Monday, December 28, 2009

Huogo I Formula

Angelica Sinensis is available for sale Solaray - Dong Quai Angelica Sinensis, 550 mg, 100 capsules

Effects of huogu I formula (I) on correlated factors of bone regeneration in chickens with steroid-induced necrosis of femoral head.

The microenvironment of a femoral head recently undergone necrosis may differ from an adult femoral head.

"Forty-eight healthy female Leghorn chickens were randomly divided into control group, model group and Huogu I group, and each group consisted of 16 chickens. At the meantime of model establishment, chickens of the Huogu I group were administrated with decoction, while the model and control group with distilled water by gavage. At the 8th and 16th week after medication, blood samples were obtained for blood lipid detection while both sides of femoral head were harvested for the rest of examinations. .
Compared with the control group, serum levels of total cholesterol (TC), triglyceride (TG) and low-density lipoprotein cholesterol (LDL-C) in the model group rose significantly. Positive cell counting of BMP2, TGFβ(1), Smad4 and OPG in femoral head of the model group dropped prominently. Positive cell counting of Smad7 and RANKL increased dramatically. In contrast with the model group, levels of TC, TG and LDL-C in Huogu I group reduced significantly. Positive cell counting of BMP2, TGFβ(1), Smad4 and OPG in femoral head of the Huogu I group increased prominently. Indices of Smad7 and RANKL both decreased significantly. Especially at the 8th week, these variations were more significant.
Huogu I formula is effective in promoting repair of necrotic femoral head by regulating the expressions of BMP2, TGFβ(1), Smads and OPG/RANKL of osteoclast in femoral head."

"Huogu Ⅰ Formula (consisting of Poria Cocos, Atractylodes Rhizome, Codonopsis Pilosula, prepared Pinellia tuber, Radix Paeonia Rubra, Ramulus Cinnamoni, Angelica Sinensis, Ligustici Chuanxiong and prepared Rhizome Rehmannia) provided by Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, was decocted into solution which contained crude drug in 1 g/mL. Methyl prednisolone sondium succinate"

The model group refers to the osteonecrosis group, the control had no stimulation, and the Huogo I group is Huogo I +necrosis.

"Eight weeks after steroid administration, the model group showed several histomorphological changes as compared with the control group: thin trabecula, increased number of empty lacuna and adipocyte proliferation and hypertrophia. At the 16th week, collapsed bone trabeculae, necrosis accompanied by hyperplasia in some regions, increased empty lacuna were observed. Moreover, the fat area in marrow cavity was higher than that in the normal group. At the 8th and 16th week after treated by Huogu Ⅰ Formula, trabeculae were arranged in regular and compact shapes, large amount of osteoclast and few osteoclast distributed around them, and the empty lacuna rate and fat area in medullary channel were significantly lower than those in the model group"

"The expressions of BMP2 and TGFβ1 positive staining were observed in chondrocye and osteocyte of femoral head in the control group. At the 8th week, the expressions of BMP2 and TGFβ1 in the model group were significantly lower than that in the control group. Additionally, the difference was even more obvious at the 16th week. Compared with the model group, the expressions of BMP2 and
TGFβ1 in Huogu Ⅰ group were significantly higher. At the 8th week, the increasing trend of the two factors in Huogu Ⅰ group was drastic and even approximate to counterparts in the control group{but not greater than the control group which is what we would hope if Huogo I formula is height increasing}"

"Rhizoma Chuanxiong is effective in improving microcirculation and reducing the blood viscosity; Atractylodes Macrocephala Koidz, Angelica Sinensis, and prepared Pinellia Tuber possess similar effects and may improve the condition of hyperlipidemia and prevent fat from accumulating in marrow cavity."

"Angelica Sinensis possesses effects of up-regulating expressions of BMP2, cbfal, insulinlike growth factors (IGF), and other related genes of osteoblast{many of these genes are pro-chondrogenic also}. We believe that, on one hand, Huogu Ⅰ Formula acts on osteoblast and bone marrow stromal cell to enhance the expression of related genes of osteogenesis. On the other hand, it regulates the lipid metabolism and improves the condition of abnormal blood rheology, leading to the promotion of blood supply of femoral head."<-So Angelica Sinensis is the most promising compound.

Study to establish the role of JAK2 and SMAD1/5/8 pathways in the inhibition of hepcidin by polysaccharides from Angelica sinensis., states that Angelica Sinensis may inhibit the SMAD1/5/8 pathway.  Smad1/5/8 is the BMP pathway signaling.

Role of Bone Morphogenetic Proteins-7 (BMP-7) in the Renal Improvement Effect of DangGui (Angelica sinensis) in Type-1 Diabetic Rats., states that Angelica sinensis may directly activate BMP-7.

Sunday, December 27, 2009

Static compression on growth plates

Static compressive loading reduces the mRNA expression of type II and X collagen in rat growth-plate chondrocytes during postnatal growth.

"This in vitro study investigated the effects of loading on the mRNA expression pattern of key molecular components of the growth-plate related to the extracellular matrix (type II and type X collagen) and the PTH-PTHrP feedback loop. Short-term static compressive loading was applied to rat proximal tibial growth-plate explants. Four age groups at specific developmental stages were investigated. The spatial variation in the mRNA expression was compared among loaded explants, their contralateral sham controls, and uncultured growth plates from normal animals. Basic cell metabolism (18S rRNA) was unaffected by load. Results indicated a narrower spatial distribution of mRNA expression of type II collagen throughout the growth plate; similarly, a narrowed distribution of expression of type X collagen was noted in the lower hypertrophic zone of the growth-plate. Mechanical compression influences chondrocytes of the hypertrophic zone to alter their expression of specific genes encoding proteins of the extracellular matrix, while PTH-PTHrP receptor mRNA, a regulatory protein, remained unaffected by loading. The effects of compression were similar at the different stages of growth, suggesting that additional factors may be involved in the clinical progression of skeletal deformities observed during growth spurts."

"Age group (days old) Body weight (g) Growth-plate thickness (μm) Static load (N) Plug area (mm2) Stress (kPa)
21 50.2 ± 5.0   741.9 ± 69.0 0.27 ± 0.03 22.4 ± 3.2 12.2 ± 1.3
35 143.3 ± 8.1 634.4 ± 76.6 0.77 ± 0.04 39.9 ± 3.9 19.5 ± 2.0
56 203.6 ± 9.4 470.9 ± 89.6 1.10 ± 0.05 44.1 ± 5.4 25.2 ± 3.4
80 251.8 ± 20.0 207.5 ± 20.5 1.36 ± 0.11 46.0 ± 5.2 29.8 ± 4.1"

Here's an image of the growth plates with and without load:

"load did not appreciably perturb the basic metabolism of chondrocytes and cell viability was preserved"

A study in vivo of the effects of a static compressive load on the proximal tibial physis in rabbits.

"Static compressive loads (10 N or 30 N) were applied for two or six weeks across one hind limb proximal tibial physis of thirteen-week-old female New Zealand White rabbits (n = 18). The contralateral hind limb in all rabbits underwent sham surgery with no load to serve as an internal control. Harvested physes were divided into portions.
Compared with unloaded shams, physes loaded at 10 N or 30 N for two weeks and at 10 N for six weeks showed histological changes in cells and matrices. Physes loaded at 30 N for six weeks were decreased in thickness and had structurally disorganized chondrocyte columns, a decreased extracellular matrix, and less intense type-II and X collagen immunohistochemical staining. Quantitative reverse transcription-polymerase chain reaction analysis of loaded samples compared with unloaded shams yielded a significantly decreased gene expression of aggrecan and type-II and X collagen and no significant changes in the matrix metalloprotease-13 gene expression with increasing load.
Compressed rabbit physes generate biochemical changes in collagens, proteoglycan, and cellular and tissue matrix architecture. Changes potentially weaken overall physeal strength, consistent with the Hueter-Volkmann principle, and lend understanding of the causes of pathological conditions of the physis."

"Compared with unloaded shams, six-week, 30-N-loaded physes were decreased in thickness and stained less intensely for collagen. The 30-N specimens also had structurally altered cells, lacunae, and chondrocyte columns (chondrons) as well as decreased extracellular matrix areas between cells and chondrons in proliferating and hypertrophic tissue zones. On enlargement compared with unloaded shams, structural changes in 30-N physes were marked by apparently fewer chondrocytes in all physeal regions, enlarged cell lacunae, irregularly aligned chondrons shortened in height, and disorganized cell arrangements within chondrons. The latter included individual chondrocytes in 30-N samples that varied in size and shape and failed to stack in an orderly, linear manner, one on the other, as did chondrons of unloaded shams"

This provides evidence that the growth plates physically push the bone apart as they are affected by load on it.  Although epiphyseal distraction helps the bones push apart and does not result in adult height gain.  Although epiphyseal distraction cannot help the growth plates with the ossification stage.

Chronic in vivo load alteration induces degenerative changes in the rat tibiofemoral joint.

"A varus loading device was attached to the left hind limb of mature rats to apply increased compression to the medial compartment and decreased compression to the lateral compartment of the tibiofemoral joint of either 0% or 100% body weight for 0, 6 or 20 weeks.
Increased compression in the medial compartment produced significant degenerative changes consistent with the development of osteoarthritis (OA) including a progressive decrease in cartilage aggregate modulus (43% and 77% at 6 and 20 weeks), diminished cellularity (38% and 51% at 6 and 20 weeks), and increased histological degeneration. At 20 weeks, medial compartment articular cartilage thickness decreased 30% while subchondral bone thickness increased 32%{consistent with endochondral ossification} and subchondral bone modulus increased 99%. Decreased compression in the lateral compartment increased calcified cartilage thickness, diminished region-specific subchondral bone thickness and revealed trends for reduced cellularity and decreased articular cartilage thickness at 20 weeks."

"Chronic increased load of 44% BW applied to the rabbit knee for 12 h/day over 12 weeks resulted in increased articular cartilage thickness and permeability with minimal fibrillation of the articular surfaces"

"9-month-old, male, Sprague–Dawley rats (weight: 666 ± 32 g)"

Strain-dependent recovery behavior of single chondrocytes.

"In this study, the compressibility and recovery behaviors of single chondrocytes were determined as a function of compressive strains from 6 to 63%. Bovine articular chondrocytes from the middle and deep zones were subjected to this range of strains, and digital videocapture was used to track changes in cell dimensions during and after compression. The normalized volume change, apparent Poisson's ratio, residual strain after recovery, cell volume fraction after recovery, and characteristic recovery time constant were analyzed with respect to axial strain. Normalized volume change varied as a function of strain, demonstrating that chondrocytes exhibited compressibility. The mean Poisson's ratio[the negative ratio of transerse to axial strain] of chondrocytes was found to be 0.29 +/- 0.14, and did not vary with axial strain{a value below 0.5 means the cell is compressible}. In contrast, residual strain, recovered volume fraction, and recovery time constant all depended on axial strain. The dependence of residual strain and recovered volume fraction on axial strain showed a change in behavior around 25-30% strain, opening up the possibility that this range of strains represents a critical value for chondrocytes."

"The average height and diameter of chondrocytes were found to be 8.4 ± 1.8μm and 11.1 ± 0.8μm, respectively. Chondrocytes were either elliptical (n = 16) or hemi-elliptical (n = 18) in appearance. All parameters determined in this study were insensitive to cell shape. The average cell volume was calculated to be 430 ± 251μm^3."

"the recovery behavior of single chondrocytes, but not their compressibility, depended on axial strain."

"At low strain levels (< 20%), cells recovered relatively quickly, and recovery was generally over 90% of the cell’s original height. At intermediate strains (20–30%), some cells exhibited similar recovery behavior, while others did not recover as quickly and showed significant residual strain"

"Above [25-30%] strain level, chondrocytes showed an impaired ability to recover, as evidenced by increased residual strain and decreased volume fraction after recovery, which differed significantly from chondrocytes compressed less than 25%. Below 25% axial strain, residual strain and recovered volume fraction showed weaker or non-existent dependence on axial strain. This suggests that 25–30% compressive strain may represent a threshold akin to the yield strain, above which some plastic deformation may occur."<-Maybe above 30% tensile strain produces similar changes.  Are these plastic changes good or bad for growth.

"Chondrocytes decreased in volume with increasing axial strain"<-could they increase in volume with increasing tensile strain?

"Though the individual components that comprise the cell, e.g., cytoskeletal filaments, cytoplasm, and plasma membrane, may be intrinsically incompressible, fluid exudation through the plasma membrane would result in a net volume loss."

It is important to note that these chondrocytes were not in the ECM.

The effects of mechanical loading on the mRNA expression of growth-plate cells.

"This in vitro study investigated the effects of mechanical loading on the mRNA expression pattern of key molecular components of the growth-plate. Short-term static loading was applied to rat proximal tibial growth-plate explants. Various age groups at specific developmental stages were investigated. In situ hybridization was used to assess the mRNA expression of the cells in different zones of the growth-plate. Four key components were investigated: 18s (basic cell metabolism), type II collagen (major extracellular matrix component), type X collagen (matrix component in hypertrophic zone) and PTH-PTHrP receptors (pre-hypertrophic chondrocytes). The spatial variation in the mRNA expression between loaded explants and their contralateral controls was compared to establish: -the sensitivity of the different growth-plate zones to mechanical loading; -the sensitivity of the different developmental stages to loading. Preliminary results indicated that static loading on the growth plate of 80 d.o. rats affects type II and X collagen gene expressions while PTH-PTHrP remains insensitive to static loading."

Couldn't get full study.

Friday, December 18, 2009


Retinol-binding protein 4 is expressed in chondrocytes of developing mouse long bones: implications for a local role in formation of the secondary ossification center.

"Retinol-binding protein 4 (Rbp4) is the major carrier of retinol in the bloodstream, a retinoid whose metabolites influence osteogenesis, chondrogenesis and adipogenesis. Rbp4 is mainly produced in the liver where it mobilizes hepatic retinol stores to supply other tissues. Rbp4 was present in a variety of locations in developing embryonic and postnatal mouse hindlimbs. Rbp4 was present in a restricted population of epiphyseal chondrocytes and perichondral cells correlating to the future region of secondary ossification. With the onset of secondary ossification, Rbp4 was detected in chondrocytes of the resting zone and in chondrocytes that bordered invading cartilage canals and the expanding front of ossification. Rbp4 was less abundant in proliferating chondrocytes involved in primary ossification."

"Serum retinol, complexed to Rbp4, is taken up into cells via the Rbp4 receptor, Stra6. In the cell, retinol metabolites (collectively known as retinoids) can interact with retinoic acid/rexinoid receptor (RAR/RXR) heterodimers to activate gene transcription. Abnormal retinoid levels impact bone growth"

"Rbp4 was detected in chondrocytes toward the middle of the epiphysis, in the region where the secondary ossification center will form"

"ucleated cells with small amounts of cytoplasm did not contain Rbp4 whereas Rbp4 was abundant in the cytoplasm where the nucleus had begun to deteriorate and was still present in cells without nuclei. With complete breakdown of the cell, Rbp4 was no longer detected in empty lacunae"

"Rbp4 is localized to chondrocytic cells in three regions of embryonic tibia; two relating to secondary ossification and the third relating to primary ossification. Primary ossification contributes to elongation of the limbs, whereas secondary ossification mediates expansion and mineralization of the epiphyses. Formation of the secondary ossification center is preceded by invagination of the perichondrium and invasion of the epiphyseal cartilage by vascularised canals"

Link N

The effect of Link N on differentiation of human bone marrow-derived mesenchymal stem cells.

"MSCs isolated from the bone marrow of three osteoarthritic patients{the gene expression of OA MSCs is different but it usually favors osteogenic rather than chondrogenic differentiation} were cultured in chondrogenic or osteogenic differentiation medium without or with Link N for 21 days.
Link N alone did not promote MSC chondrogenesis. However, after MSCs were supplemented with Link N in chondrogenic differentiation medium the quantity of GAG secreted into the culture medium, as well as aggrecan, COL2A1 and SOX9 gene expression, increased significantly. The gene expression of COL10A1 and osteocalcin (OC) were down-regulated significantly. When MSCs were cultured in osteogenic differentiation medium, Link N supplementation led to a significant decrease in mineral deposition, and alkaline phosphatase (ALP), OC and RUNX2 gene expression.
Link N can enhance chondrogenic differentiation and down-regulate hypertrophic and osteogenic differentiation of human MSCs."

"Link N (DHLSDNYTLDHDRAIH) is the N-terminal peptide of link protein, a glycoprotein that stabilizes the non-covalent interaction between an aggrecan G1 domain and hyaluronate"

Link N stimulates Col2a1 and decreases Col10a1 in normal human MSCs.

"MSCs were obtained from aspirates of the intramedullary canal of three osteoarthritic patients (40-60 years of age) undergoing total hip replacement"

"Since the DNA content of the cultures did not change significantly between days 3 and 21 of culture, the enhanced GAG synthesis is likely the result of increased production by each cell rather than a consequence of more cells due to cell proliferation"

"Link N alone does not directly stimulate chondrogenesis in a manner analogous to TGFβ, but rather enhances ongoing chondrogenesis"

"The ability of Link N to decrease the expression of ALP and OC may be through down-regulating the transcription regulator RUNX2"

The Effect of Link N on the Differentiation of Human Mesenchymal Stem Cells

"MSCs were isolated from the bone marrow of osteoarthritis (OA) patients. The cells were cultured in 24-well plates (3000 cells/well) in chondrogenesis differentiation medium (Invitrogen, Canada) according to the manufacturer's instructions. Link N was dissolved in the media with a final concentration of 0.1 µg/mL and 1 µg/mL, respectively. Medium without Link N was applied as a control. The media were changed every 3 days, and the used media were collected and stored at −20°C for GAG analysis. For gene expression analysis, the cells were cultured for 7, 14, and 21 days.
With the concentrations of 0.1 and 1.0 µg/mL, no toxic effect of Link N on MSCs was observed. After MSCs were cultured for 7 days and 14 days, the expression of aggrecan (ACAN), collagen II (COL2A1) and the transcription regulator SOX9 increased significantly in the media with 0.1 µg/mL or 1.0 µg/mL Link N. No significant differences were observed at day 21. When cells were cultured with 0.1 or 1.0 µg/mL Link N, the quantity of GAG in the media increased significantly at day 9, 12, and 15, but no major difference was observed at day 3 and 6 and at day 18 and 21. When MSCs were cultured for 7, 14, and 21 days, no significant effect of Link N on the expression of alkaline phosphatase (ALP) was observed. Link N significantly inhibited osteocalcin (OC) gene expression after culturing MSCs for 21 days."

We need to find some studies on Link N on normal human bone marrow.

Monday, December 14, 2009

Grow Taller by Delaying Puberty?

The effects of delayed puberty on the growth plate.

"Many athletes are beginning intense training before puberty, a time of increased bone accrual when up to 25% of total bone mineral accrual occurs. Female athletes experiencing late or delayed pubertal onset may have open epiphyseal plates that are vulnerable to injury. This investigation's purpose was to determine whether a delay in puberty (primary amenorrhea) affects the growth plate immediately postpuberty and at maturity.
Forty-eight female Sprague-Dawley rats (23 d old) were randomly assigned to 4 groups (n=12); short-term control (C-ST), long-term control (C-LT), short-term GnRH antagonist (G-ST), and long-term GnRH antagonist (G-LT). At 25 days of age, daily gonadotropin-releasing hormone antagonist (GnRH-a) injections were administered delaying pubertal onset. Left tibias were analyzed. Stained frontal slices of proximal tibia (5 µm thick) were analyzed in hypertrophic, proliferative, and reserve zones for total height, zone height, and cell/column counts.
Growth plate height was 19.7% wider in delayed puberty (G-ST) group and at maturity was 27.9% greater in G-LT group compared with control (C-LT). No significant differences were found in short-term or long-term growth plate zone heights or cell/column counts between groups. Growth plate zone height normalized to total height resulted in 28.7% larger reserve zone in the short-term GnRH-a group but the proliferative zone was 8.5% larger in the long-term group compared with the control group. Normalized to growth plate height a significant decrease was found in column counts in proliferative zones of the short-term and long-term GnRH-a groups.
Current data illustrate that delayed puberty using GnRH-a injections results in significant growth plate height and decreases proliferative column counts and zone height, thus potentially contributing to decreases in bone mass at maturity.
Growth plate height increases indicate increased potential for growth and bone accrual. However, previous models report decreased bone volume following delayed puberty via GnRH-a injections that may have detrimental effects in the long term."

"Estrogen in low doses stimulates growth hormone and results in the chondroblast progenitor cells in the reserve zone beginning clonal expansion. Suppressed estrogen levels during delayed puberty and secondary amenorrhea may have a direct negative effect on the growth plate and thus bone development"

"The GnRH-a model significantly delays the onset of puberty resulting in suppressed estradiol levels during growth"

"proximal growth plates of the tibia of IGF-1-deficient and acid-labile subunit knockout mice (LID+ALSKO) were smaller in total height and in the height of the proliferative and hypertrophic zones of chondrocytes compared with controls"

"delayed pubertal onset in the GnRH-a groups resulted in increased height[growth plate height not body height] but bone bridging would indicate that even though statistically growth plates are at different heights senescence was near with growth plate fusion at a time point similar to control animals. Delayed growth plate senescence following the dexamethasone injections was indicated by the lack of growth plate fusion in the experimental animals. After the 16-week period, the control group had 74% more fused growth plates in comparison with the dexamethasone group."

According to the author, there was no statistically significant differences in bone length between either the long term or short term groups.

"Means were very similar between groups at both time points."

Benefit of postponing normal puberty for improving final height

"In children with central precocious puberty, a GnRH analog (GnRHa) alone is efficacious in increasing final height, but in other conditions a combination of growth hormone (GH) and GnRHa is needed. In GH-deficient children with early onset of puberty and poor height prediction, the combination of GH and GnRHa increases final height by 1.0-1.3 s.d. In children with idiopathic short stature and persistent short stature after intrauterine growth retardation, the combination also appears to be beneficial. Potential side effects include weight gain, a negative effect on bone mineralization, and psychosocial consequences. More data on long-term safety have to be collected before the combination of GH and GnRHa in children with idiopathic short stature should be considered for clinical use outside clinical trials. "

"within the normal range, late developers have a greater leg length:sitting height ratio than early developers"

Syndecan 4

Syndecan 4 is upregulated by LSJL.

Bone fracture repair, but not fetal skeletal development is supported by syndecan-4.

"We used Sdc4-/- mice to analyze the functional role of Scd4 in endochondral ossification of mouse embryos and in adult fracture repair. In Sdc4-/-/LacZ knock-in animals, Sdc4 promoter activity was detectable in all stages of chondrocyte differentiation, and Sdc4 deficiency inhibited chondrocyte proliferation. Aggrecan turnover in the uncalcified cartilage of the epiphysis was decreased transiently in vivo, but this did not lead to a growth phenotype at birth. By contrast, fracture healing in adult mice was markedly delayed in Sdc4-/- animals and accompanied by increased callus formation. Blocking inflammation during fracture healing with a TNFα inhibitor reduced these changes in Sdc4-/- animals to WT levels. Analysing the discrepancy between the mild embryonic and the severe adult phenotype, we found a compensatory up-regulation of Sdc2{down} in the developing cartilage of Sdc4-/- mice that was absent in adult tissue. Stimulation of chondrocytes with Wnt3a in vitro, led to an increased expression of Sdc2, while stimulation with TNFα resulted in an up-regulation of Sdc4 but a decreased expression of Sdc2. TNFα stimulation decreased Sdc2- and increased Sdc-4 expression even in presence of Wnt3a, suggesting a strong effect of inflammation on the regulation of Sdc expression."

The upregulation of Sdc4 and downregulation of Sdc2 is consistent with the inflammatory role in fracture repair thus providing evidence that inflammation is involved in the LSJL response.

"Expression of Sdc1, -2 and -4 has been detected in chondrocytes and progenitor cells during development of rat mandibular condyles. Sdc3 has been implicated in regulating the size of skeletogenic condensations and growth factor-mediated proliferation of chondrocytes during limb development and growth. In addition, Sdc2 and -4 are involved in osteoblast cell adhesion and survival"

"At the cellular level, Sdc4 promoter activity was seen throughout the cartilage of the epiphysis and in resting, proliferating, prehypertrophic and hypertrophic chondrocytes within the epiphyses"

"a substantially reduced staining for ADAMTS-4{up in LSJL} protein was detected throughout the cartilage in Sdc4-/- E16.5 tibia in comparison with wild type cartilage"

"Sdc2 expression during development is most likely induced by Wnt3a, a growth factor that is involved in the transition of mesenchymal cells into chondroprogenitor cells"

"stimulation of WT chondrocytes with 100 ng/ml Wnt3a in vitro led to increased Sdc2 mRNA levels (3 fold up-regulation). In contrast TNFα stimulation decreased Sdc2 mRNA levels in a dose dependent manner by up to 50 %"

Sdc4 and Sdc2 regulation are likely highly involved in the LSJL response.
Sdc2 mRNA levels in a dose dependent manner by up to 50 %"

Friday, December 4, 2009


Biological effects of the plant-derived polyphenol resveratrol in human articular cartilage and chondrosarcoma cells.

"RSV is chondroprotective for articular cartilage in rabbit models for arthritis{Can this translate to height benefits?}. Effects of RSV on human articular cartilage homeostasis were studied by assessing production of matrix-degrading enzymes (MMP-13, ADAMTS4, and ADAMTS5), as well as proteoglycan production and synthesis. The counteractions of RSV against catabolic factors (e.g., FGF-2 or IL-1β) were examined by in vitro and ex vivo using monolayer, three-dimensional alginate beads and cartilage explants cultures, respectively. RSV improves cell viability of articular chondrocytes and effectively antagonizes cartilage-degrading protease production that was initiated by catabolic and/or anti-anabolic cytokines in human articular chondrocytes. RSV significantly enhances BMP7-promoted proteoglycan synthesis. RSV inhibits the activation of transcription factors involved in inflammation and cartilage catabolic signaling pathways, including direct downstream regulators of MAPK (e.g., AP-1, PEA3) and NFκB. RSV selectively compromises survival of human chondrosarcoma cells, but not primary articular chondrocytes, revealing cell-specific activity of RSV on non-tumorigenic versus tumor-derived cells. RSV exerts its chondroprotective functions, in part, by deactivating p53-induced apoptosis in human primary chondrocytes, but not human chondrosarcoma."

"signaling cascades generated by inflammatory cytokines (e.g., IL-1) or fibroblast growth factor-2 (FGF-2 or basic FGF) favor catabolism by stimulating protease production and inhibiting proteoglycan deposition in human adult articular cartilage or intervertebral disc tissue via ERK/MAPK activation"

"FGF-2 mediates striking antagonistic effects on cartilage anabolic activity in conjunction with IGF-1 and BMP7, and both FGF-2 and IL-1 modify chondrocyte gene expression when stimulated by mechanical injury "

"p53 DNA binding activity is significantly stimulated by IL-1β (10 ng/ml) along with AP-1, AP-2, Ets1/PEA3, NFκB, p53, Sp1, and multiple STATs that are critical for cytokine signals"

Thursday, December 3, 2009


Gpr177, a novel locus for bone-mineral-density and osteoporosis, regulates osteogenesis and chondrogenesis in skeletal development.

"Gpr177 exhibits an ability to modulate the trafficking of Wnt similar to the Drosophila Wls/Evi/Srt.  To overcome the early lethality associated with the inactivation of Gpr177 in mice, conditional gene deletion is utilized to assess its functionality. Here we report the generation of four different mouse models with Gpr177 deficiency in various skeletogenic cell types. The loss of Gpr177 severely impairs development of the craniofacial and body skeletons, demonstrating its requirement for intramembranous and endochondral ossifications, respectively. Defects in the expansion of skeletal precursors and their differentiation into osteoblasts and chondrocytes suggest that Wnt production and signaling mediated by Gpr177 cannot be substituted. Because the Gpr177 ablation impairs the secretion of Wnt proteins, we therefore identify their sources essential for osteogenesis and chondrogenesis. The intercross of Wnt signaling between distinct cell types is carefully orchestrated and necessary for skeletogenesis.  Gpr177 controls skeletal development through modulation of autocrine and paracrine Wnt signals in a lineage-specific fashion"

"formation of the calvarial, maxillary and mandibular bones mediated by intramembranous ossification is defective or completely missing in the Gpr177Dermo1 embryos"

Loss of GPR177 delays chondrocyte hypertrophy.

"The chondrogenic deletion of Gpr177 significantly reduced bone mineralization, and interfered with chondrocyte maturation"

"The number of cells undergoing mitotic division is significantly reduced in the columnar zone, but not epiphyses of the Gpr177Dermo1 and Gpr177Col2 humeruses, suggesting that expansion of the proliferating and prehypertrophic but not the resting chondrocytes was affected by the loss of Gpr177"

"disruption of endochondral ossification starts at chondrocyte maturation, and the subsequent events, including ECM remodeling, vascular invasion and osteoblastogenesis, are impaired in the Gpr177Dermo1 mutants."  Chondrocyte maturation is where Beta Catenin begins to take over for Sox9.

"Gpr177 is dispensable in the osteoblasts during intramembranous and endochondral ossifications. Our findings suggest that the impairment of osteoblast differentiation in the Gpr177Dermo1 and Gpr177Col2 limbs is attributed to delay in chondrocyte maturation but not intrinsic defects of the osteoblasts."

Adipose Tissue Stem Cells to Chondrocytes

High plasticity of pediatric adipose tissue-derived stem cells: too much for selective skeletogenic differentiation?

"We have used different methods to establish stem cells from adipose tissue (adipose-derived stem cells [ADSCs], adipose explant dedifferentiated stem cells [AEDSCs]) from several pediatric patients and investigated their phenotype and differentiation potential using monolayer and micromass cultures. We have also addressed the overlooked issue of selective induction of cartilage differentiation. ADSCs/AEDSCs from different patients showed a remarkably similar behavior. Pluripotency markers were detected in these cells, consistent with ease of reprogramming to induced pluripotent stem cells. Significantly, most ADSCs expressed markers of tissue-specific commitment/differentiation, including skeletogenic and neural markers, while maintaining a proliferative, undifferentiated morphology. Exposure to chondrogenic, osteogenic, adipogenic, or neurogenic conditions resulted in morphological differentiation and tissue-specific marker upregulation. These findings suggest that the ADSC "lineage-mixed" phenotype underlies their significant plasticity, which is much higher than that of chondroblasts we studied in parallel. Finally, whereas selective ADSC osteogenic differentiation was observed, chondrogenic induction always resulted in both cartilage and bone formation when a commercial chondrogenic medium was used; however, chondrogenic induction with a transforming growth factor β1-containing medium selectively resulted in cartilage formation."

"ADSCs expressed the mesenchymal markers CD44 (hyaluronic acid receptor), CD90 (Thy-1), and CD105 (endoglin)"

" with the exception of SOX2, transcripts for the pluripotency markers c-MYC, OCT4, NANOG, KLF4, and DNMT3B were detected in ADSCs from all patients tested"  Surprisingly, ADSC's do not express type II collagen by default.  They do express Aggrecan.  Type II collagen was induced by TGF-Beta1.

Chondroblasts in contrast to ADSC's express ColII and ALP by default.

Monday, November 30, 2009

EPH receptors

LSJL downregulates EphrinB3.  It upregulates Epha3 and Epha5

EphB/ephrin-B interactions mediate human MSC attachment, migration and osteochondral differentiation.

"EphB and ephrin-B molecules were expressed by ex vivo expanded human MSC populations and within human bone marrow trephine samples. To elucidate the contribution of EphB/ephrin-B molecules in MSC recruitment, we performed functional spreading and migration assays and showed that reverse ephrin-B signalling inhibited MSC attachment and spreading by activating Src-, PI3Kinase- and JNK-dependent signalling pathways. Forward EphB2 signalling promoted MSC migration by activating the Src kinase- and Abl-dependent signalling pathways.  Activation of ephrin-B1 and/or ephrin-B2 molecules expressed by MSC was found to increase osteogenic differentiation, while ephrin-B1 activation promoted chondrogenic differentiation. These observations suggest that EphB/ephrin-B interactions may mediate the recruitment, migration and differentiation of MSC during bone repair."

"EphB2, EphB4 and ephrin-B1 expressions appeared most pronounced within the human bone, localising to bone lining cells, "

"ephrin-B3 was expressed by a large number of bone marrow cells"<-the downregulation of ephrinB3 in LSJL could be a sign of bone marrow cell differentiation.

"Blocking the EphB2 receptor, significantly inhibited GAG synthesis"

"the formation of a PDZ complex containing NHERF1, PTPN13 and TAZ with ephrin-B1 results in the dephosphorylation of TAZ, which allows its translocation into the nucleus and subsequent activation of osteogenic specific transcription factor, Osterix"

"In mice that lack the Hoxa13 gene, the mesenchymal condensations that form the autopod skeletal elements are poorly resolved, resulting in missing digit, carpal and tarsal elements. In addition, mesenchymal and endothelial cell layers of the umbilical arteries (UAs) are disorganized, resulting in their stenosis and in embryonic death.  We generated a loss-of-function allele in which the GFP gene was targeted into the Hoxa13 locus. This allele allowed FACS isolation of mesenchymal cells from Hoxa13 heterozygous and mutant homozygous limb buds. Hoxa13(GFP) expressing mesenchymal cells from Hoxa13 mutant homozygous embryos are defective in forming chondrogenic condensations in vitro. Analysis of pro-adhesion molecules in the autopod of Hoxa13 mutants revealed a marked reduction in EphA7 expression in affected digits, as well as in micromass cell cultures prepared from mutant mesenchymal cells. Finally, antibody blocking of the EphA7 extracellular domain severely inhibits the capacity of Hoxa13(GFP) heterozygous cells to condense and form chondrogenic nodules in vitro, which is consistent with the hypothesis that reduction in EphA7 expression affects the capacity of Hoxa13(-/-) mesenchymal cells to form chondrogenic condensations in vivo and in vitro. EphA7 and EphA4 expression were also decreased in the mesenchymal and endothelial cells that form the umbilical arteries in Hoxa13 mutant homozygous embryos."

"the addition of the anti-EphA7 antibody dramatically reduced both cell adhesion and chondrogenic nodule formation of Hoxa13GFP heterozygous cells."

MicroRNA-34a regulates migration of chondroblast and IL-1β-induced degeneration of chondrocytes by targeting EphA5.

"We investigated the role of miRNA-34 during chondrogenic differentiation of chick limb mesenchymal cells. Expression of miR-34a increased upon chondrogenic inhibition. Blockade of miR-34a via PNA-based antisense oligonucleotides (ASOs) recovered the chondro-inhibitory actions of JNK inhibitor on migration of chondrogenic progenitors and the formation of precartilage condensation. EphA5 is a relevant target of miR-34a during chondrogenesis. MiR-34a was necessary and sufficient to down-regulate EphA5 expression, and up-modulation of EphA5 is sufficient to overcome inhibitory actions of miR-34 inhibition on cell migration and condensation of chick limb mesenchymal cells on collagen substrate. miR-34a is a negative modulator of chondrogenesis, particularly in migration of chondroblasts, by targeting EphA5 and resulting inhibition of cellular condensation during chondrogenesis of chick limb mesenchymal cells."

"In articular chondrocytes, Wnt-3a caused dedifferentiation of chondrocytes by upregulating the expression of c-Jun and its phosphorylation by JNK, resulting in activation of the c-Jun/activator protein. Treatment with transforming growth factor-β superfamily members promoted cartilage-specific gene expression during in vitro chondrogenic differentiation of mesenchymal progenitor cells from bone marrow and trabecular bone through activation of the mitogen-activated protein (MAP) kinases, p38, ERK-1, and JNK"

"miR-34a is one of key modulators in JNK-induced chondrogenic differentiation"

"EphA5 increases spreading and migration of chondroprogenitors in collagen substrate"

"the protein level of type II collagen decreased when cells were exposed to JNK inhibitors"

"miR-34a was significantly up-regulated by IL-1β and silencing of miR-34a significantly prevented IL-1β-induced down-regulation of type II collagen in rat chondrocytes. Increased expression level were observed in human articular chondrocytes exposed to IL-1β and human chondrocytes were prepared from osteoarthritic zone suggesting the involvement of miR-34a in the dedifferentiation of human articular chondrocytes by IL-1β. Co-treatment of miR-34a inhibitor induced the recovery of EphA5 level and type II collagen protein level which were suppressed by treatment of IL-1β. The expression level of EphA5 and type II collagen was significantly decreased in human chondrocytes were prepared from osteoarthritic zone"

Sunday, November 29, 2009

Where the Load is

In a previous entry, I talked about the reasoning which states that if weight lifting has the ability to produce height increase(via bone size increase) then why aren't bodybuilders gaining height? I reasoned that weightlifters(powerlifters, bodybuilders, strongman, and misc) are in fact gaining height but that it is not large enough to be unable to be dismissed by measurement error. Look at how extreme a bodybuilders shoulder and bicep development is relative to how it is compared to non-specific weight training. A normal weight lifter will not have a bodybuilders physique and proportions. Bodybuilders do highly specialized routines to get their disproportionately large arms and shoulders.

So too must people who want to train for height increase via bone increase. Imagine the standard back squat movement. At the top of the movement, the bar is putting most of the load on the bone. In the middle of the movement, most of the load is on the muscle. At slightly below parallel most of the load is on the tendons and ligaments. Same with bench press: Arms locked or near locked out bone, in between muscle, at the chest tendons and ligaments. If you are very flexible, you might have to do dumbell bench to get the load onto the tendons and ligaments.

How to build bone? Perform exercises where most of the load is on the bone(some load will always be on the muscle). Bodybuilders stress how important ROM after all. Bodybuilders want to keep their ROM in the muscle building range, we height builders want to keep our ROM in the bone building range.

Saturday, November 28, 2009


EIF2a-p is important for PKR inhibition of Cyclin D1.

Double-stranded RNA-dependent protein kinase regulates insulin-stimulated chondrogenesis in mouse clonal chondrogenic cells, ATDC-5.

"Double-stranded RNA-dependent protein kinase (PKR) is an interferon-induced protein that has been identified and characterized as a translational inhibitor in an interferon-regulated antiviral pathway. PKR inactivation suppresses osteoblast calcification and osteoclast formation. PKR is required for the in vitro differentiation of the mouse clonal chondrogenic cell line ATDC-5. ATDC-5 cells treated with insulin differentiated into chondrocytes and produced an alcian-blue-positive cartilage matrix. The protein expression of signal transducers and activators of transcription (STAT) peaked at day 7 of differentiation, whereas the expression of SRY-box-containing gene 9 (Sox-9), which is a transcription factor for chondrocyte differentiation, increased gradually. When the cells were treated with a PKR inhibitor (2-aminopurine), the cartilage matrix formation decreased. The protein expression of STAT1{Stat1 interacts with Cyclin D1} continued to increase up to day 21, whereas the expression of Sox-9 was low and did not increase. PKR was localized to a marginal region of the mandibular condyle cartilage in mouse embryos. PKR has important functions in the differentiation of chondrocytes through the modulation of STAT1 and Sox-9 expression."

Since PKR is important to chondroinduction maybe EIF2a-p and therefore Salubrinal are too.

"PKR/eIF-2α cascade has been implicated as a general transducer of apoptosis"

"2-Aminopurine (2-AP), which interacts with the ATP-binding site of PKR, is a potent inhibitor of PKR  and blocks tumor necrosis factor-α (TNF-α) and C2-ceramide [activities]"

"ATDC-5 cells show characteristics of undifferentiated prechondrogenic cells in the growth phase and maintain a potential for chondrogenesis. When treated with insulin, ATDC-5 cells undergo cellular condensation in the post-confluent phase and acquire the phenotype of chondrocytes, i.e., proteoglycan synthesis and type II collagen expression"

"the level of STAT1 protein was higher in the cells treated with insulin, especially at day 7 of culture and inhibition of PKR resulted in the sustained expression of STAT1. PKR interacts with STAT1 and PKR-STAT1 complexes interfere with the transactivation capacity"

"PKR inhibition sustains STAT expression, which results in the inhibition of chondrogenesis."

Wednesday, November 25, 2009

Growth Taller via Autophagy?

Autophagy may be a way to resist aging related degenerations.  Autophagy, unlike apoptosis, allows for recycling of cellular components so if all the hypertrophic cells in the growth plate underwent autophagy over apoptosis it may be possible to grow taller indefinitely.

Regulation of autophagy in human and murine cartilage: hypoxia-inducible factor 2 suppresses chondrocyte autophagy.

"HIF-2 was expressed abundantly by cells in human and murine articular cartilage and in the cartilage of mineralizing vertebrae from neonatal mice. Protein levels were reduced in articular cartilage from older mice, in end-plate cartilage from mice, and in chondrocytes from human osteoarthritic (OA) cartilage. HIF-2 was robustly expressed in the prehypertrophic cells of mouse growth cartilage. When HIF-2alpha was silenced, the generation of reactive oxygen species was found to be elevated, with a concomitant decrease in catalase and superoxide dismutase activity. Suppression of HIF-2 was associated with decreased Akt-1{pAkt was decreased as well} and mammalian target of rapamycin activities, reduced Bcl-xL expression, and a robust autophagic response, even under nutrient-replete conditions. In these silenced chondrocytes, HIF-1 expression was elevated. Decreased HIF-2 expression was associated with autophagy in OA tissues and aging cartilage samples. The autophagic response of chondrocytes in HIF-2alpha-knockout mouse growth plate showed an elevated autophagic response throughout the plate.
HIF-2 is a potent regulator of autophagy in maturing chondrocytes. HIF-2 acts as a brake on the autophagy-accelerator function of HIF-1."

"The maturing cells in [growth plate] cartilage undergo a series of phenotypic changes which include secretion of a unique sets of proteins into the avascular extracellular matrix, up-regulation of alkaline phosphatase and the release and subsequent mineralization of matrix vesicles. Prior to deletion from the plate, the mature hypertrophic chondrocyte becomes glycolytic and undergoes functional and immunohistochemical changes that are characteristic of autophagy. HIF-1, a transcription factor that responds to the tissue oxemic state, promotes chondrocyte autophagy"

"upregulation of HIF-2 lowers intracellular ROS levels by promoting the activities of the dismutating proteins, catalase and superoxide dismutase. The observation that HIF-2 knockout animals are small suggests that there may be an increased rate of chondrocyte apoptosis that serves to impede normal long bone growth."<-so increased autophagy may not increase height however this may be due to other effects like reduced Akt and MTor.

"When we compared neonatal cartilage in the mineralizing vertebrae with end plate cartilage of older mice a dramatic decrease in HIF-2α expression is seen."

HIF-2 reduces reactive oxygen species.  It's possible that autophagy results in the recycling of everything including things that would reduce height growth.

Oxygen may reduce HIF-2 levels providing the signal for epiphyseal mineralization.

"TOR plays a critical role in autophagy serving as the sensor that integrates metabolic and growth factor signals.  Terminal chondrocytes exhibit autophagic characteristics. Morphologically, the arrested cells contain double membrane vacuoles; there is a loss of membrane structure, limited staining and organelle destruction. Since the life history of the growth plate chondrocyte is very short, even minor disturbances in the metabolic state can result in gross impairment of growth. The induction of the autophagic response, permits the terminally differentiated cells to survive the brief rigors of the harsh local microenvironment."

"in the maturing zone of the growth plate, the central core of cells are most removed from oxygen and may be depleted of growth factors and critical nutrients.  In this hypoxic microenvironment, gradients in nutrients and specific growth factors would be countered to some extent by upregulation of ATP generation through the glycolytic pathway.  The loss of growth factor signaling and nutrient stress would suppress mTOR
and activate autophagy."

The life history of the growth plate chondrocyte is 1-2 days.

"Chondrocytes [express] the energy sensor AMPK-1 and [activation increases] with maturation. Thapsigargin treatment activated AMPK and autophagy in a HIF-1-dependent manner. Using serum-starved AMPK-silenced cells, we demonstrated that AMPK was required for the induction of the autophagic response. We also noted a change in chondrocyte sensitivity to apoptogens, due to activation of caspase-8 and cleavage and activation of the pro-apoptotic protein, BID. To test the hypothesis that AMPK signaling directly promoted autophagy, we inhibited AMPK activity in mTOR silenced cells and showed that while mTOR suppression induced autophagy, AMPK inhibition did not block this activity. Because of the micro-environmental changes experienced by the chondrocyte, autophagy is activated by AMPK in a HIF-1-dependent manner."

"AMPK activity is responsive to hypoxia and hyperosmotic stress. The kinase is sensitive to the AMP/ATP ratio: binding of AMP activates AMPKand induces phosphorylation by the tumor suppressor LKB1. When bound, AMP inhibits it’s dephosphorylation by Protein Phosphatase 2C. AMPK functions as an energy sensor that triggers catabolic pathways that produce ATP, while inhibiting energy-consuming anabolic activities."

"autophagy is stimulated by HIF-1 and that in concert with chondrocyte maturation, there is a profound decrease in ATP and a concomitant rise in AMP, key regulators of AMPK"

Mice growth plate cartilage cells were used.

"epiphyseal chondrocytes express AMPKa1".  AMPKa2 is expressed by osteoblasts but not chondrocytes.

"Thapsigargin is an inhibitor of sarco/endoplasmic reticulum Ca2+ ATPases (SERCA). It raises the cytosolic calcium concentration by blocking the ability of the cell to pump calcium into the sarcoplasmic and endoplasmic reticula (ER; as a result, these stores become depleted, resulting in ER stress and ultimately cell death. In a number of tissues, AMPK is activated both by a decrease in energy charge and by an increase in intracellular calcium flux."

"AMPK regulates the induction of autophagy by modulating the association between Bcl-2 and Beclin-1"

"AMPK suppression leads to a significant activation of Akt-1."

"While AMPKa1 positive cells were seen throughout the growth plate, the most densely stained cells were localized to the pre-hypertrophic and hypertrophic regions of the epiphysis. Remarkably, the location of the maximally positive chondrocytes was similar to that described for both Beclin-1 and LC-3, two major indicators of autophagic vacuole formation"

" when HIF-1 was silenced, AMPK activation was blocked; in contrast, there robust AMPK activation when control chondrocytes were treated with thapsigargin."

"autophagy can serve to delay the onset of apoptosis"

How does Salubrinal affect autophagy?

Endoplasmic reticulum stress triggers autophagy in malignant glioma cells undergoing cyclosporine A-induced cell death.

"Autophagy is a conserved, self-digestion process that is activated in response to nutrient limitation but acting also as an alternative death mechanism under certain conditions. It is accompanied by the progressive formation of vesicle structures from autophagosomes to autophagolysosomes orchestrated by autophagy effectors (Atg proteins) and modulators (that is, mTOR-mammalian target of rapamycin as a negative regulator). We demonstrate that cyclosporine A (CsA, an immunophilin/calcineurin inhibitor) induces cell death with some apoptotic features but also accompanied by the appearance of numerous cytoplasmic vacuoles, immunostained for endoplasmic reticulum (ER) and autophagy markers. The induction of ER stress in glioma cells by CsA was evidenced by detection of unfolded protein response activation (phosphorylation of PERK, accumulation of IRE1α) and accumulation of ER stress-associated proteins (BIP and CHOP). Formation of the acidic vesicular organelles, increase of autophagic vacuoles, GFP-LC3 punctation (microtubule-associated protein light chain 3) and LC3-II accumulation upon CsA treatment confirmed activation of autophagy. Decrease of phosphorylation of 4E-BP1, p70S6K1 and its downstream target S6 ribosomal protein demonstrate inhibition of mTOR signaling by CsA. Salubrinal and silencing of PERK and IRE1α partially blocked CsA-induced accumulation of LC3-II{So Salubrinal may inhibit autophagy}. It suggests that ER stress precedes CsA-induced autophagy. Surprisingly, silencing of autophagy effectors ULK1, Atg5 or Atg7 increased the level of active caspases 3, 7 and PARP degradation in CsA-treated cells. CsA induces both apoptosis and autophagy in malignant glioma cells via induction of ER stress and inhibition of mTOR/p70S6K1 pathway, however autophagy is cytoprotective in this context."

"In mammalian cells, PERK (protein kinase-like endoplasmic reticulum kinase), IRE1 (inositol-requiring enzyme 1) and ATF6 (activating transcription factor 6) sense accumulation of unfolded proteins in the ER lumen and activate specific cellular response. PERK phosphorylates the α subunit of the translation initiation factor, eIF2α, which attenuates general protein synthesis and initiates the selective translation of some stress-responsive transcripts. IRE1 is a serine/threonine kinase and also an endoribonuclease, which catalyzes the splicing of the mRNA encoding the transcription factor XBP-1 (X-box binding protein 1). PERK mediates transcriptional activation of LC3 and Atg5 proteins under hypoxic conditions via the transcription factors ATF4 (activating transcription factor 4) and CHOP (C/EBP homologous protein), respectively."

According to Effects of salubrinal on cadmium-induced apoptosis in HK-2 human renal proximal tubular cells., Salubrinal protected from apoptosis but not autophagy.  This may be due to the fact that in this study Salubrinal did not increase ATF4 levels.

"Apoptosis signal-regulating kinase 1 (ASK1) is activated by various types of stress, including, endoplasmic reticulum (ER) stress. ASK1 executes apoptosis and/or autophagy under ER stress. [We used] SH-SY5Y cells overexpressing wild-type (WT) ASK1. We show an important autophagic response and an acceleration of the paraquat (PQ)-induced autophagy with hallmarks as accumulation of autophagic vacuoles, activation of beclin-1, accumulation of LC3 II, p62 degradation, and mammalian target of rapamycin dephosphorylation. Inhibition of autophagy caused an exacerbation of the apoptosis induced by WT ASK1 overexpression{So the question is which would increase height more autophagy or apoptosis?} with or without PQ.  Autophagic response could have a protector role. We found an increase in the phosphorylation of the proteins such as IRE1 and eIF2α in response to both the overexpression of WT ASK1 and pesticide exposure.WT ASK1 overexpression-induced autophagy is an event that occurs in parallel with ER stress activation. The importance of ER stress in the autophagy induced by ASK1 and/or PQ was confirmed with salubrinal, a selective inhibitor of eIF2α dephosphorylation. PQ induces an early ER stress response that is correlated with the activation of autophagy as a protective response, which is accelerated in cells that overexpress WT ASK1."

"Apoptosis signal-regulating kinase 1 (ASK1) is a member of the mitogen-activated protein kinase family (MAP3K) that activates the c-jun N-terminal kinase (JNK) and p38 pathways by directly phosphorylating, and thereby activating, their respective MAP2Ks (also called mitogen activated kinase kinase [MKK]), MKK4(SEK1)/MKK7, and MKK3/MKK6"

"preincubation with [salubrinal] rescued cells from the apoptotic event (release of cytochrome c from mitochondria into the cytosol, caspase-3 activation, and nuclear fragmentation) induced by exposure to PQ, the overexpression of WT ASK1, and the combination of both"

Misc Stem Cell types to chondrocytes

Chondrogenic Differentiation of Amniotic Fluid Stem Cells and Their Potential for Regenerative Therapy.

"Chondrocytes constituting articular cartilage show some functional and structural differences from growth plate chondrocytes. Whereas the latter undergo highly dynamic changes as long bone growth persists, articular cartilage chondocytes provide continuous function throughout life. Articular chondrocytes originate from a pool of growth differentiation factor 5 (GDF5) positive progenitor cells and that growth plate chondrocytes are derived from GDF5 negative cell populations"

"GDF5 expression is first evident in the mesenchymal interzone of early joints. The interzone is an essential regulator region and it has been widely assumed that it is responsible for formation of joint structures including articular cartilage, ligaments and synovial lining. Interzone cells express a number of genes able to control joint development, like: GDF5, WNT5A, WNT4, GLI3{up in LSJL}, CD44, ERG{up} and NOGGIN. GDF5 has chondrogenic activity, whereas WNT ligands and NOGGIN are anti-chondrogenic"

"Joint cavitation involves differential hyaluronan synthesis under the influence of mechanical stimuli. The hyaluronan receptor, CD44, is expressed at the joint interzone and at developing articular surfaces and it induces cell adhesion as well as cell separation, depending on the concentration of hyaluronan"

"Mature articular cartilage consists of three zones: the superficial zone, where small and flat chondrocytes reside and collagen fibrils are orientated parallel to the surface, the transitional zone, representing the thickest part of cartilage, where collagen fibers are less organized, and the radial zone, where fibers are orientated perpendicular to the surface and oxygen tension is below 1 %. Underneath is the tidemark, which delineates hyaline articular cartilage from calcified cartilage."

" Upon hypertrophy, growth plate chondrocytes downregulate SOX9 and type II collagen expression, whereas RUNX2, type X collagen, alkaline phosphatase and VEGFA are upregulated"

"[Amniotic Fluid Stem Cells] can be expanded in cell culture and show expression of CD29, CD44, CD73, CD90, CD105 and SSEA4 with over 90 % of cells being positive for OCT4"

"supplementation of chondrogenesis promoting factors is necessary for successful manipulation of AFS cells and that the isolation of c-kit positive cells also enhances the differentiation process."

"GDF5 over-expression results in cartilage overgrowth and joint fusion in vivo"

"WNT-4, WNT-14, WNT-16 and the WNT signaling mediator β-catenin also show anti-chondrogenic activity during joint formation"

"up regulation of HIF1α (hypoxia-inducible factor) and HIF2α is essential for SOX9 expression"

"In articular cartilage autophagy plays an important role, since it protects chondrocytes from age related cell death and preserves homeostasis within the tissue{perhaps in growth plate chondrochytes as well}. During aging-related intracellular changes, biochemical stimuli as well as mechanical stress can lead to OA and during this process the competence to undergo autophagy is lost"<-mTor is involved in the regulation of growth plate autophagy.  Proteoglycan sulfation can determine the efficience of chondrocyte autophagy.

"The induction of autophagy is able to prevent ageing related degenerations."<-maybe autophagy can prevent epiphyseal fusion since epiphyseal fusion is an aging related degeneration.

Saturday, November 21, 2009

Analysis of Common Height Increase Methods

Hypnosis-You have to denature the bone in order to gain height. Hypnosis doesn't do that.  It is possible that if hypnosis increases the amount of growth hormone in your body for your to grow taller via greater periosteal width of short and irregular bones.  Also, if you are hypnotized while laying on your back you can gain temporary height by temporarily decompressing the spine(laying on your back results in the height gain not the hypnosis).

Stretching-Stretching usually affects the muscle not the bone(perhaps the bone in a minor way). You can have the longest muscles in the world but they will look stocky because they are resting on short bones.  You need to apply stretching forces on your bones for example by putting weights on the ends of the bones and longitudinally stretching them.

MMA, Boxing, Football-There have been reports of people having fractures of bones and denaturing the bone during the healing and thus being taller. However, their are risks in these sports, it's the physical act of being tackled in football that causes the fractures needed to denature the bone and there is no guarantee that the damage to yourself will not be too severe.

As for boxing/kickboxing, the opponents are not aiming for your bones. They are aiming for your liver or your brain.  Heavy bag punching may increase your height and hand size.  Height via the twisting forces that are applied by throwing a proper punch causing shearing forces on the spine thereby increasing periosteal width.  Hand size as a result of shearing forces along the carpal bones.

Pitching-It's been documented that a pitchers throwing arm is about three inches longer than his other arm. Pitching puts spiral forces on the bone. Now people have retorted that having one longer arm than the other may cause people to be better pitchers but since I don't have access to pitchers before and after there's nothing I can do to prove it. But say that pitching can improve height, if you can get that significant denaturing of bone with the spiral forces of pitching imagine how much you can get if you add the other three forces.

Chiropractor/Rolfing-I have done both of these and each resulted in a temporary half an inch height gain.  It went away very quickly however and both can be expensive.  Self performed chiropractic and massage techniques tend to be less effective.  They both work as a result of increasing body alignment.  They are only temporary measures and don't significantly affect the bone length.

Sky of's method of causing microfractures via sprinting with ankle weights and then sitting with ankle weights-I think this method is correct in theory but sitting with ankle weights does not provide enough distraction(stretching) forces on the bone.  The theory of stretching the bone coupled with microfractures is what I have based the majority of my height increase research on.  The best way to stretch the bone is to put a weight at the end of the bone to stretch the bone throughout it's length not to sit with ankle weights.

Growth Hormone-An increase in growth hormone levels in children without a growth hormone mutation has only been shown to increase growth rate not final bone length.  Growth Hormone may effectively increase periosteal width however without any growth hormone mutation.

Diet-This is a whole bag of worms.  Certain proteins are necessary for growth which may explain why in certain decades individuals were shorter.  Growth hormone in meat products may result in individuals with longer torsos due to an increase in bone width(bone width increases by the same mechanism as bone length in short and irregular bones).  Chemical factors affect bone growth but chemical factors that could increase height are subject to negative feedback mechanisms whereas mechanical factors that could increase height are better able to get around negative feedback(if a bone is broken it's going to heal negative feedback or no negative feedback).  Chemical factors that reduce height are not as susceptible to positive feedback mechanisms so it may be worthwhile to make sure you have all your nutrition in check.


Tacrolimus is available by prescription only.

Predominant promotion by tacrolimus of chondrogenic differentiation to proliferating chondrocytes.

"Tacrolimus (FK506) has been used as a therapeutic drug beneficial for the treatment of rheumatoid arthritis in humans. In this study, we investigated the effects of FK506 on cellular differentiation in cultured chondrogenic cells. Culture with FK506 led to a significant and concentration-dependent increase in Alcian blue staining for matrix proteoglycan at 0.1 to 1,000 ng/ml, but not in alkaline phosphatase (ALP) activity, in ATDC5 cells, a mouse pre-chondrogenic cell line, cultured for 7 to 28 days, while the non-steroidal anti-inflammatory drug indomethacin significantly decreased Alcian blue staining in a concentration-dependent manner, without altering ALP activity. FK506 significantly increased the expression of mRNA for both type II and type X collagen, but not for osteopontin, in ATDC5 cells. Similar promotion was seen in chondrogenic differentiation in both mouse metatarsals and chondrocytes cultured with FK506. However, FK506 failed to significantly affect transcriptional activity of the reporter construct for either sry-type HMG box 9 (Sox9) or runt-related transcription factor-2 (Runx2), which are both transcription factors responsible for chondrocytic maturation as a master regulator. These results suggest that FK506 may predominantly promote cellular differentiation into proliferating chondrocytes through a mechanism not relevant to the transactivation by either Sox9 or Runx2 in chondrogenic cells."

"FK506 preferentially inhibits the production of inflammatory cytokines, such as tumor necrosis factor (TNF)-α and interleukin (IL)-1β, through suppression of T-cell activation in human peripheral blood mononuclear cells, without markedly affecting proliferation and differentiation of bone marrow cells"

"The immunosuppressant drugs FK506 and cyclosporine A inhibit the activity of the ubiquitously expressed Ca2+/calmodulin–dependent protein phosphatase, calcineurin, through an interaction between the calcineurin subunit A and different immunophilins such as FK506-binding protein for FK506 and cyclophilins for cyclosporine A, which consequently leads to the inhibition of calcineurin-mediated dephosphorylation of the nuclear factor of activated T cells in T-lymphocyte cells"

"In chondrogenic ATDC5 cells, chondrogenic differentiation is promoted by FK506, but not by cyclosporine A, in terms of matrix proteoglycan production and type II collagen (Col II) expression."

Insulin increased proteoglycan matrix accumulation by ATDC5 cells.

"FK506 would predominantly promote cellular differentiation from pre-chondrocytes to proliferating chondrocytes, without markedly altering that from proliferating to hypertrophic chondrocytes or from hypertrophic to calcified chondrocytes, in cultured metatarsals ex vivo." See Figure 9 for a great graphic explaining the different stages of chondrogenic differentiation.

The immunosuppressant FK506 promotes development of the chondrogenic phenotype in human synovial stromal cells via modulation of the Smad signaling pathway.

"hSSCs were isolated from synovium of the knee joint and 2x10(5) cells were subjected to pellet culture in chondrogenic culture medium for 3 weeks with or without growth factors [bone morphogenetic protein 2 (BMP2) or transforming growth factor beta(1) (TGFbeta(1))] and +/- addition of FK506 in chondrogenic culture media was evaluated. Chondrogenesis was assessed by the size of the pellet, the production of proteoglycans, and messenger RNA (mRNA) levels for chondrogenic markers. Furthermore, levels and intracellular location of phosphorylated Smad proteins related to BMP signaling and TGFbeta signaling were evaluated following exposure to FK506.
FK506 enhanced the differentiation of hSSCs toward a chondrogenic phenotype in a dose-dependent manner associated with increases in glycosaminoglycan synthesis and increased mRNA levels for chondrogenic marker genes. Additionally, FK506 further enhanced chondrogenesis of synovial stromal cells (SSCs) induced by BMP2 and TGFbeta(1), also in a dose-dependent manner. Notably, phosphorylation of Smad1/5/8 and Smad3 was significantly increased by FK506. Also, the ratio of nuclear translocation to cytoplasmic levels of phosphorylated Smad1/5/8 and Smad3 were increased following exposure of SSCs to FK506. Moreover, inhibition of Smad signaling significantly abrogated FK506-induced chondrogenic differentiation of SSCs."

"The immunosuppressive effect of FK506 is believed to be related to its ability to inhibit calcineurin, an enzyme involved in the activation of the nuclear factor of activated T cells (NFAT)"<-inhibiting calcineurin may help make you taller.  Although some NFATs seem to pro-chondrogenic whereas others are anti-.

"SSCs were isolated from human synovium which was harvested from the knee joint of four patients during arthroscopic surgery"<-Ages 19 to 57.

"To assess chondrogenic differentiation, a pellet culture system was used24. Approximately 2 × 105 cells (passages 3–5) were placed in a 15-ml polypropylene tube, and centrifuged at 500g for 10 min. The pellets were cultured at 37°C with 5% CO2 in 500 μl of chondrogenic culture medium that contained HG-DMEM with 10% FBS, supplemented with 50 μg/ml ascorbate-2-phosphate, 100 μg/ml sodium pyruvate, and 50 mg/ml insulin, transferrin, and selenious acid (ITS) + Premix"

Inhibition of the ERK, PI3K, and the p38 pathways did not affect Tacrolimus' enhancement of chondrogenesis.


Human skeletal dysplasia caused by a constitutive activated transient receptor potential vanilloid 4 (TRPV4) cation channel mutation.

"The transient receptor potential vanilloid 4 (TRPV4) cation channel, a member of the TRP vanilloid subfamily, is expressed in a broad range of tissues where it participates in the generation of Ca2+ signals and/or depolarization of the membrane potential. Regulation of TRPV4 abundance at the cell surface is critical for osmo- and mechanotransduction. Defects in TRPV4 are the cause of several human diseases, including brachyolmia type 3; also known as brachyrachia or spondylometaphyseal dysplasia Kozlowski type, and metatropic dysplasia; also called metatropic dwarfism or parastremmatic dwarfism. These bone dysplasia mutants are characterized by severe dwarfism, kyphoscoliosis, distortion and bowing of the extremities, and contractures of the large joints. These diseases are characterized by a combination of decreased bone density, bowing of the long bones, platyspondyly, and striking irregularities of endochondral ossification with areas of calcific stippling and streaking in radiolucent epiphyses, metaphyses, and apophyses. We emphasize how the constitutive active TRPV4 mutant affects endochondral ossification with a reduced number of hypertrophic chondrocytes and the presence of cartilage islands within the zone of primary mineralization."

I couldn't find this whole paper anywhere.

Sunday, November 15, 2009

Why it is necessary to couple muscle building with bone growth in many grow taller methods

For several methods to build bone and the methods applicable for growing taller it's almost impossible to build up bone without building some muscle up as well. It's possible, to provide the forces needed to build bone without weight via "tapping"(hitting the bone with weights or a hammer) or limb lengthening surgery but then you aren't using all the possible forces on bone you can to cause microfractures(compression, strain, spiral, and impact). And in any case many of the bones are "blocked" by muscle in order to tap the bone you also have to tap the muscle as well(tapping is a bit of an underhyperbole as you want to tap as hard as you can without causing nerve damage).

Now, via Lateral Synovial Joint Loading you are putting load on the ends of the bones lateral to the joint area to provide a stretching force along the entire bone.  But, you need muscle in order to control the weight that you're using to provide the downward forces or you need muscle to help protect your tendons and ligaments from injury if it's an external force providing a stretch with downward pressure on the ends of the bones.

In addition, it is also possible to grow taller via an increase in periosteal width in the short and irregular bones.  You need to use weight to create the shearing forces on the short and irregular bones to increase the periosteal width and you need muscle to stabilize your body so you don't, for example, herniate a disc due to a lack of support.

Also, another method for growing taller is via knee microfracture(and microfracture in other areas) in order to release stem cells that exist within bone marrow that stimulate cartilage growth when provided with blood.  The more muscle you have the more force you can generate to create knee microfractures.

In regards to nerve damage, yes their are nerves that are inside the bone but it doesn't necessarily mean you are damaging them. Imagine a wall with electrical wires inside of it, if you bang on the wall you only hurt the wall and not the electrical cables inside of it. The skeletal tissue of the bone will absorb the impact. It's important that you do any tapping yourself, the punch that hurts the most is the one you don't see coming as they say and if you tap your own bones you learn how hard you can tap safely.

Let's address two possible ways to provide forces on bone: Strain fractures via hanging and compression fractures by holding a weight on your back (i.e. in a squat position). If you try to take a weight that you can't handle the golgi tendon will send a signal to your muscle telling it to relax(this is the basis behind massage) and you will not be able to do the "exercise" for very long and you won't cause very many microfractures. You need to do an exercise with a weight that your body will allow you to handle(sometimes this can be quite heavy) so you can continue to progress in weight and cause more microfractures which are necessary to grow via the articular cartilage method and may be necessary to grow taller via distraction forces on the long bones.

We want to try to maximize the forces we place on the bone when we perform exercises designed to increase their size. So, for example when he hang we should also twirl around which generates spiral and shearing forces on the irregular bones of the spine. And, when we perform a walkout in the squat rack to generate compression forces on the irregular bones we should also stomp to provide additional impact and shearing forces on the knee and the bones of the spine.

If your muscles aren't strong enough for the golgi tendon to allow them to go, you'll only be able to perform the exercises for 2 to 3 seconds rather than the perhaps 20 if your muscles(and CNS) are strong enough to allow the exercises to be fully performed. In addition, your muscles(and CNS) won't adapt and you won't be able to move on to higher weights.

Muscular conditioning is also important. You need to train frequently enough so your muscles adapt and are harder to damage thus more of the forces you want can be targeted to the bone and less to the muscle. We want inclusion of bone not exclusion of muscle. There is no such thing as an "isolation" exercise.

We are just trying to specialize our training to gain height rather than excluding forces that damage the muscle entirely.

It's not an LSJL lengthening study but it still provides insights into LSJL and it might help provide more insight into how to perform LSJL.  Although the loads are axial loads rather than lateral loads.  Also, remember that providing pressure on the muscle increases hydrostatic pressure in the bone.

Mechanical loading, damping, and load-driven bone formation in mouse tibiae.
"Loading can activate anabolic genes.  We investigated the damping capacity of bone, joint tissue, muscle, and skin using a mouse hindlimb model of enhanced loading in conjunction with finite element modeling to model bone curvature. Our hypothesis was that loads were primarily absorbed by the joints and muscle tissue, but that bone also contributed to damping through its compression and natural bending. To test this hypothesis, fresh mouse distal lower limb segments were cyclically loaded in axial compression in sequential bouts, with each subsequent bout having less surrounding tissue. A finite element model was generated to model effects of bone curvature in silico. Two damping-related parameters (phase shift angle and energy loss) were determined from the output of the loading experiments. Interestingly, the experimental results revealed that the knee joint contributed to the largest portion of the damping capacity of the limb, and bone itself accounted for approximately 38% of the total phase shift angle. Computational results showed that normal bone curvature enhanced the damping capacity of the bone by approximately 40%, and the damping effect grew at an accelerated pace as curvature was increased. Although structural curvature reduces critical loads for buckling in beam theory, evolution apparently favors maintaining curvature in the tibia. Histomorphometric analysis of the tibia revealed that in response to axial loading, bone formation was significantly enhanced in the regions that were predicted to receive a curvature-induced bending moment[maybe there's less damping effect for lateral rather than axial loads which could be why lateral loads induce different gene expression]."

"Damping is the potential of the whole musculoskeletal system to effectively dissipate energy associated with loading."

"Loads of 5 N at 0.5, 2, and 20 Hz were applied to this FE model for 10 s"

"The ratio between the phase shift angle of stage V (bone alone) and stage I (intact hindlimb) demonstrated the influence of the tibia alone. This ratio was 43% at 0.5 Hz, 38% at 2 Hz, and 28% at 20 Hz. Removal of the ankle, skin, and muscle exhibited the smallest effects on phase shift angle, contributing to approximately 14% of the total phase shift at each frequency."<-So most of the load absorption is by the bone.

"While similar trends in energy dissipation are seen in response to 2 and 20 Hz waveforms, the magnitude of the energy loss in each sample configuration decreased monotonously as the frequency was increased."

"Skin was found to contribute to approximately 12% of the total dissipated energy at all frequencies, and the remaining energy was lost with removal of the muscle and foot."

"it has been proposed that the curved shape of long bones, while reducing overall axial strength, increases predictability and better distributes stresses within the bone during loading"<-maybe axial loading  increases bone curvature thereby reducing height?

The role of muscle cells in regulating cartilage matrix production.

"We tested the hypothesis that muscle cells directly regulate cartilage matrix production by analyzing chondrocytes cocultured with muscle cells in 2D or 3D conditions. We found that chondrocytes cultured with C2C12 muscle cells exhibited enhanced alcian blue staining and elevated expression of collagen II and collagen IX proteins. Although nonmuscle cells did not promote cartilage matrix production, converting them into muscle cells enhanced their pro-chondrogenic activity. Furthermore, muscle cell-conditioned medium led to increased cartilage matrix production, suggesting that muscle cells secrete pro-chondrogenic factors. Taken together, our study suggests that muscle cells may play an important role in regulating cartilage gene expression."

"Mouse mutants that lack muscle-specific proteins such as dystrophin/utrophin or myogenin also exhibited skeletal abnormalities such as a curved spine or a reduced size of the skeleton"

"C2C12 muscle cell have an elongated, fibroblast-like morphology, while RCS chondrocytes are rounded in shape. When co-cultured, DiI-positive cells still have the fibroblast-like morphology, while all unlabeled cells maintain a round morphology. This indicates that muscle cells and chondrocytes do not change their morphology upon co-culturing"

"upon co-culturing, muscle cells herd the chondrocytes into clusters and the chondrocytes assume a rounder phenotype. Furthermore, chondrocytes (RCS) co-cultured with mouse C2C12 muscle cells expressed higher levels of collagen II and collagen IX proteins"

"muscle secretes a variety of growth factors or cytokines that can be carried away by blood or interstitial fluid. Among them is the known pro-chondrogenic factor IGF-I"

Bone, muscle, and physical activity: structural equation modeling of relationships and genetic influence with age.

"Quantitative trait locus (QTL) analysis was used to identify regions of chromosomes that simultaneously influenced skeletal mechanics, muscle mass, and/or activity-related behaviors in young and aged B6xD2 second-generation (F(2)) mice of both sexes. SEM was used to further study relationships among select QTLs, bone mechanics, muscle mass, and measures of activity. The SEM approach provided the means to numerically decouple the musculoskeletal effects of mechanical loading from the effects of other physiological processes involved in locomotion and physical activity. Muscle mass was a better predictor of bone mechanics in young females, whereas mechanical loading was a better predictor of bone mechanics in older females. An activity-induced loading factor positively predicted the mechanical behavior of hindlimb bones in older males; contrarily, load-free locomotion (i.e., the remaining effects after removing the effects of loading) negatively predicted bone performance. QTLs on chromosomes 4, 7, and 9 seem to exert some of their influence on bone through actions consistent with Wolff's Law."

Here's a diagram of how muscle may interact with bone:

Although body length is used as an independent predictor.

MyoD1 and IGF1R are two genes mentioned as being associated with muscle and bone.

Saturday, November 14, 2009



"Snail1[or Snai1] gene contributes to FGFR3 receptor signal transduction, which contributes to chondrodysplasias (achondroplasia (ACH), thanatophoric dysplasia (TD) and hypochondroplasia (HCH)). The exemplary embodiments identify Snail1 as a therapeutic and diagnostic target for chondrodysplasia, as well as the use of inhibitors thereof as drugs for the treatment of these diseases."

"Chondrocytes and osteoblasts have a mesenchymal origin, whereas osteoclasts arise from the haematopoietic lineage."

"the long-bone shortening phenotype [of being FGFR3 transgenic] is due to a disorganization and shortening of the proliferative chondrocyte columns and to delayed differentiation. Defects in the proliferative chondrocytes are due to the activation of Stat1, which is responsible for the induction of cell cycle inhibitor p21, and delayed differentiation is due to the activation of the MAPK signaling cascade, which causes a reduction in the area of hypertrophic chondrocytes, in both animal and human models. On the contrary, the de-activation of Fgfr3 in mice causes prolonged endochondral growth, resulting in a "long-bone" phenotype, which is accompanied by an expansion of the proliferative chondrocyte region in the growth plate. All these data give FGFR3 a significant role as a negative regulator of the proliferation of chondrocytes. This inhibition of proliferation by the FGF pathway is unique to chondrocytes and is mediated by transcription factor STAT1, which increases the expression of cell cycle inhibitor p21, the final agent responsible for the interruption of the proliferation induced by this signaling pathway. [p21] levels can be considered to be a reflection of the activation of the FGFR3-mediated signaling pathway in the growth plate. "

"Snail1 [is] a direct repressor of type II collagen, which is characteristic of proliferative chondrocytes, and disappears when the latter cease to proliferate and differentiate into hypertrophic chondrocytes, the cell population that expresses type X collagen. In a completely independent context, it was observed that the presence of Snail attenuated the proliferation of epithelial cells in culture and evolved with an increase in p21 levels and an increase in the phosphorylation of ERK1 and ERK2."

"The use of natiuretic peptide CNP inhibits the FGF-mediated MAPK activation in the growth plate"

"Snail1 is sufficient for FGFR3 signaling in the bone."

"Exemplary inhibitory compounds include, without limitation, antisense oligonucleotides described in, without limitation, US Patent Publication No. US20060003956 and Kajita et al., 2004 or siRNAs that inhibit the expression of Snail1 such as those described in without limitation, Peinado et al., 2005 and Tripathi et al., 2005 all of which are incorporated by reference herein. Additionally, any published nucleotide sequences or those published in the future that inhibit the expression of Snail1 are incorporated as embodiments herein, as potentially useful therapeutic compounds for the preparation of drugs designed to treat a chondrodysplasia process. Gene inhibition techniques and, more specifically, transport of compounds including, without limitation, antisense oligonucleotides, siRNA, ribozymes or aptamers--can be performed using, without limitation, liposomes, nanoparticles or other carriers that increase the success rate of the transfer to the interior of the cell, in one exemplary embodiment the cell nucleus (see, without limitation, Lu and Woodle, 2005 and Hawker and Wooley, 2005 which are incorporated by reference herein). In principle, Snail1 mRNA translation inhibitors can be used which bind both to the encoding region and/or the non-encoding region, for example, in front of the 3' non-encoding area. "

Compensatory regulation of the Snai1 and Snai2 genes during chondrogenesis.

"Several genes involved in cartilage and bone development have been identified as target genes for the Snail family of zinc finger transcriptional repressors, and a gain-of-function study has demonstrated that upregulation of Snai1 activity in mouse long bones caused a reduction in bone length. The Snai1 and Snai2[also known as slug] genes function redundantly during embryonic long bone development in mice. Deletion of the Snai2 gene, or limb bud-specific conditional deletion of the Snai1 gene, did not result in obvious defects in the skeleton. However, limb bud-specific Snai1 deletion on a Snai2 null genetic background resulted in substantial defects in the long bones of the limbs. Long bones of the Snai1/Snai2 double mutants exhibited defects in chondrocyte morphology and organization, inhibited trabecular bone formation and delayed ossification. Chondrocyte proliferation was markedly reduced, and transcript levels of genes encoding cell cycle regulators, such as p21(Waf1/Cip1) , were strikingly upregulated in the Snai1/Snai2 double mutants, suggesting that during chondrogenesis Snail family proteins act to control cell proliferation by mediating expression of cell cycle regulators. Snai2 transcript levels were increased in Snai1 mutant femurs, while Snai1 transcript levels were increased in Snai2 mutant femurs. In addition, in the mutant femurs the Snai1 and Snai2 genes compensated for each other's loss not only quantitatively, but also by expanding their expression into the other genes' normal expression domains. The Snai1 and Snai2 genes transcriptionally compensate temporally, spatially, and quantitatively for each other's loss, and demonstrate an essential role for Snail family genes during chondrogenesis in mice."

"mean bone length of femurs of the Snai1/Snai2 DM embryos was reduced approximately 20%, whether the measurements were taken of total bone length, or the length of trabecular bone"

"in Snai1/Snai2 DM growth plates the pattern of well-aligned columnar chondrocytes was disorganized, and chondrocyte morphology was altered. Proliferating chondrocytes in Snai1/Snai2 DM growth plates were more compact, and exhibited an elliptical shape with a higher chondrocyte to lacuna ratio"

"statistically significant increase in the length of the hypertrophic chondrocyte zone in Snai1/Snai2 DM growth plates"

"Snai1 and Snai2 genes are required to maintain the high rate of chondrocyte proliferation in the rapidly growing long bone"

"Cdkn1a RNA expression was approximately six-fold higher in Snai1/Snai2 DM femurs than in littermate controls"

"several markers were upregulated in Snai1/Snai2 DM femurs, including Col1a1, Col2a1, Col10a1, Sox9, and Acan"<-all these genes were upregulated by LSJL.

"genes encoding the matrix metalloproteases Mmp9 and Mmp13 were downregulated"

"Progression into chondrocyte prehypertropy and hypertrophy appeared to be delayed in Snai1/Snai2 DM growth plates. At E14.5, expression of both Col10a1, a hypertrophic chondrocyte marker, and Indian hedgehog (Ihh), a prehypertrophic chondrocyte marker, was markedly delayed in double mutant femurs"

"Blood vessel formation and penetration into the trabecular region of the femur was observed in both double mutant and controls"

"Despite the increase in Trp53 expression [in double mutants], we did not observe increased chondrocyte cell death in these embryos"

Snai1 mutant knockout mice had a slight non-signficiant increase in bone length.  At E16.5, Snai mutant knockout mice had a slight non-significant increase in Col10A1 and MMP9 levels.