoplasm. Immediate early response (c-jun) and catabolic genes (MMP3, ADAMTS-4) were up-regulated in Wnt3A stimulated chondrocytes. With load and Wnt3A there was an additive up-regulation of c-fos, MMP3 and ADAMTS-4 transcription, whereas there was a synergistic interplay on c-jun, Lef1 and ADAMTS-5 transcription.
Our data suggest that load and Wnt, in combination, can repress transcription of chondrocyte matrix genes, whilst enhancing expression of catabolic mediators[keep in mind this is tensile strain which is the primary load induced by LSJL]. Future studies will investigate the respective roles of abnormal loading and genetic predisposition in mediating cartilage degeneration."

So tensile strain may inhibit chondrogenesis but that doesn't mean that tensile strain won't play some role in height growth as objects tend to become longer when stretched even bone.  I owned a Stretch Armstrong and you could apply enough stretch such that the arms did not retract.

"In weight-bearing areas of the β-catenin cAct mice the articular cartilage surface was missing"

"After recovery, elevated mRNA levels of c-fos (1.65-fold: P < 0.01) and c-jun (1.5-fold: P < 0.05) were observed in cells subjected to tensile strain. An additive effect of Wnt3A and tensile strain was observed on c-fos transcription after recovery (2.2-fold; P < 0.01 compared to Wnt3A). In contrast, c-jun mRNA levels were significantly elevated in cells irrespective of treatment. Wnt3A treatment independently increased Lef1 mRNA levels after recovery (1.7-fold; P < 0.01), and the synergistic induction of Lef1 transcription remained in cells post-cessation of load (relative to untreated 2.4-fold: P < 0.001 or Wnt3A P < 0.05)."

"Applying a physiological strain induced the transcription of type II collagen (col2a1), aggrecan (acan) and SOX9 (SOX9), which are all markers of the chondrocyte phenotype; the mechano-responsive nature of these matrix genes in chondrocytes has been previously reported. Using this loading regime (in the absence of Wnt3A), β-catenin protein levels, as detected by Western blotting, were unaffected compared to expression in the untreated cells."<-So you don't want Wnt3A if you want to encourage chondrogenesis.  Wnt3A is the main factor to blame and not tensile strain.

"In bone, strain-induced β-catenin translocation (2% strain, 3600 cycles) was shown to result from inhibition of GSK3β activity, which was suggested to be mediated via activation of the Akt pathway"<-phosphorylation of GSK3Beta inhibits it.

How do we get rid of the confounding factors in tensile strain(like muscle fatigue) and effectively apply tensile strain?


Undifferentiated human mesenchymal stem cells (hMSCs) are highly sensitive to mechanical strain: transcriptionally controlled early osteo-chondrogenic response in vitro.

"hMSCs from 10 donors were intermittently stimulated by cyclic tensile strain (CTS) at 3000 mustrain for a period of 3 days. Differential gene expression of strained and unstrained hMSCs was analysed by real-time RT-PCR for several marker genes, including the transcription factors FOS, RUNX2, SOX9, and others. Additionally, alkaline phosphatase activity (ALP) was determined kinetically.
The application of CTS significantly stimulated the expression levels of the early chondrogenic and osteogenic marker genes (SOX9{up in LSJL}, LUM{UP}, DCN; RUNX2, SPARC, SPP1, ALPL); this was accompanied by stimulation of ALP activity (+38%+/-12 standard error of mean, P<0.05). Matrix analysis revealed that the osteo-chondrogenic response followed a coordinated expression pattern, in which FOS{up} was attributed to early osteogenic but not chondrogenic differentiation.
Undifferentiated hMSCs are highly sensitive to mechanical strain with a transcriptionally controlled osteo-chondrogenic differentiation response in vitro."

"Differentiation processes in hMSCs were found to be not synchronized throughout the cell populations, and several pheno- and genotypic investigations suggested that early differentiation may be regulated, at least in vitro, by stochastic[unorganized] mechanisms, while gene expression programs underlying late events in cell differentiation appear to be more fixed"

"no dependences were found between FOS messages of unstrained stem cells and either the chondrogenic or the osteogenic response"

"the activity of AP-1 may be involved in the early stage of MSC chondrogenesis"


Nanoscale Deformation Mechanisms in Bone


We would want the tensile strain to be all in one direction.

"bone deformation is not homogeneous but distributed between a tensile deformation of the fibrils and a shearing in the interfibrillar matrix between them."

"bone consists at the nanometer level of type I collagen molecules (300 nm long, 1.1−1.5 nm wide) interspersed with irregularly shaped nanocrystal platelets of carbonated apatite (3−5 nm thick and a lateral size of  50 nm). A composite of these two constituents forms the mineralized collagen fibril, with typically a diameter of 100 nm. The fibrils are hierarchically organized into lamellae and further on into the compact bone material, which, in combination with a cellular trabecular bone material, forms the organ bone."

"Because the collagen molecules are staggered axially along the fibril, a periodic electron density profile exists along the fibril axis (the D-period ≈ 64−67 nm), with the less dense regions referred to as the “gap” zones. Mineral particles nucleate and form first in the gap zones."

"the mineral density along the fibril axis is a step function (step length ≈ 0.46D^20), which results in a series of Bragg reflections with period 2π/D ≈ 0.094−0.098 nm-1. When a fibril is stretched, the gap zones move apart, and the fibril strain is measured from the percentage shift in D, as measured from the SAXS pattern."

"The typical load−deformation curve of bone showed an initial elastic range up to 0.5−0.6% strain, followed by a slower rate of stress increase with strain following the elastic/inelastic transition (in the postyield regime)."<-So you have to have over 0.7% strain in the longitidunal direction to get a permanent longitudinal increase in bone length.  A 0.7% strain would be a temporary increase of 0.7% in length.

Shear and compression differentially regulate clusters of functionally related temporal transcription patterns in cartilage tissue.

"we subjected intact cartilage explants to 1-24 h of continuous dynamic compression or dynamic shear loading at 0.1 Hz. We then measured the transcription levels of 25 genes known to be involved in cartilage homeostasis using real-time PCR and compared the gene expression profiles obtained from dynamic compression, dynamic shear, and our recent results on static compression amplitude and duration. Using clustering analysis, we determined that transcripts for proteins with similar function had correlated responses to loading. However, the temporal expression patterns were strongly dependent on the type of loading applied. Most matrix proteins were up-regulated by 24 h of dynamic compression or dynamic shear, but down-regulated by 24 h of 50% static compression, suggesting that cyclic matrix deformation is a key stimulator of matrix protein expression. Most matrix proteases were up-regulated by 24 h under all loading types. Transcription factors c-Fos and c-Jun maximally responded within 1 h to all loading types. Pre-incubating cartilage explants with either a chelator of intracellular calcium or an inhibitor of the cyclic AMP pathway demonstrated the involvement of both pathways in transcription induced by dynamic loading."

" Application of a transient, radially unconfined compressive deformation (using displacement or load control) induces an initial build up of hydrostatic pressure within the tissue and concomitant intratissue fluid flow and flow-induced electrical streaming potentials. Mechanical stress relaxation (at constant displacement) or creep relaxation (at constant load) then leads to a new static equilibrium compressed state of the tissue at which fluid exudation has ceased. In contrast, dynamic compression leads to cyclic changes in pressure, deformation, and fluid flow within the tissue. For the case of unconfined dynamic compression of cylindrical cartilage explant disks using impermeable compression platens, theoretical models have predicted frequency-dependent increases in the dynamic amplitude of the hydrostatic pressure and radial strain at the explant center, with radially directed fluid flow velocities greatest at the explant periphery. Dynamic tissue shear in the “simple shear” induces the cyclic matrix strain in a nearly uniform manner throughout the explant disk with minimal fluid flow or increased hydrostatic pressure. Normal joint motion in vivo produces a superposition of all these components of cartilage loading. "


FIGURE 1.
These are for cartilage but they should apply to the bone marrow as well and the growth plate if it exists.

"The expression of COX-2 and most matrix proteases increased by 100–200% by 24 h, although MMP3, MMP9, MMP13, and COX-2 were mainly suppressed during earlier time points"

Dynamic compression:

"Immediate early genes c-Fos and c-Jun and signaling genes mitogen-activated protein kinase-1 (MAPK1), and TNFα were transiently up-regulated by 100–200% after 1 h, returning to control levels after 8 h of loading"

"Sox9, interleukin 1β, and HSP70 were mildly up-regulated only at the 4-h time point"<-this differs from LSJL where Sox9 was significantly upregulated.

LSJL gene expression took place at 49 hours with 1 hour after the third loading.  So genes upregulated at both 1 hour after the time point and 24 hours may be shown in those results.

Genes upregulated at 1 or 24 hours by Dynamic Compression(over 2 fold) also upregulated by LSJL:
Aggrecan
MMP3
c-Fos
c-Jun
ADAMTS4
Col1a1

Col2a1 and Sox9 were upregulated by LSJL but not significantly upregulated by dynamic compression.  0.1Hz of 3% strain was used.  LSJL used 5Hz but far less strain.  The greater frequency may have made the difference.  Or the upregulation of Col2a1 and Sox9 could be due to ectopic chondroinduction of MSCs which is what we're hoping for.

Dynamic Tissue Shear:

"Similar to dynamic compression, c-Fos and c-Jun were transiently up-regulated by 2–3-fold within 1 h in response to dynamic shear, and c-Jun was also up-regulated by 2-fold after 24 h. TGFβ and interleukin 1β were mildly transiently up-regulated at early time points, and ribosomal 6-phosphate was unaffected. MAPK1 was up-regulated ∼150% after 1 and 24 h"

Genes upregulated at 1 or 24 hours by Dynamic Compression(over 2 fold) also up by LSJL:
Aggrecan
Col2a1
COL1A1
ADAMTS4
MMP3
TIMP1
Sox9
c-Fos
c-Jun
PTGS2(aka COX-2)

Static Compression:
Aggrecan
Col1a1
Col2a1
MMP3
ADAMTS4

Co-regulation of LPS and tensile strain downregulating osteogenicity via c-fos expression.

"Cultures of MC3T3-E1 osteoblasts were pre-treated with conditioned medium from RAW264.7 macrophages exposed to 100ng/ml Porphyromonas gingivalis (Pg)-LPS. Conditioned medium was analyzed by ELISA for interleukin-1 beta (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-α). Osteoblasts were then subjected to tensile strain (0.5Hz; 1000μ or 3000μ) for 0min, 5min, 15min, 30min, 1h, 3h, and 6h. The cultures were analyzed for mRNA and protein levels of c-fos. Cells were also analyzed for alkaline phosphatase (ALP) activity.
(Pg)-LPS stimulated the secretion of all three cytokines from RAW264.7 cells in a dose- and time-dependent manner. Medium from (Pg)-LPS stimulated cells induced a 10-fold increase in c-fos expression, which decreased to a 4-fold plateau after 3h. In contrast, ALP activity of control osteoblasts decreased during the first 60min, then recovered over the next 4h. Pretreatment with conditioned medium generated the same initial decrease during tensile strain but prevented the recovery.
Our study showed, for the first time, that the inhibitory effect of inflammation and tensile strain on osteogenicity is associated with the upregulation in c-fos expression{LSJL upregulates c-fos}. In addition, inflammation may reduce the ability of osteoblasts to restore their osteogenic capacity during sustained tensile stress and contribute to periodontium damage."

"LPS can indirectly mediate inflammation-induced bone remodeling through the induction of TNF-α released from macrophages"

"onditioned medium from Pg-LPS-stimulated macrophages contains all three major pro-inflammatory cytokines: TNF-α, IL-1β, and IL-6."

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