Chondrocyte-specific transcription factors

This post will be confusing to most and normally I would bury it in the blog for reference purposes but I'm hoping that a transcription factor expert could look these over and find any evidence that LSJL induces ectopic chondroinduction.

Chondrogenic transcription factors up in LSJL:

Sox9

GATA4

TRPS1{downstream of GDF5}

Barx2{downstream of GDF5}
Gli3
Prrx1
Prrx2
Pax1
Lhx8

It's predicted that GDF5 is involved in LSJL.

Other transcription factors involved in LSJL.

Up:
Fos
ATF3
Meox2
Hey2
Hoxd10
WWTR1
Dbx2
Hoxb1
Jun
Phox2b

Down(bold are related to chondrogenesis):
E2F6
Smad1
Gtf2i
Npas4
Dbx1
Cdx1
Foxb1
HNF4A
Hoxb13
Tcf7{According to Regulation of Tcf7 by Runx2 in chondrocyte maturation and proliferation. knockout of Tcf7 causes dwarfism}
Tcf12
Mxd4{According to Novel late response genes of PTHrP in chondrocytes., Mxd4 is downregulated by PTHrP}
Btf3
Mafb
Nrf1{Mentioned at a growth plate meeting: Meeting Report:The 7th ESPE Growth Plate Working Group Symposium - EUROGROP June 27th 2007, Helsinki, Finland. but I cannot get the details}
Tcfec
Mxd3
Tfdp2
E2F1

Placenta to cartilage: direct conversion of human placenta to chondrocytes with transformation by defined factors.

"mouse and human fibroblasts can be reprogrammed to a pluripotent[iPSCs] state with a combination of four transcription factors. We hypothesized that combinatorial expression of chondrocyte-specific transcription factors could directly convert human placental cells into chondrocytes. Starting from a pool of candidate genes, we identified a combination of only five genes (5F pool)-BCL6, T (also called BRACHYURY), c-MYC, MITF, and BAF60C (also called SMARCD3)-that rapidly and efficiently convert postnatal human chorion and decidual cells into chondrocytes. The cells generated expressed multiple cartilage-specific genes, such as Collagen type II α1, LINK PROTEIN-1, and AGGRECAN, and exhibited characteristics of cartilage both in vivo and in vitro. Expression of the endogenous genes for T and MITF was initiated, implying that the cell conversion is due to not only the forced expression of the transgenes, but also to cellular reprogramming by the transgenes."

MyoD is the gene responsible for transdifferentiation into different cell types.

"Murine chondrocytes can be converted from fetal fibroblasts by the direct reprogramming method using the cartilage-specific transcription factors Sox9, c-Myc, and Klf4"

"expression of placenta-associated genes such as GATA3, CD200{CD200r3 is downregulated by LSJL}, PDCD1LG2, OLR1, TEK, HSD17B2, and FOXF1 was lost in the infected cells{chondrogenic differentiation induced cells}."

"T{BRACHYURY} is necessary for chondrogenic conversion"

"c-Myc is a cell cycle driver, and Klf4 is involved in the down-regulation of p53"

"T and MITF may act as inducers of chondrogenic fate determinant, and BAF60C may act as epigenetic modifier"

BAF60C may permit binding of GATA4{up in LSJL} to chondrocytes.

Identification of a 3Kbp Mechanoresponsive Promoter Region in the Human Cartilage Oligomeric Matrix Protein Gene.

"the proximal 3 Kb of the human cartilage oligomeric matrix protein promoter is sufficient to mediate a mechanoresponse in human articular chondrocytes and stem cells, and that the magnitude of mechanoresponse correlates to the regulation of the endogenous gene at the RNA and protein level."

"the growth plate aligns itself with the axis of compressive mechanical force."

"there was no difference in the viability of chondrocytes or BMSC subject to 5 days of dynamic compression and their respective uncompressed controls "

FoxA family members are crucial regulators of the hypertrophic chondrocyte differentiation program.

"FoxA factors are induced during chondrogenesis, bind to conserved binding sites in the collagen X enhancer, and can promote the expression of a collagen X-luciferase reporter in both chondrocytes and fibroblasts. In addition, we demonstrate by both gain- and loss-of-function analyses that FoxA factors play a crucial role in driving the expression of both endogenous collagen X and other hypertrophic chondrocyte-specific genes. Mice engineered to lack expression of both FoxA2 and FoxA3 in their chondrocytes display defects in chondrocyte hypertrophy, alkaline phosphatase expression, and mineralization in their sternebrae and, in addition, exhibit postnatal dwarfism that is coupled to significantly decreased expression of both collagen X and MMP13 in their growth plates."

"Runx2 and Runx3 are expressed in chondrocytes as they initiate differentiation, and loss of these factors (in genetically engineered mice) severely delays or blocks chondrocyte hypertrophy in a number of developing bones"

"ectopic expression of Runx2 in immature chondrocytes drives premature cellular maturation and induces expression of collagen X and other hypertrophic markers"

MEF2C and MEF2D{down in LSJL} promote Runx2 expression.

"FoxA2{activates Col10 which was up in LSJL} and FoxA3 are expressed in developing cartilages"

" FoxA factors, which are present in chondrocytes yet absent from fibroblasts, may confer competence for Runx2, Smad1{down}, and MEF2C to activate expression of the collagen X luciferase reporter in the former cell type."

"FoxA3−/− mice were smaller, with a specific deficiency of cells in the hypertrophic zone but not in the proliferating zone "

"both FoxA2 and FoxA3 are necessary to promote high-level expression of several hypertrophic chondrocyte markers in the growth plate, including collagen X, alkaline phosphatase, and MMP13."

"Sox transcription factors, which are expressed in immature chondrocytes and downregulated in hypertrophic chondrocytes, may act to compete for FoxA factor binding to the collagen X enhancer and thereby inhibit precocious expression of collagen X and/or other hypertrophic markers in immature chondrocytes. Indeed, others have noted that mutation of a Sox9 binding site within the murine Col10a1 enhancer is required to repress expression of a transgene driven by this enhancer in nonhypertrophic chondrocytes "

Control of mesenchymal lineage progression by microRNAs targeting skeletal gene regulators Trps1 and Runx2.

"seven RUNX2-targeting miRNAs (miR-23a, miR-30c, miR-34c, miR-133a, miR-135a, miR-205, and miR-217) also regulate the chondrogenic GATA transcription factor tricho-rhino-phalangeal syndrome I (TRPS1){up}. Although the efficacy of each miRNA to target RUNX2 or TRPS1 differs in osteoblasts and chondrocytes, each effectively blocks maturation of precommitted osteoblasts and chondrocytes. Furthermore, these miRNAs can redirect mesenchymal stem cells into adipogenic cell fate with concomitant up-regulation of key lineage-specific transcription factors."

TRPS1 is a Zn finger transcription factor that promotes chondrogenesis.

"five RUNX2-targeting miRNAs potentially target the Kruppel-like transcription factor KLF12, a principal transcriptional repressor of the activator protein-2 α (AP-2 α) gene, which controls vertebrate development"

"miR-30c dramatically up-regulates expression of early-stage ECM genes when administered prior to differentiation of MC3T3 osteoblasts, including three collagen genes (Col1A1, Col1A2, and Col3A1) and matrix metalloproteinases 2 and 9 (Mmp2 and Mmp9). In addition, late-stage ECM genes, including OP, osteocalcin (OC-BGLAP), and Dmp1 expression, are suppressed by miR-30c"

"miR-30c modulates expression of genes encoding ligands and receptors of TGFβ, FGF, and IGF signaling and that this modulation is strikingly different during differentiation in ATDC5 chondrocytes and MC3T3 osteoblasts"

"TGFβs, TGFβRs, and SMADs are clearly down-regulated by miR-30c in chondrocytes but up-regulated in osteoblasts. Also, expression of genes for Fgf2, Fgfr1, Fgfr2, and Igf1r is clearly stimulated in MC3T3 cells, whereas expression of Fgf1, Fgf3, Fgfr1, and Igf1 genes is suppressed in ATDC5 cells."<-Despite the presence of TRPS1, FGF2 and FGFR1 were upregulated.

Trps1 plays a pivotal role downstream of Gdf5 signaling in promoting chondrogenesis and apoptosis of ATDC5 cells.

"Tricho-rhino-phalangeal syndrome (TRPS) is an autosomal dominant skeletal disorder caused by mutations of TRPS1. Based on the similar expression patterns of Trps1 and Gdf5, we hypothesized a possible functional interaction between these two molecules. Using a chondrogenic cell line (ATDC5), we investigated the association of Gdf5-mediated signaling pathways with Trps1 and the phenotypic changes of ATDC5 cells due to over-expression or suppression of Trps1. Treatment of cells with Gdf5 enhanced Trps1 protein levels and phosphorylation of p38 mitogen-activated protein kinase (MAPK) in a dose-dependent manner. Nuclear translocation of Trps1 was also induced by Gdf5. These effects were blocked by a dominant negative form of activin-linked kinase 6 (dn-Alk6) and by SB203580, an inhibitor of the p38 MAPK pathway. Conversely, Gdf5 expression was suppressed by the over-expression of Trps1. Trps1-overexpressing ATDC5 (O/E) cells differentiated into chondrocytes more quickly than mock-infected control cells, whereas cells transfected with dn-Alk6 showed slower differentiation. On the other hand, O/E cells showed an increase of apoptosis along with the up-regulation of cleaved caspase 3 and down-regulation of Bcl-2, whereas dn-Alk6 cells showed suppression of apoptosis. In conclusion, Trps1 acts downstream of the Gdf5 signaling pathway and promotes the differentiation and apoptosis of ATDC5 cells."

"Trps1 is a transcriptional repressor, which interacts with the dynein light chain and RING finger protein 4 (Rnf4) in the nucleus"

"Trps1 is mainly expressed in the joints and in the limb growth plate cartilages during late embryogenesis. Mice with Trps1 deficiency die of respiratory failure soon after birth and show changes such as skeletal deformities that resemble those seen in human TRPS patients"

"Expression of Trps1 by ATDC5 cells was not observed in the absence of insulin, while it gradually increased in the presence of insulin "

"Trps1 mRNA levels were not significantly enhanced by Gdf5 compared with Trps1 protein levels"

"expression of Bcl-2 mRNA was significantly decreased by the over-expression of Trps1, whereas transfection with dn-Alk6 resulted in elevated Bcl-2 expression"

"enhancement of apoptosis in Trps1-overexpressing cells"

"Trps1 might negatively regulate a transcriptional repressor of the type II and type X collagen genes. It has been reported that the other transcription factor downstream of Gdf5, Barx2{up in LSJL}, binds to the intronic regulatory region of the Col2a1 gene during chondrogenesis. We found that Trps1 binds to two GATA binding sites in the promoter of the Stat3 gene and represses the expression of Stat3"

Trps1, a regulator of chondrocyte proliferation and differentiation, interacts with the activator form of Gli3.

"Trps1 interacts with Indian hedgehog (Ihh)/Gli3 signaling and regulates chondrocyte differentiation and proliferation. We demonstrate that Trps1 specifically binds to the transactivation domain of Gli3{up in LSJL} in vitro and in vivo, whereas the repressor form of Gli3 does not interact with Trps1. A domain of 185aa within Trps1, containing three predicted zinc fingers, is sufficient for interaction with Gli3. Using different mouse models we find that in distal chondrocytes Trps1 and the repressor activity of Gli3 are required to expand distal cells and locate the expression domain of Parathyroid hormone related peptide. In columnar proliferating chondrocytes Trps1 and Ihh/Gli3 have an activating function. The differentiation of columnar and hypertrophic chondrocytes is supported by Trps1 independent of Gli3. Trps1 seems thus to organize chondrocyte differentiation interacting with different subsets of co-factors in distinct cell types."

"In the presence of Hh signals, Gli2 and Gli3 are thought to enter the nucleus to activate target genes, whereas in absence of Hh signals, Gli3 and, to a lesser extend, Gli2 are proteolytically processed into a short, N-terminal repressor form that inhibits the expression of Hh target genes"<-LSJL increases Dhh signaling.

"Measurements of Trps1−/− ulnae revealed a reduction to 80% in length and 82% in diameter"  Gli3 knockout decreases height as well.

"The zone of proliferating chondrocytes can further be subdivided into distal chondrocytes, which express Fibroblast growth factor receptor 1 (Fgfr1){up} and Unique cartilage matrix-associated protein (Ucma), and columnar chondrocytes, which are characterized by the expression of Bone Morphogenetic Protein 7 (Bmp7), Fgfr3 and the lack of Ihh expression"

"in absence of Ihh signals Gli3 acts as a strong repressor of proliferation"

Trps1 regulates proliferation and apoptosis of chondrocytes through Stat3 signaling.

"disrupted Trps1 gene develop a chondrodysplasia characterized by diminished chondrocyte proliferation and decreased apoptosis in growth plates. Our analyses revealed that Trps1 is a repressor of Stat3 expression, which in turn controls chondrocyte proliferation and survival by regulating the expression of cyclin D1 and Bcl2. Our conclusion is supported (i) by siRNA-mediated depletion of Stat3 in Trps1-deficient chondrocytes, which normalized the expression of cyclin D1 and Bcl2, (ii) by overexpression of Trps1 in ATDC5 chondrocytes, which diminished Stat3 levels and increased proliferation and apoptosis, and (iii) by mutational analysis of the GATA-binding sites in the Stat3 gene, which revealed that their integrity is critical for the direct association with Trps1 and for Trps1-mediated repression of Stat3."

"a strong reduction in cyclin D1 expression in freshly isolated Trps1−/− chondrocytes"

The homeobox transcription factor Barx2 regulates chondrogenesis during limb development.

"Barx2 is necessary for mesenchymal aggregation and chondrogenic differentiation. In accordance with these findings, Barx2 regulates the expression of several genes encoding cell-adhesion molecules and extracellular matrix proteins, including NCAM and collagen II (Col2a1) in the limb bud. Barx2 bound to elements within the cartilage-specific Col2a1 enhancer, and this binding was reduced by addition of Barx2 or Sox9 antibodies, or by mutation of a HMG box adjacent to the Barx2-binding element, suggesting cooperation between Barx2 and Sox proteins. Moreover, both Barx2 and Sox9 occupy Col2a1 enhancer during chondrogenesis in vivo. We also found that two members of the BMP family that are crucial for chondrogenesis, GDF5 and BMP4, regulate the pattern of Barx2 expression in developing limbs."

"Dlx2 acts downstream of BMP signal"

"Barx2 is involved in regulation of Ca+2-independent and Ca+2-dependant adhesion."

"Barx2 constructs induced chondrogenesis in micromass cultures prepared from the proximal region of the limb bud, whereas micromass cultures made from the distal mesenchyme of the same limb bud showed a fivefold increase in chondrogenesis after overexpression of Barx2. These results suggest that Barx2 can promote chondrogenesis only in the distal mesenchyme of the limb bud, which contains mostly uncommitted cells "

"Barx2 and GDF5 are co-expressed during limb development"

"application of GDF5 or BMP4-soaked beads induced ectopic expression of Barx2 all around the bead"

"Barx2 is also necessary for chondrogenesis and interacts functionally with both BMPs and Sox9."

" GDF5 can induce chondrogenesis in mesenchymal cells that have not yet condensed, while BMPs induce chondrogenic differentiation only after condensation"

Hdac-mediated control of endochondral and intramembranous ossification.

"Several Hdacs contribute to the molecular pathways and chromatin changes that regulate tissue-specific gene expression during chondrocyte and osteoblast specification, maturation, and terminal differentiation. In this review, we summarize the roles of class I and class II Hdacs in chondrocytes and osteoblasts. The effects of small molecule Hdac inhibitors on the skeleton are also discussed."

"Hdac1 associates with Nkx3.2, an essential transcriptional repressor governing the formation of the cartilage anlagen during endochondral ossification. Furthermore, Hdac1 represses Smad-dependent signaling to control responses to Bmp2, a critical chondrogenic factor.29 Hdac4 and Hdac5 also regulate Smad signaling downstream of Tgf-β and thus may participate in controlling the process of chondrogenesis."

"Hdac4 binds to Runx2 and represses Runx2-induced hypertrophy"

Directed differentiation of human embryonic stem cells toward chondrocytes.

GATA4, Sox9, and KDR are up in LSJL.

The role of NELL-1, a growth factor associated with craniosynostosis, in promoting bone regeneration.

"NELL-1, which is a potent growth factor that is highly specific to the osteochondral lineage, and has demonstrated robust induction of bone in multiple in vivo models from rodents to pre-clinical large animals. NELL-1 is preferentially expressed in osteoblasts under direct transcriptional control of Runx2, and is well-regulated during skeletal development. NELL-1/Nell-1 can promote orthotopic bone regeneration via either intramembranous or endochondral ossification, both within and outside of the craniofacial complex. Unlike BMP-2, Nell-1 cannot initiate ectopic bone formation in muscle, but can induce bone marrow stromal cells (BMSCs) to form bone in a mouse muscle pouch model, exhibiting specificity that BMPs lack. In addition, synergistic osteogenic effects of Nell-1 and BMP combotherapy have been observed, and are likely due to distinct differences in their signaling pathways. NELL-1's unique role as a novel osteoinductive growth factor makes it an attractive alternative with promise for future clinical applications. [Note: NELL-1 and NELL-1 indicate the human gene and protein, respectively; Nell-1 and Nell-1 indicate the mouse gene and protein, respectively.]"

" A 100 ng/mL quantity of NELL-1 protein can significantly activate the ERK1/2 and JNK1/2/3 kinases in primary rat fetal calvarial (RFC) cells after only 10 min of stimulation"

Relaxin to augment height?

Relaxin may play a role in the observed height increase and foot size increase in some pregnances.  Relaxin may be used as a novel way of weakening the type I collagen in the bone enabling the bone to be less resistant to stretch.

Pelvic development as affected by relaxin in three genetically selected frame sizes of beef heifers.

"Purified porcine relaxin was administered into the cervical os on Day 278 of gestation to determine its effects on pelvic development in three genetically selected frame sizes of primiparous beef heifers. Heifers were categorized as small, medium and large frame based upon their genetic composition. Pelvic height, pelvic width and cervical dilatation were determined from Day 270 to 2 days postpartum. On Day 270, heifers were assigned at random to one of three treatments: vehicle control, n = 16; relaxin once (3,000 U), n = 14; and relaxin twice (2 times 3,000 U 12 h apart), n = 17. Each heifer-frame size was represented in each treatment. Relaxin caused marked increases in pelvic height and width, as well as in the rate of linear increase (cm/day) of these parameters (p less than 0.05). These linear increases in pelvic height were 510, 264 and 204%, and pelvic width, were 280, 213 and 204% of the respective pretreatment rates for small, medium and large heifers. The rate of linear increase in pelvic width was greater than pelvic height in all heifers, but maximal in small-frame heifers; relaxin attenuated these intrinsic differences. For small heifers, the rate of linear increase in pelvic width was 121 and 145% of increases for medium and large heifers, respectively, before treatment, and 160 and 200% after treatment. The rate of postpartum involution of pelvic width was -0.03, -0.36 and -0.50 cm/day and, for pelvic height, -0.02, -0.27 and -0.29 cm/day in small, medium and large heifers, respectively."

"pelvic area in beef heifers increases normally from 200 to 250 cm^2."

"During gestation, the pelvis grows linearly at about 0.5 cm^2 /day."

"All heifers were bred by artificial insemination at estrus (Day 0); pregnancy lasts approximately 283 days. Heifers calved at an average age of 25 ± 1 mo (± SE)."

Relaxin was injected directly into the cervix.

Relaxin increases human endothelial progenitor cell NO and migration and vasculogenesis in mice.

"The ovarian peptide hormone, relaxin, circulates during pregnancy, contributing to profound maternal vasodilation through endothelial and nitric oxide (NO)-dependent mechanisms. Circulating numbers of bone marrow-derived endothelial cells (BMDECs), which facilitate angiogenesis and contribute to repair of vascular endothelium, increase during pregnancy. Relaxin enhances BMDEC NO production, circulating numbers, and function. Recombinant human relaxin-2 (rhRLX) stimulated PI3K/Akt B-dependent NO production in human BMDECs within minutes, and activated BMDEC migration that was inhibited by L-N(G)-nitroarginine methyl ester. In BMDECs isolated from relaxin/insulin-like family peptide receptor 2 gene (Rxfp2) knockout and wild-type mice, but not Rxfp1 knockout mice, rhRLX rapidly increased NO production. Similarly, rhRLX increased circulating BMDEC number in Rxfp2 knockout and wild-type mice, but not Rxfp1 knockout mice as assessed by colony formation and flow cytometry. Relaxin effects BMDEC function through the RXFP1 receptor. Both vascularization and incorporation of GFP-labeled BMDECs were stimulated in rhRLX-impregnated Matrigel pellets implanted in mice. Relaxin is a regulator of BMDECs number and function, which has implications for angiogenesis and vascular remodeling."

"cause morphologic changes in endothelial cells of endometrial blood vessels consistent with hypertrophy and hyperplasia, and enlargement of arterioles and capillaries"<-maybe Relaxin can cause hypertrophy and hyperplasia in bones and chondrocytes too.

"Humans have 3 relaxin genes, designated relaxin-1, -2, and -3. Rats and mice each have 2 relaxin genes designated relaxin-1 and -3. Human relaxin-2, as well as rat and mouse relaxin-1 gene products, are true orthologs, insofar as they are secreted by the corpus luteum during pregnancy and circulate. Humans, rats, and mice have 1 relaxin receptor, the LGR7 (leucine rich repeat-containing G protein coupled) receptor recently renamed relaxin/insulin-like family peptide 1 receptor, RXFP1. Although human relaxin may also bind to the LGR8 receptor (RXFP2), albeit with reduced affinity, the preferred ligand for RXFP2 is insulin-like 3 (INSL3). Recently, 2 new receptors have been described for relaxin-3, GPCR135 and 142, although GPCR142 is a pseudogene in rats."

Relaxin stimulates osteoclast differentiation and activation. states that Relaxin induces osteoclast differentiation.

Relaxin affects the dentofacial sutural tissues.

"Relaxin might modulate the remodeling of connective tissue within the craniofacial sutures and periodontal tissues. Relaxin is produced by the pregnant female. It is responsible for the relaxing of the pubic symphysis; the birth canal is widened for parturition. It has also [has] effects on other areas of the body, including ligaments and regions containing collagen and fibroblastic activity. Twenty-one Swiss retired-breeder mice were used to: 1) demonstrate the presence of relaxin within the sutures; 2) demonstrate its effects on the integrity of the suture-like tissues; and 3) assay its effects on protease activity. Relaxin in concentrations of 250 and 500 ng/ml was used in the treated samples and allowed to incubate in complete tissue culture for 24 h. Relaxin [is present] within the cranial suture. [There were] definite changes in the collagen fibril arrangement in the PDL[periodontal ligament] - from being dense and highly organized with a perpendicular direction between tooth and bone to randomly organized and loose, lacking any direction between tooth and bone. An elevation in the protease activity was evident in the relaxin-treated samples."

"In the pregnant female, [relaxin] interferes with the collagen types I and III gene expression, reduces total collagen, and increases the collagenase activity."

"Relaxin has also been found to be produced in the male, the primary source being the prostate gland, though it has not been possible to demonstrate its release into the peripheral blood"

"Relaxin-treated samples had collagen fibrils that were disorganized and dispersed.  The cervices became soft and more extensible."<-so maybe relaxin allows for bone stretching.

"relaxin was shown to inhibit collagen accumulation in a dose-dependent manner"

Effect of relaxin on the phenotype of collagens synthesized by cultured rabbit chondrocytes.

"The effect of porcine relaxin on rabbit articular and growth plate chondrocytes in primary culture was investigated by measurement of total collagen production and analysis of the phenotypes of newly synthesized collagen chains. A 24-h treatment of monolayer articular and multilayer growth plate chondrocytes with 2 micrograms per ml relaxin had no effect on total DNA and did not significantly modify the amount of [3H]proline-labelled collagen chains secreted by the cells. However, polyacrylamide gel electrophoresis demonstrated relevant modifications in relaxin treated chondrocytes. A significant increase was observed in the proportion of type III collagen and in the intensity of the band corresponding to alpha 2I chains. Two-dimensional peptide mapping of CNBr-cleaved molecules indicated that the band that was identified as alpha 1II on monodimensional gels contained a significant proportion of alpha 1I collagen chains, as demonstrated by the presence of alpha 1I cyanogen bromide-digested peptides. The intensity of this band was increased by relaxin treatment. Furthermore, total RNA analysis by slot blot and Northern blot techniques showed a dose-dependent stimulation of alpha 1I and alpha 1III mRNA levels after incubation with increased relaxin concentrations, but no change in the amount of alpha 1II mRNA. These results suggested that when added to cartilage cells in vitro, relaxin modulated the expression of type I, type II and type III collagen genes by amplifying the dedifferentiation process."

So you don't want relaxin with active growth plates but maybe with closed growth plates relaxin causes dedifferentiation of osteoblasts and bone collagen allowing for stretching and formation of new growth plates.

"relaxin acts on the pubic symphisis before and during parturition by softening and lengthening the pubic cartilage and the interpubic ligament"

Effect of pregnancy and obesity on arch of foot.

"Endocrine changes[like relaxin] occurring during pregnancy result in increased laxity of the ligaments of the foot. This may lead to gradual collapse of the foot arches. [We] determine whether pregnancy and body mass index (BMI) had a role in affecting the foot arches at long term.

A collapsed arch results in widening of the feet, thus altering the foot size. The control group included nulliparous women, while the study group included women who had been pregnant at least once. The groups were stratified secondarily by obesity according to BMI. We reviewed over 1000 charts. The age, BMI, and shoe size in an athletic shoe were recorded.

There were 40 subjects in the control group and 70 in the study group. 19/40 women in control and 46/70 in study group experienced a change in shoe size (P = 0.06). Of those affected, the non-obese control group experienced a 9.7% change in shoe size while the obese study group experienced a 15.5% change.

There was neither a change in size between women who had been pregnant and the nulliparous, nor was there a difference between the obese and non-obese. However, there was a statically significant difference between those affected who were both non-obese and nulliparous and those who had been pregnant and who are obese. Individually, the effect of pregnancy and BMI are highly suggestive and clinically relevant."

[Changes in shape and size of the foot during pregnancy].

"Many women report an increase in foot size during their pregnancy. In an initial survey of 21 mothers in 2 Münster nursery schools we found a tendency towards an increase in foot size during pregnancy. We  measure changes in foot length, width, height and volume. A total of 40 women recruited from the antenatal clinic of the University Hospital of Münster and a participating practice were seen three times during their pregnancy. We found a statistically significant increase in foot length, width and volume, whereas foot height decreased slightly. This difference was, however, not significant."

"the overall contact surface of the foot in pregnant women was greater by 12% than in the control group."<-maybe due to collapsed arches?

"a consequence of an altered weight bearing (half a body weight to the body weight), the extension of the foot by 0.7%, the widening of the forefoot by 3.1% and the broadening of the hindfoot by 2.8%, with the width of the rear foot increased by 8.7% compared to the unloaded condition and the forefoot by 6%. The average weight gain of a woman at the end of pregnancy is about 12 kg, which are distributed more or less equally to an increase in fat deposits and water retention"

"Changes in foot size may be due to fluid retention during pregnancy. Occurring edema of the ankles and legs are partly due to the compression of the venous system through the uterus"<-like hydrostatic pressure via LSJL.

Relaxin induces matrix-metalloproteinases-9 and -13 via RXFP1: induction of MMP-9 involves the PI3K, ERK, Akt and PKC-ζ pathways.

"Relaxin treatment of cells in which RXFP1 was silenced resulted in diminished induction of MMP-9 and -13 by relaxin, whereas overexpression of RXFP1 potentiated the relaxin-induced expression of these proteinases. Suppression or overexpression of RXFP2 resulted in no changes in the relaxin-induced MMP-9 and -13. Studies using chemical inhibitors and siRNAs to signaling molecules showed that PI3K, Akt, ERK and PKC-ζ and the transcription factors Elk-1, c-fos and, to a lesser extent, NF-κB are involved in relaxin's induction of MMP-9."

"The human relaxin H2 activates both RXFP1 and RXFP2 resulting in an increase in intracellular cAMP concentrations"

"relaxin H2 induces MMP-9 and -13 in fibrochondrocytes through the RXFP1 receptor, and that relaxin’s modulation of MMP-9 occurs via PI3K–AKT–PKCζ–ERK1/2 signaling pathway and involves Elk-1 and c-fos transcription factors."

"In addition to activating ERK1/2, PI3K and Akt, relaxin treatment also enhances phosphorylation of PKC and PKC-ζ [which can prevent the degradation of NFKb by IKb"

Grow with CLA?

MRM CLA 1250 High Potency,180 Softgels

CLA will help you grow taller with open growth plates due to IGF-1 and CTGF.  However, with trying to induce new growth plates by LSJL it's unclear as CLA has anti-inflammatory effects and inflammatory factors are likely needed to induce new growth plate formation.  But with open plates, chondrocyte hypertrophy is the predominant determinant of height increase so any stimulatory effects on chondrocyte hypertrophy outweigh any other inhibitory effects on other stages.  However with closed plates without first stages like mesenchymal condensation and initial differentiation into chondrocytes, you won't grow taller despite chondrocyte hypertrophy benefits.

Gene expression profiles in rat mesenteric lymph nodes upon supplementation with conjugated linoleic acid during gestation and suckling.

"Human milk contains conjugated linoleic acid (CLA), a fatty acid.
The expression profile of mesenteric lymph nodes from animals supplemented with CLA during gestation and suckling through dam's milk (Group A) or by oral gavage (Group B), supplemented just during suckling (Group C) and control animals (Group D) was determined with the aid of the specific GeneChip(®) Rat Genome 230 2.0 (Affymettrix). {We identify} 89 genes differentially expressed in all three dietary approaches. Several genes, such as connective tissue growth factor (Ctgf){CTGF binds to Nov and CTGF has pro chondrogenic effects}, tissue inhibitor of metalloproteinase 1 (Timp1), galanin (Gal), synaptotagmin 1 (Syt1), growth factor receptor bound protein 2 (Grb2), actin gamma 2 (Actg2) and smooth muscle alpha actin (Acta2), [are] highly interconnected nodes of [a] resulting network."

"Ctgf, Timp1, Gal and Syt1 are genes modulated by CLA supplementation."

"Ctgf promotes DNA synthesis in chondrocytes, osteoblasts and fibroblasts"

This next study provides explanation as to whether CLA is needed when one is already taking colostrum.  Remember CLA is in human milk and not bovine milk.

Effects of conjugated linoleic acids fed to dairy cows during early gestation on hematological, immunological, and metabolic characteristics of cows and their calves.

"The aim of the present experiment was to test the stimulation ability of peripheral blood mononuclear cells (PBMC) expressed as stimulation index (SI) of newborn calves and of their dams fed a control fat supplement (CON) or 50 and 100g/d of a CLA-containing fat supplement (CLA50 and CLA100, respectively) during the preceding lactation period for 182 d after calving. The total intake of cis-9,trans-11 and trans-10,cis-12 CLA by groups CLA50 and CLA100 amounted to 4 and 8 g/d each, respectively. For this purpose, blood was collected immediately after parturition from calves before and after colostrum intake, and from cows after parturition and 21 d later. The SI was related to the fatty acid composition of erythrocyte and milk lipids and to various hematological and clinical-chemical parameters. Retrospective evaluation revealed that depletion time (i.e., the individual period elapsed between the day of terminating the feeding of the experimental diet in the preceding lactation period and the day of calving) ranged from 190 to 262 d, which corresponded to fetal exposure times of 19 to 102 d. The SI from cows increased significantly by 77 and 55%, within 21 d after calving according to the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) and Alamar Blue assays, respectively.  Feeding of 50 g of the CLA product failed to demonstrate this increase. Moreover, SI was significantly lower for calves whose dams belonged to the CLA50 group, whereas stimulation ability was comparable for the PBMC from calves whose mothers were treated with CON and CLA100. Plasma metabolites (total bilirubin, total cholesterol, glucose, nonesterified fatty acids, 3-β-hydroxybutyrate, total protein, and albumin) and hematological parameters (hematocrit, white blood cell profile) were not significantly influenced by dietary treatments of the cows in the preceding lactation period. Although the fatty acid pattern of erythrocyte lipids of cows remained uninfluenced, that of calves showed alterations due to the feeding type of their dams. For example, C16:0 increased significantly from 14.4 to 16.9% of total fatty acid methyl esters, whereas cis-9,trans-11 CLA increased slightly from 0.11 to 0.15% at the same time in calves when their mothers were fed the CLA100 instead of the CON diet. Fatty acid profile of colostrum was significantly different from that of milk after 3 wk for most of the detected fatty acids, but was not influenced by diet type.  feeding a CLA-containing fat supplement during the preceding lactation and gestation period exerted effects on the stimulation ability of PBMC from cows and calves for the subsequent parturition."

"[There were] long-term effects of CLA feeding on cows and their offspring."<-but we don't know the effects on chondrocytes and bone.

Conjugated linoleic acid and bone biology.

"Inflammatory cytokines inhibit chondrocyte proliferation and induce cartilage degradation for which part of the response is mediated by PGE2. Excess production of PGE2 is associated with bone and proteoglycan loss. PGE2 influences the IGF-I/IGFBP axis to facilitate bone and cartilage formation. Growing rats given butter fat and supplements of CLA demonstrated an increased rate of bone formation and reduced ex vivo bone PGE2 production, respectively. The supplements of CLA isomers resulted in their enrichment in lipids of various bone compartments of animals. The effects of CLA on bone biology in rats (IGF action and cytokines) appear to be dependent on the level of n-6 and n-3 fatty acids in the diet;  CLA [also decreases] ex vivo bone PGE2 production and in osteoblast-like cultures.  CLA, may be beneficial in moderating cyclooygenase 2 (COX-2) activity or expression (influencing PGE2 biosynthesis)."

Inflammation may be pro-differentiation.  Distraction Osteogenesis involves inflammation after all.

"CLAs occur naturally in ruminant food products (beef, lamb and dairy) because of the process of bacterial biohydrogenation of linoleic acid in the rumen"

"The anabolic effects of PGE2 may occur through stimulation of osteoblast endogenous IGF-I production or by increasing bone cell responsiveness to IGF-I."

"chondrocytes are either sensitive to excess n-6 fatty acids or to an overproduction of PGE2. Growth cartilage in children and young animals contains small amounts of n-6 fatty acids, but a relatively high concentration of 20:3(n-9) (Mead acid)"

CLA reduced liver IGF-1 levels possibly a negative feedback mechanism.

Evaluation of the mechanism of action of conjugated linoleic acid isomers on reproduction in dairy cows. stated that CLA increased circulating IGF-1 levels.  Supplementation occurred for 37 days.  cis-12 CLA (CLA 50:50) resulted in greatest IGF-1 levels.

Growth Plate X-ray's

This should refute the recent post on Natural Height Growth.

Comparison study of growth plate fusion using MRI versus plain radiographs as used in age determination for exclusion of overaged football players.

"150 healthy male football players were grouped into five age groups ranging from 15 to 19 years old. Each participant had coronal T1-weighted MRI and plain radiograph of the left wrist. The degree of distal radius fusion was rated randomly by three radiologists using a six-stage grading system proposed by the FIFA Research Centre.

MRI assessment of distal radial growth plate fusion has good correlation with plain radiograph fusion (r=0.949). The mean of x-ray grading is higher than the MRI grading in the 15, 16, 17, 18 and 19 years old age groups with p=0.443, 0.001, 0.009, <0.001 and 0.003, respectively, using Wilcoxon signed ranked test. Intraobserver and interobserver correlations were high (r=0.9). T1 MRI correlation with chronological age (r=0.771) was close to plain radiographs (r=0.821) with p value of <0.001.

X-rays significantly overrate the grading of fusion in this age group and therefore should not be used to exclude overaged players as is occurring now to the distress of many genuinely eligible players."

"Grade 2.(A)[X-ray:] There is disruption of the lucent band noted in the distal radial epiphysis suggesting early fusion. (B)[MRI:] This correlates to the intermediate high signal area causing ‘fuzziness’ of the low signal band of the growth plate."

"Grade 3 growth plate. (A,B) Less than 50% fusion on plain radiographs of the radial epiphysis both on x-ray and T1-weighted MRI. Arrows point to areas of fusion."<-A would appear to be totally fused except for one tiny opening on the fibula.

"Grade 4 growth plate. (A) There is more than 50% fusion of the growth plate on the plain radiograph. (B) Similar correlating appearance on T1 MRI weighted MRI of the same patient. Arrow shows absence of epiphysial plate fusion of more than 5 mm in length." Grade 4A actually looks more fused than Grade 3A.

"Grade 5 growth plate. (A) There is less than 5 mm fusion left of the fusion of the growth plate in plain radiograph. (B) Similar correlating appearance on T1-weighted MRI of the same patient (arrows)."<-And 5A looks even less fused. 5B however does look more fused than 4B.

"Grade 6 growth plate. (A,B) Complete fusion of the growth plate seen on both x-ray and T1-weighted MRI."<-in 6B you can still see a line in the tibia and faintly in the fibula.

"[The MRI] has superior soft tissue contrast and cross sectional imaging ability that would allow the epiphysial growth plate and epiphysial–metaphyseal fusion to be beautifully demonstrated."

"Grading system for assessing the fusion of the distal radial growth plate on PA plain radiographs of left wrist

Grade Description

1 No fusion seen of the growth plate. There are two or three continuous broad lucent lines traversing the growth plate horizontally (figure 1)

2 There is disruption of one or two of the continuous bands above (figure 2)

3 There is evidence of fusion across the growth plate but less than 50% of the total width of the radius (figure 3)

4 There is more than 50% fusion across the growth plate. The unfused portions on one side of the growth plate must exceed 5 mm from any one side across the growth plate (figure 4)

5 There is almost complete fusion with only the presence of a notch measuring less than 5 mm on the radial or ulna side or both sides of the growth plate (figure 5)

6 There is complete fusion of the growth plate. There is no evidence of any notch (figure 6)"

Note that there is evidence of partial fusion starting at Grade 2 which is when boys arms are still getting longer.  Thus you can increase bone length without complete opening of the growth plate.  Thus you can grow taller with LSJL partial growth plates theory.

Bony Bridge and Growth Arrest

Bony Bridges are a model of LSJL driven bone growth.  It's unrealistic to assume that LSJL can cause the spontaneous regeneration of the growth plate at once.  A more reliastic model is to assume that LSJL forms mini-growth plates and these growth plates results in angular growth but also longitudinal growth that eventually stabilizes as the growth plates acquire a more normal distribution.

I have included many fragments here that I was not able to get full studies on but I've included this stub as it is important to the mechanism of LSJL action.  Please help if you can.  Studies with exclamation points definitely need full studies for. There are two studies that show that bone growth can occur despite partial closure via osseous bridge and that the bone can spontaneously correct itself.

This leads weight to the view that LSJL will induce mini growth plates that individually induce angular growth but over time the growth plates will lead to uniform longitudinal growth.

[Osseous bridge after physeal-injury to the distal tibia with spontaneous resolution].!

"The risk of osseous bridge development after certain types of physeal injury is well established. Once formed, the bridge continues to grow and results in a progressive deformity. The authors present an unusual case of a five-year-old girl who had a Salter-Harris Type-IV fracture of the distal tibial epiphyseal plate, with subsequent osseous bridge formation and deformity development. The bridge resolved spontaneously in 16 months, and joint mechanical axis alignment was gradually restored with normal growth of the distal tibia."

Patterns of premature physeal arrest: MR imaging of 111 children.

"If the bridge is large and centrally located in the physis, growth is slowed or stopped and limb shortening results."<-In the center of the physis and not at the ends of the physis where we're worried about being able to induce growth with LSJL.

"If the bridge is small but peripheral, growth is tethered and angular deformity develops."

"Peripheral bone bridges in the distal tibia frequently caused angular deformity and, not surprisingly, were strongly associated with tethered growth recovery lines. Growth recovery lines, also called Park or Harris lines, represent disks of transversely oriented, rather than the normal longitudinally oriented, bony trabecula. These disks form at the physis during slowed growth because of injury, immobilization, or illness. As growth resumes, the physis migrates away from the line that remains in the metaphysis. A growth recovery line angled relative to the physis indicates tethering of physeal migration by a bony bridge "

"Cartilage persists in the metaphysis when enchondral ossification is disrupted as the result of metaphyseal vascular disruption"

Premature partial closure and other deformities of the growth plate: MR imaging and three-dimensional modeling.

"If the growth plate is affected eccentrically, tethering will cause angular deformity. If the growth plate is affected centrally, growth at the periphery causes cupping of the metaphysis with shortening of the bone"

"portions of cartilage from the growth plate become fixed in the metaphysis [after growth plate trauma] while growth of bone continues. This results in several patterns: deep intrusions of the growth plate into the metaphysis, persistence of a band of cartilage in the metaphysis, or widening and irregularity of the growth plate. Small islands of cartilage may also be trapped in the epiphysis"

Growth arrest of the distal radius following a metaphyseal fracture: case report and review of the literature.

"five reported case of distal radial metaphyseal fractures not invloving the physis leading to growth arrest."

Fracture through a Harris growth arrest line: a case report.

Spontaneous correction of partial physeal arrest: report of a case and review of the literature.!

"This study describes the rare phenomenon of partial physeal arrest spontaneous correction. It concerns a case of a 3.5-year-old girl who suffered from a Salter-Harris IV fracture of the distal tibial epiphysis, which was managed conservatively. After fracture healing an osseous bridge was formed at the medial part of the physis, leading to a varus deformity. The parents refused the operation, but 6 years later, both the ankle's deformity and the shortening of the extremity had been spontaneously corrected. It seems that the growth potential of the physis healthy portion is able to break the already transformed osseous bridge."

18F-FDG uptake in metaphyseal growth arrest lines: a case study.

"Growth arrest lines (also referred to as Harris lines) refer to dense metaphyseal trabecular lines, perpendicular to the long axis of the bone. The phenomenon is seen only in patients with immature skeletal structure after periods of nutritional insufficiency, illness, and prolonged immobilization or bisphosphonates administration during which bone growth is inhibited. With resumption of bone growth, a dense trabecular line becomes visible, typically at the metaphyses of rapidly growing long bone."

18F-FDG is a supstance used for imaging.

"Over time, the growth arrest lines migrate toward the diaphysis as bone growth continues at the physes."

"Bone scan 2 years after treatment with chemotherapy and bisphosphonate demonstrating symmetrical band of increased MDP uptake immediately proximal to the femoral physes representing the initial migration of growth arrest lines proximally"

Continued Growth After Limited Physeal Bridging.

"After any physeal injury, the primary concern is the possibility of some pattern of growth alteration, particularly transphyseal bridging that may cause lasting deformities and impact subsequent patient care. Small areas of physeal bridging, however, may be associated with continued growth, rather than impairment.{this may give us an idea of how much a growth plate has to be open in order to grow}

Seven patients with small central physeal bridges of the distal femur were identified. 

Radiography identified small, relatively centrally located transphyseal osseous bridging that was associated with a linear (longitudinal) region of osseous density extending from the physeal bridge proximally into the metaphysis. This linear striation disappeared at the metaphyseal/diaphyseal gradation, an area of progression proximally from metaphysis to diaphysis. Only 1 patient had a significant leg length inequality. Magnetic resonance imaging confirmed the intrametaphyseal linear sclerotic bone and its disappearance with diaphyseal remodeling.

Small, central transphyseal osseous bridges may form after radiologically confirmed acute physeal injury. Normal physiological (hydrostatic) growth forces can be sufficient to overcome such limited central bridging and allow continued, essentially normal, longitudinal growth."

The scientists state that an unobstructed region of the Zone of Ranvier is essential for normal longitudinal growth.  The Zone of Ranvier is the groove that provides resting zone cells into the growth plate.

"The zone of Ranvier consists of cells lying laterally and circumferential around the upper zones of the physis whose responsibility it is to provide latitudinal growth of the physis and thereby provide for a wider bone at the level of the growth plate."

Towards a better understanding of bone bridge formation in the growth plate - an immunohistochemical approach.

"From the clinical experience it is known that in some patients this bone bridge eventually disappears during the growth process.  The aim of this study was to investigate the spatial and temporal protein level of molecules potentially involved in these processes, i.e. RANKL, OPG, DKK-1, Coll 10, BMP-2 and IL-6, in an experimental rat model using an immunohistochemical approach. The results from our study suggest that bone bridge formation might be an early event starting immediately after growth plate injury and involving several pro-osteoblastic molecules, i.e. IL-6, BMP-2 as well as OPG and Coll X. In the late studied time points 3 and 9 month post injury expression of anti-osteoblastic proteins, i.e. DKK1 and RANKL, was increased. This indicates that bone bridge dissolution might be late event and potentially linked to WNT signaling inhibition and RANK/RANKL signaling activation."

Couldn't get full study yet.

Increase longitudinal bone growth with Colostrum?

Colostrum contains lactoferrin. Symbiotics Colostrum Plus, 240 Capsules is one source of colostrum.  Colostrum may help increase height during development by increasing IGF-1, HGH, and TGF-Beta1 levels.  Eating casein with colostrum may help increase the bio availability of those proteins.  Colostrum may need to be cycled based on lack of benefits on serum levels past 14 days but mice showed no decrease in efficiency to colostrum over time.

Bovine colostrum supplementation and exercise performance: potential mechanisms.

"Bovine colostrum (BC) is rich in immune, growth and antimicrobial factors, which promote tissue growth and the development of the digestive tract and immune function in neonatal calves. Although the value of BC to human adults is not well understood, supplementation with BC is becoming increasingly popular in trained athletes to promote exercise performance. The combined presence of insulin-like growth factors (IGF), transforming growth factors, immunoglobulins, cytokines, lactoferrin and lysozyme, in addition to hormones such as growth hormone, gonadotrophin-releasing hormone, luteinizing hormone-releasing hormone and glucocorticoids. A review of studies investigating the influence of BC supplementation on exercise performance suggests that BC supplementation is most effective during periods of high-intensity training and recovery from high-intensity training, possibly as a result of increased plasma IGF-1, improved intramuscular buffering capacity, increases in lean body mass and increases in salivary IgA. The bioavailability of the active constituents in BC [has not been fully] determined."

"In vivo human studies have demonstrated a significant increase in gastrointestinal villus height and depth following epidermal growth factor administration in neonates with congenital microvillus atrophy"<-so maybe egf has an effect on bone too.

"A reduction in gastrointestinal damage following BC administration in rats has been observed following TGF-β administration. When administered alone, TGF-β is susceptible to digestion with fasting gastric juices, but in the presence of casein, enzyme inhibitors and other peptides contained in BC, the breakdown of growth factors can be prevented, maintaining their activity following ingestion."<-So maybe TGF-Beta in colostrum can affect the bones too.

"[In] rats 9% of IGF-1 ingested alone appears in the bloodstream, while 67% of IGF-1 survives digestion when administered with casein."  Colostrum supplementation increased IGF-1 levels by 25% though this may be due to an increase in exogenous production and not due to the exogenous IGF-1 in colostrum.

"BC supplementation in humans has not been associated with increased plasma nutrient concentrations"

"athletes [were supplemented] with 60 g/day of colostrum and collected blood prior to and following 4 weeks of supplementation for the analysis of IGF-1 and associated binding protein, IGF binding protein-3."  No significant increase in IGF-1 was detected other long term 6-8 weeks have reported no elevation in IGF-1 levels.  Maybe colostrum has to be cycled.  Durations of 8-14 days have resulted in positive increases in IGF-1 but not longer periods.

Effect of a Growth Protein-Colostrum Fraction on bone development in juvenile rats.

"A bovine colostrum 1-30 kDa fraction, Growth Protein-Colostrum (GP-C), was administered to juvenile rats as a dietary supplement to determine effects on growth and development. GP-C enhanced the growth and mineralization of the femur as evidenced by increased serum osteocalcin and bone mineral density. Increased levels of serum growth hormone and insulin-like growth factor-1 suggest that the mechanism of enhanced growth is partially controlled by endocrine factors. GP-C was also found to increase osteoblast proliferation in vitro"

Rat femurs from colostrum fed rats were about 5% longer.  The duration of colostrum feeding was 6.2 weeks.  Rats were 3 week old sprague-dawley rats weighing 50g.  The rats that had 5% of their diet as GP-C gained the most length.  GP-C also seemed to delay senescence as growth rate slowed more in control group than GP-C fed groups.  GP-C effectively increased serum GH and IGF-1 levels.  Serum levels were taken at the conclusion so serum levels remained elevated without a negative feedback mechanism.

The production of GP-C was special and you can read about it in the materials section(the study doesn't let you copy and paste).  GP-C is essentially higher quality than normal colostrum.  Casein was supplemented with the GP-C.

Colostrum also contains miRNA.

Bovine milk contains microRNA and messenger RNA that are stable under degradative conditions.

"In total, 102 miRNA were detected in bovine milk by microarray analysis (100 in colostrum and 53 in mature milk; 51 were common to both). Among these miRNA, we selected several immune- and development-related miRNA, including miR-27b, miR-34a, and miR-130a. These miRNA were detected in bovine milk by quantitative PCR, and each of these miRNA was significantly more highly expressed in colostrum than in mature milk. We also confirmed the presence of some mRNA in bovine milk. Nevertheless, synthesized miRNA spiked in the raw milk whey were degraded, and naturally existing miRNA and mRNA in raw milk were resistant to acidic conditions and RNase treatment. The RNA molecules in milk were stable. We also detected miRNA and mRNA in infant formulas purchased from Japanese markets."

Although none of the miR seemed directly related to bone growth.

Gaining Stature with Bone Morphogenic Protein-2?

Benzoic Acid a possible BMP-2 stimulator is available for sale: Source Naturals PABA 100mg, 100 Tablets (Pack of 3).

Bone Morphogenic Protein-2 is important for normal height development and could potentially be used for gaining stature.  It is involved in a number of signaling pathways in height development and stimulates stem cells to differentiate into osteoblasts(which can increase height at the top of the head or feet and could conceivably increase height on the top of long bones based on bone deposition patterns).  BMP-2 also helps stem cells differentiate into chondrocytes.  TGF-Beta does not typically encourage osteogenic differentiation which is why it is a better signal for potential chondrogenic differentiation.  How can we utilize Bone Morphogenic Protein-2 to increase our stature?

Effects of low-intensity pulsed ultrasound, dexamethasone/TGF-beta1 and/or BMP-2 on the transcriptional expression of genes in human mesenchymal stem cells: chondrogenic vs. osteogenic differentiation. 

"hMSCs [human Mesenchymal Stem Cells] were subjected to LIPUS [Low Intensity Pulsed Ultrasound] with or without dexamethasone/transforming growth factor-beta1 (TD) or bone morphogenetic protein-2 (BMP-2). TD-treated hMSCs exhibited characteristic chondrogenic morphology and increased messenger RNA (mRNA) expression of chondrogenic markers and LIPUS enhanced the chondrogenic differentiation of hMSCs treated with TD. The expression of Runx2, an osteogenic transcription factor was not altered in either TD treatment group[so TGF-Beta1 did not enhance osteogenesis]; however, a significant increase was detected in the LIPUS only group[LIPUS does enhance osteogenesis]. The osteogenic appearance exhibited 3 days after LIPUS and/or BMP-2 treatment. Increases in the mRNA expression levels of osteogenic markers, Runx2 and ALP were also detected. There was no additive or altered effect with combined LIPUS and BMP-2 treatment. LIPUS alone can increase osteogenic differentiation of hMSCs and LIPUS enhances TD-mediated chondrogenic differentiation of hMSCs[so LIPUS encourages osteogenic differentiation of MSCs but it can enhance chondrogenic differentiation of MSCs so if you want to grow taller you want to upregulate TGF-Beta1 in MSCs by a method like LSJL before applying LIPUS]." 

Ultrasounds are available for purchase: 1MHz Portable Ultrasound Therapy Machine w/ 1 gallon Ultrasound Gel.  From this study, it seems that BMP-2 mainly encourages stem cell differentiation into osteoblasts but BMP-2 has been reported to induce chondrogenic differentiation.

"Chondrogenic differentiation of both animal and human MSCs is enhanced by the application of LIPUS"<-LIPUS can help you grow taller if the cells are encourage to a chondrogenic phenotype such as by TGF-Beta1.

"1 MHz, pulsed 1:4 (2 ms “on” and 8 ms “off”) with a repetition rate of 100 Hz had an optimal effect on osteoblast differentiation. A 200 μs tone burst repeating at 1.0 kHz, 30mW/cm2 may better stimulate aggrecan synthesis. An intensity of 200 mW/cm2 [may result] in more pronounced MSC chondrogenesis in vivo"<-Higher intensities are more likely to result in chondrogenic differentiation.  Dexamethasone and TGF-Beta1 helped encourage more Type II collagen(cartilage) whereas LIPUS increased Type I and Type II Collagen in near equal quantities.  So LIPUS would work better in synergy with another height increasing method specifically targeted to chondrogenesis rather than merely LIPUS alone.

BMP-2 and LIPUS in conjunction seemed to encourage osteogenic markers.  LIPUS seems to act to heighten existing cell stimulants rather than controlling differentiation on it's own.  However, LIPUS was not applied to the whole bone where the effects of LIPUS on the periosteum may increase levels of TGF-Beta1 encouraging more chondrogenic differentiation.  It is unclear whether TGF-Beta or BMP-2 are more important for height increase.  However, TGF-Beta is much easier to induce via mechanical means whereas to increase BMP-2 levels requires chemical means.

Quantitative, structural, and image-based mechanical analysis of nonunion fracture repaired by genetically engineered mesenchymal stem cells. 

"Murine nonunion fractures can be repaired by implanting MSCs over-expressing recombinant human bone morphogenetic protein-2 (rhBMP-2). Nanoindentation studies of bone tissue induced by MSCs in a radius fracture site indicated similar elastic modulus compared to intact murine bone, eight weeks post-treatment.  We investigate temporal changes in microarchitecture and biomechanical properties of repaired murine radius bones, following the implantation of MSCs.  [Analysis] was performed 10 and 35 weeks post MSC implantation. regenerated bone tissue remodels over time, as indicated by a significant decrease in bone volume, total volume, and connectivity density combined with an increase in mineral density[This could be bad as bone volume includes bone height]. the axial stiffness of limbs repaired with MSCs was 2-1.5 times higher compared to the contralateral intact limbs, at 10 and 35 weeks post-treatment. These results could be attributed to the fusion that occurred in between the ulna and radius bones. MSCs induce bone formation, which exceeds the fracture site [but] significant remodeling of the repair callus occurs over time.  Limbs treated with an MSC graft demonstrated superior biomechanical properties." 

Long bones ordinarily grow by chondrogenesis rather than osteogenesis but long bones could grow by Mesenchymal Stem Cells over-expressing BMP-2 by injecting these stem cells close to the very ends of the bones.  Preferably the over-expressing BMP-2 cells would be injected at the end of the cortical bone in the long bones or right beneath the periosteum in the short/irregular ones.  There's the problem of remodeling occurring but we don't know why it occurs presumably so that the bone is proportional.  There would have to be a way to get around this remodeling. 

Bone morphogenetic protein-2 delivered by hyaluronan-based hydrogel induces massive bone formation and healing of cranial defects in minipigs. 

"The authors developed a hydrogel that is formed in situ by the cross-linking of multifunctional hyaluronic acid and polyvinyl alcohol derivatives mixed with hydroxyapatite nanoparticles, in the presence of BMP-2. [We] evaluate the suitability of the hydrogel as a carrier for BMP-2 in repairing critical size cranial defects in a minipig model. Cranial defects (2 x 4 cm) were created in 14 minipigs. The experimental groups were as follows: group 1, craniotomy and application of 5 ml of hydrogel with 1.25 mg of BMP-2; group 2, craniotomy and application of 5 ml of hydrogel without BMP-2; and group 3, craniotomy with no further treatment. After 3 months, examinations were performed. There was spontaneous ossification in the untreated group, but the healing was incomplete. The hydrogel alone demonstrated no further effects. The addition of 1.25 mg of BMP-2 to the hydrogel induced a greater than 100 percent increase in bone volume and complete healing of the defects.  Compact lamellar bone [was seen] in the BMP group without intertrabecular fibrous tissue, as was seen in the other groups. The hydrogel was resorbed completely within 3 months and caused no inflammatory reaction." 

"successful bone formation can be obtained using various hydrogels that resemble the natural extracellular matrix in terms of high water content and structural stability."

"The volumes of newly formed bone in animals treated with hydrogel and BMP-2 were 23.4 ± 6.3 cm3 (119 percent ossification of the defects)"<-19% is huge.  Assuming a limb is 12 inches long that would result in a final bone of 14.28.  Over two inches in height from a single bone.

"Hyaluronan [induces] the expression of its own receptor, CD44, specifically in mesenchymal stem cells."<-more benefits of hyaluronic acid supplementation.

"The hyaluronan/CD44 interaction induces adhesion and migration of mesenchymal stem cells to hyaluronan, which suggests a dual capacity of hyaluronan-based biomaterials by functioning as both a matrix for attraction of mesenchymal stem cells and as a carrier and protective container for differentiation factors."

Clearly, BMP-2 use can initiate supernatural height growth.  BMP-2 is involved in chondrogenesis, which increases height in the long bones. 

Effect of TGF beta1, BMP-2 and hydraulic pressure on chondrogenic differentiation of bovine bone marrow mesenchymal stromal cells. 

"Bioactive factors, such as TGF beta and BMP-2, as well as mechanical factors i.e. compressive loading and hydraulic pressure, have been shown to induce and/or modulate chondrogenesis of bone marrow derived mesenchymal stromal cells (BMSCs). Since these factors are intracellularly transduced through different mechanisms, TGF beta, BMP-2 and hydraulic pressure act synergistically on chondrogenic differentiation of BMSCs. Aggregates of bovine BMSC were cultured in the presence of 10 ng/ml TGF beta1, 50 ng/ml BMP-2 or both. Half of the samples were loaded for 4 hours per day with 0.5-3 MPa cyclic hydraulic pressure at 1 Hz. After 14 days of culture/loading, gene expression of chondrogenic genes was assessed. DNA as well as glycosaminoglycan (GAG) content of the pellets were analysed. Neither pressure nor BMP-2 had an influence on GAG/DNA content. cells responded to the presence of TGF beta1 with an up-regulation of chondrogenic genes and GAG/DNA of the aggregates increased compared to controls demonstrating the cells ability to respond to external stimuli. The used concentrations of BMP-2 and parameters for pressure were neither able to induce nor modulate chondrogenesis of bovine BMSCs." 

BMP-2 may be involved in chondrogenesis but it doesn't seem to induce it in this case.  Hydraulic pressure is similar to the fluid pressure generated during LSJL.  So, for LSJL to be effective the pressure should be more than 1 Hz(anyway to measure the pressure?).  However LSJL may operate by mechanisms other than increasing fluid pressure(like trabecular microfracture, this study was performed in petri dishes so increase in fluid pressure could have had other effects not noticeable outside a living host).   Hydraulic pressure alone may not upregulate chondrogenic genes but LSJL has also been shown to upregulate TGF-Beta1 which is effective in upregulating chondrogenic genes.

"cyclic compressive loading promoted chondrogenic differentiation of rabbit BMSCs in the absence of TGFβ emphasising the inductive role of the mechanical environment."<-LSJL is like cyclic compressive loading.  So too is LSJL likely effective in promoting chondrogenic differentiation in the absence of TGF-Beta.

"TGF β, BMP-2 and mechanical factors influence chondrogenic differentiation of BMSCs via different mechanisms. TGF β and BMP-2 belong both to the TGF β superfamily of ligands. These ligands bind to a type II receptor which phosphorylates a type I receptor. Type I receptor phoshorylates receptor-regulated SMAD (R-SMAD) which then bind to common-partner SMAD 4 (co-SMAD 4). Finally these complexes act as transcription factors in the nucleus. Although TGF β and BMP-2 belong to the same superfamily of ligands, they bind to different receptors (TGF β receptor and BMP-2 receptor, respectively) which lead to activation of different R-SMAD molecules: SMAD 2 and 3 are TGF β receptor-associated SMAD and SMAD 1, 5 and 8 are BMP-associated SMAD[SMAD 2 and 3 versus SMAD 1/5/8 affects terminal differentiation of chondrocytes, however it is likely that achieving terminal differentiation of chondrocytes may be beneficial given various knockout studies]. All activated SMAD bind to the co-SMAD 4. Binding of either ligand can result in an up-regulation of so-called chondrogenic genes Sox-9, collagen type II and aggrecan. For the mechanical factors, cyclic compressive loading leads to up-regulation of endogenous TGF β gene expression as well as upregulation of type I and type II and TGF β receptors. TGF β signal transduction seems to be involved in chondrogenesis of BMSCs induced by compressive loading[which can be exemplified by the evidence that TGF-Beta1 is upregulated during LSJL]. bioactive factors and mechanical factors act through similar but different pathways to influence chondrogenesis of BMSCs"

BMP-2 had chondrogenic effects in conjunction with TGF-Beta but osteogenic effects without TGF-Beta.

Mesenchymal Stem Cells hyper-expressing BMP-2 may be a new mechanism of increasing height growth in the future.  They could be injected at where the cortical bones end to induce new height growth.

However, in already active growth plates BMP-2 accelerates terminal differentiation.  You may only want BMP-2 when you're trying to induce new chondrogenic differentiation of stem cells like with LSJL.

Interaction of TGFβ and BMP signaling pathways during chondrogenesis.

"TGFβ and BMP signaling pathways exhibit antagonistic activities during the development of many tissues. We generated hypomorphic mouse models of cartilage-specific loss of BMP and TGFβ signaling to assess the interaction of these pathways in postnatal growth plate homeostasis. We used the chondrogenic ATDC5 cell line to test effects of BMP and TGFβ signaling on each other's downstream targets. conditional deletion of Smad1{LSJL downregulates Smad1 but the shortening of the growth plate was not observed} in chondrocytes resulted in a shortening of the growth plate[remember Smad1 phosphorylation affects height in active growth plates(Phosphorylation in this case refers to activation)]. The addition of Smad5 haploinsufficiency led to a more severe phenotype with shorter prehypertrophic and hypertrophic zones and decreased chondrocyte proliferation. The opposite growth plate phenotype was observed in a transgenic mouse model of decreased chondrocytic TGFβ signaling that was generated by expressing a dominant negative form of the TGFβ receptor I (ΔTβRI) in cartilage. Histological analysis demonstrated elongated growth plates with enhanced Ihh expression, as well as an increased proliferation rate with altered production of extracellular matrix components[But does this increase final height?  Just because there were no TGF-Beta receptors in cartilage doesn't mean there were none in stem stems which is where they are needed for chondrogenic differentiation].  in chondrogenic ATDC5 cells, TGFβ was able to enhance BMP signaling, while BMP2 significantly reduces levels of TGF signaling. During endochondral ossification, BMP and TGFβ signaling can have antagonistic effects on chondrocyte proliferation and differentiation in vivo."

 "TGFβ or BMP ligands bind to specific type II receptors to recruit the corresponding type I receptor to initiate a cascade of events leading to phosphorylation of their specific receptor-Smads (R-Smads). Generally, TGFβ signaling depends on Smad2 and Smad3, while BMP signaling depends on Smad1, 5 and 8"<-remember we don't want Smad 1/5/8 phosphorylation(deactivation) but we do want Smad 2 and 3 phosphorylation in active growth plates(when the skeleton is still developing).  So we don't want fully functional TGF-Beta signaling(unless we're trying to form new growth plates like with LSJL).  Remember when TGF-Beta signaling was decreased an elongated growth plate was the result.

"TGFβ/BMP signaling can also be mediated by noncanonical MAPK pathways, such as P38, JNK and Erk1/2 signaling pathways, during chondrogenesis"

"In vitro BMPs can promote mesenchymal cells to differentiate into chondrocytes in high-density cultures in part by inducing Sox9 gene expression[remember though you want Beta-Catenin levels to be higher than Sox9 levels]"

"Loss of Noggin, a potent BMP antagonist, leads to overgrowth of skeletal elements in mice"<-so transgenic expression of BMP-2 may increase height as Noggin a BMP antagonist decreases height.

"BMPs promote the differentiation of proliferating chondrocytes to hypertrophic chondrocytes, the chondrocyte specific expression of constitutively active Bmpr-1a in transgenic mice accelerated the maturation and hypertrophy of proliferating chondrocytes"<- you would think accelerating maturation would decrease height

"reduced BMP signaling in proliferating chondrocytes leads to a shortening of the growth plate in part due to decreased cell proliferation, reduced TGFβ signaling results in an increased proliferation rate and an elongated growth plate"<-so maybe we want to enhance BMP signaling while inhibiting TGF-Beta signaling.

"BMP2 can inhibit TGFβ signaling, while TGFβ1 enhances BMP signaling in-vitro"<-Both BMP-2 and TGF-Beta can induce chondrogenic differentiation but BMP-2 is better than TGF-Beta since BMP-2 inhibits TGF-Beta anyways you'd want to increase BMP-2 levels to grow taller.  All the chondrogenic benefits for TGF-Beta may be due to BMP-2 signaling enhancement and not TGF-Beta itself.

However, in the previous study only TGF-Beta1 upregulated chondrogenic genes whereas BMP-2 had no effect.

Bezafibrate may be a way to upregulate BMP-2 expression.

Bezafibrate enhances proliferation and differentiation of osteoblastic MC3T3-E1 cells via AMPK and eNOS activation.

"MC3T3-E1 cells, a mouse osteoblastic cell line, were used. Cell viability and proliferation were examined. NO production was evaluated. The mRNA expression of ALP, collagen I, osteocalcin, BMP-2, and Runx-2 was measured. [We] detect the expression of AMPK and eNOS proteins.
Bezafibrate increased the viability and proliferation of MC3T3-E1 cells in a dose- and time-dependent manner. Bezafibrate (100 μmol/L) significantly enhanced osteoblastic mineralization and expression of the differentiation markers ALP, collagen I and osteocalcin. Bezafibrate (100 μmol/L) increased phosphorylation of AMPK and eNOS, which led to an increase of NO production by 4.08-fold, and upregulating BMP-2 and Runx-2 mRNA expression[this increase in BMP-2 could help you grow taller by helping LSJL start that initial differentiation of chondrocytes]."

"Bezafibrate, a dual ligand for peroxisome proliferator-activated receptors α (PPARα) and PPARβ, is a lipid-lowering drug widely used to treat hypertriglyceridemia"

"After addition of bezafibrate, BMP-2 mRNA expression was increased to 1.80- and 1.74-fold of the control group by bezafibrate at d 7 and 14, respectively"

Identification of Novel 2-((1-(Benzyl(2-hydroxy-2-phenylethyl)amino)-1-oxo-3-phenylpropan-2-yl)carbamoyl)benzoic Acid Analogues as BMP-2 Stimulators.

"10 new chemical entities (NCEs) have shown BMP-2 stimulation and osteoblast differentiation. Among these, 2-((1-(benzyl(2-hydroxy-2-phenylethyl)amino)-1-oxo-3-phenylpropan-2-yl)carbamoyl)benzoic acid (11) was the most effective while its analogue 13 also showed good activity in inducing osteoblast BMP-2 production. Compound 11 induced osteoblast differentiation in vitro, and this effect was abrogated by a physiological BMP-2 inhibitor, noggin."

"Piceatannol stimulates osteoblast differentiation and is associated with the increase in BMP-2 production. Recently, some proteasome inhibitors, viz. epoximycin (a natural product) and bortezomib, showed increased bone formation rates in vitro, with the participation of BMP-2 signaling in osteoblasts.  Wwo series of substituted benzothiophene and benzofuran derivatives [are] BMP-2 up-regulators."

BMP2 induces segment-specific skeletal regeneration from digit and limb amputations by establishing a new endochondral ossification center

"BMP treatment can induce a regeneration response in mouse digits amputated at a proximal level of the terminal phalangeal element (P3) "

"the regeneration-inductive ability of BMP2 extends to amputations at the level of the second phalangeal element (P2) of neonatal digits, and the hindlimb of adult limbs[really 8-10 week old mice]."

"BMP2-induced regeneration is associated with a localized proliferative response and the transient expression of established digit blastema marker genes. This is followed by the formation of a new endochondral ossification center at the distal end of the bone stump. The endochondral ossification center contains proliferating chondrocytes that establish a distal proliferative zone and differentiate proximally into hypertrophic chondrocytes. Skeletal regeneration occurs from proximal to distal with the appearance of osteoblasts that differentiate in continuity with the amputated stump. Using the polarity of the endochondral ossification centers induced by BMP2 at two different amputation levels, we show that BMP2 activates a level-dependent regenerative response indicative of a positional information network."

"BMP7 induces bone growth when applied to neonatal limb amputations in mice"

"BMP2-induced skeletal elongation occurs by the regeneration of an endochondral ossification center at the amputated stump that forms an apical growth zone that directs skeletal elongation. "

"BMP2 [induces] regeneration of the amputated distal region of the P2 element."

"Bone formation during digit development involves endochondral ossification and the differentiation of Col2a1 expressing chondroctyes into Col10a1 expressing hypertrophic chondrocytes establishes the polarity of skeletal elongation."

"mammalian tissues that lack regenerative ability still possess and/or are able to re-acquire positional information that is necessary to participate in a functional regenerative response that is appropriate for the amputation level."

"at 1 DPI we found [TWO] blastemal markers were expressed in the distal mesenchyme. Msx1 transcripts were expressed primarily by cells closely associated with the BMP2 bead, whereas Pedf expressing cells were scattered throughout the BMP2 treated amputation wound and not restricted to the region surrounding the BMP2 bead"

"On the other hand amputated limbs treated with BMP2 displayed directed skeletal elongation and a regenerative response of both the tibia and fibula"<-Note the wounds were allowed to heal first and that these were adult amputees.

"differentiation occurs distal to proximal during P3 regeneration and proximal to distal in P2 regeneration. This finding demonstrates that BMP2 is not acting to pattern the regenerative response, but rather appears to activate a level-specific regenerative potential."

"The stimulation of an apical zone of proliferating chondrocytes represents a foundation for exploring segment-specific de novo skeletal regeneration by effectively regenerating a growth plate."

BMP-2 was able to induce endochondral ossification after wound healing had occurred in 8 to 10 week old mice amputations.

Gene expression profiling following BMP-2 induction of mesenchymal chondrogenesis in vitro.

"The experimental system consists of micromass cultures of C3H10T1/2 cells, a murine multipotential embryonic cell line, treated with the chondroinductive growth factor, bone morphogenetic factor-2 (BMP-2). In this system, chondrogenic differentiation characterized by both morphological changes and cartilage matrix gene expression has been shown to be completely dependent upon BMP-2 treatment and the high cell plating density of micromass cultures."

"chondrogenesis can be induced at ectopic extra-skeletal sites by the implantation of demineralized bone matrix led to the isolation of chondroinductive factors"

BMP-2 induced col2a & Col2b however Col2a was already present in controls.  a-actin disrupts some B-catenin activities.

BMP-2 upregulated genes also upregulated by LSJL:
Osteopontin

BMP-2 downregulated genes also downregulated by LSJL:

"BNIP3 is a pro-apoptotic protein that heterodimerizes with Bcl-2 and Bcl-XL. Induction of BNIP3 may be required to control cell number during chondrogenesis.  BNIP3 is induced in this C3H10T1/2 system. hypertrophy [occurs] in late stages of the chondrifying micromass cultures"

This next study involves osteoblasts but I believe information can be inferred to chondrocytes and stem cells:

BMP2 and mechanical loading cooperatively regulate immediate early signalling events in the BMP pathway.

"We use a three-dimensional bioreactor system. Time-dependent phosphorylation of Smad, mitogen-activated protein kinases and Akt in human fetal osteoblasts was investigated under loading and/or BMP2 stimulation conditions. The phosphorylation of R-Smads is increased both in intensity and duration under BMP2 stimulation with concurrent mechanical loading.  The synergistic effect of both stimuli on immediate early Smad phosphorylation is reflected in the transcription of only a subset of BMP target genes, while others are differently affected. Regulation of osteogenesis is guided by both signalling pathways.

Mechanical signals are integrated into the BMP signalling pathway by enhancing immediate early steps within the Smad pathway, independent of autocrine ligand secretion. This suggests a direct crosstalk of both mechanotransduction and BMP signalling, most likely at the level of the cell surface receptors."

"roughly 1,000 times the normal physiological concentration has to be administered [for various BMP2 and BMP7 applications]"

"Id1 expression was slightly induced by BMP2 stimulation after 90 minutes"

"Dlx5, [under] both under loading and non-loading conditions, BMP2 led to enhanced gene expression"

"Id2{down} expression was also induced by BMP2 but the enhancing effect of mechanical loading was not present. c-fos{up} and osteopontin{up} expression was strongly up-regulated by mechanical loading, while BMP treatment exhibited no effect. By contrast, Runx2 expression, that was induced after 90 minutes, was down-regulated by mechanical loading after 24 hours. Gene expression of members of the Distal-less homeobox family, Dlx2 and Dlx3, was induced by BMP2 but expression was not significantly enhanced by concurrent mechanical loading. Dlx5 expression after 24 hours of stimulation was not regulated by the different treatments."

"Both BMP type I and type II receptors co-localize with αvβ integrins"

"BMP4 and BMP7 tend to be down-regulated under loading conditions, BMP6 expression was positively affected by mechanical loading, even more so when BMP2 was present"

Assessment of gene regulation by bone morphogenetic protein 2 in human marrow stromal cells using gene array technology.

"we used a conditionally immortalized human marrow stromal cell line (hMS) and a gene expression microarray containing probes for a total of 6800 genes to compare gene expression in control and BMP-2-treated cultures. A total of 51 genes showed a consistent change in messenger RNA (mRNA) frequency between two repeat experiments. Seventeen of these genes showed a change in expression of at least 3-fold in BMP-2-treated cultures over control cultures. These included nuclear binding factors (10 genes), signal transduction pathway genes (2 genes), molecular transport (1 gene), cell surface proteins (2 genes) and growth factors (2 genes). Of particular interest were four of the nuclear binding factor genes ID-1, ID-2{down}, ID-3, and ID-4. These encode dominant negative helix-loop-helix (dnHLH) proteins that lack the nuclear binding domain of the basic HLH proteins and thus have no transcriptional activity. They have been implicated in blocking both myogenesis and adipogenesis. Other transcription factors up-regulated at least 3-fold by BMP-2 included Dlx-2, HES-1{UP}, STAT1, and JunB{up}."

"core binding factor beta (CBFbeta), AREB6, and SOX4, showed changes in expression of between 2- and 3-fold with BMP-2 treatment."

Vcam1 which was downregulated by LSJL had a rendency to be downregulated by BMP-2 treatment.  IL6 tended to be downregulated by BMP-2 but was upregulated by LSJL.


Chondrogenesis of human bone-marrow derived mesenchymal stem cells is modulated by complex mechanical stimulation and adenoviral-mediated overexpression of bone-morphogenetic protein 2.

"This study strived to investigate the combined effect of complex mechanical stimulation and adenoviral-mediated over-expression of bone morphogenetic protein 2 (BMP-2) on hMSC chondrogenesis. hMSCs were encapsulated in a fibrin hydrogel and seeded into biodegradable polyurethane (PU) scaffolds. A novel three-dimensional transduction protocol was used to transduce cells with an adenovirus encoding for BMP-2 (Ad.BMP-2). Control cells were left un-transduced. Cells were cultured for 7 or 28 days in chondropermessive medium, which lacks any exogenous growth factors. Thereby, the in vivo situation is mimicked more precisely. hMSCs in fibrin-PU composite scaffolds were either left as free-swelling controls or mechanically stimulated using a custom built bioreactor system which is able to generate joint-like forces. Outcome parameters measured were BMP-2 concentration within the culture medium, biochemical and gene expression analysis. Mechanical stimulation resulted in an up-regulation of chondrogenic genes. Further, GAG/DNA ratios were elevated in mechanically stimulated groups. Transduction with Ad.BMP-2 led to a pronounced up-regulation of the gene aggrecan and an up-regulation of Sox9 message after 7 days. Furthermore, a synergistic effect in combination with mechanical stimulation on collagen 2 message was detected after 7 days. This synergistic increase was more than 8-fold if compared to the additive effect of the application of each stimulus on its own. However, BMP-2 over-expression consistently resulted in a trend towards decreased GAG/DNA ratios in both mechanical stimulated and unloaded groups."

"[Gene transfer] is based on the delivery of cDNA (encoding a specific transgene) to a target cell. This procedure enables the cell to produce the desired transgene."

"Chondropermessive medium: Serum-free DMEM, 1% insulin-transferrin selenium premix, 50 μg/ml ascorbate-2-phosphate, 10-7 M dexamethasone and 1% MEM non-essential amino acids. P/S was replaced with 100 μg/ml of Primocin. This was necessary in order to prevent possible contaminations as the bioreactor system is not a closed system. A further 5 μM of 6-aminocaproic acid was added, in order to prevent fibrin degradation"

"Mechanical stimulation was executed for either 7 or 28 days using a custom built bioreactor system 37 and a ceramic hip ball with a diameter of 32 mm. Unconfined, dynamic compression strain was generated by pressing the ceramic hip ball onto the cell-seeded fibrin-PU scaffolds. Shear stresses were generated by rotating the ball around an axis perpendicular to the scaffold axis. Both stimuli were superimposed on a static offset strain of 0.4 mm. The following loading protocol was used: Compression 1Hz 0.4-0.8 mm; Rotation 1 Hz ± 25°"  The lowest MSCs used were from a 41 year old.

"In the unloaded groups, a trend towards a decrease in BMP-2 release into the culture medium was monitored if compared to the loaded groups. However, this trend did not reach statistical significance. Furthermore, BMP-2 concentrations steadily increased until week 4 in the loaded groups."

"The peak BMP-2 concentrations were 130±37.3.4 ng/ml in the unloaded groups and 339.8±186 ng/ml in the loaded groups."

Osteogenic genes Collagen I and Runx2 were unresponsive to mechanical loading but were upregulated by BMP-2.  Collagen X is mechanically upregulated but BMP-2 counteracts this mechanical upregulation.

"[A] synergistic effect of [mechanical and BMP-2] stimuli was detected on day 7. The n-fold up-regulation in Col 2 message was 2655±7417 (control loaded), 936±1785 (BMP unloaded) respectively 29878±86410 (BMP-2 loaded)."

BMP-2 but not loading seem to upregulate Sox9.  Aggren is upregulated by BMP-2 and loading independently but not synergestically.

"A trend towards an increased GAG release in loaded vs. unloaded groups was detected."

"Mechanical stimulation is known to lead to the paracrine production of TGF-β1, inducing chondrogenesis of hMSCs in [a] fibrin-PU composite system"

"in the unloaded group, the synthesized BMP-2 can leave the scaffold only by means of passive diffusion. In the loaded group however, application of mechanical forces will likely squeeze out most of the BMP-2 which is retained within the scaffold."

"An up-regulation in gene expression does not always correlate to elevated protein levels."

Latexin is involved in bone morphogenetic protein-2-induced chondrocyte differentiation.

"Latexin is the only known carboxypeptidase A inhibitor in mammals. BMP-2 significantly induced latexin expression in Runx2-deficient mesenchymal cells (RD-C6 cells), during chondrocyte and osteoblast differentiation. In this study, we investigated latexin expression in the skeleton and its role in chondrocyte differentiation. Proliferating and prehypertrophic chondrocytes expressed latexin during skeletogenesis and bone fracture repair. In the early phase of bone fracture, latexin mRNA expression was dramatically upregulated. BMP-2 upregulated the expression of the mRNAs of latexin, Col2a1, and the gene encoding aggrecan (Agc1) in a micromass culture of C3H10T1/2 cells. Overexpression of latexin additively stimulated the BMP-2-induced expression of the mRNAs of Col2a, Agc1, and Col10a1. BMP-2 treatment upregulated Sox9 expression, and Sox9 stimulated the promoter activity of latexin."

"The distribution of latexin-positive cells was similar to that of Sox9-positive cells"

Comparative Analysis of Osteogenic/Chondrogenic Differentiation Potential in Primary Limb Bud-Derived and C3H10T1/2 Cell Line-Based Mouse Micromass Cultures.

"we provide a detailed comparative analysis of the differentiation potential of micromass cultures established from either BMP-2 overexpressing C3H10T1/2 cells or mouse embryonic limb bud-derived chondroprogenitor cells, using micromass cultures from untransfected C3H10T1/2 cells as controls. Although the BMP-2 overexpressing C3H10T1/2 cells failed to form chondrogenic nodules{learning why they failed to form chondrogenic nodules could be key}, cells of both models expressed mRNA transcripts for major cartilage-specific marker genes including Sox9, Acan, Col2a1, Snorc, and Hapln1{all upregulated by LSJL except for Snorc} at similar temporal sequence, while notable lubricin expression was only detected in primary cultures. Furthermore, mRNA transcripts for markers of osteogenic differentiation including Runx2, Osterix, alkaline phosphatase, osteopontin and osteocalcin were detected in both models, along with matrix calcification. Although the adipogenic lineage-specific marker gene FABP4 was also expressed in micromass cultures, Oil Red O-positive cells along with PPARγ2 transcripts were only detected in C3H10T1/2-derived micromass cultures. Apart from lineage-specific marker genes, pluripotency factors (Nanog and Sox2) were also expressed in these models, reflecting on the presence of various mesenchymal lineages as well as undifferentiated cells. This cellular heterogeneity has to be taken into consideration for the interpretation of data obtained by using these models."

"the cell line-based colony exhibited a substantially different morphology than the primary model. Cells in the [embryonic] limb bud-derived HDC[High Density Culture] formed numerous nodules with multiple cell layers, while cellular density remained low in the internodular areas. The fact that differentiation of chondroprogenitor cells into chondroblasts primarily occurs within these foci was confirmed by staining with DMMB: on day 3, metachromatic cartilage matrix could only be detected within the aggregates and no metachromasia was visible in the internodular areas. By contrast, the central region of the C3H10T1/2-based culture was densely populated with complete lack of foci and internodular areas. Furthermore, cellular behaviour in terms of migratory characteristics was also different in the two models: although some cells have also migrated to the periphery of the primary HD culture, this area remained relatively sparse compared to the cell line-based HDC in which a very high number of cells have spread from the centre and they even formed dense, multiple cellular layers on the periphery."<-Formation of condrogenic foci and internodular areas could be key to neo-growth plate formation.  You can look at figure 1 for more analysis.

"there is an increasing tendency in the amount of metachromatic[color stained] ECM areas as differentiation proceeds."

"While we observed extensive metachromatic areas within cartilaginous nodules (but not in the internodular areas) in 3-day-old primary HDC, mainly orthochromatic[no color change] staining was visible in the cell line based models on the same culturing day. By days 6 and 10 of culturing the disparity became even more pronounced between primary HDC and the C3H10T1/2 models. Nonetheless, the BMP-2 overexpressing cell line-based cultures presented higher amounts of metachromatic ECM areas in comparison with the control ones. After 15 days of culturing, the ECM was exclusively metachromatic and enlarged, presumably hypertrophic chondrocytes were also visible in the primary HDC. The size of metachromatic matrix areas also increased in the BMP-2 overexpressing C3H10T1/2 colonies{So in the cell line culture it just takes longer}, but it failed to reach the amount detected for primary cultures of the same age. In contrast, no metachromatic territories could be identified in micromass cultures of control C3H10T1/2 cells even on day 15 of culturing. It is of note that the appearance of metachromatic territories in the embryonic limb bud-derived HDC and that in cultures established from the BMP-2 overexpressing variant of C3H10T1/2 was different after staining with DMMB; while the former model exhibited distinct, heavily metachromatic regions that corresponded to cartilaginous nodules, a weaker but relatively homogenous metachromasia was observed in the latter one. Furthermore, considerable orthochromatic territories were also visible throughout the culturing period in colonies of the BMP-2 overexpressing C3H10T1/2."

The embryonic cell culture had higher expression of Prg4, Col10a1, and Acan than the cell line overexpressing BMP2.