Grow Taller with Mechanotransduction?

Hiroki Yokota has not tried injecting mesenchymal stem cells into the epiphyseal bone marrow.  He is also studying mechanotransduction which is the study of how interstitial fluid flow activates various molecular pathways and alters gene expression.  Now the reason why H. Yokota chose the name lateral synovial joint loading over epiphyseal loading is that the cartilage has a much different genetic expression than bone and that loading of the cartilage may be necessary to achieve results with Lateral Synovial Joint Loading.  But, the epiphyseal ends of the bones are also covered with articular cartilage.

If H. Yokota injects dedifferentiated mesenchymal stem cells(formerly chondrogenic) into the epiphyseal bone marrow and chondrocyte proliferation does not occur then we know it is not solely MSCs that are responsible for bone growth.  It may be like the pump in your muscle which creates an anabolic environment.  An increased anabolic environment as a result of increased intramedullary pressure may be necessary for the stem cells to re-initiate chondrocyte proliferation and differentiation.

How can load change the expression of genes in a way as to increase height?

Osteoblast responses one hour after load-induced fluid flow in a three-dimensional porous matrix.

"When bone is loaded, substrate strain is generated by the external force and this strain induces fluid flow that creates fluid shear stress on bone cells. Our current understanding of load-driven gene regulation of osteoblasts is based primarily on in vitro studies on planer two-dimensional tissue culture substrates. However, differences between a flat layer of cells and cells in 3-dimensional (3D) ECM are being recognized for signal transduction. Proliferation and differentiation of osteoblasts are affected by substrate geometry. Here we developed a novel 3D culture system that would mimic physiologically relevant substrate strain as well as strain-induced fluid flow in a 3D porous collagen matrix. The system allowed us to evaluate the responses of osteoblasts in a 3D stress-strain environment similar to a mechanical field to which bone is exposed. Using MC3T3-E1 osteoblasts grown in the 3D collagen matrix with and without hydroxyapatite deposition, we tested the role of strain and the strain-induced fluid flow in the expression of the load-responsive genes such as c-fos, egr1, cox2, osteopontin, and mmp1B involved in transcriptional regulation, osteogenesis, and rearrangement of ECM[Extracellular matrix]. Strain-induced fluid flow was visualized with a microspheres approximately 3 microm in diameter in real time, and three viscoelastic parameters were determined. The results obtained by semi-quantitative PCR, immunoblot assay, enzymatic activity assays for collagenase and gelatinase, and mechanical characterization of collagen matrices supported the dominant role of strain-induced fluid flow in expression of the selected genes one hour after the mechanical treatment."

It's possible that those genes: c-fos, egr1, etc. caused the height gain from Lateral Synovial Joint Loading but it is not osteogenesis that increases height but rather chondrogenesis.  C-fos and egr1 were upregulated by LSJL.   The rearrangement of Extracellular Matrix could be involved though in helping to align the growth plate.  Transcription factors are also involved as well.

"Connective tissue cells such as chondrocytes, osteoblasts, and osteocytes alter gene expression in response to mechanical loads[what about mesenchymal stem cells], but alteration of mRNA and protein levels as well as enzyme activities differs depending on loading conditions such as stress elements (tensile/compressive vs. fluid shear), intensity and duration"<-so we need to know how to change the stress, intensity, and duration so as to alter the mRNA and protein levels to increase height.

"an oscillatory compressive loading at 1 Hz with a peak-to-peak magnitude of 3000 μstrain was chosen as an input."<-this is much higher than loads in LSJL which uses 0.5Hz and much less microstrain.

"Expression of cox2 and release of prostaglandin E₂ are linked to mechanically induced bone formation, and osteopontin (bone sialoprotein) is shown to be responsive to fluid shear in 2D cell cultures. Mmp1B is a collagenase involved in rearrangement of ECM and its expression is sensitive to mechanical strain and fluid flow"

C-fos and egr-1 are definitely indicative of shear strain according to the study.  So it would be a good measurement tool of whether the method of LSJL induces shear strain.

Model-based comparative prediction of transcription-factor binding motifs in anabolic responses in bone.

"Transcription-factor binding motifs [=] (TFBMs). Mechanical loading and administration of bone morphogenic proteins (BMPs) are two known treatments to strengthen bone.  Is there any TFBM that stimulates both "anabolic responses of mechanical loading" and "BMP-mediated osteogenic signaling"? Although there is no significant overlap among genes in the two responses, a comparative model-based analysis suggests that the two independent osteogenic processes employ common TFBMs, such as a stress responsive element and a motif for peroxisome proliferator-activated receptor (PPAR). The post-modeling in vitro analysis using mouse osteoblast cells supported involvements of the predicted TFBMs such as PPAR, Ikaros 3 [zinc finger protein], and LMO2 [LIM domain transcription regulator 2] in response to mechanical loading."

"The linkage analysis through mapping to TRANSFAC also identified other known motifs and transcription regulators, such as HEN1 (basic helix-loop-helix protein), Helios A (Ikaros-related protein), NRSF (neuron-restrictive silencer factor), major Tantigen binding site, GCNF (germ cell nuclear factor), STAT6 (signal transducer and activator of transcription 6), ER (estrogen receptor), and ERRα (estrogenrelated receptor α)"

Long-term dynamic loading improves the mechanical properties of chondrogenic mesenchymal stem cell-laden hydrogel.

"Mesenchymal stem cells (MSCs) are an attractive cell source for cartilage tissue engineering given their ability to undergo chondrogenesis in 3D culture systems{chondrogenesis in the epiphyseal bone marrow = growing taller}. Mechanical forces play an important role in regulating both cartilage development and MSC chondrogenic gene expression. We applied long-term dynamic compression to MSC-seeded constructs and assessed whether varying pre-culture duration, loading regimens and inclusion of TGF-beta3 during loading would influence functional outcomes and these phenotypic transitions. Loading initiated before chondrogenesis decreased functional maturation, although chondrogenic gene expression increased. In contrast, loading initiated after chondrogenesis and matrix elaboration further improved the mechanical properties of MSC-based constructs, but only when TGF-beta3 levels were maintained and under specific loading parameters{Is lateral loading one of those parameters?}. Although matrix quantity was not affected by dynamic compression, matrix distribution, assessed histologically and by FT-IRIS analysis, was significantly improved on the micro- (pericellular) and macro- (construct expanse) scales. Further, whole genome expression profiling revealed marked shifts in the molecular topography with dynamic loading. Dynamic compressive loading initiated after a sufficient period of chondro-induction and with sustained TGF-beta exposure enhances matrix distribution and the mechanical properties of MSC-seeded constructs."

Increase in chondrogenic gene expression in the growth plate could have nice effects.

"In the presence of TGF-β superfamily members, MSCs in 3D culture express cartilage specific markers an deposit a extracellular matrix (ECM) rich in proteoglycans and collagen type II"<-TGF-Beta1 seems to be the member most commonly found in mechanical loading studies.  Thus TGF-Beta1 is the most likely target gene to induce chondrogenic differentiation of MSCs.

"[There] may be due to an intrinsic limitation in TGF-β mediated chondrogenesis, in the absence of additional stimuli."<-This limitation of TGF-Beta chondrogenesis may affect LSJL results.

"During development, inhibition of muscle contraction and forces acting on skeletal elements results in abnormal joint formation.  After birth, loading-induced remodeling of articular cartilage leads to dramatic changes in tissue structure (particularly of the collagen network) and increases in mechanical properties; in the absence of loading, this remodeling is not observed. In addition, normal joint loading has also been implicated in the maintenance of the chondrogenic phenotype of articular chondrocytes within cartilage"<-But can joint loading cause differentiation of MSCs into chondrocytes?

"Loading of human MSCs increases expression of aggrecan and collagen type II"

"The presence/absence of TGF-β also dictates the response of MSCs to compressive loading. Loading in the absence of TGF-β improves proteoglycan synthesis levels of equine MSCs relative to free swelling controls while in cultures loaded in the presence of TGF-β, matrix synthesis levels diminish. Dynamic compression modulates MSC chondrogenic differentiation and that the presence/absence of TGF-β influences this process."<-So perhaps Tgf-Beta1 competes with mechanical stimulation for stimulating chondrogenesis or TGF-Beta1 inhibits the response of MSCs to loads?  Although several conflicting data to this point are mentioned in the study.

"Dynamic compression alone, in the absence of TGF-β3, failed to induce chondrogenesis; in fact, after three weeks of loading, GAG content decreased in loaded groups, though chondrogenic gene expression increased"<-So perhaps a source of TGF-Beta3 or possibly 1 is needed for LSJL to induce chondrogenesis?

"loading initiated after chondrogenesis and matrix elaboration in the presence of TGF-β3 consistently improved the mechanical properties of MSC-seeded constructs"<-So maybe LSJL is most effective after growth plates are formed.  But this study took place in a medium.  Articular cartilage and periosteum may provide signals like those in MSCs in media that already has chondrogenesis and matrix elaboration.  Articular cartilage may send signals to stem cells within the epiphyseal bone marrow to help them differentiate.

"mechanotransduction pathways initiated by dynamic compression may be fundamentally different between chondrocytes and undifferentiated or chondrogenically differentiated MSCs."

"one potential mechanism [for load improving mechanical properties of chondrocytes] may be facilitated nutrient/growth factor transport with dynamic deformation. Theoretical models of dynamic compression of porous permeable materials indicate that solute transport into constructs may be improved by dynamic loading and that higher frequencies enhance this phenomenon.  The transport of large solutes (with molecular weights similar to that of growth factors such as TGF-β) is facilitated by dynamic compression and is dependent on loading duration"<-This doesn't really help us grow taller as we need new chondrogenesis not acceleration of existing chondrogenesis.

"Loading in the absence of exogenous TGF-β3 (after chondrogenic pre-culture) failed to elicit any changes in mechanical properties."<-this indicating the possibility that dynamic loading only increased uptake of TGF-Beta3 without causing any change in properties on it's own.

Effect of dynamic compressive loading and its combination with a growth factor on the chondrocytic phenotype of 3-dimensional scaffold-embedded chondrocytes.

"Three-dimensionally (3D-) embedded chondrocytes maintain the chondrocytic phenotype. Mechanical stress and growth factors have been found to be capable of enhancing cell proliferation and ECM synthesis. We investigated the effect of mechanical loading and growth factors on reactivation of the 3D-embedded chondrocytes.
Freshly isolated chondrocytes from rat articular cartilage were grown in monolayer cultures and then in collagen gel. Analysis for aggrecan and type II and type I collagen was performed to evaluate their chondrocytic activity. Then, the 3D-embedded chondrocytes were cultured under either mechanical loading alone or in combination with growth factor. The dynamic compression (5% compression, 0.33 Hz) was loaded for 4 durations: 0, 10, 60, and 120 min/day. The growth factor administered was either basic fibroblast growth factor (bFGF) or bone morphogenetic protein-2 (BMP-2).
Mechanical loading statistically significantly reactivated the aggrecan and type II collagen expression with loading of 60 min/day as compared to the other durations{This is consistent with axial loading which increased Aggrecan and COL2A1 but not Sox9}. The presence of BMP-2 and bFGF clearly enhanced the aggrecan and type II collagen expression of 3D-embedded chondrocytes. Unlike reports using monolayer chondrocytes, however, BMP-2 or bFGF did not augment the chondrocytic phenotype when applied together with mechanical loading{So BMP-2 and FGF-2 may mimic some of the signaling of 3D cultures}. Dynamic compression effectively reactivated the dedifferentiated chondrocytes in 3D culture. However, the growth factors did not play any synergistic role when applied with dynamic compressive loading, suggesting that growth factors should be administered at different time points during regeneration of the transplantation-ready cartilage."

"static compression has been shown to reduce ECM synthesis, whereas dynamic compression at low amplitude (1–5% compression loading, 0.01–1 Hz) stimulates the synthesis "<-maybe loading for LSJL needs to be lighter load but more dynamic.  For example, constant very light clamping and unclamping or using a foam roller.

"Col2 mRNA expression was also upregulated with loading of 60 min/day as compared to that with loading of 0 and 10 min/day (p < 0.01) "<-This was chondrocytes in a 3D scaffold with no serum(so no TGF-Beta).  So maybe LSJL has to be done with a light load for 60 min/day.  However, Sox9 was not measured so we don't know if it enhanced Sox9 as effectively as LSJL.  60minutes a day upregulated Aggrecan more than 120 minutes a day.

"indicate that chondrocytic differentiation promoted by the mechanical stress may not coexist with cell proliferation driven by the growth factors."<-Now LSJL managed to increase height in active mice growth plates.  And, in adult growth plates there aren't many growth factors in cells or otherwise people would still be growing taller(although it's possible that there are growth factors and another cause prevents height growth).

"simultaneous application of growth factor and mechanical stress suppressed AGC and Col2 gene expression "

"mechanical loading clearly stimulated dedifferentiated chondrocytes "

So the effect of mechanical stimulation on chondrocytes is unclear but it is still clear that LSJL increased height in mice with some mechanism.

The P2X7 nucleotide receptor mediates skeletal mechanotransduction.

"The P2X7 nucleotide receptor (P2X7R) is an ATP-gated ion channel expressed in many cell types including osteoblasts and osteocytes. Mice with a null mutation of P2X7R have osteopenia in load bearing bones. Skeletal sensitivity to mechanical loading was reduced by up to 73% in P2X7R null (knock-out (KO)) mice. Release of ATP in the primary calvarial osteoblasts occurred within 1 min of onset of fluid shear stress (FSS). After 30 min of FSS, P2X7R-mediated pore formation was observed in wild type (WT) cells but not in KO cells. FSS increased prostaglandin (PG) E2 release in WT cells but did not alter PGE2 release in KO cells. Studies using MC3T3-E1 osteoblasts and MLO-Y4 osteocytes confirmed that PGE2 release was suppressed by P2X7R blockade, whereas the P2X7R agonist BzATP enhanced PGE2 release."

It's possible this ATP signaling can affect chondroinduction as well.

"P2 signaling also modulates cyclo-oxygenase-2 expression , PG release, and c-fos expression in osteoblasts and other cell types."

"P2Y receptors are G protein-linked receptors that in many cases are coupled through phospholipase C to the release of Ca2+ from intracellular stores. "

"The P2X7R forms a complex with several proteins including β2 integrin, receptor-like tyrosine phosphatase (RPTP), α-actinin, phosphatidylinositol 4-kinase, membrane-associated guanylate kinase, and several heat shock proteins"

"RPTP is involved in force-dependent formation of focal adhesion complexes, and α-actinin links β integrins with the actin cytoskeleton promoting transduction of mechanical forces"

"No differences in femoral length were found between KO and WT in female (p = 0.63) or male (p = 0.62) mice [with P2X7R KO]."

"When primary osteoblasts from WT mice were subjected to steady laminar fluid flow at 12 dynes/cm2 FSS, release of ATP was also observed within 1 min. Primary bone cells from KO mice released similar amounts of ATP compared with WT cells"

Maybe P2X7R decreases in sensitivity in response to load and reduces LSJL effectiveness with conditioning.

Is skeletal mechanotransduction under genetic control?

"An immobilized femur will develop with a fairly round cross-section, rather than the typical elliptical shape. The high bone mass C3H/HeJ (C3H) strain of mice has long bones that are largely unresponsive to mechanical loading.  A tibia of a C3H mouse has a rounded cross-sectional shape, rather than the more triangular shape seen in mice from the C57BL/6J (B6) strain. The rounded C3H tibia resembles a rat’s tibia that developed without mechanical loading, suggesting that the C3H tibia did not adapt its shape to loading patterns during growth."

"The study involved twelve BXH RI strains of mice and the progenitor strains C3H and B6. All mice used in the study were female and 8 months old."

An inhibitor of the stretch-activated cation receptor exerts a potent effect on chondrocyte phenotype.

"Rat chondrosarcoma (RCS) cells are unusual in that they display a stable chondrocyte phenotype in monolayer culture. This phenotype is reflected by a rounded cellular morphology with few actin-containing stress fibers and production of an extracellular matrix rich in sulfated proteoglycans, with high-level expression of aggrecan, COMP, Sox9, and collagens type II, IX, and XI [without expression of collagen type I]. In the absence of any mechanical stimulation, treatment of RCS cells with gadolinium chloride (Gd3+), a stretch-activated cation channel blocker, caused the cells to undergo de-differentiation, adopting a flattened fibroblast phenotype with the marked appearance of actin stress fibers and vinculin-containing focal contacts. This change was accompanied by a dramatic reduction in the expression of aggrecan, Sox9, collagen types II, IX, and XI, with a corresponding increase in the expression of collagen type I and fibronectin. These effects were found to be reversible by simple removal of Gd3+ from the medium. Gd3+ also had a similar effect on expression of chondrocyte marker genes in freshly isolated human chondrocytes. Mechanoreceptor signaling plays a key role in maintenance of the chondrocyte phenotype, even in the absence of mechanical stimulation."

"The chondrocytes in articular cartilage exist in an unusual environment where cell–cell contact is extremely limited and the cells display a very rounded morphology. The cells at the superficial layer, however, are elongated and fibroblastic in shape. The cells within the middle and deep layers of articular cartilage are in an acidic environment, with significantly reduced oxygen tension and lack of a blood supply"

"De-differentiation [caused by Gd3+ addition] is reversible by simple removal of Gd3+ from the medium."

"IHH expression in chondrocytes is tied to mechanotransduction signaling. Treatment of chondrocytes under mechanical load with Gd3+ abolished IHH expression"

"Sox9 couples with the transcriptional activator CBP, which combines to aid in the activation of the collagen II promoter.  CBP RNA levels drop appreciably in Gd3+ treated cells."

"the effect of Gd3+ on RCS cells is likely not because of an effect on other cation channels but is likely specific for the SACC[Stretch-Activated Cation Channel]."

"The Gd3+ effect on cell morphology is very similar in human chondrocytes to RCS cells."

"Compression of chondrocytes by mechanical pressure, membrane deformation or fluid shear leads to activation of the SACC, setting off internal signaling cascades and propagating Ca2+ waves, ultimately resulting in the continued expression of chondrocyte marker genes"

Signaling networks and transcription factors regulating mechanotransduction in bone.

"Mechanoreceptors, such as integrins, G protein-coupled receptors, receptor protein tyrosine kinases, and stretch-activated Ca(2+) channels, together with their downstream effectors coordinate the transmission of load-induced signals to the nucleus and the expression of bone-related genes."

"Upon mechanical stretching, prostaglandin synthase (PGES), and COX-1/2 mediate production of PGE2 from arachidonic acid (AA). COX1/2 is induced through the ERK and PKA pathway on the focal adhesions formed. Notably, COX-2 can also be activated by other mechanoresponsive cascades, namely PI3K/Akt and Wnt/β-catenin. Mechanical loading on bone cells activates the L-VSCC, facilitating the entrance of extracellular Ca2+ into the cell cytoplasm, where it induces further release of Ca2+ from the intracellular stores, synthesis of NO and activation of ERKs. Ca2+ is also released by the internal Ca2+ stores and the inositol triphosphate (IP3) signaling pathway.  {These steps seem to happen in LSJL} NO inhibits induction of the JNK. Mechanical stress or extracellular growth factor (ligand) binding on receptor protein tyrosine kinase (RPTK) leads to the dimerization and autophosphorylation of tyrosine residues. Binding of the adaptor protein Grb2 to a specific phosphorylated tyrosine evokes conformational changes in Grb2 that facilitate its connection with SOS and activation of the membrane-associated GTPase Ras. The latter then activates MAPKK kinases (MAPKKKs), which phosphorylate and thus activate MAPKKs (MEK1/2, MKK3/6, MKK4/7) that in turn potentiate ERK1/2, p38 and SAPK/JNK MAPKs. These kinases translocate to the nucleus where they activate transcription factors (Runx2 and AP-1) augmenting osteoblast-specific gene transcription. Alterations in osteoblast microenvironment are sensed by integrin receptors that recruit non-receptor kinases such as FAK and Src family kinases. The complex that is formed induces several molecular phenomena including ERK phosphorylation/activation and intracellular Ca2+ release via the activation of PLC–IP3 circuit. (E) Mechanical load stimulates G protein activation. This induces PLC–IP3 and PLC–DAG–PKC systems that eventually facilitate Ca2+ release and ERK activation. (F) Mechanical stimulation promotes the formation of the Wnt–LRP5/6–FZD complex, which promotes the activation of Dsh and the association of Axin, APC, GSK-3β and other proteins to the cytoplasmic tail of LRP5. In the absence of Wnt binding, the complex of Axin, APC, GSK-3β and β-transducin repeat-containing protein (β-TrCP) promotes β-catenin ubiquitination and thus its degradation. Activated Dsh induces GSK-3β inactivation, which, in turn, enables β-catenin stabilization and translocation to the nucleus, where it forms a complex with LEF/TCF inducing the transcription of specific mechanoresponsive target genes."

"functional COX-2 is not required for the response of bone to mechanical loading. This discrepancy could be explained, at least in part, by the induction of COX-1 after mechanical stimulation of COX-2–/– mice elbow bones."

"Loading experiments on tibias of LRP5G171V-transgenic mice and on MC3T3-E1 cells revealed increased expression of Wnt10B, SFRP1, cyclin D1, FZD2{up in LSJL}, WISP2{up}, and connexin 43, supporting the role of the canonical Wnt pathway in bone mechanosensory phenomena."

"Wnt/β-catenin cascade genes Wnt1, Wnt3a, Wnt5a, Lrp5, Ctnnb1, Lef1, and Axin were upregulated [in a mice cell line that showed higher levels of Beta-Catenin]"

" In normal cartilage, α1β1, α5β1, α10β1, and ανβ5 are the major integrin heterodimers. The extracellular domain of integrins provides the ligand-binding site, whereas the cytoplasmic tail of the β subunit interacts with intracellular signaling molecules and the actin cytoskeleton"

"NOS function is principally regulated by free intracellular Ca2+, the phosphatidylinositol 3-kinase (PI3K)/Akt pathway and estrogen. Downstream NO signaling depends upon activation of guanylate cyclase, which, in turn, catalyzes the production of cyclic guanosine monophosphate (cGMP) from GTP. In addition, NO can directly nitrosylate proteins as has been shown for NO action to decrease the RANKL/OPG ratio in stromal cells."

"Mechanical tension stress is associated with over-expression of the BMP-2 and BMP-4 genes"

"Increased condylar cartilage stress was associated with higher cellular levels of FGFR-3 at the proliferating and early hypertrophic zone."

Mechanosomes carry a loaded message.

"Adhesion complexes at the surface of the sensor cell activate multiprotein complexes (mechanosomes) that include both proteins involved in adhesion and transcription factors that move to the nucleus and regulate transcriptional activity of target genes. [One] mechanotransduction complex [consists] of nitric oxide (NO), cyclic guanosine monophosphate (cGMP), protein kinase G II, SHP-1, and SHP-2-that associates with β₃ integrins through Src. This complex regulates gene expression in response to fluid flow."

"the ATF nuclear matrix protein 4 (Nmp4) as a general inhibitor of bone anabolism that inhibits fluid flow–induced nuclear translocation of β-catenin"

"Fluid flow activated extracellular signal–regulated kinase (ERK) through a NO-cGMP-PKGII pathway and so induced expression of several genes, including c-fos, fra-1, fra-2, and fosB. Fluid flow–induced dephosphorylation of Src by the phosphatase SHP-1 or SHP-2 is a key regulatory step in the assembly of this complex. PKGII can directly phosphoryl-ate SHP-1 to trigger phosphatase activity and, compared with cells from wild-type mice, cells from PKGII-null mice exhibit attenuated flow-induced ERK-dependent gene expression."

"p130Cas is a mechanosensor that associates with focal adhesions. Mechanosensitivity of focal adhesions is dependent on conformational changes in p130Cas and phosphorylation by Src family kinases"

"The absence of FAK function compromises multiple responses to oscillatory fluid flow, including activation of ERK1 and 2; increased abundance of c-Fos, cyclooxygenase-2, and osteopontin; and release of prostaglandin E2. Furthermore, activation of nuclear factor B (NF-B) signaling by fluid flow also requires FAK. However, FAK is not required for tumor necrosis factor––induced activation of NF-B in osteoblasts"

"FAK does localize to the nucleus during mouse development, where it appears to act as a scaffold that enhances p53 degradation, which promotes cell survival. In addition, in the nucleus of muscle cells, FAK associates with methyl CpG binding domain protein 2, which results in the remodeling of heterochromatin and directly or indirectly mediates the regulation of expression of Myog {down in LSJL} (which encodes myogenin or myogenic factor 4) and muscle differentiation"

Tactile and Kinesthetic Stimulation (TKS) intervention improves outcomes in weanling rat bone in a neonatal stress model.

"Preterm infants are born with low bone mineral. Neonatal stress further impedes bone mineralization. Clinical evidence suggests that tactile and kinesthetic stimulation (TKS) improves bone phenotype and decreases stress response. Clinical and translational studies indicate the IGF-1 axis, responsible for postnatal growth and bone mineralization, is a key player. We hypothesized that TKS would attenuate the negative impact of neonatal stress on bone phenotype and the IGF-1 axis in weanling rats{would this stimulation help in individuals not exposed to neonatal stress?}. Neonatal stress (STRESS) or TKS (STRESS + 10min TKS) was administered from D6 to D10. Control animals received standard care. Tissue was harvested on D21. Dual energy x-ray absorptiometry (DXA) and bone morphometry were performed and serum osteocalcin, type I procollagen N-terminal propeptide (PINP), tartrate-resistant acid phosphatase (TRAP), and bone and liver mRNA levels of IGF-1, IGF-1 receptor (IGF-1R), and growth hormone receptor (GHR) were measured. Neonatal stress increased bone mineral content (BMC), area (BA), growth plate width{one might think that increasing growth plate width would be beneficial for height growth}, liver IGF-1 mRNA, and serum IGF-1. TKS maintained areal bone mineral density (aBMD) and bone specific IGF-1 and IGF-1R mRNA while STRESS decreased compared to controls. Neonatal stress results in apparent accelerated growth response. TKS differed from STRESS with improved tibia aBMD and increased bone specific IGF-1 mRNA."

Premature human children have smaller less mineralized bones.

"10 minutes of tactile stimulation with a soft camel hair brush to the ventral and dorsal body and kinesthetic movement that involves a range of motion movement to the fore and hind limbs"<-This was just one 10 minute stimulation period immediately after birth.

Neither the Control, tactile stimulation, or preterm infant stress simulation infant group had a difference in tibial length.

Growth plate width was higher for the preterm stress stimulated group and further wider for the tactile stimulation group.

"the pup received a needle puncture, a hypoxic and a hyperoxic challenge, with maternal separation lasting for a total of 60 minutes."

"neonatal stress groups appeared to be in a state of accelerated bone growth."