TNF-alpha

Conditional Inactivation of TNFα-Converting Enzyme in Chondrocytes Results in an Elongated Growth Plate and Shorter Long Bones.

" we generated a Tace[aka ADAM17, Membrane-bound proteolytic TNF-a converting enzyme] mutant mouse in which Tace is conditionally disrupted in chondrocytes under the control of the Col2a1 promoter. These mutant mice were fertile and viable but all exhibited long bones that were approximately 10% shorter compared to those of wild-type animals. Tace mutant mice exhibited a longer hypertrophic zone in the growth plate, and there were fewer osteoclasts at the chondro-osseous junction in the Tace mutant mice than in their wild-type littermates. we found an increase in osteoprotegerin transcripts and a reduction in Rankl and Mmp-13 transcripts in the TACE-deficient cartilage, indicating that dysregulation of these genes is causally related to the skeletal defects in the Tace mutant mice.  phosphorylation of EGFR was significantly reduced in the cartilage tissue lacking TACE, and that suppression of EGFR signaling increases osteoprotegerin transcripts and reduces Rankl and Mmp-13 transcripts in primary chondrocytes. In accordance, chondrocyte-specific abrogation of Egfr{up in LSJL} in vivo resulted in skeletal defects nearly identical to those observed in the Tace mutant mice. Taken together, these data suggest that TACE-EGFR signaling in chondrocytes is involved in the turnover of the growth plate during postnatal development via the transcriptional regulation of osteoprotegerin, Rankl, and Mmp-13."

So the upregulation of Egfr may play a role in the LSJL lengthening effects.

" the growth plate in adult[8 weeks of age] Tace/Col2 mice appeared normal"

129Sv and C57Bl/6 mice were used.

It's also possible that the reduction of the TNFa converting enzyme increased circulating levels of TNF-alpha.

Finding the right exercises to increase height

Building bone is the key to increasing height. Bone is the limiting factor. Things like spinal fluid and perhaps cartilage in the knee may contribute to height and if those areas are not in check(for example a sublaxed disc) then it may result in a reduction in height but bone is by far the biggest determinant of height. Tendons and ligaments do not heal nearly as fast as bone and if the bone is growing then it is natural to assume that the tendons and ligaments will adapt as well.

In terms of muscle, the longest muscle in the world will look squatty on a short bone.

The height increase sites state that you must first cause microfractures and then you must stretch out these microfractures(mimicing the limb lengthening procedure). But is stretching microfractures really necessary? Certainly causing microfractures is at least a necessary but perhaps not sufficient condition so if you're not causing microfractures any height increase routine will fail. But, what if we define the second part of limb lengthening surgery not as stretching microfractures but as perhaps causing new microfractures or "keeping" microfractures.

If you cause a fracture that creates space for new bone to grow(microfractures are the same as fractures but on a much smaller scale). Fractures fundamentally denature the bone(you don't want to fracture the bone so severly as to say have a chunk of bone chip off) creating space for new bone to grow.

So, all forms of causing microfractures will lead to height increase. The ways of causing microfractures are impact, compression, strain(stretching), and twisting. It may be possible to cause these forces by hand but it is much easier to have a weight do it to you than to try to do it yourself.

Tapping has to be done by hand(can be done with a weight or hammer) and has the advantage that it can be done on short bones(bones that tend to be shaped weird and do not tend to be as load bearing as the long bones such as the head or feet).

You can do jumping but the body will try to absorb most of the impact with the muscle(the more the knees are bent the more impact that is absorbed by the muscle with the exception of the knee being bent to a ridiculous degree in which case the impact begins to be taken by tendons/ligaments).

Impact can be covered by stomping(muscle will always absorb the impact to some degree) and you can use more weight if you stomp.

Strain is your typical hanging from a bar with weights. I find though that holding weights on a decline bench works better than an inversion table.

Compression is putting a bunch of weight on your back or via a leg press(I've found that other positions aren't really designed to take a high amount of load).

Twisting is hard to pull off because a lot of the load goes on the muscle.

Vitamin K

Effects of vitamin K on the morphometric and material properties of bone in the tibiae of growing rats.

"Suboptimal vitamin K nutriture is evident during rapid growth. We aimed to determine whether vitamin K(2) (menaquinone-4 [MK-4]) supplementation is beneficial to bone structure and intrinsic bone tissue properties in growing rats. Male Wistar rats (5 weeks old) were assigned to either a control diet or an MK-4-supplemented diet (22 mg d(-1) kg(-1) body weight). After a 9-week feeding period, we determined the serum concentration ratio of undercarboxylated osteocalcin to γ-carboxylated osteocalcin and the urinary deoxypyridinoline level. All rats were then euthanized, and their tibiae were analyzed. Neither body weight nor tibia length differed significantly between the 2 groups. Dietary MK-4 supplementation decreased the ratio of undercarboxylated osteocalcin to γ-carboxylated osteocalcin but did not affect deoxypyridinoline, indicating a positive effect on bone formation but not bone resorption. Trabecular volume fraction and thickness were increased by MK-4. Neither the cortical pore structure nor mineralization was affected by MK-4. On the other hand, MK-4 increased mineral crystallinity, collagen maturity, and hardness in both the anterior and posterior cortices."

Tibia length was 35.6 for the K supplement group and 35.5 for control.

Effects of vitamin K on the morphometric and material properties of bone in the tibiae of growing rats.

“ Vitamin K is a liposoluble vitamin. The predominant dietary form, phylloquinone or vitamin K1, is found in plants and green vegetables; whereas menaquinone, or vitamin K2, is endogenously synthesized by intestinal bacteria and includes several subtypes that differ in side chain length. Aside from its established role in blood clotting, several studies now support a critical function of vitamin K in improving bone health. Vitamin K is in fact required for osteocalcin carboxylation that in turn regulates bone mineral accretion; it seems to promote the transition of osteoblasts to osteocytes and also limits the process of osteoclastogenesis. Several observational and interventional studies have examined the relationship between vitamin K and bone metabolism, but findings are conflicting and unclear. This systematic review aims to investigate the impact of vitamin K (plasma levels, dietary intake, and oral supplementation) on bone health with a particular interest in bone remodeling, mineral density and fragility fractures.”

Couldn’t get full

Effects of the combination of vitamin K and teriparatide on the bone metabolism in ovariectomized rats

“ The purpose of the present study was to evaluate the combined effects of vitamin K (VK) and teriparatide (TPTD) on bone mineral density (BMD), mechanical strength and other parameters for bone metabolism using a rat ovariectomized osteoporosis model. Ovariectomized female Sprague‑Dawley rats were administered with VK (an oral dose of 30 mg/kg/day), TPTD (a subcutaneous dose of 30 µg/kg, three times a week) or a combination for 8 weeks. Thereafter, serum levels of γ‑carboxylated osteocalcin (Gla‑OC) were quantitated by ELISA; BMD and mechanical strength were measured by computed tomography and biomechanical testing, respectively at the femoral metaphysis. Additionally, histomorphometry was performed using the toluidine blue‑stained coronal sections of distal femur. The combination of VK and TPTD clearly increased the serum levels of Gla‑OC (a specific marker for bone formation) and osteoblast surface (the number of osteoblasts attaching with the surface of cancellous bone), compared to VK or TPTD alone. In addition, the combination of the two agents improved the BMD and bone strength of the femur in the ovariectomized rats, compared to VK or TPTD alone. Taken together, these findings suggest that the treatment with VK and TPTD may have a therapeutic advantage over VK or TPTD monotherapy for postmenopausal osteoporosis, possibly by enhancing the bone formation through the actions on OC and osteoblasts.”

An insight into the role of vitamins other than D on bone

https://scholarworks.utrgv.edu/cgi/viewcontent.cgi?article=1060&context=hbs_fac

“ Vitamins are essential micronutrients for normal development. Great emphasis has been placed on vitamin D for bone development and maintenance. However, other vitamins also influence bone health. While some of them are more beneficial to bone and increase bone mass by increasing bone formation, calcium deposition and stimulate osteoblastogenesis, higher concentrations of others have deleterious effects causing fragile bones and increasing the risk of fractures. Knowledge about the effects of these vitamins will help in better maintenance of bone. This review focuses on the information available on vitamins A, B, C, E and K on bone health. Existing information supports vitamin C and K to play a role in bone formation and calcification. Vitamin E in low amounts and some of the B vitamins may also be beneficial to bone. There is very limited data supporting the favorable effects of vitamin A”

“ RA treatment of growth plate chondrocytes, induced annexins II, V and VI to form Ca2+ channels and influx calcium into the cells. ”

Versican

Versican is upregulated in LSJL.

Versican/PG-M regulates chondrogenesis as an extracellular matrix molecule crucial for mesenchymal condensation.

"Mesenchymal cell condensation is an essential step for cartilage development. Versican/PG-M, a large chondroitin sulfate proteoglycan, is one of the major molecules expressed in the extracellular matrix during condensation. However, its role, especially as an environment for cells being condensed, has not been elucidated. Here we showed several lines of evidence for essential roles of versican/PG-M in chondrogenic condensation using a new chondrocytic cell line, N1511. Chondrogenic stimuli (treatment with parathyroid hormone, dexamethasone, 10% serum) induced a marked increase in the transcription and protein synthesis of versican/PG-M. Stable antisense clones for versican/PG-M, depending on suppression of the expression of versican/PG-M, showed different capacities for chondrogenesis, as indicated by the expression and deposition of aggrecan, a major chondrocytic cell product. The cells in the early stages of the culture only expressed V0 and V1 forms, having more chondroitin sulfate chains among the four variants of versican/PG-M, and treatment of those cells with chondroitinase ABC suppressed subsequent chondrogenesis. Treatment with beta-xyloside, an artificial chain initiator of chondroitin sulfate synthesis to consequently inhibit the synthesis on the core proteins, suppressed chondrogenesis. In addition, forced expression of the variant V3, which has no chondroitin sulfate chain, disrupted the deposition and organization of native versican/PG-M (V0/V1) and other extracellular matrix molecules known to be expressed during the mesenchymal condensation and resulted in the inhibition of subsequent chondrogenesis. Versican/PG-M is involved in positively regulating the formation of the mesenchymal matrix and the onset of chondrocyte differentiation through the attached chondroitin sulfate chains."

"[Versican modifies] cell-extracellular matrix molecule interactions so as to influence the cell shape."

"High level expression of versican/PG-M [occurs] in the cells at high density. The chondrogenic effects of the treatment and cell density on N1511 cells involve the strong expression and deposition of versican/PG-M in the early phase."

B-xylosides inhibited chondroitin sulfate synthesis and in chick embryos decreased growth rate.

" a marked expression and deposition of versican/PG-M in the mesenchymal ECM are required for subsequent chondrocytic gene expression. The V0 and V1 forms of versican/PG-M are important for this activity, and their chondroitin sulfate chains are the crucial molecular portions."

Periosteal/Perichondrial gene expression versus LSJL

Differential gene expression in the perichondrium and cartilage of the neonatal mouse temporomandibular joint.

"Genes with higher expression in the PC sample related to growth factor ligand-receptor interactions [FGF-13 (6.4x), FGF-18 (4x), NCAM (2x); PGDF receptors, transforming growth factor (TGF)-beta and IGF-1], the Notch isoforms (especially Notch 3 and 4) and their ligands or structural proteins/proteoglycans [collagen XIV (21x){Col14a1 up in LSJL}, collagen XVIII (4x), decorin (2.5x)]. Genes with higher expression in the C sample consisted mostly of known cartilage-specific genes [aggrecan (11x){up}, procollagens X (33x){up}, XI (14x){up}, IX (4.5x){up}, Sox 9{up} (4.4x) and Indian hedgehog (6.7x)]. However, the functional or structural roles of several genes that were expressed at higher levels in the PC sample are unclear [myogenic factor (Myf) 9 (9x), tooth-related genes such as tuftelin (2.5x) and dentin sialophosphoprotein (1.6x), VEGF-B (2x) and its receptors (3-4x) and sclerostin (1.7x)]. FGF, Notch and TGF-beta signalling may be important regulators of MCC proliferation and differentiation; the relatively high expression of genes such as Myf6 and VEGF-B and its receptors suggests a degree of unsuspected plasticity in PC cells."

C stands for Cartilage.  PC stands for periochondrium.

Genes upregulated in perichondrium versus cartilage also up in LSJL:
Col4a1
Col18a1
Cdh13
Cdh15{down}
Bgn
Col6a1
MMP2
VCAM1{down}
Kdr

Down:
Osteopontin{up}
Bsp{up}

"chondrogenic stimulation by BMP-2 up-regulates VEGF-B"


Immunohistochemical analysis of Sox9 expression in periosteum of tibia and calvaria after surgical release of the periosteum.

"The purpose of the present study was to analyze histologically the bone formation in surgically released and repositioned periosteum, and to determine expression of Sox9 and type-2 collagen in periosteal bone formation of tibia and calvaria. After surgery, the released tibial periosteum formed ectopic cartilage. At 7 days, a combination of endochondral and intramembranous ossification was apparent. Some fibroblasts derived from the released periosteum showed Sox9 expression. Chondrocytes and cartilage matrix both displayed type-2 collagen expression. At 7 days, an additional new bone was formed on the calvaria. Osteoblasts and fibroblasts derived from released calvarial periosteum did not express Sox9 or type-2 collagen. Sox9 was not expressed throughout the process periosteal bone formation on the calvaria. Sox9 and type-2 collagen expression in periosteal cells after periosteum release and that the generative potential of periosteal cells of calvaria is different from that of tibia."

The surgery: "An incision was made on the periosteum and the flap was gently released. The periosteum and skin flaps were repositioned on the bone surface and sutured"  The periosteal flap was turned over.

Tibial periosteum:

"At 7 days, periosteum had undergone changes at the release site, displaying loose connective tissue. New bone formation by endochondral ossification was observed in this inflammatory tissue. These periosteal cells of the fibrous layer were markedly more abundant and had differentiated into chondroblasts and chondrocytes. Fibroblasts derived from periosteal cells displayed differentiation and proliferation. These chondrocytes and chondroblasts had formed a cartilage mass. At 14 days after surgery, periosteal bone formation appeared as a combination of intramembraneous and endochondral ossification. Chondrogenic cells derived from the periosteum had formed a cartilage mass. Hypertrophic and mature chondrocytes formed new external callus cartilage, which in turn was replaced by newly formed trabeculae of cancellous bone undergoing endochondral ossification. At 28 days after surgery, all newly formed cartilage had been changed into trabecular bone"

"At 3 days after surgery, periosteal cells proliferated and changed into fibroblasts. These fibroblasts showed Sox9 expression but no type-2 collagen expression. At 7 days after surgery, a small number of fibroblasts from the released periosteum were immunostained for Sox9. Sox9 immunostaining was observed in fibroblasts surrounding ectopic cartilage and differentiated chondrocytes. Chondrogenic cells from the released periosteum were positive for type-2 collagen. At 14 days after surgery, Sox9 immunostaining was observed in fibroblasts and chondrocytes, but not in hypertrophic chondrocytes. Type-2 collagen was expressed around chondrocytes and hypertrophic chondrocytes"

Calvarial[skull cap] periosteum:

"At 3 days, inflammatory tissue and rich vascularization were found. Periosteal cells were attached to the bone surface at the release site and were connected to adjacent cells. At 7 days after surgery, periosteum contained loose connective tissue. Additional bone formation started on the calvarial bone surface. Many osteoblasts were localized on the bone surface and induced bone matrix formation. Fibroblasts derived from the periosteum were concentrated on the bone surface and had differentiate into osteoblasts. At 14 days after surgery, bone formation was continuing by intramembraneous ossification. New trabecular bone contained many osteocytes. Osteoblasts on calvarial bone produced extracellular bone matrix and induced calcification of the bone. At 28 days after surgery, additional bone formation was continuously apparent. New trabecular bone consisted of osteocytes, osteoblasts and extracellular bone matrix "

"Expression of Sox9 or type-2 collagen was not detected in cells derived from the periosteum throughout the experiment"

"Microscopical (A, B) and electron microscopical (C, D) observations of tibial (A, C) and calvarial (B, D) periosteum. Periosteum (P) is connective tissue covering the bone surface (Bone). It comprises of a fibrous layer (FL) and an osteogenic layer (OL). The comparatively thick outer fibrous layer is made up of dense connective tissue. The less well-defined inner osteogenic layer contains osteogenic cells. Scale bars A, B: 20mm; C, D: 5mm."

"Photomicrograph of released periosteum after toluidine blue staining. (A) Released tibial periosteum (RP) consisting of the fibrous layer and part of the osteogenic layer. The fibrous layer is released from the bone (Bone), and the osteogenic layer is damaged by the surgical procedure. Few osteogenic cells are present in the inner layer. Osteogenic cells are not present on the surface of the cortex bone. (B) Released calvarial periosteum (RP) consisting the fibrous layer containing a few fibroblasts and collagen fibers. Released calvarial periosteum is thinner than tibial periosteum. Osteogenic cells are not present on the surface of cortical bone (Bone)."

Identification of unique molecular subdomains in the perichondrium and periosteum and their role in regulating gene expression in the underlying chondrocytes.

"Both the perichondrium and the periosteum are reported to have two morphologically distinct layers. While the inner layer is proposed to contribute to appositional growth of cartilage and bone the outer fibroblastic layer is thought to perform a structural role by offering attachment sites for tendons and ligaments "

"Fgf18[produced by the perichondrium and regulated by Runx2 and TGFb1 is required both for chondrocyte proliferation and for osteogenesis, acting through two distinct receptors"


12 day chick tibia showing the distinction between the periosteum and perichondrium.

"localized to this inner cambial layer of the perichondrium, including Tsp2[upregulated in perichondrium downregulated in periosteum], MafB[perichondrium specific gene]{down}, Dkk3{up}, THBS2{UP}, and galectin-1[perichondrium specific gene]. We also observed that cellular retinoic acid binding protein-I (CRABP-I){up as Crabp1} and Undulin (COL14A1){up} are expressed in the outer layer of the perichondrium"

Genes specific to periosteum(inner layer): "B-Tubulin{LSJL upregulates Tubulin Beta 6}, Neuronal protein 3.1 (P311), Y-box binding protein 1 (YBX-1), Type V collagen(Col5a1), Retinoic acid induced 14 (RAI14), alpha2 chain of type I collagen and Decorin "


EFEMP1 a gene associated with stature was reported to be sporadically detected in the periosteum and perichondrium.  matn4 is upregulated by LSJL.

MAML1

MAML1 Enhances the Transcriptional Activity of Runx2 and Plays a Role in Bone Development.

"Mastermind-like 1 (MAML1) is a transcriptional co-activator in the Notch signaling pathway. MAML1 enhances the transcriptional activity of runt-related transcription factor 2 (Runx2), a transcription factor essential for osteoblastic differentiation and chondrocyte proliferation and maturation. MAML1 significantly enhanced the Runx2-mediated transcription of the p6OSE2-Luc reporter, in which luciferase expression was controlled by six copies of the osteoblast specific element 2 (OSE2) from the Runx2-regulated osteocalcin gene promoter. A deletion mutant of MAML1 lacking the N-terminal Notch-binding domain also enhanced Runx2-mediated transcription. Inhibition of Notch signaling did not affect the action of MAML1 on Runx2, [thus] activation of Runx2 by MAML1 may be caused in a Notch-independent manner. Overexpression of MAML1 transiently enhanced the Runx2-mediated expression of alkaline phosphatase, an early marker of osteoblast differentiation, in the murine pluripotent mesenchymal cell line C3H10T1/2. MAML1(-/-) embryos at embryonic day 16.5 (E16.5) had shorter bone lengths than wild-type embryos. The area of primary spongiosa of the femoral diaphysis was narrowed. At E14.5, extended zone of collagen type II alpha 1 (Col2a1) and Sox9 expression, markers of chondrocyte differentiation, and decreased zone of collagen type X alpha 1 (Col10a1) expression, a marker of hypertrophic chondrocyte, were observed{but we can't know if this results in an increase or decrease of adult height}. Chondrocyte maturation was impaired in MAML1(-/-) mice. MAML1 enhances the transcriptional activity of Runx2 and plays a role in bone development."

MAML2 also enhances RUNX2 transcriptional activity.

" the expression of Sox9, a transcription activator of collagen type II, was upregulated by Notch activation and this activation of Notch signaling thereby promoted differentiation of proliferative and prehypertrophic chondrocytes"

Wip1

The modulation of the oxidative stress response in chondrocytes by Wip1 and its effect on senescence and dedifferentiation during in vitro expansion.

"Rapid senescence or dedifferentiation during in vitro expansion results in loss of chondrocyte phenotype and the formation of fibrous cartilage replacement tissue, rather than hyaluronic cartilage, after transplantation. Wild-type p53-inducible phosphatase (Wip1), a well-established stress modulator, was highly expressed in early-passage chondrocytes, but declined rapidly during in vitro expansion. Stable Wip1-expressing chondrocytes generated by microporation were less susceptible to the onset of senescence and dedifferentiation, and were more resistant to oxidative stress. The increased resistance of Wip1 chondrocytes to oxidative stress was due to modulation of p38 mitogen-activated protein kinase (MAPK) activity. Importantly, chondrocytes expressing Wip1 maintained their innate chondrogenic properties for a longer period of time, resulting in improvements in cartilage regeneration after transplantation. Chondrocytes from Wip1 knockout (Wip1(-/-)) mice were defective in cartilage regeneration compared with those from wild-type mice."

Could upregulating Wip1 in GP chondrocytes be a way to grow taller?

"Antioxidants such as N-acetyl cysteine (NAC) and ascorbic acid delay chondrocyte senescence and degeneration by attenuating ROS insult"

"Low oxygen tension protects cells from the onset of senescence. Oxidative stress in chondrocytes induces telomere genomic instability, expression of matrix metalloproteinases (MMPs), and downregulation of Sirt1, a mammalian Sir2 ortholog, through p38 MAPK activation"

"Wip1 is encoded by PPM1D (for protein phosphatase 1D Mg2+-dependent, delta isoform) and is a type 2C protein phosphatase (PP2C). A number of stress mediators such as p53, p38, Chk1/2, ATM, and γ-H2AX are dephosphorylated and inactivated by Wip1. "

"late-passage chondrocytes, defined as passage five chondrocytes (P5-Chon), showed morphological changes typical of cells undergoing senescence, including an enlarged nucleus and a flat, spread-out morphology"

"The expression of collagen II (Col II) and aggrecan (AGG) was markedly suppressed in P5-Chon compared with that in control cells, while the expression of collagen X (Col X) was highly increased"

"Levels of mitochondrial ROS were higher in P5-Chon. Telomere length in P5-Chon was unaltered"

"To mimic this stressful environment, chondrocytes were exposed to hydrogen peroxide (H2O2) to induce acute oxidative stress in the presence or absence of β-mercaptoethanol (BME), a strong reducing agent that is used to assess the specific effects of H2O2-induced oxidative injury. Exposure to H2O2 markedly increased SA-β-gal activity in chondrocytes, and this effect was highly attenuated by supplementation with BME"

" In both early- and late-passage chondrocytes (P2- and P8-Chon), supplementation with BME significantly reduced the number of SA-β-gal positive cells. Furthermore, Col X expression decreased whereas Col II expression increased"

"The area of cartilage tissue in Wip1−/− mice was approximately 1.68-fold lower (0.16 ± 0.0034 mm2) than that in wild-type mice (0.27 ± 0.0032 mm2). The cartilage tissue from Wip1−/− mice was also much thinner than that from wild-type mice (86.75 ± 9.3913 μm vs 119.49 ± 3.8486 μm)."

The Restrictions on Bone Growth after Puberty are mechanical not chemical

The growth plate phenomenon which purports that growth is no longer possible after puberty only restricts chemical(HGH) not mechanical(limb lengthening surgery) growth. You can send an infinite number of signals to grow bone but if the mechanical tools are not there then no growth will be possible.

The mechanical tools to influence bone are:

1) Impact(tapping something against the bone or jumping)
2) Strain(pulling apart the bone)
3) Compression
4) Torsion, Spiral, twisting, etc.

We have to perform these forces strong enough to create the mechanical space to build the bone(via microfractures) but not strong enough so as to denature the bone(say a multiple compound fracture).

Endochondral ossification merely means that the epiphyseal cartilage cells stop duplicating and where growth once occurred is now what is referred to as a growth plate. This growth plate in no way inhibits growth, it merely means that natural growth is done.

The same process of endochondral ossification which is used to grow the long bones is the same that is used to heal fractures.  Microfractures are smaller fractures but that don't create quite the same microenvironment for cartilagenous growth as larger fractures.

The key is merely finding the best exercises to cause microfractures.  And make sure that other stimuli is added to produce a similar microenvironment as larger fractures or that the fracture is large enough to produce a similar microenvironment.

Mechanical Regulation of Skeletal Development.

"Spatially organized differentiation is vital to the production of functionally appropriate tissues contributing to precise, region specific morphologies, for example transient chondrogenesis of long bone skeletal rudiments, which prefigures osteogenic replacement of the cartilage template, compared with the production of permanent cartilage at the sites of articulation."

"mechanical forces generated by embryonic muscle contractions are required for normal skeletogenesis."

"marking of cells that express Matrilin 1 (within the rudiments) shows that they do not contribute to articular cartilage"

"mature articular cartilage cells can be induced to undergo chondrogenic maturation and hypertrophy by treatment with 5-azacytidine"

"Hydrostatic Pressure (HP) has been largely associated with chondrogenic differentiation whereas tensile strain and fluid induced shear stress are generally shown to induce osteogenesis"

STAT1

Identification of STAT-1 as a molecular target of IGFBP-3 in the process of chondrogenesis.

"Using the RCJ3.1C5.18 chondrogenic cells, which in culture progresses from undifferentiated to terminally differentiated chondrocytes, we have shown that IGFBP-3 has an IGF-independent, antiproliferative effect in undifferentiated and early differentiated but not in terminally differentiated chondrocytes. In the present study, cDNA microarray analysis was used to screen for genes: 1) that were regulated by IGFBP-3 in early but not in terminally differentiated chondrocytes; 2) that were regulated specifically by IGFBP-3, but not by IGF-I; and 3) whose regulation was abolished by coincubation of IGFBP-3 with IGF-I. Signal transducer and activator of transcription (STAT)-1 was the gene that, fulfilling the screening criteria, exhibited the greatest up-regulation by IGFBP-3 (>40-fold). STAT-1 gene up-regulation was confirmed by Northern analysis of cells treated with IGFBP-3 or transfected with an IGFBP-3 expression vector. Remarkably, similar results were obtained when cells were transfected with an IGFBP-3 mutant unable to bind IGFs, definitively demonstrating the IGF-independent action of IGFBP-3. Consistent with the up-regulation of STAT-1 mRNA, IGFBP-3 also increased STAT-1 protein expression. Furthermore, both IGFBP-3 and the IGFBP-3 mutant induced STAT-1 phosphorylation and its nuclear localization. An antisense STAT-1 oligonucleotide abolished the IGF-independent cell apoptosis induced by IGFBP-3. STAT-1 is a major intracellular signaling and transcriptional target of the IGF-independent apoptotic effect of IGFBP-3 in chondrogenesis."

"IGFBP-3 binds to the extracellular matrix and to cell membrane receptors and is transported in the nucleus by the importin β subunit, where it binds to the retinoid X receptor-α "

"IGFBP-3 induced apoptosis in chondroprogenitors."

"In chondrocytes, STAT-1 has been implicated as a key signaling molecule that mediates the antiproliferative and apoptotic activity of FGFR-3"

Folate

Solgar - Folate (Metafolin)800 Mcg, 100 tablets

Folate modulates Hox gene-controlled skeletal phenotypes.

"Essential nutrient folate modulates genetically induced skeletal defects in Hoxd4 transgenic mice. Chondrocytes require folate for growth and differentiation and that they express folate transport genes, providing evidence for a direct effect of folate on skeletal cells."

"overexpression of Hoxc8 in transgenic mice impairs cartilage formation through a delay in chondrocyte maturation. When a Hoxd4 transgene is expressed in exactly the same fashion as Hoxc8, the Hoxd4 transgenic animals also exhibit profound cartilage defects: the cartilaginous portions of the ribs lack tensile strength, and the vertebral column is very flexible and unstable due to reduced or absent intervertebral cartilages"

"skeletons prepared from folate-supplemented Hoxd4 transgenic embryos exhibit restored Alcian Blue staining in rib and vertebral cartilage."

"Hoxd4 transgenic chondrocytes have a specific requirement for folate early in chondrogenesis."

"the effective time window for nutritional supplementation coincides with the early phase of cartilage formation."

"Utilization of folate requires uptake into cells, mediated by specific folate transport proteins, the GPI-anchored Folate receptors (Folate binding proteins in mouse) and the integral membrane protein Reduced folate carrier 1 (Rfc1)."

LSJL upregulates Folr2 the Folate receptor but downregulates Skiv2l2 and Ercc3 which are involved in Folate biosynthesis.

"cells plated at low density—which leads to dedifferentiation into fibroblastoid cells—do not exhibit Folbp gene expression [in contrast to differentiation chondrocytes]"

"Chondrocytes [do] not proliferate in the absence of folate"

"Folate supplementation was done by gavage administration of folinic acid, the biologically active and more stable form of folate"