If Fibrous tissue is not currently encouraged by LSJL and fibrous tissue production is key to endochondral ossification related processes and therefore growing taller then finding mechanisms to stimulating fibrous tissue production in the bone marrow may help individuals grow taller. LSJL alters Fibronectin related proteins. It upregulates Fank1 and FNDC4.
Fibronectin- and collagen-mimetic ligands regulate bone marrow stromal cell chondrogenesis in three-dimensional hydrogels.
"While adhesion to RGD peptides[cells containing RGD peptides inhibit attachment and induce apoptosis] has been shown to inhibit in vitro chondrogenesis, the effects of extracellular matrix (ECM)-mimetic ligands with complex secondary and tertiary structures are unknown. This study aimed to determine whether collagen- and fibronectin-mimetic ligands would retain biologic functionality in three-dimensional (3D) hydrogels, whether different ECM-mimetic ligands differentially influence in vitro chondrogenesis, and if effects of ligands on differentiation depend on soluble biochemical stimuli. A linear RGD peptide, a recombinant fibronectin fragment containing the seven to ten Type III repeats (FnIII7-10) and a triple helical, collagen mimetic peptide with the GFOGER motif were covalently coupled to agarose gels using the sulfo-SANPAH crosslinker, and bone marrow stromal cells (BMSCs) were cultured within the 3D hydrogels. The ligands retained biologic functionality within the agarose gels and promoted density-dependent BMSC spreading. Interactions with all adhesive ligands inhibited stimulation by chondrogenic factors of collagen Type II and aggrecan mRNA levels and deposition of sulfated glycosaminoglycans. In medium containing fetal bovine serum, interactions with the GFOGER peptide enhanced mRNA expression of the osteogenic gene osteocalcin whereas FnIII7-10 inhibited osteocalcin expression. In conclusion, modification of agarose hydrogels with ECM-mimetic ligands can influence the differentiation of BMSCs in a manner that depends strongly on the presence and nature of soluble biochemical stimuli."
"integrin-mediated adhesion to fibronectin is required for precartilage condensation of limb bud cells"<-Fibronectin may be critical for chondrogenesis. Demineralized bone matrix binds Fibronectin. Fibronectin may also be key in the hypertrophic chondrocyte to allow for differentiation into osteoblasts. If the fibronectin is not exposed in the chondrocyte, then no osteogenic differentiation occurs and thus a chondrogenic phenotype is maintained. RGE was the most chondrogenic of all the ligands. RGE is the non-adhesive version of the RGD integrin. So we want to find ways to make RGD non-adhesive.
"Our data provide additional support for the role of the cytoskeleton and suggest that cell shape, more
so than the specific integrin ligand, is a key regulator of chondrogenesis"<-this is good for LSJL as hydrostatic pressure may induce a more pro-chondrogenic cell shape.
It may be that Fibronectin is key but that it's key for it to be non-adhesive to allow for chondrogenesis. Perhaps, the adhesive form of Fibronectin is produced post cessation of endochondral ossification and that's what inhibits growth.
TGF-Beta1 may stimulate fibronectin and TGF-Beta1 is very pro-chondrogenic. We know that Fibronectin is important in the pre-condensation stage of MSCs before chondrogenesis is initiated.
Fibronectin regulates proteoglycan production balance in transforming growth factor-β1-induced chondrogenesis.
"Transforming growth factor (TGF)-β and bone morphogenetic protein (BMP) induce a cartilage-specific extracellular matrix (ECM) gene, aggrecan, in a chondrogenic cell line, ATDC5. The results of our recent study show that TGF-β1, but not BMP-4, strongly induces an ECM gene, fibronectin, during chondrogenesis. Our results showed that TGF-β1, but not BMP-4, led to versican-dominant proteoglycan production during chondrogenesis of ATDC5 cells. siRNA-mediated reduction of fibronectin and interference in the liaison between fibronectin and integrins by the Arg-Gly-Asp-Ser (RGDS) peptide increased aggrecan expression, and decreased versican expression by TGF-β1 stimulation[so fibronectin affects the aggrecan/versican balance in chondrogenic regions, dependent on whether versican or aggrecan is better for height growth, inhibiting or stimulating fibronectin could be used to grow taller]. These data suggest that fibronectin is a critical mediator for TGF-β-specific production balance of 2 major proteoglycans, aggrecan and versican, during chondrogenesis."
"These data suggest that increased expression of versican in the cartilage correlates with fibrosis and decreases the water-holding capacity, and TGF-βs, which induce versican expression, may be key molecules that increase the degree of fibrosis during chondrogenesis."<-So Aggrecan is likely better than versican for height growth as more water equals a larger growth plate. Fibrosis usually refers to thickening and scarring of cartilagenous tissue. So this study indicates that inhibting fibronectin may help grow taller.
Fibroconectin signaling may be modulated by magnetic fields. PEMFs have been discussed before.
Mechanical integrin stress and magnetic forces induce biological responses in mesenchymal stem cells which depend on environmental factors.
"Because integrin receptors function as mechanotransducers, we applied drag forces to β1 integrins on the apical surface of adherent human MSC. In addition to mechanical forces, the technique we used involved also the exposure of the cells to an inhomogeneous magnetic field. In order to assess the influence of the substrate on cell adhesion, cells were cultured on plain tissue culture polystyrene (TCP) or on coated well plates, which allowed only adhesion to embedded fibronectin or RGD peptides[RGD peptides inhibiting attachment and fibronectin allowing it]. We found that the expression of collagen I, which is involved in osteogenesis, and VEGF, a factor which stimulates angiogenesis, increased as a result of short-term mechanical integrin stress. Whereas, collagen I expression was stimulated by mechanical forces when the cells were cultured on fibronectin and RGD peptides but not on TCP, VEGF expression was enhanced by physical stimulation on TCP. The study further revealed that magnetic forces enhanced Sox 9 expression, a marker of chondrogenesis, and reduced the expression of ALP[magnetic forces help enhance Sox9 which helps you grow taller]. Concerning the intracellular mechanisms involved, we found that the expression of VEGF induced by physical forces depended on Akt activation. Together, the results implicate that biological functions of MSC can be stimulated by integrin-mediated mechanical forces and a magnetic field. However, the responses of cells depend strongly on the substrate to which they adhere and on the cross-talk between integrin-mediated signals and soluble factors."
"Mesenchymal stem cells are also able to sense the elasticity of a substrate, which determines the direction of differentiation or maintains their quiescence"<-We can manipulate this. Remember, bone becomes more elastic when exposed to acid for instance. How to do this safely is another question.
"The forces subjected to one bead were adjusted to 2 × 10−10 N. Because of the varying number and location of beads attached to one cell, differential strains across the cell can occur. A cyclic stress of 1 Hz (0.5 s on, 0.5 s off) was applied for 15 min."<-this seems pretty small so it's promising for potential application.
"On TCP, cells can adhere to a mix of extracellular matrix proteins both produced by the cells and contained in the culture medium" The increase in SOX9 was only observed on Tissue Cultured Polystyrene. So SOX9 was only induced when cells were free not to bind to Fibronectin and RGD peptides.
DLK1 may inhibit chondrogenic differentiation by interacting with Fibronectin.
Delta-like 1/fetal antigen-1 (Dlk1/FA1) is a novel regulator of chondrogenic cell differentiation via inhibition of the Akt kinase-dependent pathway.
"Delta-like 1 (Dlk1, also known as fetal antigen-1, FA1) is a member of Notch/Delta family that inhibits adipocyte and osteoblast differentiation. We overexpressed Dlk1/FA1 in mouse embryonic ATDC5 cells and tested its effects on chondrogenic differentiation. Dlk1/FA1 inhibited insulin-induced chondrogenic differentiation as evidenced by reduction of cartilage nodule formation and gene expression of aggrecan, collagen Type II and X. Similar effects were obtained either by using Dlk1/FA1-conditioned medium or by addition of a purified, secreted, form of Dlk1 (FA1) directly to the induction medium. The inhibitory effects of Dlk1/FA1 were dose-dependent and occurred irrespective of the chondrogenic differentiation stage: proliferation, differentiation, maturation, or hypertrophic conversion. Overexpression or addition of the Dlk1/FA1 protein to the medium strongly inhibited the activation of Akt, but not the ERK1/2, or p38 MAPK pathways, and the inhibition of Akt by Dlk1/FA1 was mediated through PI3K activation. Interestingly, inhibition of fibronectin expression by siRNA rescued the Dlk1/FA1-mediated inhibition of Akt, suggesting interaction of Dlk1/FA1 and fibronectin in chondrogenic cells. Our results identify Dlk1/FA1 as a novel regulator of chondrogenesis and suggest Dlk1/FA1 acts as an inhibitor of the PI3K/Akt pathways that leads to its inhibitory effects on chondrogenesis."
"suppression of fibronectin expression in ATDC5-Dlk1 cells, rescued the inhibition of insulin-induced Akt activation. Interestingly, these effects were not related to the fibronectin receptor integrin beta 1 (Itgb) or the canonical target of integrin signaling, FAK (focal adhesion kinase) which is one of the upstream regulators of Akt kinases. These results suggest that Dlk1/FA1 may exert its effects through interaction with fibronectin, but that the signaling is not mediated though Fn/Itgb/FAK1"
So Dlk1 may not exert it's height decreasing effects through Fibronectin/integrin binding and you may want to inhibit Fibronectin to grow taller and not just disrupt the binding of fibronectin and integrin.
Fibronectin regulates proteoglycan production balance in transforming growth factor-β1-induced chondrogenesis.
"Transforming growth factor (TGF)-β and bone morphogenetic protein (BMP) induce a cartilage-specific extracellular matrix (ECM) gene, aggrecan, in a chondrogenic cell line, ATDC5. The results of our recent study show that TGF-β1, but not BMP-4, strongly induces an ECM gene, fibronectin, during chondrogenesis. However, the role of fibronectin in chondrogenesis is unclear. In the current study, our results showed that TGF-β1, but not BMP-4, led to versican-dominant proteoglycan production during chondrogenesis of ATDC5 cells. siRNA-mediated reduction of fibronectin and interference in the liaison between fibronectin and integrins by the Arg-Gly-Asp-Ser (RGDS) peptide increased aggrecan expression, and decreased versican expression by TGF-β1 stimulation. These data suggest that fibronectin is a critical mediator for TGF-β-specific production balance of 2 major proteoglycans, aggrecan and versican, during chondrogenesis."
"Aggrecan expression levels increased by both TGF-β1 (7.4-fold) and BMP-4 (7.5-fold) stimulation. However, versican was more strongly induced by stimulation with TGF-β1 (16.2-fold) than by BMP-4 (2.1-fold)"
"Versican is a large chondroitin sulfate proteoglycan expressed by human fibroblasts. The expression levels of versican are much lower than those of aggrecan in human articular cartilage"
"Focal adhesion kinase(FAK) is one of the upstream mediators of MAP kinase, and FAK/Src signaling suppresses early chondrogenesis, including the induction of aggrecan, Sox6, and Ccn2 in mesenchymal cells by connective tissue growth factor gene regulation in micromass culture. On the other hand, versican expression is regulated by activator protein-1 (AP-1) and T cell factor (TCF) transcriptional factors. Moreover, fibronectin induces AP-1 activation through integrin α5β1-dependent Akt, extracellular signal regulated kinase (ERK), and Jun-aminoterminal kinase (JNK) signaling pathways in endothelial cells"
So Fibroconectin may be a key to growing taller but not by stimulating it. The breaking of the bone may loosen fibrous tissue and decrease the integrin binding to fibroconectin allowing for chondrogenesis. Acidifying the bone or applying magnetic fields may be possible ways to weaken integrin signaling to allow for chondrogenesis.
Fibronectin and stem cell differentiation - lessons from chondrogenesis.
"The principal integrins for collagen binding are integrin a1b1 and integrin a2b1"
"Integrin receptors that bind laminins include integrins a3b1, a6b1 and a7b1"
"fibronectin binds to integrins a5b1, a4b1, and avb3"
"Tenascin-C binds to fibronectin and modulates cell adhesion"
"During embryogenesis, prechondrogenic mesenchymal cells that reside within the ECM of the limb bud rearrange into condensed cell aggregates"
"Condensed cells are readily detected by staining with labeled peanut agglutinin – which binds to a galactose moiety that is upregulated in cell aggregates during chondrogenic differentiation in vivo and in vitro"
"Condensation is a prerequisite for chondrogenic differentiation and depends on the expression of the cell–cell adhesion proteins N-cadherin and neural cell adhesion molecule (NCAM)."
"SOX9 is not required during condensation."<-But Sox9 enhances condensation.
"Undifferentiated mesenchymal cells and the condensing mesenchyme deposit a fibronectin-rich matrix that also contains versican {upregulated by LSJL}, type I collagen, and hyaluronan"
"Tenascin-C is expressed during condensation and, as condensed cells undergo differentiation, levels of tenascin-C and collagen I decrease while a cartilaginous matrix consisting of collagen types II and IX, and the proteoglycans aggrecan and versican develops during this stage"<-LSJL upregulates Tenascin N or Tnn. This stage is most consistent with LSJL gene expression.
"Increased expression of fibronectin during condensation would result in upregulation of the fibronectin matrix, thus generating additional contact sites between the cells and fibronectin that could positively influence cell proliferation"
"Activation of focal adhesion kinase (FAK) and paxillin occurs during pre-cartilage condensation"
"binding of preadipocyte factor 1 (Pref-1, also known as DLK1, a transmembrane protein that contains EGF-like repeats) to fibronectin [activates ERK]" which upregualtes Sox9.
"reduced sulfation of proteoglycans and downregulation of the cell-surface proteoglycan syndecan-2 substantially reduces fibronectin matrix assembly"<-Syndecan 2 is downregulated by LSJL.
DTDST affects sulfate transport and mutations can decrease height.
"exostosin 1 (EXT1) is involved in heparan sulfate synthesis" Knockout of EXT1 or EXT2 impairs chondrogenic differentiation.
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