Tuesday, March 27, 2012

Grow taller with open-wedge osteomies

Osteomies are sometimes used to lengthen or shorten a bone but other times the bone is cut just to adjust it's alignment.  This would seem to be similar to limb lengthening surgery however unlike limb lengthening surgery the bone is not cut completely and the bone is not forcibly held apart.

Change in limb length after high tibial osteotomy using computer-assisted surgery: a comparative study of closed- and open-wedge osteotomies.

"Limb length changes were evaluated after closed- and open-wedge high tibial osteotomies (HTOs) using computer-assisted surgery.
Closed- and open-wedge HTOs were performed. The changes in limb length were evaluated on a navigation system and radiographs. The correction angle was defined as the difference between the pre and postoperative mechanical axis on the navigation system. The change in limb length with respect to the correction angle was analyzed.
Following the closed-wedge HTOs, the mean changes in limb length based on the navigation system and radiographs were -1.3 ± 1.9 and -1.3 ± 10.7 mm, respectively, versus 6.2 ± 2.6 and 7.8 ± 2.9 mm after the open-wedge HTOs. The mean correction angle was 11.6 ± 3.2° for closed-wedge HTOs and 11.5 ± 1.9° for open-wedge HTOs. The correction angle did not affect the change in limb length after closed-wedge HTO, while the larger the correction angle required, the greater the increase in limb length after open-wedge HTO.
The change in limb length was negligible after closed-wedge HTO, while the limb length was increased slightly after open-wedge HTO. The possibility of limb lengthening must be considered carefully when determining whom to perform open-wedge HTO on, especially when a large correction angle is required."

In a closed wedge osteomy, a piece of the bone is removed and medal plates or pins are used to pin the bone together.  In open-wedge osteomy, the surgeon cuts open a wedge and puts in a bone graft to hold the wedge open.  Now unlike limb lengthening surgery the bone is not fractured completely.  The force of growth in the bone graft must be such to cause expansion of the bone to a slightly longer length.  The bone must after all grow into and within the bone graft.  All the mechanical reactions required to do this may produce force that causes overall bone expansion.

Unlike limb lengthening surgery where the bone is held out at a longer length, the only stimulation to increase length is the force of bone growing in and around the wedge itself.  Thus a force inside the bone like LSJL induced growth plates may be able to cause expansion in the bone increasing length.  There is no cut like with open-wedge osteomy but there is already a wedge that exists called the epiphyseal bone marrow. LSJL induced chondrogenesis or even osteogenesis may produce that wedge within the epiphyseal bone marrow.  An osteogenic wedge though may only produce the millimeters of height gain reported in this study.  A chondrogenic wedge may produce more height gain.

Tuesday, March 13, 2012

Gain some height with ATF4?

ATF4 induces endogenous IHH expression.  And Ihh(with FGF-2) has been shown to be an inducer of human mesenchymal chondrogenesis. Therefore, ATF4 may be an inducer of human mesenchymal chondrogenesis.  Salubrinal increases levels of ATF4.

EIF2alpha phosphorylation can increase ATF4 so that maybe a reason to take Salubrinal in conjunction to performing LSJL as LSJL downregulates EIF2alpha phosphorylation.

Concentrated Bovine Milk Whey Active Proteins Facilitate Osteogenesis through Activation of the JNK-ATF4 Pathway., states that Bovine Milk Whey can increase ATF4 levels. Alpha-lactalbumin and beta-lactoglobulin are two proteins that make up a large portion of this milk. Lactalbumin is available for sale: Hydrolyzed Lactalbumin Protein (Pure Whey Protein) 18 oz.  I could not find lacoglobulin.  However, ATF4 is a key molecule linking food intake and skeletal development., found that any high protein diet could rescue ATF4 knockout.

Eukaryotic initiation factor 2 phosphorylation and translational control in metabolism.

"In response to stresses, such as nutrient deprivation and accumulation of misfolded proteins in the endoplasmic reticulum, phosphorylation of the α subunit of eukaryotic initiation factor 2 (eIF2α~P) reduces general translation initiation while facilitating the preferential translation of transcripts that [encode] activating transcription factor 4 (ATF4), a transcriptional activator of genes subject to the integrated stress response (ISR)."

Hedgehog proteins stimulate chondrogenic cell differentiation and cartilage formation.

"Implantation of dermal fibroblasts expressing hedgehog proteins into nude mice[mice with no immune system; triggering IHH expression within the body shouldn't trigger an immune response] induces ectopic cartilage and bone formation. Ectopic tissues derived largely if not exclusively from host cells. Treatment of clonal prechondrogenic RMD-1 and ATDC5 cells in culture with Ihh[The cell lines were already prochondrogenic] or recombinant amino half of Shh (recombinant N-terminal portion of Shh [rShh-N]) induced their differentiation into chondrocytes. Induction of RMD-1 cell differentiation by Ihh or rShh-N was synergistically enhanced by cotreatment with bone morphogenetic protein 2 (BMP-2) but was blocked by cotreatment with fibroblast growth factor 2 (FGF-2)[interesting considering in another study FGF-2 was used in the serum to make the cells prechondrogenic]. ."

In the study, The impaired bone anabolic effect of PTH in the absence of endogenous FGF2 is partially due to reduced ATF4 expression., FGF-2, which LSJL upregulated, increased ATF4 expression so perhaps in this study FGF-2 pushed ATF4 expression above equilibrium but in the other it didn't.

"FGF-4 blocks the induction of chondrogenesis by BMP-4"

Phenoytoin[prescription only] may be a way to increase Ihh levels via ATF4 as well:

Phenytoin stimulates chondrogenic differentiation in mouse clonal chondrogenic EC cells, ATDC5.

"We evaluated the effects of DPH(Phenytoin) on cartilage formation in a model system using ATDC5 cells, a clonal murine chondrogenic cell line. The ATDC5 cells demonstrated enhanced cartilage formation in cultures with DPH. During promoted chondrogenic differentiation, DPH increased the mRNA expression of TGF-beta1, BMP-4, Ihh, and Ptc, in a dose-dependent manner on Days 5 to 15[other epileptic drugs did not have this effect]."

DPH shares a common receptor with glucocorticoids.  DPH may also interact with the thyroid receptor.

According to Inactivation of Patched1 in the mouse limb has novel inhibitory effects on the chondrogenic program., continuous activation of Ihh by inhibiting the hedgehog receptor Ptc-1 inhibited chondrogenesis so there is likely an equilibrium level of Ihh for chondrogenesis.

"a certain level of hedgehog signaling is required for optimal chondrogenesis, pathway activation can lead to inhibition of chondrogenesis in certain contexts."

According to Activating transcription factor 4., hypoxia also upregulates ATF4 so salubrinal may serve to get around a possible inability to generate hypoxia in adult bone.

Transcriptional regulation of bone formation.

"Major transcription factors including Sox9, Runx2, and Osterix, which are active in chondrocytes or osteoblasts or even in both cells, are indispensable for chondrocyte and/or osteoblast differentiation"

"Sox9-overexpressed mice in chondrocytes display chondrodysplasia by the inhibition of chondrocyte proliferation"<-too much Sox9 inhibits Cyclin D1 unless the Sox9 is methylated by CARM1.

The study notes that ATF4 is mainly expressed by osteoblasts.

"Investigations of the Atf4(-/-);Col2a1-Atf4 mouse model, in which Atf4 is selectively overexpressed in chondrocytes in an Atf4-null background, demonstrate that chondrocyte-derived Atf4 regulates osteogenesis during development and bone remodeling postnatally. Atf4 overexpression in chondrocytes of the Atf4(-/-);Col2a1-Atf4 double mutants corrects the reduction in stature and limb in Atf4(-/-) embryos and rectifies the decrease in Ihh expression, Hh signaling, proliferation and accelerated hypertrophy that characterize the Atf4(-/-) developing growth plate cartilages[So ATF4 only needs to be expressed in chondrocytes for bone to develop normally]. This genetic manipulation also restores the expression of osteoblastic marker genes, namely Ocn and bone sialoprotein, in Atf4(-/-) developing bones. In Atf4(-/-);Col2a1-Atf4 adult mice, all the defective bone parameters found in Atf4(-/-) mice, including bone volume, trabecular number and thickness, and bone formation rate, are rescued. In addition, the conditioned media of ex vivo cultures from wild-type or Atf4(-/-);Col2a1-Atf4, but not Atf4(-/-) cartilage, corrects the differentiation defects of Atf4(-/-) bone marrow stromal cells and Ihh-blocking antibody eliminates this effect."

If ATF4 based Ihh induction increased mesenchymal chondrogenesis then it wouldn't be likely that only stimulating ATF4 expression in chondrogenesis would rescue all symptoms of stature. Maybe, however that there's only a certain level of ATF4 required for Ihh to induce chondrogenesis and it didn't matter whether it was specifically in chondrocytes that the ATF4 was produced. Also, maybe Ihh can signal mesenchymal chondrogenesis even from chondrocytes as chondrocytic ATF4 can signal osteoblast differentiation too.

According to Combinatorial control of ATF4-dependent gene transcription in osteoblasts., dimerization with the CEPB family occurs.

Positive regulation by GABAB receptor subunit-1 of chondrogenesis through acceleration of nuclear translocation of activating transcription factor-4.

"In cultured murine costal chondrocytes, mRNA was constitutively expressed for metabotropic GABA(B) receptor subunit-1 (GABA(B)R1), but not for GABA(B)R2. The predominant expression of GABA(B)R1 by prehypertrophic to hypertrophic chondrocytes in tibial sections of newborn mice. The GABA(B)R agonist baclofen failed to significantly affect chondrocytic differentiation while newborn mice knocked out of GABA(B)R1 (KO) showed a decreased body size[length of long bones was significantly shorter whereas short bones were about the same size] and delayed calcification in hyoid bone and forelimb and hindlimb digits. Delayed calcification was also seen in cultured metatarsals from KO mice with a marked reduction of Indian hedgehog (Ihh) expression. Introduction of GABA(B)R1 led to synergistic promotion of the transcriptional activity of activating transcription factor-4 (ATF4) essential for normal chondrogenesis, in addition to facilitating ATF4-dependent Ihh promoter activation. Although immunoreactive ATF4 was negligibly detected in the nucleus of chondrocytes from KO mice, ATF4 expression was again seen in the nucleus and cytoplasm after the retroviral introduction of GABA(B)R1 into cultured chondrocytes from KO mice. In nuclear extracts of KO chondrocytes, a marked decrease was seen in ATF4 DNA binding. GABA(B)R1 positively regulates chondrogenesis through a mechanism relevant to the acceleration of nuclear translocation of ATF4 for Ihh expression in chondrocytes."

So ATF4 may affect height through a GABABR1 and Ihh related mechanism.

"GABABR1 subunit [could] play a role in chondrogenesis through upregulation of Ihh expression"

The eIF2 kinase PERK and the integrated stress response facilitate activation of ATF6 during endoplasmic reticulum stress., states ATF4 is involved in ATF6 activation and that ATF6 deletion does not result in any changes in mouse phenotype.

"Vimentin{LSJL upregulates Vimentin}, a leucine zipper-containing intermediate filament protein, suppresses ATF4-dependent osteocalcin (Ocn){LSJL upregulates Ocn below 2-fold} transcription and osteoblast differentiation. TGFβ inhibits ATF4-dependent activation of Ocn by upregulation of vimentin expression. Osteoblasts lacking Atf4 (Atf4-/-) were less sensitive than wild-type (WT) cells to the inhibition by TGFβ on alkaline phosphatase activity, Ocn transcription and mineralization. The anabolic effect of a monoclonal antibody neutralizing active TGFβ ligands on WT mice was blunted in Atf4-/- mice. ATF4 is required for TGFβ-related suppression of Ocn transcription and osteoblast differentiation in vitro and in vivo. TGFβ did not directly regulate the expression of ATF4; instead, it enhanced the expression of vimentin, a negative regulator of ATF4, at the post-transcriptional level.
Knockdown of endogenous vimentin in 2T3 osteoblasts abolished the inhibition of Ocn transcription by TGFβ, confirming an indirect mechanism by which TGFβ acts through vimentin to suppress ATF4-dependent Ocn activation. Inhibition of PI3K/Akt/mTOR signaling, but not canonical Smad signaling, downstream of TGFβ, blocked TGFβ-induced synthesis of vimentin and inhibited ATF4-dependent Ocn transcription in osteoblasts. TGFβ stimulates vimentin production via PI3K-Akt-mTOR signaling, which leads to suppression of ATF4-dependent Ocn transcription and osteoblast differentiation."

"TGFβ targets Runx2 via a canonical Smad signaling pathway to achieve its inhibition of both Runx2 and Ocn transcription, thereby suppressing osteoblast differentiation"

"Wortmannin, an inhibitor of Akt phosphorylation by PI3K; rapamycin (1 and 5 nM), an inhibitor of S6K phosphorylation by mTOR; and SB505124, an inhibitor of Smad2/3 phosphorylation downstream of the type I TGFβ receptor, were used to treat ROS17/2.8 cells."

"Both wortmannin and rapamycin (1 and 5 nM) drastically reduced vimentin protein expression induced by TGFβ, which correlated with a strong decrease in Akt and S6K phosphorylation." LSJL increases Akt phosphorylation.

"SB505124 (0.1 and 0.2 nM) effectively decreased TGFβ-induced Smad2 phosphorylation but not TGFβ-induced vimentin expression"

"silencing endogenous vimentin by siRNA-Vim attenuated the effects of TGFβ"

Positive regulation by γ-aminobutyric acid B receptor subunit-1 of chondrogenesis through acceleration of nuclear translocation of activating transcription factor-4.

y-aminobutyric acid is also known as GABA.

"In cultured murine costal chondrocytes, mRNA was constitutively expressed for metabotropic GABA(B) receptor subunit-1 (GABA(B)R1), but not for GABA(B)R2. Immunohistochemical analysis revealed the predominant expression of GABA(B)R1 by prehypertrophic to hypertrophic chondrocytes in tibial sections of newborn mice. The GABA(B)R agonist baclofen failed to significantly affect chondrocytic differentiation determined by Alcian blue staining and alkaline phosphatase activity in cultured chondrocytes, whereas newborn mice knocked out of GABA(B)R1 (KO) showed a decreased body size and delayed calcification in hyoid bone and forelimb and hindlimb digits. Delayed calcification was also seen in cultured metatarsals from KO mice with a marked reduction of Indian hedgehog gene (Ihh) expression. Introduction of GABA(B)R1 led to synergistic promotion of the transcriptional activity of activating transcription factor-4 (ATF4) essential for normal chondrogenesis, in addition to facilitating ATF4-dependent Ihh promoter activation. Although immunoreactive ATF4 was negligibly detected in the nucleus of chondrocytes from KO mice, ATF4 expression was again seen in the nucleus and cytoplasm after the retroviral introduction of GABA(B)R1 into cultured chondrocytes from KO mice. In nuclear extracts of KO chondrocytes, a marked decrease was seen in ATF4 DNA binding. GABA(B)R1 positively regulates chondrogenesis through a mechanism relevant to the acceleration of nuclear translocation of ATF4 for Ihh expression in chondrocytes."

"The introduction of ATF4 alone significantly increased the luciferase activity of a reporter vector containing six tandem copies of ATF4 binding site (6XOSE1) and Ihh promoter fragment"

"Abnormal endochondral bone formation and mineralization are shown in mice conditionally deleted of Ihh from Col2a1-expressing cells"

"GABABR1 subunit alone is constitutively expressed by chondrocytes to positively regulate chondrogenesis during skeletal development after acceleration of nuclear translocation of ATF4 toward up-regulation of Ihh expression in a manner irrelevant to the role as a dimeric partner for the canonical functional GABABR composed of both GABABR1 and GABABR2 subunits."

Height with Taurine?

Now Foods Taurine 1000Mg, 100-Capsules

Taurine postponed the replicative senescence of rat bone marrow-derived multipotent stromal cells in vitro.

"Taurine exhibited anti-replicative senescence effect on rat BMSCs by promoting colony forming unit-fibroblast formation and cell proliferation, shortening cell population doubling time, enormously inhibiting senescence-associated beta-galactosidase activity and slowing the loss of differentiation potential, while having no significant effect on the maximum passage number and total culture time, and slight influences on the cell surface CD molecules expressions. Taurine is a quite safe antioxidant and nutrient extensively used in food addition and clinical treatment. Taurine is a promising anti-replicative senescence additive for ex vivo expansion of BMSCs in experimental and clinical cell therapies."

"As a cellular redox-controlling molecule, taurine protect the molecular constituents of cells against reactive species (SP)-induced damages"

"The addition of 100–2,000 mg/l taurine afforded virtually complete protection, in terms of both cell viability and of cell swelling. The maximal effect of taurine on neural progenitor cell proliferation was at 1,000 mg/l, though a significant increase was observed already at 100 mg/l"

Taurine may increase the number of mesenchymal stem cells available for chondrogenic differentiation thus helping you grow taller.

However Taurine may decrease chondrocyte cell volume:

The role of a swelling-activated taurine transport pathway in the regulation of articular chondrocyte volume.

"Swelling articular chondrocytes by reducing osmolarity stimulates a taurine transport pathway, which is implicated in regulatory volume decrease (RVD) in various cell types. The present study investigated factors controlling the activity of this pathway in chondrocytes, in particular (1) the effects of the acute (seconds) and chronic (hours) exposure of chondrocytes to anisotonic media, and (2) whether there is a role for metabolites from the arachidonic acid cascade in activating the taurine transport pathway. For in situ and isolated chondrocytes, the point at which swelling-activated [14C]taurine efflux was stimulated (the "set-point") corresponded closely to the osmolarity of the incubation medium (180, 280 or 380 mosmol/l). However, the volume of chondrocytes isolated into these media and measured by confocal microscopy was not different ( congruent with 645 microm3){so taurine did not reduce volume}. Activity of the swelling-activated taurine transport pathway was inhibited by REV5901 (an inhibitor of steps of the arachidonic acid cascade; K0.5 8+/-4 microM), NDGA (a general lipoxygenase inhibitor; K0.5 28+/-5 microM), or MK886 (an inhibitor of the 5-lipoxygenase-activating protein; 91% inhibition at 10 microM), but weakly by the more potent 5-lipoxygenase inhibitor REV5901 para (K0.5 350+/-100 microM). Addition of the leukotriene (LT) B4 or D4 receptor antagonists, CP-105,696 and L660,711 respectively, or of the leukotrienes LTB4, LTC4, LTD4 and LTE4 or lipoxins (hepoxylin A3 or B3) had no effect on the activity of the pathway in isotonic or hypotonic media. The role of the pathway in RVD was determined in isolated calcein-loaded chondrocytes using fluorescence imaging. RVD was observed and inhibited by REV5901 (50 microM) and by NDGA (75 microM). Despite chronic exposure of chondrocytes to anisotonic media, the cells maintain a pre-determined volume that is the "set-point" for the activation of the taurine transport pathway following acute hypotonic challenge{so the volume of chondrocytes is set by other compounds and not by taurine itself}."

"The proteoglycans surrounding the chondrocytes of articular cartilage carry a high density of immobile negative charges, resulting in high concentrations of free cations (e.g. Na+, 250-350 mM) and low concentrations of free anions(e.g. Cl- 60-90 mM)"

"[Regulatory Volume Decrease] is stimulated following hypotonicity, and leads to osmolyte loss and recovery of cell volume."

"Although the volume of chondrocytes isolated into 180, 280 or 380 mosmol/l media are the same, matrix synthesis rates are very different"

"when osmolarity was altered over 280-480 mosmol/l and matrix synthesis measured using 35SO4 incorporation over 2 h, the maximum rate was 380 mosmol/l, i.e. close to the in situ osmolarity. However, when synthesis was determined over 2 h following 24 h of incubation in 280 mosmol/l, the optimal rate was then at 280 mosmol/l."<-So the optimal matrix synthesis is based on the mean default osmolarity.

Monday, March 12, 2012

Height Growth Genes and Aging

LSJL age related genes altered:


Genes and gene expression modules associated with caloric restriction and aging in the laboratory mouse.

"[Caloric Restriction] increased expression of Tsc22d3{downregulated by LSJL}, Zbtb16, BC055107, Sult1a1, Per1, Timp3, S3-12, Pnpla2, Ppargc1a and Cp, as well as decreased expression of Ifitm3{downregulated by LSJL}, Lasp1, Tia1, 5830428H23Rik, Hsp90b1, Hist1h2bc, Cxcl12, Col6a2{downregulated by LSJL} and Dock4{downregulated by LSJL}"

"CR increased expression of genes encoding the anti-oxidant metallothioneins (Mt1{upregulated in LSJL} and Mt2{upregulated in LSJL}), the NF-κB inhibitor IκBα (Nfkbia), as well as the tumor suppressor P21 (Cdkn1a)"

"CR increased expression of phosphatase and tensin homolog (Pten), which is a tumor suppressor that inhibits IGF-1 signals by preventing activation of the PI3K/Akt signaling pathway"

CR upregulated Sgk1 which is upregulated when the PI3K pathway is inhibited.  Surprisingly, Sgk is upregulated in LSJL.  It should be noted that no caloric restriction analysis was done specifically on the bone, bone marrow, or cartilage.

It should be remembered that mice do have complete growth plate resorption that occurs in humans.

Notable genes upregulated by aging in bone marrow:
Anxa3{downregulated in LSJL}

Notable genes downregulated by aging in bone marrow:
Vim{Upregulated by LSJL}

Age-Related Changes of Chondrogenic Growth Factors in Platelet-Rich Plasma

"PRP[Platelet-Rich Plasma] contains many growth factors that play critical roles in chondrogenesis, including insulin-like growth factor 1 (IGF-1), human growth hormone, transforming growth factor beta 1, basic fibroblast growth factor, and bone morphogenetic proteins 2, 4, and 7. PRP was isolated from 40 healthy volunteers between 20 and 60 years of age, and concentrations of bone morphogenetic proteins 2 and 4, basic fibroblast growth factor, human growth hormone, IGF-1, insulin-like growth factor-binding proteins 2 and 3, platelet-derived growth factor BB (PDGF-BB), transforming growth factor beta 1, and vascular endothelial growth factor were analyzed by enzyme-linked immunosorbent assay. Significant differences with respect to age were detected between subjects less than 30 and >30 years of age for PDGF-BB, insulin-like growth factor-binding protein 3, and IGF-1. A significant difference was also detected between subjects <40 and >40 years of age for PDGF-BB. Concentrations of other growth factors did not vary significantly across age-groups, suggesting that the molecular contents of PRP are similar for patients aged 20-60 years."

"BMP-7 [levels decline] throughout the human aging process"

"Accumulation of cartilage matrix by micromass cultures in vitro is stimulated by IGF-1 but inhibited by IGFBP-2. Elevated expression of IGFBP-3 inhibits the effects of IGF-1 and decreases proteoglycan synthesis. Increased IGFBP-3 levels are associated with degenerative cartilage diseases, induction of mesenchymal stem cell apoptosis, and antagonism of TGF-β1 chondroinductive effects in chondroprogenitor cells."

"PRP contains large quantities of IGFBP-3 (mean concentration 451 ng/mL); however, the mean concentration decreased significantly in patients older than 30 years of age (20-30 age-group = 752 ng/mL vs >30 years age-group = 350 ng/mL), suggesting that the antichondrogenic effect of IGFBP-3 may decrease during the later decades of life."

"PDGF-BB is secreted from platelet α-granules and is a major mitogenic factor for mesenchymal cells. PDGF-BB stimulates collagen synthesis and plays a role in redifferentiation of the autologous dedifferentiated chondrocytes toward chondrogenic lineage."

" The mean PDGF-BB concentration of 39.5 ng/mL in the 20-30 age-group significantly decreased to 24.9 ng/mL among all other groups. However, effective concentrations of PDGF-BB have historically varied from 4.7 to 300 ng/mL in vitro, with many studies reporting [chondroinduction] effects with concentrations below 40 ng/mL"

"In our study, the mean value of bFGF over all age-groups was 6.3 ng/mL. Previous studies illustrate that a concentration of 1 ng/mL of bFGF increased the potential for the chondrogenic differentiation of hBMSCs after 15 days in culture."

"The mean concentration [of TGF-B1] we were able to detect throughout all age-groups in our study was 54.7 ng/mL. Doses between 0.3 and 20.0 ng/mL stimulated in vitro chondrogenesis of mesenchymal cells."

"this investigation detected an average of 285 pg/mL (BMP-2) and 277 pg/mL (BMP-4) across all ages, with no significant difference among age-groups. Although our results show consistently elevated levels of BMP-2 and BMP-4, chondrogenic differentiation media typically contains 50-100 ng/mL BMP-2, suggesting that either BMP-2 is not present in sufficient concentration for chondroinduction in PRP or that picogram doses have not been adequately studied."

So older platelet rich plasma may be better for chondroinduction due to lower IGFBP-3 levels.  PRP contains sufficient levels of PDGF-BB, bFGF, and TGF-B1 for chondroinduction but not sufficient levels of BMP-2 and BMP-4.

Generating platelet rich plasma involves increasing the concentration of platelets by five-fold.  Platelet rich plasma injections are legal but can only be done by doctors.

Wednesday, March 7, 2012

Possible alternative loading device to induce adult height growth?

The key to adult height growth is to induce in vivo chondrogenesis in the bone marrow to get height growth from new endochondral ossification.  LSJL is one method to do such that.  However, there may be another device capable of inducing in vivo chondrogenesis in the bone marrow and therefore other devices capable of adult height growth.

A combination of shear and dynamic compression leads to mechanically induced chondrogenesis of human mesenchymal stem cells.

"Articular motion is a combination of compressive, tensile and shear deformations; therefore, one can presume that compression alone is unlikely to be a sufficient mechanical signal to generate a cartilage-like tissue in vitro[so compressive, tensile, and shear deformations are likely needed to induce chondrogenesis in bone marrow]. we aimed to determine the role of shear on the fate of stem cell differentiation[LSJL induces fluid based shear strain]. Specifically, we investigated the potential enhancing effect of surface shear, superimposed on cyclic axial compression, on chondrogenic differentiation of human bone marrow-derived stem cells. Using a custom built loading device[could this device be an alternative to LSJL?] we applied compression, shear or a combination of both stimuli onto fibrin/polyurethane composites in which human mesenchymal stem cells were embedded, while no exogenous growth-factors were added to the culture medium. Both compression or shear alone was insufficient for the chondrogenic induction of human mesenchymal stem cells. However, the application of shear superimposed upon dynamic compression led to significant increases in chondrogenic gene expression[LSJL should imply both shear and compressive forces.  Axial loading should only apply compressive forces as there is no pressure gradient hence why axial loading does not induce height growth]. Histological analysis detected sulphated glycosaminoglycan and collagen II only in the compression and shear group."

The device is basically a ball placed directly on the mesenchymal stem cells.  I assume to be like the ball of the knee joint.  So the purpose of the ball was for it to oscillate to generate fluid shear strain.

"Group A was the free-swelling (unloaded) control. Group B was exposed to unconfined dynamic compression at 1 Hz with 0.4 mm sinusoidal strain, superimposed on a 0.4 mm static offset strain, resulting in a strain amplitude of 10-20 % of the scaffold height at the centre of the construct. Group C was exposed to ball oscillation of ±25° at 1 Hz, superimposed on a 0.4 mm static compression offset strain. Group D was a combination of both loading regimes. Mechanical load was applied during 1 h a day for 5 consecutive days per week over 3 weeks"<-LSJL loading applies load for slightly over 1 minute which is much less than 1 hour.

"There was a trend towards higher total GAG/DNA values in the loaded groups which included shear[so shear strain is very important for inducing chondrogenesis, axial loading induces no shear strain thus no chondrogenesis], but no significant difference over the compression only group was detected. All individual donors demonstrated the same trend[so all individual bone marrow sources were capable of chondrogenesis]"

Compression and Sox9 groups expressed much more Sox9 than just shear groups alone.  So compressive forces are still important to chondrogenesis.  If you look at Figure 6D in the study you can almost see a growth plate.

What we can take from this study is that maybe the LSJL clamping routine is not dynamic enough at inducing fluid based shear strain.  Perhaps rapid clamping and unclamping would be a way to make LSJL induce more shear forces.

The ball device is not something that could be used as you'd need to put the ball directly against the bone marrow.  Maybe surgically implanting a ball in the bone marrow may be a possibility for the future.

Monday, March 5, 2012

Gene expression of normal growth plate

Microarray analysis of perichondral and reserve growth plate zones identifies differential gene expressions and signal pathways.

"Normal Sprague-Dawley rat growth plate chondrocytes from the perichondral zone (PC)[the layer of fibrous connective tissue that envelops cartilage] and reserve zone (RZ) were isolated by laser microdissection and then subjected to microarray analysis. In order to most comprehensively capture the unique features of the two zones, we analyzed both the most highly expressed genes and those that were most significantly different from the proliferative zone (PZ) as a single comparator. Confirmation of the differential expression of selected genes was done by quantitative real-time RT-PCR. A total of 8 transcripts showing high expression unique to the PC included translationally-controlled tumor protein (Tpt1), connective tissue growth factor (Ctgf), mortality factor 4 (Morf4l1), growth arrest specific 6 (Gas6), type V procollagen (Col5a2){upregulated by LSJL}, frizzled-related protein (Frzb), GDP-dissociation inhibitor 2 (Gdi2) and Jun D proto-oncogene (Jund). In contrast, 8 transcripts showing unique high expression in the RZ included hyaluronan and proteoglycan link protein 1 (Hapln1){upregulated by LSJL}, hemoglobin beta-2 subunit, type I procollagen (Col1a2), retinoblastoma binding protein 4 (LOC685491), Sparc-related modular calcium binding 2 (Smoc2) {Upregulated by LSJL}, and calpastatin (Cast). Other genes were highly expressed in cells from both PC and RZ zones, including collagen 2a1, collagen 9a3 {both upregulated by LSJL}, ND3, catenin (cadherin associated protein) beta 1, Eef1a1{Eef1a1 was downregulated by LSJL}, HGMB1, Rpl35a, Mtap7, Rcn1, Thbs1, Rsp6, Cpe, Cpt1a, Sparc, plexin B2 (Plxnb2), and gGja1. Functional classification of the most highly expressed transcripts were analyzed, and the pathway analysis indicated that ossification, bone remodeling, and cartilage development were uniquely enriched in the PC whereas both the PC and RZ showed pathway enrichment for skeletal development, extracellular matrix structural constituent, proteinaceous extracellular matrix, collagen, extracellular matrix, and extracellular matrix part pathways."

"RNAs highly expressed within both the PC and RZ were 21 genes, including RGD 1308977 (similar to RIKEN cDNA), RGD 1306734 (similar to hypothetical protein FLJ32743), and LOC 685491 (similar to retinoblastoma binding protein 4), Mgp (matrix Gla protein)."

"Col9a3 may contribute to the three-dimensional integrated structure of type II collagen molecules"

"Matrix GLA protein (Mgp) is a mineral binding extracellular matrix protein synthesized by growth plate cartilage chondrocytes"

"Reticulocalbin (Rcn1), which is distributed predominantly in endocrine and exocrine organs, is one member of the Ca2+-binding proteins in the secretory pathway "

"Ctgf deficiency leads to skeletal dysmorphisms as a result of impaired chondrocyte proliferation and extracellular matrix composition within the hypertrophic zone"

"Frzb-1 [is] strong in prehypertrophic chondrocytes."

Spatial and temporal regulation of gene expression in the mammalian growth plate.

"we used microdissection to collect individual growth plate zones from proximal tibiae of 1-week rats and the PZ and early hypertrophic zones of growth plates from 3-, 6-, 9-, and 12-week rats"

"candidate markers for the resting zone [include Sfrp5], bone morphogenetic protein 3 (Bmp3) and gremlin 1 (Grem1) two genes involved in BMP signaling, epidermal growth factor-containing fibulin-like extracellular matrix protein 1 (Efemp1), and collagen type 14 alpha 1 (Col14a){up in LSJL}."

Other Resting Zone Markers:

Percentage of total upregulated in LSJL: 17.5%
Downregulated: 5%

Proliferating Zone Markers:

Hypertrophic Zone:
Cmkor1{up as Cxcr7}
Col6A2{up and down, counted twice in percentages}
Col10A1(up in LSJL)
IBSP(up in LSJL as BSP)
Igsf4a{down as Cadm1}

Percentage of total upregulated by LSJL: 11.6%
Downregulated: 5.4%

Genes upregulated with youth:

Percentage upregulated of total: 4.76%
Downregulated: 2.38%

Genes upregulated with age:
Col6a2{up and down}

Upregulated: 18%
Downregulated: 4.16%

"Some important transcription factors were being significantly regulated between the resting and proliferative zones (Dlx5, c-Fos{up in LSJL}, c-Maf, Nrf2, Runx2, Runx3, Sox6, upregulation; Hif1α, Shox2, downregulation), or between the proliferative and hypertrophic zones (Atf3{up}, c-Fos{up}, c-Maf, MafB{down}, Mef2c, Mef2d, Runx2, Sox6, upregulation; Hif1α, Runx3, Shox2, downregulation)"

"several transcription factors showed significant upregulation (Hif1α, Jun{up in LSJL}) or downregulation (Mef2c, Runx2, Runx3, Sox9{up in LSJL}) with age."

"Gli3{up in LSJL} represses the transition from round into columnar chondrocytes"

Temporal and spatial expression of a growth-regulated network of imprinted genes in growth plate.

"We characterized expression of the network during postnatal growth in microdissected metaphyseal bone and growth plate zones of 1-, 3-, and 9-week-old rats using real-time PCR. The expression pattern of the network is modified in growth plate. Similar to the coordinated decline previously observed in kidney, lung, liver, and heart, expression of all genes, except Gtl2, decreased with age in metaphyseal bone. On the contrary, Mest, Dlk1, H19{up}, and Gtl2 decreased, and Cdkn1c, Grb10, and Slc38a4{up} increased with age in growth plate. During differentiation from resting to hypertrophic zone, Mest, Dlk1, Grb10, and Gtl2 expression decreased, whereas Slc38a4 expression increased. In particular, developmental changes in the expression of growth-promoting genes, Mest, Dlk1, Gtl2, and growth-inhibitory genes, Cdkn1c, [Ndn], and Grb10, may contribute to the decline in longitudinal bone growth that occurs with age."

"Targeted ablation of either Dlk1, Mest, Gtl2, Igf2, Plagl1, or Peg3 results in reduced body size at birth or earlier"

"Dlk1, Mest, Gtl2, and Igf2 ablation also negatively affect postnatal growth"

"Disruption of Grb10 in mice results in overgrowth of mutant embryos"

Expression profiling of human fetal growth plate cartilage by EST sequencing.

"Here, we describe the sequences of 4,748 clones from a human growth plate cartilage cDNA library generated from 20 weeks prenatal-2 years postnatal specimens. In silico analysis of these sequences revealed 1,688 individual transcription units, corresponding to known (1,274) and to novel, yet uncharacterised potential genes (414). The tissue specificity of the library was reflected by its corresponding EST profile representing a total of approximately 10% proteins already shown to be involved in cartilage/bone development or homeostasis. The EST profile also reflects the developmental stage of the tissue with significant differences in the expression of matrix proteins compared to corresponding EST profiles from 8-12 and 12-20 week human fetal cartilage."

"As expected, the most prevalent transcript encodes the type II procollagen (COL2A1) with 103 ESTs (56,6% of collagen clones). The next most highly expressed transcripts corresponded to COL9A1 with 15 ESTs (8.24%), COL9A3 with 12 ESTs (6.59%), COL11A2 with 9 ESTs (4.95%), and COL9A2 with 4.47% of the collagen, while COL6A2, COL16A1 and COL3A1 represented 2.75%, 2.2% and 1.65%, respectively. The remaining identified collagen transcripts are represented by less than 3 ESTs (< 1.5% of the total collagen clones). It has to be emphasized that the number of ESTs for the different chains of collagen I and IX does not reflect the expected molar ratios of COL1A1 / COL1A2 (2 : 1) and COL9A1 / COL9A2 / COL9A3 (1 : 1 : 1), respectively"

"collagen XVI is strongly expressed in differentiating chondrocytes, where it is a component of small heterotypic D-banded fibrils, mainly occurring in the territorial matrix of chondrocytes. Based on in vitro studies, COL16A1 has been suggested to play a role in later chondrogenesis, which would match with the present data."<-Col16a1 is upregulated in LSJL.

"transcripts encoding 11 different individual proteoglycans were identified (38 ESTs), some of them with high expression levels, like biglycan (BGN) with 9 ESTs, fibromodulin (FMOD), chondroitin sulfate proteoglycan 4 (CSPG4) and aggrecan 1 (AGC1) with 6 ESTs."<-All but FMOD were upregulated in LSJL.

Syndecan 4{up} and 2{down} also had significant EST transcripts.  HAPLN1 another EST significantly observed was upregulated in LSJL.

Non-Collagen ESTs observed altered by LSJL:

" transcripts of seven different matrix-metabolising genes were identified (64 ESTs). The most prevalent transcript in this group was matrix metalloproteinase-3 (MMP3) with 34 ESTs, which encodes a proteoglycanase, followed by MMP1 with 22 ESTs and MMP13 with 4 ESTs, both representing collagenases. MMP3 and MMP13 were also reported to be the most strongly expressed MMPs in 12–20 week fetal cartilage."<-MMP3 was upregulated by 3.6 fold.

Profiling genes expressed in human fetal cartilage using 13,155 expressed sequence tags.

8-12 week old fetal cartilage.

Genes expressed whose expression was altered in LSJL:
HTRA1(as PRSS11){up}
TIMP1{up very highly}

"The fact that IGF-II was the most abundant growth factor identified in fetal cartilage suggests that both glypican 3 and IGF-II are important in fetal cartilage development."

Gene expression during endochondral bone development: Evidence for coordinate expression of transforming growth factor β1 and collagen type I

"Subcutaneous implatation of demineralized bone particles (DBP) into rats induces the formation of a bone ossicle by a tightly controlled sequence of chondro- and osteo-inductive events which are directly comparable to those which occur in normal endochondral bone development.  To examine the expression of genes in this system, RNA was isolated from implants every 2 days over a time course spanning 3 to 19 days after implantation of DBP into rats. Cellular levels of mRNA transcripts of cell-growth-regulated and tissue-specific genes were examined by slot blot analysis and compared to the morphological changes occuring during formation of the ossicle. Analysis of the mRNA levels of histone H4 and c-myc, markers of proliferative activity, revealed several periods of actively proliferating cells, corresponding to (1) production of fibroprogenitor cells (day 3), (2) onset of bone formation (day 9), and (3) formation of bone marrow (day 19). The mRNA levels of collagen type II, a phenotypic marker of cartilage, peaked between days 7 and 9 post-implantation, corresponding to the appearance of chondrocytes in the implant, and rapidly declined on day 11 (to 5% of maximum value) when bone formation was observed. The peak mRNA levels of collagen type I, found in fibroblasts and osteoblasts, occurred first with the onset of bone formation (days 7–10) and again during formation of bone marrow (day 19). This study has demonstrated that the temporal patterns of mRNA expression of cartilage type II and bone type I collagens coincide with the morphological sequence in this model of endochondral bone formation. Further, the mRNA levels of transforming growth factor β1 (TGFβ) were compared to those of collagen types I and II; a direct temporal correlation of TGFβ mRNA levels with that of collagen type I was found throughout the developmental time course. This observation of a tightly coupled relationship between TGFβ and type I collagen mRNA levels is consistent with a functional role for TGFβ in extracellular matrix production during in vivo bone formation."

"The time points in the present study were selected to illustrate the progressive stages of endochondral bone formation in the DBP implants. By day 3 the early stage of recruitment of fibroprogenitor cells around the particles of the implant was apparent. By day 7 chondroblasts were observed in the areas adjacent to the bone particles. On day 9, more of the implant had undergone chondrogenesis and the cartilage matrix had mineralized.  Bone formation, noted by the presence of osteoblasts and newly formed osteoid, was observed on day 11. Completion of bone formation and the onset of the bone remodeling stage were observed by day 15, and marrow formation and hematopoeisis were evident at day 18. Thus, the DBP implants exhibited the chondro- and osteogenic events which normally occur during endochondral bone formation"

Histone H4, c-Myc, Col1a1, TGFB, and Col2a1 increased in expression.

"Bone powder was prepared from long bones of adult rats (8-10 weeks). The diaphyses of the long bones were cleaned, pulverized in liquid nitrogen, sieved to particle size of 75-250 pm, and then acid demineralized. Fifty milligrams of DBP was implanted in bilateral subcutaneous pockets on the thoracic region in 28-day-old male Sprague Dawley CD strain rats"