Friday, September 28, 2012

MMP9 and height

MMP9 regulates the cellular response to inflammation after skeletal injury.

"To assess the role of inflammation on skeletal cell differentiation, we used mouse models of fracture repair that stimulate either intramembranous or endochondral ossification. In the first model, fractures are rigidly stabilized leading to direct bone formation, while in the second model, fracture instability causes cartilage and bone formation. We compared the inflammatory response in these two mechanical environments and found changes in the expression patterns of inflammatory genes and in the recruitment of inflammatory cells and osteoclasts. We then exploited matrix metalloproteinase 9 (MMP9) that is expressed in inflammatory cells and osteoclasts, which is a potential regulator of cell fate decisions during fracture repair. Mmp9-/- mice heal stabilized fractures via endochondral ossification, while wild type mice heal via intramembranous ossification[So to grow taller being MMP knockout may be better]. In parallel, we observed increases in macrophages and T cells in the callus of Mmp9-/- compared to wild type mice. To assess the link between the profile of inflammatory cells and skeletal cell fate functionally, we transplanted Mmp9-/- mice with wild type bone marrow, to reconstitute a wild type hematopoietic lineage in interaction with the Mmp9-/- stroma and periosteum. Following transplantation, Mmp9-/- mice healed stabilized fractures via intramembranous ossification and exhibited a normal profile of inflammatory cells. Mmp9-/- periosteal grafts healed via intramembranous ossification in wild type hosts, but healed via endochondral ossification in Mmp9-/- hosts. We observed that macrophages accumulated at the periosteal surface in Mmp9-/- mice, suggesting that cell differentiation in the periosteum is influenced by factors such as BMP2 that are produced locally by inflammatory cells. MMP9 mediates indirect effects on skeletal cell differentiation by regulating the inflammatory response and the distribution of inflammatory cells, leading to the local regulation of periosteal cell differentiation."

The mice were 12-16 weeks old.  Mast cells(connective tissue cell) typically produce MMP9.  Fractures were produced by three point bending loading.

"Inflammatory mediators such as tumor necrosis factor-α (TNFα) are required for bone formation but can also impair later stages of repair by stimulating cartilage degradation"

"The role of Cox2 is counterbalanced by 5-lipoxygenase (5-LO) and leukotriene inhibitors can stimulate cartilage and bone formation in the early phase of repair through direct actions on chondrocytes"<-Grow taller with leukotriene inhibitors?  Pharmacological inhibition of 5-lipoxygenase accelerates and enhances fracture-healing. states that 5-LO inhibition via AA-861 increased COL2, COX2, COL10, and osteopontin.  Celecoxib[COX2 inhibitor] also increased COL2.

"MMP9−/− but not wild type skeletal progenitors differentiate into chondrocytes in both non-stabilized and stabilized fractures"

"the presence of Mmp9−/− inflammatory cells alone was not sufficient to induce chondrogenic cell differentiation at the fracture site. "

"Osteoclast recruitment was impaired in Mmp9−/− fracture calluses and bone marrow transplants rescued the cartilage remodeling defects in non-stabilized fractures via providing MMP9-expressing osteoclasts. This result was in concordance with the rescue of the growth plate phenotype in Mmp9−/− developing long bones"

"the recruitment of T cells and macrophages was affected [in] Mmp9−/− fracture calluses"

"This indirect effect of MMP9 on periosteal cell fate was via the regulation of macrophage localization. Only macrophages were located within the periosteum to locally influence chondrogenic and osteogenic cell differentiation. CD4 + T cells were found away from the periosteum, and so are unlikely to have an effect on chondrogenesis and osteogenesis."

"MMP9 acts indirectly at the level of the periosteum"

"Ectopic localization of macrophages in the periosteum of Mmp9−/− stabilized fractures may be responsible for the induction of endochondral ossification as we found an increase in BMP2 expression."

"MMP9 indirectly regulates angiogenesis by regulating the bioavailability of VEGF"

"the lack of MMP9 in both the periosteum and inflammatory cells was responsible for the cartilage phenotype."

Genes upregulated in Unstablized versus Stabilized(MMP9 knockout) at Day 7 that were also upregulated(or -down) by LSJL:

All below are signal transduction related molecules:

Glycoprotein Related:

Cell Differentiation:
ALDH1A3(down)[an enzyme produced by retinoic acid]


LSJL was highly homologous to the most chondrogenic stimulation group in the study MMP9-/- unstabilized fractgure.

Genes upregulated in stabilized versus unstabilized MMP9 KO mice that were also altered in LSJL
So these are essentially downregulated by unstabilized mice:

Unstabilized non-MMP9 knockout on day 7 versus pre-fracture gene upregulation that were also upregulated(or -downregulated) by LSJL:


At day 2 these are the genes that are also upregulated by LSJL that were not previously reported above:

LSJL produces a similar gene expression profile to MMP9-/- and Unstabilized fracture mice which were pro-chondrogenic thus providing further evidence that LSJL is pro-chondrogenic.  No change in MMP9 expression was reported due to LSJL.

Thursday, September 27, 2012

Height Gaining with mTor(leucine)?

mTor regulates growth plate autophagy.  Without mTor autophagy cannot be induced.  mTor can increase the translation of Myc.  It also increases the availability of free eIF4E.  Inhibition of PTEN increases mTor and induces skeletal overgrowth.  Cadherin 13 activates mTor.

Leucine may increase mTor levels and Leucine is available for sale: Source Naturals L-Leucine 500mg, 120 Capsules

As is Isoleucine: Supplement Direct L-isoleucine Powder 250 Grams


"The objective of this study is to identify mechanisms by which nutrient availability, specifically leucine which is the most abundant essential amino acids, affects chondrocyte proliferation, differentiation, deposition of extracellular matrix (ECM) and their interactions. Nutritional status effect linear growth by modulating GH/IGF-I axis. Recently signaling mechanism by which nutrients directly regulate cells via mammalian Target Of Rapamycin (mTOR) has been elucidated. We hypothesize that leucine directly regulates chondrogenesis directly by affecting chondrocyte proliferation, differentiation and ECM deposition, 3 key components of bone growth. Our hypothesis is based on our observations. First, mTOR directly regulates chondrogenesis. MTOR inhibition results in decreased expression of Indian hedgehog (Ihh), a key regulator of chondrogenesis. Decreased Ihh could be central to mTOR action. Second, leucine restriction and mTOR inhibition result in significant reduction of ECM mRNA expression likely via miRNA regulation. Lastly, we utilized an ex vivo metatarsal explant system to demonstrate the physiological relevance of observations we made in the chondrogenic ATDC5 cell line.

Specific Aim 1: Identify the molecular mechanisms that mediate the effect of mTOR inhibition and leucine restriction on Ihh regulation and, as a consequence, chondrocyte growth and differentiation. .
Specific Aim 2: Determine the effect of mTOR inhibition and leucine restriction on miRNA-29 regulation resulting in altered ECM expression and functional role in chondrocyte growth and differentiation Specific Aim 3: Utilizing a fetal metatarsal explant model, we will study the effect of mTOR inhibition and leucine restriction on bone growth, growth plate dynamics and chondrogenic markers, in particular Ihh and miRNA-29. We will study the effect of restoring Ihh and miRNA-29 expression in bones subjected to mTOR inhibition and leucine restriction."

Isoleucine and leucine independently regulate mTOR signaling and protein synthesis in MAC-T cells and bovine mammary tissue slices.

"Omission of L-arginine, L-isoleucine, L-leucine, or all EAA[Essential amino acids] reduced (P < 0.05) mammalian target of rapamycin (mTOR; Ser2448) and ribosomal protein S6 (rpS6; Ser235/236)[LSJL upregulates rpS6ka6 and downregulates rpSKa3] phosphorylation in MAC-T cells. Phosphorylation of mTOR and rpS6 kinase 1 (S6K1; Thr389) decreased (P < 0.05) in the absence of L-isoleucine, L-leucine, or all EAA in lactogenic mammary tissue slices. Omission of L-tryptophan also reduced S6K1 phosphorylation (P = 0.01). Supplementation of L-leucine to media depleted of EAA increased mTOR and rpS6 and decreased eukaryotic elongation factor 2 (Thr56) phosphorylation (P < 0.05) in MAC-T cells. Supplementation of L-isoleucine increased mTOR, S6K1, and rpS6 phosphorylation (P < 0.05). No single EAA considerably affected eukaryotic initiation factor 2-α (eIF2α; Ser51) phosphorylation[LSJL reduces eIF2a phosphorylation], but phosphorylation was reduced in response to provision of all EAA (P < 0.04). FSR[protein synthesis rates] declined when L-isoleucine (P = 0.01), L-leucine (P = 0.01), L-methionine (P = 0.02), or L-threonine (P = 0.07) was depleted in media and was positively correlated (R = 0.64, P < 0.01) with phosphorylation of mTOR and negatively correlated (R = -0.42, P = 0.01) with phosphorylation of eIF2α. Such regulation of protein synthesis will result in variable efficiency of transfer of absorbed EAA to milk protein and is incompatible with the assumption that a single nutrient limits protein synthesis that is encoded in current diet formulation strategies."

Leucine nutrition in animals and humans: mTOR signaling and beyond.

"After structural roles are satisfied, the ability of leucine to function as signal and oxidative substrate is based on a sufficient intracellular concentration. Therefore, leucine level must be sufficiently high to play the signaling and metabolic roles. Leucine is not only a substrate for protein synthesis of skeletal muscle, but also plays more roles beyond that. Leucine activates signaling factor of mammalian target of rapamycin (mTOR) to promote protein synthesis in skeletal muscle and in adipose tissue{and bone and growth plates}. It is also a major regulator of the mTOR sensitive response of food intake to high protein diet. Meanwhile, leucine regulates blood glucose level by promoting gluconeogenesis and aids in the retention of lean mass in a hypocaloric state."

"activation of mTOR/S6K1 pathway by AA has been shown to phosphorylate serine (Ser-1101) of insulin receptor substrate 1 (IRS-1) and suppress tyrosine phosphorylation, which results in impaired phosphatidylinositol 3-kinase (PI3K) activity"

IRS1 which inhibits PI3K is downregulated by LSJL.

So increase in p-mTor(in conjunction with increase in p-Akt) has been shown to induce overgrowth by inhibition of PTEN.  However, p-mTor has a negative feedback mechanisms where it inhibits p-Akt.

The effect of rapamycin on bone growth in rabbits.

"[Rapamycin is an mTOR inhibitor]i. [We directly infused] rapamycin into the proximal tibial growth plates of rabbits. Rapamycin or DMSO vehicle was infused directly into growth plates by an osmotic minipump for 8 weeks. At the end of the experiment, growth plates were recovered for histological analysis. Six animals were studied. Bone growth of limbs exposed to rapamycin was slower than control limbs, particularly during the period of most rapid growth. Histological analysis revealed that growth plate height in the rapamycin-infused limbs was reduced. Both the hypertrophic and proliferative zones were significantly smaller in the rapamycin-infused limbs. Direct infusion of rapamycin into proximal tibial growth plates decreased the size of the growth plate and inhibited overall long bone growth. Rapamycin appears to affect both the proliferative and hypertrophic zones of the tibial growth plate."

"One animal was excluded from these data because closure of the growth plate had occurred on the rapamycin infused side but not the control side."<-So mTor inhibition may cause adult height reduction and not just growth rate reduction.

"rapamycin affects the expression of Indian Hedgehog (Ihh)"

Chondrogenic differentiation of growth factor-stimulated precursor cells in cartilage repair tissue is associated with increased HIF-1alpha activity.

"Scaffold-bound autologous periosteal cells, which had been activated by Insulin-like Growth Factor 1 (IGF-1) or Bone Morphogenetic Protein 2 (BMP-2) gene transfer using both adeno-associated virus (AAV) and adenoviral (Ad) vectors, were applied to chondral lesions in the knee joints of miniature pigs. Six weeks after transplantation, the repair tissues were investigated for collagen type I and type II content as well as for HIF-1alpha expression. The functional role of phosphatidylinositol 3-kinase (PI3K)/mammalian target of rapamycin (mTOR) and mitogen-activated protein kinase (MEK)/extracellular signal-regulated kinase (ERK) signaling on BMP-2/IGF-1-induced HIF-1alpha expression was assessed in vitro by employing specific inhibitors.
Unstimulated periosteal cells formed a fibrous extracellular matrix in the superficial zone and a fibrocartilaginous matrix in deep zones of the repair tissue. This zonal difference was reflected by the absence of HIF-1alpha staining in superficial areas{so maybe HIF-1a isn't need to induce chondrogenesis}, but moderate HIF-1alpha expression in deep zones. In contrast, Ad/AAVBMP-2-stimulated periosteal cells, and to a lesser degree Ad/AAVIGF-1-infected cells, adopted a chondrocyte-like phenotype with strong intracellular HIF-1alpha staining throughout all zones of the repair tissue and formed a hyaline-like matrix. In vitro, BMP-2 and IGF-1 supplementation increased HIF-1alpha protein levels in periosteal cells, which was based on posttranscriptional mechanisms rather than de novo mRNA synthesis, involving predominantly the MEK/ERK pathway.
This pilot experimental study on a relatively small number of animals indicated that chondrogenesis by precursor cells is facilitated in deeper hypoxic zones of cartilage repair tissue and is stimulated by growth factors which enhance HIF-1alpha activity."

"Application of Wortmannin or Rapamycin, inhibiting PI3K or mTOR, respectively, only moderately decreased the HIF-1α levels following BMP-2 and IGF-1 stimulation. However, treatment with the MEK inhibitor UO126 nearly completely abolished HIF-1α induction by either growth factor"

"HIF-1 was shown to transactivate Sox9. The HIF-1 subunit HIF-1α [directly binds] to the Sox9 promotor region"

Leucine may increase height via activation of mTor.  However:

1) We don't know the optimal dosage of Leucine
2) p-mTor inhibits p-Akt(although LSJL increases p-Akt)

Tuesday, September 25, 2012

Can LSJL work on adults?

LSJL was tested on 14 week old male rats.  Many chondrogenic genes were upregulated and studies have shown that Adult Humans BMMSC's are capable of chondroinduction.  However, more evidence would be helpful.

The next step would be to test LSJL on adult female mouse ERalphaAF-1 knockout mice which experience growth plate fusion as described in the study The role of estrogen receptor-α and its activation function-1 for growth plate closure in female mice.  Then see if there's any length increase when LSJL is applied.  You can't apply LSJL to another species because the Yokota lab pizeoelectric loader is custom built for rats and LSJL rat growth plates don't fuse which may alter gene expression.  So by forcing fusion using ERa AF-1 knockout mice, we can test how LSJL would work on results.  Gene expression data would be interesting but length increase would be what is needed.

The transactivating function 1 of estrogen receptor α is dispensable for the vasculoprotective actions of 17β-estradiol explains how to generate ERa AF-1 knockout mice.

"ERα AF-1 was generated through a targeted deletion by using a knockin strategy, through which 441 nt of exon 1 were deleted. The truncated protein lacks the A domain and all three motifs constituting ERα AF-1 (AF-1 boxes 1–3) in the B domain, thus yielding a 451-aa-long, 49-kDa protein"

The cost of these mice is at least $5000 but it should cheaper from there on.  I'll try to do some research and see what can be done.  Then there's the confounding variable that the knockout may affect sensitivity of cells to the stimulus.

Bone mechanotransduction may require augmentation in order to strengthen the senescent skeleton.

"Age-related increases in osteocyte apoptosis is observed and holds potential to alter intercellular signaling and the function of the bone cell syncytium"

"there is a marked age-related increase in marrow adiposity"<-although adiposal stem cells are capable of differentiating into chondrocytes.

"aging is accompanied by a decline in periosteal lining cell numbers"

" Age-related increases in osteocyte apoptosis is also observed and holds potential to alter intercellular signaling and the function of the bone cell syncytium"

"attenuated biophysical stimulation could arise in part from decreases in the surface to volume ratio of bone mineral matrix and increased viscosity of interstitial fluids.  If these physical mechanisms were to occur in bone, the magnitudes of fluid flow induced secondary to skeletal loading could be reduced with age."<-since fluid flow is a vital part of the LSJL stimulus this could reduce LSJL based chondrogenesis.

"PGE2 release has been found to be substantially increased in cells derived from aged vs. young human donors. In contrast, the number of cells displaying spontaneous Ca2+ oscillations and fluid flow induced oscillations were found to be significantly lower in primary cells derived from aged animals, while the amplitudes of Ca2+ oscillations themselves were equivalent"<-Ca2+ oscillations may be critical to induce chondrogenesis.

"Activation of phosphatases and kinases downstream of second messenger signaling including Calcineurin, CAMK, and MAPK are decreased with age. Further downstream (of the phosphatases and kinases), activation and DNA binding by a variety of transcription factors such as NFAT, AP-1, and Wnt/β-catenin also display age-related deficits or suppression"<-LSJL involves both AP-1 and Wnt transcription factors.

"primary cells isolated from young and senescent mice reveals that relative increases in Cox-2 and c-fos gene expression levels in response to fluid flow (vs. no flow controls) were not altered with age "<-Cox-2 and c-fos are both upregulated by LSJL.

"The absolute Cox-2 but not c-fos levels displayed age-related declines in large part due to lower baseline levels of Cox-2 expression in cells derived from aged animals."

"aging markedly diminishes the periosteal response to mechanical stimuli"

"suppression of increased osteoclastic activity [is] associated with aging"

"Cyclosporin A (CsA)[immunosuppresent drug that is available with prescription only], at low-dosages, could be a candidate agent to address age-related deficits in NFAT (or AP-1) activation and transcription"

"senescent mice (22 months) were subjected to mechanical loading in conjunction with low-dose CsA treatment, the resulting periosteal bone formation was significantly increased compared to vehicle-treated aged matched controls subject to loading alone."

CsA inhibits p38, GSK-3beta, and Calcineurin.  Lithium is another GSK-3Beta inhibitor.

Other genes that may be differentially regulated due to aging in LSJL:

Turner's axial loading study used 20 week old mice versus 14 week old.  So we can also study whether genes were deferentially regulated in the two studies due to age rather than due to the different loading modalaties(axial vs. lateral).

MicroRNA-199a-3p, microRNA-193b, and microRNA-320c are correlated to aging and regulate human cartilage metabolism.

"The expression of two miRNAs (miR-199a-3p and miR-193b) was upregulated with age and that of one miRNA (miR-320c) was downregulated with age."

"Type 2 collagen, aggrecan, and SOX9 expression were downregulated in the miR-199a-3p mimic group[axial loading 20 week old mice upregulated COL2A1 and acan but not Sox9, whereas all three were upregulated in LSJL thus the significant upregulation of Sox9 versus axial loading is likely due to the different modalaties and not aging] but [were] upregulated in the inhibitor group. Similar results were observed for miR-193b. By contrast, ADAMTS5 expression was downregulated in the miR-320c mimic group and upregulated in the inhibitor group. Cell proliferative activity was upregulated significantly in the miR-193b inhibitor group compared with the control group. miR-199a-3p and miR-193b are involved in the senescence of chondrocytes, and miR-320c is involved in the juvenile properties of chondrocytes."

LSJL upregulates the anti aging genes Lin-28b and HMGA2.

Donor age and long-term culture affect differentiation and proliferation of human bone marrow mesenchymal stem cells.

"chondrogenic potential did not change [with aging]"

The growth of adult(25-50) MSCs was only slightly than child MSCS(0-12) but old had significantly less growth of MSCs.

Thus LSJL inducible chondrogenesis may not change due to alterations in chondrogenic potential with aging but rather due to secondary signaling from osteocytes which do experience alterations in aging.

Effects of treadmill exercise and training frequency on anabolic signaling pathways in the skeletal muscle of aged rats.

"protein levels of IRS-1 and p-mTOR as well as COX activity were reduced in advanced age."  IRS1 was deferentially expressed in the two studies up in axial loading and down in LSJL despite the axial loading rats being older.  The tissues are different, muscle versus bone, but the insulin pathway has a general anabolic effect in all tissues.

Effect of aging on the basal expression of c-Fos, c-Jun, and Egr-1 proteins in the hippocampus. found that aging decreased egr1 expression in the hippocampus.  egr1 was downregulated in axial loading whereas it was upregulated in LSJL.  The differential expressions of egr1 in the two studies could be based on aging rather than loading modality.

"The expression profile of stemness markers was altered in BM-MSCs derived from old rats. BM-MSCs from young rats (4 months) expressed Oct-4, Sox-2 and NANOG, but we failed to detect Sox-2 and NANOG in BM-MSCs from older animals (15 months). Chondrogenic, osteogenic and adipogenic potential is compromised in old BM-MSCs{chondrogenic potential is not compromised in human BM-MSCs so maybe epiphyseal fusion adult female mice would not be the best model}. Stimulation with a cocktail mixture of bone morphogenetic protein (BMP-2), fibroblast growth factor (FGF-2) and insulin-like growth factor (IGF-1) induced cardiomyogenesis in young BM-MSCs but not old BM-MSCs. Significant differences in the expression of gap junction protein connexin-43 were observed between young and old BM-MSCs. Young and old BM-MSCs fused with neonatal ventricular cardiomyocytes in co-culture and expressed key cardiac transcription factors and structural proteins. Cells from old animals expressed significantly lower levels of VEGF, IGF, EGF, and G-CSF. Significantly higher levels of DNA double strand break marker γ-H2AX and diminished levels of telomerase activity were observed in old BM-MSCs."

"Old BM-MSCs displayed spread out, flat enlarged morphology which is consistent with late passage and extensively cultured BM-MSCs"

"Human fetal bone marrow is known to contain approximately 1 in 10, 000 MSCs in comparison to 1 in 250, 000 in adults "

Bone marrow stromal cells from aged male rats have delayed mineralization and reduced response to mechanical stimulation through nitric oxide and ERK1/2 signaling during osteogenic differentiation.

"Here, we investigated the ability of bone MSCs from mature and aged rats to differentiate into osteoblasts and to respond to short and long periods of mechanical stimulation through signaling by ERK1/2, nitric oxide (NO), and prostaglandin E(2) (PGE(2)) during differentiation[It's osteoblasts and not chondrocytes but it should be indicative of the ability for MSCs to respond to NO, ERK1/2, and PGE2]. Mineralization was delayed and reduced, but extracellular matrix production appeared less affected by increased age. Differentiating MSCs from aged animals had a decreased response to short and long periods of mechanical stimulation through ERK1/2 signaling, and to long periods of mechanical loading through NO signaling early and late during differentiation. Increases in relative PGE(2) signaling were higher in MSCs from aged animals, which could compensate for reduced ERK1/2 and NO signaling. The decreased mineralization may decrease the ability of cells from aged animals to respond to mechanical stimulation through ERK1/2 and NO signaling, with increased impairment over differentiation time. Decreasing the delay in mineralization of MSCs from aging animals might improve their ability to respond to mechanical stimulation."

"Calcium concentration also increased linearly in differentiating MSCs from both aged and mature rats over the time course, but it was consistently lower in cells from aged animals; the difference was statistically significant at day 28"<-This may be applicable to chondrogenic differentiation as calcium secretion may help trigger chondrogenesis.

Aged animals had increased expression of the pro-chondrogenic protein BMP-2.

"Differentiating cells from aged rats expressed higher p21 and p53 levels at day 7 therefore senescence could play a role in the age-related changes in mineralization and response to mechanical stimulation observed."

Time course of epiphyseal growth plate fusion in rat tibiae.

"there has been a question as to when or to what extent the rat growth plate fuses. To investigate this question, we used microcomputed X-ray tomography, at voxel resolutions ranging from (5.7 micro m)(3) to (11 micro m)(3), to image the proximal epiphyseal growth plates of both male (n = 19) and female (n = 15) rat tibiae, ranging in age from 2 to 25 months. The three-dimensional images were used to evaluate fusion of the epiphyseal growth plate by quantitating the amount of cancellous bone that has bridged across the growth plate. The results suggest that the time course of fusion of the epiphyseal growth plate follows a sigmoidal pattern, with 10% of the maximum number of bridges having formed by 3.9 months in the male tibiae and 5.8 months in the female tibiae, 50% of the maximum number of bridges having formed by 5.6 months in the male tibiae and 5.9 months in the female tibiae, and 90% of the total maximum of bridges have formed by 7.4 months for the males and 6.5 months for the females. The total volume of bridges per tibia at the age at which the maximum number of bridges per tibia has first formed is 0.99 mm(3)/tibia for the males and 0.40 mm(3)/tibia for the females. After the maximum number of bridges (-290 for females, -360 for males) have formed the total volume of bridges per tibia continues to increase for an additional 7.0 months in the males and 17.0 months for the females until they reach maximum values (-1.5 mm(3)/tibia for the males and -2.2 mm(3)/tibia for the females)."

No bony bridges were observed for the female rat until 5 months.

"the growth cartilage of female Sprague–Dawley rats to be bridged by bone between 6 and 18 months of age"

"Although the tibiae from the 6-month-old rats exhibited an increase in number and volume of bridges when compared to the younger rats, the growth plates still appeared to show proliferative and hypertrophic zonest hence, there is a local limited potential for further elongation. Conversely, there seemed to be an absence of all four of the distinct zones of an active growth plate (zones of resting cells, cell proliferation, cell maturation, and lacunar hypertrophy) in the 9-, 13-, and 24-month-old male growth plates and the 8-, 12-, and 25-month-old female growth plates, as demonstrated by conventional histological evaluation "<-So you can grow taller despite some briding.

16 weeks the oldest LSJL mice age is 4 months. Note that the C57BL/6J female used have delayed senescence.  Here's the growth rate for C57BL/6J mice according to Growth of C57Bl/6 mice and the material and mechanical  properties of cortical bone from the tibia:

Because of the loss of chondrogenic differentiation potential in aged rats but not adult humans it's necessary to have more adult humans test LSJL(read: you).  It's possible that the existing growth plates in the rats inhibited chondrogenic differentiation and thus if growth plates were removed in old rats this would not be the case.  This theory is supported by research on the tide mark.  However, the stem cells were cultured outside of the rat bone and differentiation was induced there where there would be no growth plate.

Thus, there is not likely to be an age related difference in LSJL, independent on the loss of the growth plates of course, in humans but there is in rats with respect to chondrocyte differentiation potential but there may be an indirect effect due to secondary osteocyte signaling and changes in fluid viscosity and bone shape.

Impact of aging on rat bone marrow-derived stem cell chondrogenesis.

"We measured the responses of rat bone marrow-derived mesenchymal stem cells (BMSCs) to chondrogenic induction in vitro. BMSCs from immature rats (1 week old), young adult rats (12 weeks old), and old adult rats (1 year old) were analyzed for cartilage extracellular matrix (ECM) production. Histologic analysis showed strong cartilage ECM formation by BMSCs from 1-week-old rats, but not by BMSCs from 12-week-old[LSJL has been proven effective on 8 and 16 week old mice. or 1-year-old rats. Age-related declines [were observed] in messenger RNA encoding type II collagen, aggrecan, and link protein, three major cartilage ECM components. [There were] significant age-related differences in the expression of genes that influence cartilage ECM formation. These findings support the hypothesis that the chondrogenic potential of mesenchymal stem cells declines with age."

"BMSC isolates contain large, slowly replicating cells and small, rapidly growing cells, which retain multipotential differentiation status longer than do larger cells"

"The level of c-Kit, a protein tyrosine kinase receptor for stem cell factor appeared to decrease with increasing age."

Type II collagen gene expression was decreased in 1 year old rats.  Aggrecan expression decreased in twelve week old rats.  Link protein decreased in one year old rats.  Sox9 expression decreased in 1 year old rats but was still greater than that of 1 week old rats.

The decrease in Aggrecan is significant but not the decrease in collagen.  Most genes related to chondrogenesis did not change significantly in the microarray data(with the noted exception of aggrecan) except that many chondrogenic genes increased in expression between 1 week old and 12 week old mice.

Increasing Aggrecan may be a way to enhance the chondrogenic potential of MSCs in adults.

Here's a grant related to the effects of aging on Mechanotransduction:


"Mechanical loading and physical exercise hold promise for enhancing bone mass and morphology. [Previously], we examined how real-time Ca2+ signaling/NFAT pathway activation induced acutely by mechanical stimuli influences bone formation and the degraded response of bone at senescence. [Our] hypothesis [is] that age-related decline in bone formation induced by mechanical loading arises primarily via deficits in activation of the Ca2+/NFAT pathway{NFAT5 and NFATC3 are downregulated by LSJL}. We will quantify agerelated alterations in gene expression downstream of the Ca2+/NFAT pathway and their relation to deficits in cell function and bone formation. We will also demonstrate the requirement for this pathway in bone mechanotransduction by examining the impact of inhibiting (using NFATc1 knock-out mice and high dose Cyclosporine A; CsA) or enhancing its activation (using low-dose CsA). These experimental data will in-turn be used to develop multi-scale simulations for how modulating activation of the Ca2+/NFAT pathway influences the dynamics of cell function and bone formation in young adult and aged animals. In the final S. Aim, [we] will optimize activation of the Ca2+/NFAT pathway with the objective of restoring bone response to loading in the aged skeleton to levels observed in the young adult skeleton. Success of this project would clarify the Ca2+/NFAT pathway as a critical mechanism underlying the agerelated degradation in bone's ability to respond to physical stimuli, and will demonstrate the benefits that are anticipated via interventions in this pathway. Cyclosporine A (one of the proposed interventions) is currently approved for clinical use."

Here's a study detailing the properties of 17 week old female mice tibias(LSJL has induced length gain in female mice ages up to 16 weeks):

Intrinsic material properties of cortical bone.

"The G171V mutation (high bone mass, HBM) is autosomal dominant and is responsible for high bone mass in humans {The humans are transgenic for LRP5, being transgenic for LRP5 inhibits GSK-3Beta so it may help with height growth}. Transgenic HBM mice in which the human LRP5 G171V gene is inserted also show a similar phenotype with greater bone mass and biomechanical performance than wild-type mice, as determined by whole bone testing. Whole bone mechanics, however, depend jointly on bone mass, architecture, and intrinsic bone tissue mechanical properties. To determine whether the HBM mutation affects tissue-level biomechanical performance, we performed nano-indentation testing of unembedded cortical bone from HBM mice and their nontransgenic (NTG) littermates. Femora from 17-week-old mice (female, 8 mice/genotype) were subjected to nano-indentation using a Triboscope. For each femoral specimen, approximately 10 indentations were made on the midshaft anterior surface with a target force of either 3 or 9 mN{LSJL uses 0.5N of Force} at a constant loading rate of 400 mN/s. The load-displacement data from each test were used to calculate indentation modulus and hardness for bone tissue. The intrinsic material property that reflected the bone modulus was greater (48%) in the HBM as compared to the NTG mice. The greater intrinsic modulus in HBM reflects greater bone mineral content as compared to NTG (wild-type, WT) mice. The greater intrinsic property of cortical bone is derived from the greater bone mineral content and BMD, resulting in greater bone strength in HBM as compared to NTG (WT) mice."

"Elastic depth (nm, deformation) is elastic, that is, the deformation recovers when the load is removed, whereas plastic depth (nm, deformation) is plastic, or the deformation is permanent."<-plastic depth of the long bones in a longitudinal direction may way to growth taller.  And the plasticity and elasticity of the bone may effect the ability of bone to respond to deformation by newly induced growth plate.  And this plasticity and elasticity may be affected by age.

This is what a 17 week old bone looks like.  There seems to be lots of porous areas to induce new growth plates.

"the composition (mineral, collagen, etc.) of bone may be responsible for the differences in the elastic and plastic components of total deformation"<-We can alter this composition to get more permanent changes in bone growth.

Skeletal growth and the changing genetic landscape during childhood and adulthood.

"All processes of skeletal growth (longitudinal growth as well as gains and losses of bone mass) are subjected to environmental and genetic influences. These influences, and their relative contributions to the phenotype, can be asserted at any stage of life."

" The shape and content of bone will change throughout the life span with fluctuating contributions of [environmental and genetic] influences."

"in humans, long bones lengthen with age from birth to ∼18 years (or until skeletal maturity), with fluctuations in growth velocity occurring at various periods therein. Increases in total bone diameter and cortical thickness occurs from birth to approximately 30 years of age"

"Genetic influence on bone changes over the life course. The mechanisms for changes in genetic influence can include developmental timing of gene action, epigenetic modification of the bone-related gene product, or even variation in environmental factors influencing gene expression."

"Quantitative measures of bone size and shape were obtained from the second metacarpal from anteroposterior radiographs of the left hand."

"the genetic contribution to bone length remains high and is relatively consistent throughout the remainder of childhood and into young adulthood (average heritability of 0.92 from age of 7 to 29 years). In later adulthood, the genetic influence on metacarpal length again fluctuates, with heritabilities ranging between 0.64 and 0.88. "

Functional comparison of chronological and in vitro aging: differential role of the cytoskeleton and mitochondria in mesenchymal stromal cells.

"We established MSCs cultures from young (yMSCs) and aged (aMSCs) rats that were cultured for more than 100 passages[a round of cell growth and proliferation in cell culture]. These long-term MSCs cultures were non-tumorigenic and exhibited similar surface marker patterns as primary MSCs of passage 2. During in vitro expansion, but not during chronological aging, MSCs progressively lose their progenitor characteristics{so by reducing some of of these division related changes may enable stem cells to differentiate into chondrocytes once more}, e.g., complete loss of osteogenic differentiation potential, diminished adipogenic differentiation, altered cell morphology and increased susceptibility towards senescence. Long-term in vitro MSCs cultivation leads to down-regulation of genes involved in cell differentiation, focal adhesion organization, cytoskeleton turnover and mitochondria function{or loss of progenitor characteristics could be due to culture and it's possible similar changes may occur in aging by degeneration of the microenvironment}. Altered mitochondrial morphology, decreased antioxidant capacities and elevated ROS levels [occurred] in long-term cultivated yMSCs as well as aMSCs. Notably, only the MSC migration potential and their antioxidative capacity were altered by in vitro as well as chronological aging."

"Cell roundness increases during long term culture of aMSCs and yMSCs"

Comparison of genes upregulated and downregulated by long term passaging to LSJL to be done.  A number of genes downregulated by passaging are focal adhesion proteins, regulation of actin cytoskeleton, mitochondrion, wnt signaling pathway, and TGF/BMP signaling.

Age-dependent decrease in the chondrogenic potential of human bone marrow mesenchymal stromal cells expanded with fibroblast growth factor-2.

"We examined age-related changes in the chondrogenic, osteogenic and adipogenic potential of mesenchymal stromal cells from 17 donors (25-81 years old), including patients with or without systemic vascular diseases.
All stem cell lines were expanded with fibroblast growth factor-2{upregulated by lsjl} and then exposed to differentiation induction media. The chondrogenic potential was determined from the glycosaminoglycan content and the SOX9, collagen type 2 alpha 1 (COL2A1) and aggrecan (AGG) messenger RNA levels{all upregulated by LSJL}. The osteogenic potential was determined by monitoring the alkaline phosphatase activity and calcium content, and the adipogenic potential was determined from the glycerol-3-phosphate dehydrogenase activity and oil red O staining.
Systemic vascular diseases, including arteriosclerosis obliterans and Buerger disease, did not significantly affect the trilineage differentiation potential of the cells. Under these conditions, all chondrocyte markers examined, including the SOX9 messenger RNA level, showed age-related decline, whereas none of the osteoblast or adipocyte markers showed age-dependent changes.
The aging of donors from young adult to elderly selectively decreased the chondrogenic potential of mesenchymal stromal cells."

GAG content of the chondrogenic medium decreased fairly dramatically by age and by age 90 was almost non-existent.    But is still quite significant in the ages of 20-40 in life.  Sox9 and Aggrecan were non-existant by age 70.   Whereas, Col2a1 was non-existent by age 65.  Osteogenic potential on the other hand increased with age.

Vascular disease decreased chondrogenic potential whereas it increased the osteogenic potential.  So the aging trends in osteogenic vs. chondrogenic differentiation may be related to vascular issues.

"The age-dependent decrease in SOX9 expression must limit the cartilage regeneration capability of MSCs."

Human fetal and adult bone marrow derived mesenchymal stem cells use different signalling pathways for the initiation of chondrogenesis.

"Human fetal mesenchymal stem cells (MSCs) have been isolated from a range of perinatal tissues including first trimester bone marrow and have demonstrated enhanced expansion and differentiation potential. However their ability to form mature chondrocytes for use in cartilage tissue engineering has not been clearly established. Here we compare the chondrogenic potential of human MSCs isolated from fetal and adult bone marrow and show distinct differences in their responsiveness to specific growth factors. Transforming growth factor beta 3 (TGFβ3) induced chondrogenesis in adult but not fetal MSCs. In contrast, bone morphogenetic protein 2 (BMP2) induced chondrogenesis in fetal but not adult MSCs. When fetal MSCs co-stimulated with BMP2 and TGFβ3 were used for cartilage tissue engineering they generated tissue with type II collagen and proteoglycan content comparable to adult MSCs treated with TGFβ3 alone. Investigation of the TGFβ/BMP signalling pathway showed that TGFβ3 induced phosphorylation of SMAD3 in adult but not fetal MSCs. These findings demonstrate that the initiation of chondrogenesis is modulated by distinct signalling mechanisms in fetal and adult MSCs. This study establishes the feasibility of using fetal MSCs in cartilage repair applications and proposes their potential as an in vitro system for modelling chondrogenic differentiation and skeletal development studies."

"Fetal MSCs have enhanced plasticity, proliferation propensity and expansion potential compared to adult MSCs. They appear to form an intermediate cell type between adult MSCs and embryonic stem cells (ESCs) as they have active telomerase and express pluripotency markers, albeit at a considerably lower level than ESCs"

"Fetal MSCs also lack intracellular HLA class II and have lower HLA class I expression compared to adult MSCs which suggests that these cells may be immunologically inert"

"the bone morphogenetic proteins (BMPs) have been shown to have the ability to induce de novo ectopic cartilage formation in a system that recapitulates endochondral ossification during skeletogenesis. Specifically, BMP2 has been shown to promote condensation of the mesenchymal cells in the developing limb"

"Fetal and adult MSCs both induced strong phosphorylation of BMP-specific SMAD1 and SMAD5 with BMP2 treatment alone or in combination with TGFβ3. However, strikingly we detected TGFβ3-mediated phosphorylation of SMAD1/5 in adult MSCs as well as a marginal level in fetal MSCs. TGFβ-induced signalling is predominantly activated through TGFBR1 and TGFBR2 to phosphorylate SMAD2/3. However, TGFβ has also been shown to activate SMAD1/5 phosphorylation, traditionally activated by BMP signals in numerous cell types. By signalling through TGFBR1, activin A receptor type II-like 1 (ALK1) is recruited and in combination with TGFBR2, activates SMAD1/5 phosphorylation. This dual signalling results in the formation of mixed-receptor SMAD complexes, which may bind to BMP promoters and influence BMP-mediated transcriptional responses. The expression of TGFBR2 and upregulation of BMPR2 in adult MSCs stimulated with TGFβ3 indicates that TGFβ-mediated SMAD1/5 phosphorylation may signal through a receptor complex incorporating TGFBR2-BMPR2 receptors to mediate the initiation of chondrogenesis."

"phosphorylation of both SMAD2/3 and SMAD1/5/8 is essential at the onset of chondrogenic differentiation and these SMADs remain in an active state in differentiated MSCs, while only SMAD2/3 is present in native articular cartilage"

"the initiation of chondrogenic differentiation of MSCs may be mediated by either TGFβ or BMP, but can only be maintained through signalling associated with SMAD3. This may explain the need for TGFβ3 in addition to BMP2 for optimal cartilage tissue engineering by fetal MSCs even though BMP2 alone is sufficient for the initiation of chondrogenic differentiation"

Monday, September 24, 2012

LSJL increases DNA Methylation

Since DNA Methylation markers tend to be upregulated during cellular differentiation, this is further evidence that LSJL induces chondrogenic differentiation.

Endothelin is upregulated 2.258 fold by LSJL.

Localized methylation in the key regulator gene endothelin-1 is associated with cell type-specific transcriptional silencing.

"To identify key developmental regulator genes whose expression in terminally differentiated cells may be inhibited by DNA methylation, mouse dermal fibroblasts were demethylated with 5-aza-2'-deoxycytidine, and changes in gene expression monitored. Endothelin-1 (Et1 or Edn1), which encodes a cytokine with diverse regulatory functions, was among the genes upregulated following demethylation. CpG dinucleotides within a short region in intron 1 of the gene have dramatically higher levels of methylation in Et1-non-expressing fibroblasts and chondrocytes as compared to the Et1-expressing mouse cell line, mIMCD-3. Strong evolutionary conservation of this region implies its role in the cis-regulation of Et1 transcription. To confirm that should Et1 in dermal fibroblasts become aberrantly activated, it could indeed lead to the dysregulation of many downstream genes, we exposed fibroblasts to exogenous ET1 peptide and assayed for transcriptional changes by microarray. ET1 treatment resulted in significant expression changes - primarily downregulation - of a significant number of genes. In particular, Tgfbeta2 and Tgfbeta3 were among the downregulated genes, which in turn alter the expression status of their many target genes."

"inhibition of Et1 in fibroblasts and chondrocytes may involve targeted methylation of the Sp1 binding site (among other sites) in intron 1, which block Sp1 binding and contribute to Et1 silencing."

New born mice were used.

Bold genes were also altered in demethylated fibroblasts.

Genes upregulated in Fibroblasts that were treated with exogenous et1 that also upregulated(or downregulated) in LSJL:
Slc7a3(downregulated in LSJL)

Genes downregulated in exogenous et1 fibroblasts:
Asporin(up in LSJL)
cnn1(up in LSJL)
col6a2(up in one DNA region down in another)
Prrx2(up in LSJL)
col6a1(up in LSJL)
c1qtnf3(up in LSJL)
slc38a4(up in LSJL)
Ccl2(up in LSJL)
ccl7(up in LSJL)
il6(up in LSJL)
9930013L23Rik(up in LSJL)

Lrrc15(up in LSJL)

A large portion of differentially regulated genes were genes that were altered in demethylated fibroblasts.  A large portion of the similarly regulated genes were those only effected by Et1 which corresponds to the data as knee loading upregulated Et1.  So LSJL likely involves cellular methylation.

Epigenomic and microRNA-mediated regulation in cartilage development, homeostasis, and osteoarthritis.

"epigenetic derepression [IS] associated with DNA methylation loss on chondrocyte genes, including MMP3[up in LSJL], MMP9, MMP13, ADAMTS4[up in LSJL], IL-1β, and LEP"

5-Azacytidine makes human preadipocytes able to differentiate into mesoderm-derived cell lineages.

"In the present study we have evaluated whether (i) 5-azacytidine (AZA), a well-known demethylating agent, could be able to modify the phenotype of human preadipocytes and (ii) the modified cells could possess multilineage differentiation potential. Human preadipocytes at the 3rd passage were treated for 48 or 96 h with 10 μM AZA and then expanded up to passage 5. Stem cell markers, such as OCT-4, Nanog, and Sox2, were upregulated after 96 h of treatment with the demethylating treatment. Further, decreases in the expression of genes, such as adipose differentiation-related protein, characterizing the preadipocytes were noted. AZA-treated preadipocytes differentiated into cell lineages derived from mesoderm. Indeed, after incubation with inductive media for 3 weeks, osteblast-, chondrocyte-, and myoblast-like cells were detected in the cultures. Interestingly, both upregulation of stem cell markers and differentiation potential were maintained by the treated cultures expanded until the 5th passage. AZA, without the use of transduction methods, convert preadipocytes to a less differentiated state that can be induced, under suitable stimuli, to the formation of mesoderm-derived cell lineages."

Instead of trying to induce methylation you may want to induce demethylation to induce chondrogenesis.  Since LSJL increases methylation, decreasing methylation in the bone before LSJL and only then(as decreasing methylation will reduce differentiation as well) may allow for more available stem cells to differentiate chondrocytes.  However, the next study suggests that the methylation status of chondrogenic genes is already low in adult human cells.

"DNA methylation is one mechanism that regulates human chondrogenesis.  [What's] the CpG methylation status in human synovium-derived MSCs during experimental chondrogenesis?
Human synovium-derived MSCs were subjected to chondrogenic pellet culture for 3 weeks. The methylation status of 12 regions in the promoters of 10 candidate genes (SOX9, RUNX2, CHM1, FGFR3, CHAD, MATN4, SOX4, GREM1, GPR39, and SDF1) was analyzed before and after differentiation. The expression levels of these genes were analyzed. Methylation status was also examined in human articular cartilage.
10 of the 11 CpG-rich regions analyzed were hypomethylated in human progenitor cells before and after 3 weeks of pellet culture, regardless of the expression levels of the genes. The methylation status was consistently low in SOX9{up in LSJL}, RUNX2, CHM1, CHAD, and FGFR3 following an increase in expression upon differentiation and was low in GREM1 and GPR39 following a decrease in expression upon chondrogenesis. One exceptional instance of a differentially methylated CpG-rich region was in a 1-kb upstream sequence of SDF1, the expression of which decreased upon differentiation. Paradoxically, the hypermethylation status of this region was reduced after 3 weeks of pellet culture.
The DNA methylation levels of CpG-rich promoters of genes related to chondrocyte phenotypes are largely kept low during chondrogenesis in human synovium-derived MSCs."

"chondromodulin 1 (CHM1), fibroblast growth factor receptor 3 (FGFR3), [MATN4]{up in LSJL}, and chondroadherin (CHAD) for genes up-regulated in chondrogenic pellet cultures, and for genes down-regulated in chondrogenic pellet cultures, these were SOX4, Gremlin 1 (GREM1), G protein–coupled receptor 39 (GPR39), and stromal cell–derived factor 1 (SDF1)."

"DNA methylation conditions in immature mesenchymal cells are permissive of the expression of chondrocyte phenotype"

Although one study suggests that loss of DNA methylation is associated with cellular senescence.  However this may have been only a correlational association and not a causal association.  Also, DNA methylation may be a marker of cellular differentiation state which corresponds to other data rather than a cell cycle differentiation counter.

It's possible that the upregulation of DNA Methylation related genes is due to fibroblast differentiation.  However the presence of chondrogenic genes like Sox9 confirms condrogenic differentiation. 

Thursday, September 20, 2012

Grow taller by Gap Repair

Sky's operates on the bone stretching principle.  If bone is stretched, tiny gaps are formed within the cortical bone that eventually heal resulting in a taller, longer bone.  Unfortunately, Sky seems to have fallen off the face of the earth and we don't know how Limbcenter is doing.  Here's an example of gap repair taken from an article about Bone Development and fracture repair:

In this article about the periosteum, the author explains about gap repair: "When no gap at all is present between the fractured cortical ends primary bone repair occurs by dint of osteoprogenitor cells derived directly from the Haversian systems within the cortex. This repair mechanism is also termed contact repair and produces lamellar bone already oriented correctly along the long axis of the bone. Gap repair or direct transformational bone repair comes into play when a small gap is present between the fractured ends. In this form, lamellar bone is formed initially at right angles to the bone and is then remodeled to the correct orientation. In wider gaps, woven bone is first produced and transformed to lamellar bone. In neither case is there a cartilage intermediary model."  You create more and more of these gaps by stretching the bone gradually resulting in a taller self.  So, even in cortical bone with no access to the periosteum you can grow taller.  You can grow taller with microfractures.  You can grow taller with the haversian canal.

Sky's method should work.  He just needs to provide photographic evidence.

Does LSJL induce more woven or lamellar bone formation?

Differential gene expression from microarray analysis distinguishes woven and lamellar bone formation in the rat ulna following mechanical loading.

"Formation of woven and lamellar bone in the adult skeleton can be induced through mechanical loading.  [We] distinguish the molecular responses between woven and lamellar bone formation induced through mechanical loading. Rat forelimb loading was completed in a single bout to induce the formation of woven bone (WBF loading) or lamellar bone (LBF loading). A set of normal (non-loaded) rats were used as controls. Microarrays were performed at three timepoints after loading: 1 hr, 1 day and 3 days. The micorarray identified numerous genes and pathways that were differentially regulated for woven, but not lamellar bone formation. Few changes in gene expression were evident comparing lamellar bone formation to normal controls. A total of 395 genes were differentially expressed between formation of woven and lamellar bone 1 hr after loading, while 5883 and 5974 genes were differentially expressed on days 1 and 3, respectively. A strong early inflammatory response preceded an increase in angiogenic and osteogenic gene expression for woven bone formation.  At later timepoints there was evidence of bone resorption after WBF loading."

Woven bone is typically associated with fractures so by analyzing gene expression data we can see if LSJL induces microfractures and if that plays a role in LSJL height increase.

"LBF loading did not increase cell proliferation, vessel volume or expression of angiogenic genes"

"The right forelimbs of adult male rats (Fischer F344, 5 mo., 337±24 g; Harlan) were loaded in axial compression"

"WBF loading applies a cyclic, haversine (2 Hz) waveform to the right forelimbs in a single bout (18 N peak force) until a prescribed increase in displacement (1.3 mm, 65% of fracture). "

"LBF loading applies a trapezoidal waveform to the right forelimb in a single bout (0.5 s triangle load-unload to 15 N, followed by 9.5 s rest; 100 cycles). Both WBF and LBF loading waveforms have a load/unload period of 0.5 s per cycle."

"Two transverse cuts were made to isolate the central 5 mm of the ulna and surrounding periosteum"<-whereas with LSJL the whole bone gene expression was analyzed.

Genes upregulated in Woven Bone:

IL6(up in LSJL)
Ptgs2(up 7 fold in LSJL)
Cxcl1(up 6.431 fold in LSJL)
Ctsk(downregulated initially in woven bone then upregulated)
Tgfb3(initially downregulated than upregulated)
Smad1(down in LSJL)
e2f1(down in LSJL)
Junb(up in LSJL)

Genes downregulated:

Egln1(Hif-1a inhibitor)
Dvl1(down in LSJL)

There is remarkable dissimilarity between woven bone formation and LSJL gene regulation.  The genes in woven bone formation were upregulated much higher than any gene in LSJL(sometimes up to 500 fold).  As noted earlier though gap repair can occur via lamellar and not woven bone formation.

Thus, LSJL is much more likely to induce new formation via lamellar bone formation(okay for height increase via gap repair) or endochondral ossification(great for height increase).

Distal radial fractures heal by direct woven bone formation.

"Descriptions of fracture healing almost exclusively deal with shaft fractures and they often emphasize endochondral bone formation. In reality, most fractures occur in metaphyseal[the area of the bone between the epiphysis and diaphysis] cancellous bone. We studied histological biopsies from the central part of 12 distal radial fractures obtained during surgery 6-28 days after the injury, using routine hematoxylin and eosin staining. New bone formation was seen in 6 cases. It was always in the form of fetal-like, disorganized woven bone. It seldom had contact with old trabeculae and appeared to have formed directly in the marrow. Cartilage was scarce or absent. The samples without bone formation showed only necrosis, scar, or old cancellous bone. The histology suggests that cells in the midst of the marrow respond to the trauma by direct formation of bone, independently of trabecular surfaces."<-If Cartilage was even there at all it's a good sign.

Men were ages 22-77.  Cartilage was found in 3 of the 12 fractures.  One of the individuals with cartilage had woven bone formation but no necrosis or soft tissue on day 28.  Another had necrosis and woven bone formation without soft tissue on day 14.  A third has necrosis and soft tissue but no woven bone formation.

What the three individuals who formed cartilage had in common was they had no bleeding, no trabeculae, and no old osteocytes.

"Cartilage was seen in scant amounts in 3 cases, all dominated by new woven bone formation. The cartilage mainly occurred in relation to necrotic areas{is bone necrosis important to induce cartilage differentiation?}. A gradual transition from cartilage to woven bone could be seen, suggesting that a gradient in local conditions had governed tissue differentiation. Endochondral ossification was never seen, but the small amounts of cartilage observed are most likely prone to undergo this process in due time."

"The most prominent occurrence of cartilage (C) in the study. It is located between woven bone and a necrotic hematoma[blood clot]. There appears to be a gradual transition from woven bone, via fibrocartilage, to hyaline cartilage."

"Platelet activation and danger-associated molecular patterns (DAMPs) activate the inflammatory response that is thought to start healing in general"

Cilium's role in adaptation to LSJL length increase response

LSJL may reduce in it's chondroinitiating ability over time because of the actin cytosekelton or the primary cilium.

The solitary (primary) cilium--a mechanosensory toggle switch in bone and cartilage cells.

"In bone the large number of osteocytes form a vast osteointernet in which the gap junctionally interconnected members are lodged in an extensive lacunocanalicular network. The much smaller number of articular chondrocytes are not interconnected in a chondrointernet [but] are separately lodged in capsules called chondrons. The non-motile solitary (primary) cilia protruding like aerials from osteocytes (as well as osteoblasts) and chondrocytes are switches that when toggled by cyclical pulses of lacunocanalicular fluid or cartilage compression send signals such as Ca(2+) surges into the cell to trigger a cascade of events that include appropriate gene activations to maintain and strengthen bone and cartilage. The chondrocyte cilium with its Ihh(Indian hedgehog)-activated Smo receptor is a key player along with PTHrP in endochondral bone formation."

"The trabecular lattice provides an immense surface for the interaction of bone cells with various hormones and cytokines to release Ca2+ into the blood when needed. Moreover the trabeculae also provide niches (nests) carpeted with retired osteoblasts, called bone-lining cells, that anchor and regulate the long-term hematopoietic stem cells"<-thus osteoblast signaling may play a role in chondroinduction by LSJL.

"Osteocytes must have a flow-metering device that is switched on and off by the pulsing L-C fluid and triggers Ca2+ surges, the expression of the COX-2 gene for PGE2 production and induces the expression of NO synthase that makes NO"

"murine MC3T3 preosteoblasts and MLO-Y4 osteocytes express the Tg737 and Kif3a genes for cilial proteins, express the genes for the PKD1 (PC-1) and TRPP2 (PC-2) signalplex. proteins and indeed have the solitary (primary) cilium protruding from them"<-LSJL does not up- or down-regulate any of these proteins or genes above threshold so maybe the cilia does not play a role in the reduction of adaptation to LSJL over time.

"PKD1 (PC-1) [activates] the P1 promoter of the gene for the Runx2 transcription factor which targets the osteoblast’s genes for α1 (I) procollagen, osteocalcin, osteopontin, and osterix"

"Chondrocytes are encapsulated in fibrillar-walled chondrons which are embedded in an ICM (interchondronal matrix), a hydroelastic collagenous composite. The chondron is distinguished by being selectively enriched with the minor collagen VI{many forms of Col6 are upregulated by LSJL} and water-binding proteoglycans (hyaluoronan and aggrecan)"

"when the cartilage and its chondrons are strongly and cyclically compressed the water associated with the chondron proteoglycans is squeezed through the PCC (pericellular capsule) wall into the much less strained ICM (i.e., the PCC has a smaller compression modulus than the ICM; it takes less force to produce a given amount of PCC strain than ICM strain) leaving the chondron with a higher osmotic pressure because of the compression-concentrated proteoglycans and salts."

"When the compression drops during each cycle, water with chondrocyte nutrients is drawn back into the decompressed chondron because of the high osmotic pressure produced during compression"

"By straining the PCC’s fibrillar meshwork and pushing the PCC wall closer to the cell surface during the flattening of the chondron during each compression cycle the chondrocyte cilium with meshwork fibrils attached to it will be bent "

"The upper part of the cilium is decorated with plaques containing α2β1 and α3β1 integrins which are tethered to the pericellular collagen fibers"

"Cilium generation in murine growth plate chondrocytes can be prevented by disabling the gene for the Kif3a subunit of the kinesin motor that carries the various structural and functional components up along the ciliary microtubules"<-Kif3a is not affected by LSJL above threshold.

"The Ihh receptor complex Smo•Ptc on the cilia of transit-amplying chondrocytes in developing bone is part of the Ihh-PTHrP feedback mechanism that determines the length of the pre-hypertrophic zone and thus the final adult bone length. In adult bones, the cyclical compression produced by various activities sends pulses of fluid through the bones’ extensive L-C steointernet. These pulses toggle the osteocytes’ solitary (primary) cilia which send waves of Ca2+ and Ca2+-mobilizaing IP3 signals through the L-C osteointernet."<-These waves of Ca2+ may initiate chondrogenic differentiation.

Stem cells have cilia as well:

Primary Cilia Mediated Mechanotransduction in Human Mesenchymal Stem Cells.

"The primary cilium is a single sensory cellular extension, which has recently been shown to demonstrate a role in cellular mechanotransduction and MSC lineage commitment. Short periods of mechanical stimulation in the form of oscillatory fluid flow (OFF) is sufficient to enhance osteogenic gene expression and proliferation of human MSCs. Cilium mediates fluid flow mechanotransduction in hMSCs by maintaining OFF-induced increases in osteogenic gene expression and, surprisingly, to limit OFF-induced increases in proliferation. [Primary cilium play a] pro-osteogenic mechanosensory role."

"As little as 1hr of pulsatile fluid flow (PFF) was sufficient to enhance COX2 gene expression in adipose derived hMSCs"<-COX2 gene expression was upregulated in less than an hour of LSJL but that was in rats.  Maybe human cells need longer duration in addition to more load.

"An increase in proliferation of hMSCs [follows] high magnitude, short-term OFF"

"The primary cilium is a singular, immotile microtubule based cellular extension which projects from the apical surface of nearly every cell in the human body"

"Upon HH ligand binding of the Patched1 (PTCH1) receptor, PTCH1 derepresses Smoothened (SMO) which in turn translocates to the tip of the primary cilium activating the Gli transcription factors{LSJL upregulates Gli3}"

Osteogenic differentiation was favored by the media so that is likely why chondrogenic factors were not detected.

"The flow rate was chosen to yield a peak shear stress of 1.0 Pa (28ml/min). For proliferation rate studies the flow rate was adjusted accordingly to yield a peak shear stress of both 1.0 Pa (28ml/min) and 2.0 Pa (56ml/min). For all experiments, cells were exposed to 2 hrs of oscillatory fluid flow."

"Mechanical stimulation did not significantly affect the mRNA expression of RUNX2, OPN, OCLN,
ALKLP, COL1a1 or BSP at any time point."<-this is in contract to LSJL which altered mRNA expression of the majority of those genes.

"higher magnitudes of flow are required to elicit a proliferative response in hMSCs."

When the cilia was inhibited responsed to mechanical stimuli was stunted.  Maybe the cilia play a role in adaptation to stimulis.

"ATP induced proliferation activates PI3K/Akt, mTOR/p70/S6K and ERK1/2 dependent signaling pathways in fibroblasts"

"the application of 30mins of OFF was sufficient to significantly enhance cAMP production in hMSCs"

"adult bone cells which do not possess b1 integrin and/or Focal Adhesion Kinase (FAK) do not respond to fluid shear with an increase in osteogenic gene expression"

"b1 integrins have been shown to localize to the primary cilium, including chondrocytes"

"Polaris siRNA was used to inhibit the primary cilia and the mRNA levels of transcription factors Runx2, PPARgamma, [and Sox9] were measured as markers of osteogenic, adipogenic and chondrogenic differentiation, respectively. MSCs with inhibited primary cilia had significantly decreased basal mRNA expression levels of all three lineages specific transcription factors indicating that primary cilia are critical in multiple differentiation pathways. Furthermore, to determine if primary cilia play a role in the differentiation potential of MSCs, progenitor cells transfected with either scrambled or polaris siRNA were cultured in osteo-inductive, chondro-inductive, or adipo-inductive media and lineage commitment was ascertained. Interestingly, within 24 h of culture, cells transfected with polaris siRNA in both osteogenic and adipogenic media lost adhesion and released from the slides; however MSCs in chondrogenic media as well as cells transfected with scrambled siRNA did not. Primary cilium is necessary for the normal progression of chemically induced osteogenic and adipogenic differentiation. As a control, the experiment was repeated with NIH3T3 fibroblasts and none of the effects of inhibited primary cilia were observed indicating that the loss of adhesion may be specific to MSCs. Furthermore after biochemically inducing the cells to differentiate, polaris knockdown resulted in abrogation of both Runx2 and PPARgamma mRNA while SOX9 mRNA expression was significantly lower[So lack of cilia reduces chondroinduction but does not inhibit]."

So if cilia does play a role in adaptation then LSJL will still work at inducing chondrogenesis just a reduced level.

"the response of cells to the family of secreted Wnt proteins has also been shown to be regulated by the primary cilium."<-LSJL involves Wnt2.

"The Sox-9 mRNA levels were 0.86 for the polaris transfected group vs. 1.53 for the [group with functional cilia]"

"chondrogenic differentiation may be regulated by additional mechanisms other than signaling cascades involving the primary cilia."<-thus maybe the 2 week decay in adaptation to bone genes to LSJL will be different for chondrogenesis.

Ift88 regulates hedgehog signaling, Sfrp5 expression, and β-catenin activity in post-natal growth plate.

"Dysfunction of primary cilia results in pleiotropic symptoms including skeletal dysplasia. Deletion of Ift88 and subsequent depletion of primary cilia from chondrocytes [results] in disorganized columnar structure and early loss of growth plate. We compared gene expression profiles in normal and Ift88 deleted growth plates. Pathway analysis indicated that Hedgehog (Hh) signaling was the most affected pathway in mutant growth plate. Expression of the Wnt antagonist, Sfrp5, was also down-regulated. Sfrp5 was up-regulated by Shh in rib chondrocytes and regulation of Sfrp5 by Shh was attenuated in mutant cells. TSfrp5 is a downstream target of Hh and Ift88 regulates its expression. Sfrp5 is an extracellular antagonist of Wnt signaling. We observed an increase in Wnt/β-catenin signaling specifically in flat columnar cells of the growth plate in Ift88 mutant mice as measured by increased expression of Axin2 and Lef1 as well as increased nuclear localization of β-catenin. Ift88 and primary cilia regulate expression of Sfrp5 and Wnt signaling pathways in growth plate via regulation of Ihh signaling."

"Arl13b{down} [is] a cilia specific protein"

Genes downregulated in primary cilia depleted chondrocytes also downregulated by LSJL:


Intraflagellar transport is essential for endochondral bone formation

"Cilia formation requires intraflagellar transport (IFT), and mutations disrupting the IFT process result in loss of cilia and mid-gestation lethality with developmental defects that include polydactyly and abnormal neural tube patterning. We generated a conditional allele of the IFT protein Ift88 (polaris). Using the Cre-lox system, we disrupted cilia on different cell populations within the developing limb. While deleting cilia in regions of the limb ectoderm had no overt effect on patterning, disruption in the mesenchyme resulted in extensive polydactyly with loss of anteroposterior digit patterning and shortening of the proximodistal axis. The digit patterning abnormalities were associated with aberrant Shh pathway activity, whereas defects in limb outgrowth were due in part to disruption of Ihh signaling during endochondral bone formation. the limbs of mesenchymal cilia mutants have ectopic domains of cells that resemble chondrocytes derived from the perichondrium, which is not typical of Indian hedgehog mutants."

"ectopic chondrocyte-like cells were observed between the perichondrium and diaphysis [in mice lacking Cilia]."

"cells adjacent to the perichondrium resembled chondrocytes rather than osteoblasts. While it is unclear if these cells originated from the perichondrium or the growth plate of the developing bone, they appeared to be continuous with the perichondrium in some sections, suggesting that they may have originated there"

B is where the ectopic chondrocytes are located(where the green arrows highlight).

"the development of ectopic chondrocytes along the diaphysis is not seen in Ihh;Gli3 mutants. Rather, this is characteristic of defects in canonical Wnt signaling."

"mice with conditional loss of β-catenin in the developing skeleton exhibited ectopic chondrocyte differentiation in the perichondrium"

"The ectopic cells in conditional mutants expressed aggrecan and morphologically resembled chondrocytes"

LSJL gene expression versus dynamic chondrocyte compression

LSJL involves a lateral compressive force.  Since in the 14 week old rats there were growth plates in the bone, it's important to see how much of the upregulation was due to stimulus of the growth plate itself and how much was stimulation of stem cells to differentiate into chondrocytes to form new growth plates.

"We used an experimental system consisting of primary mouse chondrocytes embedded within an agarose hydrogel; embedded cells were pre-cultured for one week and subjected to short-term compression experiments. Chondrocytes maintain a differentiated phenotype in this model system and reproduce typical chondrocyte-cartilage matrix interactions. After 15 min of dynamic compression, we observed transient activation of ERK1/2 and p38 (members of the mitogen-activated protein kinase (MAPK) pathways) and Smad2/3 (members of the canonical transforming growth factor (TGF)-β pathways). A microarray analysis performed on chondrocytes compressed for 30 min revealed that only 20 transcripts were modulated more than 2-fold. A less conservative list of 325 modulated genes included genes related to the MAPK and TGF-β pathways and/or known to be mechanosensitive in other biological contexts. Of these candidate mechanosensitive genes, 85% were down-regulated. Down-regulation may therefore represent a general control mechanism for a rapid response to dynamic compression. Furthermore, modulation of transcripts corresponding to different aspects of cellular physiology was observed, such as non-coding RNAs or primary cilium."

"The main collagen-binding integrin on chondrocytes in cartilage is α10β1 integrin, whereas α11β1 integrin is more characteristic of mesenchymal tissues."

Upregulated Genes:

Fos(up in LSJL)
Egr1(up in LSJL)
Nr4a1(up in LSJL)
Fosb(up in LSJL)
Junb(up in LSJL)
Atf3(up in LSJL)
Cyr61(up in LSJL)
Zfp36(up in LSJL, may be a limiting agent against growth)
Bgn(up in LSJL)

Downregulated Genes:
Sdpr(down in LSJL)
Htra1(up in LSJL, inhibits TGFbeta signaling)
MMP3(up in LSJL)
Col11a1(up in LSJL)

Of note is that the expression of Sox9, COL2A1, Aggrecan did not appear to be altered.  Thus the induction of new growth plates by LSJL may not require existing chondrocytes.

The effects of intermittent dynamic loading on chondrogenic and osteogenic differentiation of human marrow stromal cells encapsulated in RGD-modified poly(ethylene glycol) hydrogels.

"Poly(ethylene glycol) hydrogels were fabricated with tethered cell adhesion moieties, RGD. Cell-laden hydrogels were subjected to 4 h daily intermittent dynamic compressive loading (0.3Hz, 15% amplitude strain) for up to 14 days and the cell response evaluated by gene expression and matrix deposition for chondrogenic and osteogenic markers. The three-dimensional hydrogel supported chondrogenesis and osteogenesis under free swelling conditions, as shown by the up-regulation of cartilage-related markers (SOX9, Col II, Col X, and aggrecan) and staining for type II collagen and aggrecan and osteogenically by up-regulation of ALP and staining for type I collagen and for mineralization. Under dynamic loading the expression of cartilage-related markers SOX9, Col II, Col X, and aggrecan were down-regulated, along with reduced aggrecan staining and no positive staining for type II collagen. The bone-related markers RUNX2, Col I, and ALP were down-regulated and positive staining for type I collagen and mineralization was reduced.  The selected loading regime appears to have an inhibitory effect on chondrogenesis and osteogenesis of hMSC encapsulated in PEG-RGD hydrogels after 14 days in culture, potentially due to overloading of the differentiating hMSC before sufficient pericellular matrix is produced and/or due to large strains, particularly for osteogenically differentiating hMSC."

"dynamic compressive loading at 0.1 Hz and 5% amplitude strains led to a 79% increase in glycosaminoglycan content compared with statically compressed explants"

"Intermittent loading (0.5 h on, 1.5 h off) was applied for 16 h, followed by 8 h of rest. During the loading period a dynamic compressive strain was applied from 0% to 15% with a sinusoidal waveform and at a frequency of 0.3 Hz, while the resting period experienced no strain"

"SOX9 and Col X were upregulated by day 14. Col II increased 1900-fold by day 7 and remained high (2200-fold) at day 14. ACAN increased 70-fold by day 7 and dropped to levels similar to the basal condition by day 14 [in CDM(chondrogenic differentiation medium) with no load]."

"The application of loading to CDM constructs downregulated the expression of SOX9, ACAN, and Col I by day 7"

"either reduced loading (e.g. lower strains) and/or delaying the application of loading may be important in the differentiation potential of MSC."

Mechanical signals control SOX-9, VEGF, and c-Myc expression and cell proliferation during inflammation via integrin-linked kinase, B-Raf, and ERK1/2-dependent signaling in articular chondrocytes.

"Cytokines like interleukin (IL)-1beta suppress homeostatic mechanisms and inhibit cartilage repair and cell proliferation. Matrix synthesis and chondrocyte (AC) proliferation are upregulated by the physiological levels of mechanical forces.
ACs isolated from articular cartilage were exposed to low/physiologic levels of dynamic strain in the presence of IL-1beta.
Mechanoactivation, but not IL-1beta treatment, of ACs initiated integrin-linked kinase activation. Mechanical signals induced activation and subsequent C-Raf-mediated activation of MAP kinases (MEK1/2). IL-1beta activated B-Raf kinase activity. Dynamic strain did not induce B-Raf activation but instead inhibited IL-1beta-induced B-Raf activation. Both mechanical signals and IL-1beta induced ERK1/2 phosphorylation but discrete gene expression. ERK1/2 activation by mechanical forces induced SRY-related protein-9 (SOX-9), vascular endothelial cell growth factor (VEGF), and c-Myc mRNA expression and AC proliferation. IL-1beta did not induce SOX-9, VEGF, and c-Myc gene expression and inhibited AC cell proliferation. SOX-9, VEGF, and Myc gene transcription and AC proliferation induced by mechanical signals were sustained in the presence of IL-1beta."

ERK1/2 has two phosphorylation sites and mechanical loading and IL-1B can phosphorylation different sites.

Wednesday, September 19, 2012

Oxytetracycline: Possible Height increase compound

Oxytetracycline is a pretty commonly prescribed anti-biotic.  I have taken it before as an anti-acne medication.

Identification of oxytetracycline as a chondrogenic compound using a cell-based screening system.

"we screened natural and synthetic compound libraries using a Col2GFP-ATDC5 system and identified oxytetracycline (Oxy) as a chondrogenic compound. Oxy induced cartilaginous matrix synthesis and mRNA expressions of chondrocyte markers in ATDC5 cells. In addition, Oxy suppressed mineralization and mRNA expressions of terminal chondrocyte differentiation markers in ATDC5 cells, primary chondrocytes, and cultured metatarsal bones. Oxy's induction of Col2 mRNA expression was decreased by the addition of Noggin and was increased by the addition of BMP2. Oxy increased mRNA expression of Id1, Bmp2, Bmp4, and Bmp6. Oxy induces chondrogenic differentiation in a BMP-dependent manner and suppresses terminal differentiation."

"Oxy induced mRNA expressions of chondrocyte collagens (Col2, Col9, Col11), and transcription factors regulating chondrogenesis (Sox5, Sox6, Sox9) in a dose-dependent manner"<-All of these except for Sox5 and Sox6 were upregulated above threshold in LSJL.  Sox5 and Sox6 could have been upregulated below threshold in LSJL.

"Oxy reduced von Kossa staining and mRNA expressions of terminal chondrocyte differentiation markers, Col10{upregulated by LSJL}, Opn{upregulated by LSJL}, and Mmp13 in a dose-dependent manner. Oxy also reduced Col10, Opn, Mmp13 mRNA expressions in primary chondrocytes"

"Oxy’s induction of Col2 mRNA expression was decreased by the transfection of siSox6, siSox9, and siBmp2"

According to Tetracyclines inhibit rat osteoclast formation and activity in vitro and affect bone turnover in young rats in vivo., Tetracyclines may inhibit osteoclast formation and osteoclasts may play a key role in height growth.

Effects of mannan oligosaccharide or antibiotics in neonatal diets on health and growth of dairy calves.

"Calves were fed a 20% protein, 20% fat milk replacer containing antibiotics (400 g/ton neomycin + 200 g/ton oxytetracycline), MOS (4 g of Bio-Mos/d), or no additive (control) for 5 wk"

"[No] growth differences during the experimental period."

Although hip height in the antiobitic group was not statistically significantly shorter versus control.

Unfortunately, there is hardly any research on this.  The beneficial effects of Oxy on chondrogenic proteins may be counteracted by the inhibition of osteoclasts.  There seems to be a followup study [Novel small compound in combination with cell-sheet technology for articular cartilage regeneration]. but I cannot find the full version.

Also, Oxy has only been shown so far to have effects in ATDC5 cells and ATDC5 cells have been shown to have different effects to Human BMMSC's before.  But, Oxy is available.

Does anyone have any cases of overgrowth or undergrowth due to Oxytetracycline?

Tuesday, September 18, 2012

Surpass your Natural Height by increasing Mechanosensitivity

Many people have reported a tapering off of LSJL results like our individual who grew 1/4" an inch and another who also grew 1/4" of an inch. The decrease in LSJL results over time could be a result of a loss of mechanosensativity. I have grown 1 1/2" with Lateral Synovial Joint Loading, each increase in height occurring after an increase in load Bone(and probably chondrocytes, stem cells, and periosteum) loses mechanical sensitivity after loading but recovers 98% after 24 hours.  Now, mechanical sensitivity in response to load of muscles is well documented and a number of pathways throughout the body are shared like the Wnt signaling pathway and actin filaments.

So, we can probably infer a lot about how to surpass plateau's in LSJL by how people surpass plateau's in muscle building.  Some possible methods are inhibiting myostatin, increasing the load, and strategic deconditioning.  Let's look at how we can increase mechanosensitivity in stem cells.

Mechanosensitivity of the rat skeleton decreases after a long period of loading, but is improved with time off 

"After the initial adaptation to large mechanical loads, it appears as though the skeleton's responsiveness to exercise begins to wane. To counteract the waning effects of long-term mechanical loading, “time off” may be needed to improve the responsiveness of bone cells to future mechanical signals and reinitiate bone formation{this may be true for chondrocytes and stem cells as well}. Fifty-seven female Sprague–Dawley rats (7–8 months of age) were randomized to one of following groups: Group 1 loading was applied for 5 weeks followed by 10 weeks of time off (1 × 5); Group 2 loading was applied for 5 weeks, followed by time off for 5 weeks and loading again for 5 weeks (2 × 5); Group 3 loading was applied continuously for 15 weeks (3 × 5); Group 4 age-matched control group; and Group 5 baseline control group. An axial load was applied to the right ulna for 360 cycles/day, at 2 Hz, 3 days/week at 15 N[so not lateral loading so no pressure gradient(laterally loading squeezes the bone thereby inducing fluid flow) like LSJL but still shows effects to sensitivity to load over time]. At the end of the intervention, all three loaded groups showed similar increases in bone mass, cortical area, and IMIN in response to mechanical loading. Bone formation rate of the loaded ulna was increased in the first 5 weeks of loading for all three loaded groups; however, during the last 5 weeks, it was only significantly increased in the group that had time off (2 × 5). The group that had time off (2 × 5) also showed greater improvements in work to failure compared to the group loaded for 5 weeks (1 × 5) and the entire 15 weeks (3 × 5). A second experiment showed that the waning effect of long-term loading on the skeleton is not a result of aging. mechanical loading of the rat ulna results in large improvements in bone formation during the first 5 weeks of loading, but continual loading decreases the osteogenic response. Having time off increases bone formation and improves the resistance to fracture."

Note that the bone was loaded for 3 days a week which is well within the normal deconditioning period for the actin cytoskeleton(24-48 hours).  The group that had 5 weeks off had the greatest improvements in bone parameters but that doesn't mean 5 weeks is needed to restore mechanosensativity.

"To counteract the waning effects of long-term mechanical loading, “time off” may be needed to improve the responsiveness of bone cells to future mechanical signals and reinitiate bone formation"<-the same principles may apply to chondrocytes and stem cells depending on what mechanism requires the time off.  If it is a mechanism shared by all three cell types then it is likely that all three types may need the same deconditioning.  And the mechanism of conditioning is important to know whether this adaptation to axial loading also applies to lateral loading(which increases intramedullary pressure).

"Short-term loading studies in rats suggest that rest periods ranging from 10 to 14 s in between individual loading cycles, and from 1 to 8 h in between bouts of loading enhance the osteogenic response more than continuous uninterrupted loading"<-this may be an area of difference between stem cells and osteogenic cells.

"Mechanical loading imposed a strain of 3288 ± 83 μvar epsilon at the midshaft of the ulna in the beginning of the experiment. The strain was decreased in all three loaded groups and age-matched controls due to periosteal bone apposition that occurred over the 15-week period."<-so the adaptation can be due to a loss of relative strain(which decreases due to bone apposition as the stronger the bone is the less strain that is being applied at the same load).  IF relative strain increased then adaptation would continue.  This may not apply to LSJL in which increased pressure results in chondrogenic differentiation.  However, these chondrocytes eventually undergo endochondral ossification not resulting in a permanent adaptation.  Osteoblasts become osteocytes for a longer period time than chondrocytes which are transient as they undergo apoptosis before completing endochondral ossification.

"An exercise regime that maintains the same loads for many years (i.e., long-distance running) may only be beneficial during the initial months of training. This phenomenon has already been identified in tennis players and exercise interventions in children. a period of time off may be needed to reinitiate a response."<-Strain on the bone is required for TGF-Beta release by the osteocytes.  TGF-Beta helps induce chondrogenic differentiation so time off from LSJL may be important.  Stronger clamping may be another alternative to time off.

Loading alters actin dynamics and up-regulates cofilin gene expression in chondrocytes.

"The actin cytoskeleton has been implicated in cell mechanics and mechanotransduction. Loading modulates actin dynamics and organisation with subsequent changes in gene expression for actin associated proteins. Chondrocytes were transfected with eGFP-actin, seeded in agarose and subjected to cyclic compression (10 cycles, 1 Hz, 0-15% strain). Compression resulted in a subsequent reduction in cortical eGFP-actin intensity and a reduction in fluorescence recovery after photobleaching (FRAP), suggesting net cortical actin de-polymerisation, compared to unloaded controls. Cyclic compression for 10 min up-regulated gene expression for the actin depolymerising proteins, cofilin and destrin. mechanical loading alters cortical actin dynamics, providing a potential mechanism through which chondrocytes can adapt their mechanical properties and mechanosensitivity to the local mechanical environment."

Lateral Loading is compressive loading for the stem cells to differentiate into chondrocytes.  Compressive loading reduces mechanosensitivity by upregulating actin depolymerising proteins.  If you can re-polymerise the actin you may be able to restore mechanosensitivity in the chondrocytes(and your muscles, bones, etc.).  Thus, the need for time off to allow actin to re-polymerise or increase the strength of LSJL clamping to make up for less sensative actin.

Membrane type-1 matrix metalloproteinase is induced following cyclic compression of in vitro grown bovine chondrocytes.

"Membrane type-1 matrix metalloproteinase (MT1-MMP)[MMP14 which is upregulated by LSJL] [responds] to cyclic compression of chondrocytes grown in vitro.
Cyclic compression (30min, 1kPa, 1Hz) was applied to bovine [articular] chondrocytes (6-9-month-old animals) grown on top of a biodegradable substrate within 3 days of initiating culture.
After cyclic compression, MT1-MMP showed a rapid and transient increase in gene expression. Elevated protein levels were detected within 2h of stimulation which returned to baseline by 6h. During cyclic compression, phosphorylation of the mitogen activated protein kinase ERK1/2 increased significantly. This was followed by increased gene and protein expression of the transcription factor; early growth factor-1 (Egr-1)[upregulated by LSJL] and Egr-1 binding to the MT1-MMP promoter. Blocking Egr-1 DNA binding with a decoy MT1-MMP oligonucleotide, downregulated MT1-MMP gene expression. The ERK1/2 inhibitor U0126 also reduced Egr-1 DNA binding activity to MT1-MMP promoter sequences and subsequent transcription of MT1-MMP.
cyclic compression of chondrocytes in vitro upregulates MT1-MMP via ERK1/2 dependent activation of Egr-1 binding."

Since LSJL increased EGR-1 and MT1-MMP maybe LSJL also increased ERK1/2 phosphorylation?  But we don't know Egr-1 and MMP14 binding only that they both increased in expression.

MT1-MMP contributes to the formation of cartilage canals.  Cartilage canals being very important in the formation of growth plates and in turn surpassing your natural height.  LSJL also upregulates early growth factor-1 which further accelerates MT1-MMP expression.  The mechanosensitivity of MT1-MMP may be very important in LSJL induced height growth as MT1-MMP is crucial in the development of cartilage canals a possible mechanism for how LSJL works. 

"MT1-MMP can activate MMP-13 and has been shown to be mechanosensitive"

"After 3 days of culture, cells were cyclically compressed for 30 min and 24 h later showed a significant (3.9-fold) increase in relative luciferase activity (RLU) (indicative of MT1-MMP expression) compared to unstimulated controls"<-About twice as much increase in fold as was observed in LSJL.  And LSJL was 1 hour after loading whereas this was 24 hours after loading.

"the phosphorylated form of ERK1/2 (p-ERK1/2) increased 13.1-fold within the first 15 min of mechanical stimulation "

"Displacement of Egr-1 by Sp1 only occurs after phosphorylation at several different sites on Sp1, which increases its affinity for DNA binding. Sp1 is mechanosensitive as shear stress increased Sp1 activation in endothelial cells and inhibited MT1-MMP expression"

"In human chondrocytes, induction of Egr-1 by interleukin-1β resulted in repression of transcriptional activity of type II collagen. Type II collagen expression, one of the main constituents of articular cartilage matrix is upregulated when Sp1 is bound, or inhibited when Egr-1 is bound and displacement of the Egr-1 protein by the Sp1 transcription factor reversed this inhibition"

Mechanical compression and hydrostatic pressure induce reversible changes in actin cytoskeletal organisation in chondrocytes in agarose.[Lateral Synovial Joint Loading induces both mechanical compression and hydrostatic pressure, thus LSJL induces reversible changes in actin cytoskeletal organization] 

"In numerous cell types, the cytoskeleton has been widely implicated in mechanotransduction pathways involving stretch-activated ion channels, integrins and deformation of intracellular organelles. Studies have also demonstrated that the cytoskeleton can undergo remodelling in response to mechanical stimuli such as tensile strain or fluid flow. This study utilises the well-characterised chondrocyte-agarose model to demonstrate that both static and cyclic, compressive strain and hydrostatic pressure all induce remodelling of actin microfilaments. This remodelling was characterised by a change from a uniform to a more punctate distribution of cortical actin around the cell periphery. For some loading regimes, this remodelling was reversed over a subsequent 1h unloaded period. This reversible remodelling of actin cytoskeleton may therefore represent a mechanism through which the chondrocyte alters its mechanical properties and mechanosensitivity in response to physiological mechanical loading." 

The actin skeleton of chondrocytes remodels to become less sensitive to mechanical loads.  This is reversible over time due to unloading. 

"Two different compressive loading regimes were investigated, namely cyclic strain between 0% and 15% at a frequency of 1 Hz and static strain of 15%"

"Pressure was applied in either a cyclic form between 0 and 5 MPa at 1 Hz or statically at 5 MPa. Control specimens remained unstrained and at atmospheric pressure throughout."

"Cells fixed 1 min after any of the four loading regimes exhibited notable differences in actin cytoskeletal organisation with a more pronounced punctate distribution than that observed in unloaded cells."

Static strain induced greater changes in actin cytoskeleton than cyclical strain.  Actin cytoskeleton decreased to almost control in only 60 minutes after cyclic strain.

In contrast to cyclical hydrostatic pressure actin cytoskeleton organization increased after 60 minutes.  In static hydrostatic pressure actin cytoskeleton organization decreased.

"Gross compression causes cell deformation from a spherical to an oblate ellipsoid morphology. The presence of a limited pericellular matrix at day 3 reduces the level of cell deformation"

"Cells were fixed in the unstrained spherical state to enable analysis of actin remodelling without the influence of changes in cell shap"

Mechanosensativity may also involve neural regulation:

Functional adaptation to loading of a single bone is neuronally regulated and involves multiple bones.

"Regulation of load-induced bone formation is considered a local phenomenon controlled by osteocytes [and] may be neuronally regulated. Load-induced responses in the left and right ulnas and humeri were determined after loading of the right ulna in male Sprague-Dawley rats (69 +/- 16 days of age). After a single period of loading at -760-, -2000-, or -3750-microepsilon initial peak strain, rats were given calcein to label new bone formation. Bone formation and bone neuropeptide concentrations were determined at 10 days. In one group, temporary neuronal blocking was achieved by perineural anesthesia of the brachial plexus with bupivicaine during loading. right ulna loading induces adaptive responses in other bones in both thoracic limbs compared with Sham controls and that neuronal blocking during loading [stopped] bone formation in the loaded ulna and other thoracic limb bones[bones relating to the chest]{but does this neuronal blocking stop chondrogenic induction?}. Skeletal adaptation was more evident in distal long bones{farther away from the body so the the tibia versus the femur} compared with proximal long bones. The single period of loading modulated bone neuropeptide concentrations persistently for 10 days[so the most efficient period of loading for LSJL may be once every 10 days, HIT(High Intensity Training) recommended weight lifting every ten days.  So LSJL once every 10 days may be most efficient if you're short on time but results will take much longer than if you load more frequently]. Functional adaptation to loading of a single bone in young rapidly growing rats is neuronally regulated and involves multiple bones. Persistent changes in bone neuropeptide concentrations after a single loading period suggest that plasticity[ability to undergo permanent changes] exists in the innervation of bone."

The study involves osteocytes but properties of chondrocytes can be inferred.  If there is some neurological control then different means to restore adaptation may be used.  Also, in the Lateral Synovial Joint Loading study some increase in height was detected in the non-loaded contra-lateral limb.  This indicates that there are neurological mechanisms at play in terms of stem cells and chondrocytes.  For example, perceived strain may have a role on neurological adaptation but not at more local physical levels.

If you're short on time do LSJL once every 10 days.  However, neuropeptide concentrations may still be sensitive to LSJL loading at much shorter intervals.

"The periosteoum is densely innervated with a dense net-like meshwork of nerves, suggesting the existence of a sophisticated and specialized neuronal regulatory mechanism optimized for detection of mechanical distortion of periosteum and bone. Peptidergic nerves are arranged into networks on the surface of bones, and are most numerous in the epiphysis and metaphysis."<-So it's possible that load on the fibrous capsule can induce a functional response in the epiphysis.

"Neuropeptides have pleiotrophic effects on bone cells, which express receptors for a range of neurotransmitters. In vitro, CGRP-α and SP act anabolically on bone cells, where as CGRP-β is not anabolic, and in vivo, CGRP+ and SP+ sensory nerves and sympathetic nerves may influence bone mass."<-Studying neurotransmitters such as SP may be a way to induce new height growth.

"In-vivo load-induced bone formation was associated with persistent decreases in bone CGRP, suggesting that the regulatory effects of CGRP+ sensory nerves on functional adaptation of bone are complex[CGRP may play a role in why the body may become less susceptible to LSJL over time]. Strain-dependent effects on bone concentrations of neuropeptides were most evident with CGRP. As initial peak strain during loading increased, significant depression of bone CGRP concentrations occurred in as little as one hour after loading at high initial peak strain[CGRP concentration plays a role in mechanical sensitivity to load, of course we are looking for the neuropeptide associated with stem cells and chondrocytes]. This suggests that the peptideric innervation of bone is mechanosensitive and capable of a sophisticated response to changes in the loading environment of bone. In contrast, changes in SP and VIP concentrations in bone appeared much less mechanically sensitive. As initial peak strain increased, bone concentrations of these neuropeptides were higher, particularly at 10 days after loading. Such pleiotropic responses to mechanical loading by the innervation of bone again suggest the existence of a sophisticated mechanosensitive physiological pathway."<-We have to find out what neuropeptide is associated with stem cell/chondrogenic adaptation to loading.  It's unlikely to be CGRP.  However, Calcitonin[The C in CGRP stands Calcitonin] does induce responses in chondrocytes.  However, it's unknown whether is an actual receptor chondrocytes or the effects of calcitonin are collateral affects from calcitonin on bone.  But, if it is collateral effects that induce height growth than the mechanosensativity to CGRP is something worth pursuing.

A melacorticon[ACTH] may be the neuropeptide that is mechanosensative in chondrocytes.

A possible role for melanocortin peptides in longitudinal growth.

"The elevation of the melanocortin peptide, ACTH [is associated with] longitudinal growth. Overproduction of ACTH in familial glucocorticoid deficiency (FGD) is associated with increased growth and ACTH increases the differentiation of chondrocytes along the endochondral pathway in vitro[more differentiation of stem cells to chondrocytes = more height growth]. Using the leptin-deficient obese (ob/ob) mouse along with lean control littermates (n = 9-10), we investigated the effects of adrenalectomy[removal of the adrenal glands] (ADX)-induced elevated ACTH with and without peripheral administration of the MC3-R-specific agonist, gamma2-melanocyte stimulating hormone (gamma2-MSH), on longitudinal growth. Naso-anal and tibial growth were measured together with growth plate parameters; both total and zonal heights together with the proliferative index. ADX significantly increased naso-anal length in lean mice and ADX plus gamma2-MSH administration significantly increased naso-anal length above ADX alone in ob/ob mice. gamma2-MSH administration to ADX lean and ob/ob mice significantly increased tibial length. In ob/ob mice, these changes occurred in the context of reduced food intake. Analysis of total and zonal growth plate heights suggest an increase in hypertrophic differentiation and an overall increase in growth plate turnover in ADX lean and ob/ob mice. ADX enhances linear growth and the results of gamma2-MSH treatment suggest that the melanocortin system plays a role in linear growth."

ACTH is sold as a homeopathic remedy.  The effectiveness is unclear.Adrenal Corticot. (Potency: 12C)  Maybe Adult Height Increase should test it as part of their monster trial?

"ACTH also evokes transient elevations in intracellular free calcium [Ca2+]i and increases basal [Ca 2+]i and differentiation of chondrocytes along the endochondral pathway is associated with an incremental increase in basal [Ca2+]i"

So, ACTH may involve Calcium ions as well. Maybe CGRP and ACTH play a role in enhancing longitudinal growth with Calcium ions being the main factor.

L-Type Voltage Gates may play a role in mechanosensativity to these calcium ions.

Bone strength: current concepts.

"Bone is a multiphase material made up of a tough collagenous matrix intermingled with rigid mineral crystals. The mineral gives bone its stiffness. Without sufficient mineralization, bones will plastically deform under load[we are looking for this deformation to increase hydrostatic pressure in the epiphysis to induce chondrogenic differentiation]. Collagen provides toughness to bone making it less brittle so that it better resists fracture. Bone adapts to mechanical stresses largely by changing its size and shape. Tissue is added in regions of high mechanical stress.   bone tissue possesses a mechanosensing apparatus.  Several pathways [for mechanosensing] including membrane ion channels, ATP signaling, second messengers, such as prostaglandins and nitric oxide, insulin-like growth factors, and Wnt signaling."

It's possible that numerous pathways play a role in bone cell response to mechanical loading.

"In cultured osteoblastic cells, fluid flow increases intracellular calcium within minutes and this response is suppressed by gadolinium, a blocker of the stretch-activated calcium channel[Fluid flow is initiated by LSJL]. In addition, the L-type voltage-operated calcium channel probably plays a role in bone cell mechanotransduction. Studies using bone explants showed that gadolinium abolished loading-related responses in osteocytes, while a blocker of L-type calcium channels inhibited loading-related responses in osteoblasts. In addition, two blockers of L-type calcium channels, verapamil and nifedipine, strongly suppress mechanically induced bone formation in rats."<-Chondrocytes do seem to be receptive to calcium ions with Calcium ions encouraging MMP synthesis.  So the L-type calcium channel sensitivity may play a role in chondrocyte driven height growth.

"Prostaglandins and nitric oxide are released from bone cells within minutes after dynamic mechanical loading. Blockade of prostaglandin synthesis using nonsteroidal anti-inflammatory drugs (NSAID) suppresses mechanically induced bone formation in vivo, as does the nitric oxide synthesis inhibitor L-NAME."<-This is likely true of chondrocytes as well.

"In cultured osteoblasts, PTH(1-34) sensitizes cells to mechanical forces possibly by enhancing the mobilization of intracellular Ca2+."PTH(1-34) is teraparatide.

"Estrogen is another hormone that may interact with mechanical loading pathways. Mechanical loading increases estrogen receptor alpha phosphorylation in osteoblasts through activation of ERK 1. In addition, mice deficient in estrogen receptor alpha expression show suppressed osteogenic responsiveness to mechanical loading. In contrast, others have shown that estrogen suppresses the anabolic effect of mechanical loading. These observations might be reconciled by considering that the effects of estrogen on the skeleton are site specific. Estrogen suppresses bone resorption on trabecular and endocortical bone surfaces, thus preserving bone mass. Conversely, estrogen suppresses bone formation on the periosteal surfaces"<-These effects wouldn't seem to affect height growth.  The suppression of bone resorption within the trabecular bone may be a problem for growth plate modeling.

"Pressure in the marrow cavity and/or fluid shear forces on marrow stromal cells (MSC) may stimulate nitric oxide synthase (NOS) activity and nitric oxide (NO) release"<-LSJL definitely increases pressure in the epiphyseal marrow cavity.

Staurosporine and cytochalasin D induce chondrogenesis by regulation of actin dynamics in different way.

"Actin cytoskeleton has been known to control and/or be associated with chondrogenesis. Staurosporine and cytochalasin D modulate actin cytoskeleton and affect chondrogenesis[So straurosporine and cytochalasin D may have height increase applications].  We investigate the effect of staurosporine and cytochalasin D on the actin dynamics as well as possible regulatory mechanisms of actin cytoskeleton modulation. Staurosporine and cytochalasin D have different effects on actin stress fibers in that staurosporine dissolved actin stress fibers while cytochalasin D disrupted them in both stress forming cells and stress fiber-formed cells[so they two compounds are complementary of each other]Increase in the G-/F-actin ratio either by dissolution or disruption of actin stress fiber is critical for the chondrogenic differentiation[Thus staurosporine and cytochalasin D can help us grow taller]. Cytochalasin D reduced the phosphorylation of cofilin, whereas staurosporine showed little effect on cofilin phosphorylation. Either staurosporine or cytochalasin D had little effect on the phosphorylation of myosin light chain (MLC). These results suggest that staurosporine and cytochalasin D employ different mechanisms for the regulation of actin dynamics and provide evidence that removal of actin stress fibers is crucial for the chondrogenic differentiation."

Now just need to find a supplement that involves staurosporine and cytochalasin D.  If you take these supplements you might not need a deconditioning period.

"Dynamic actin cytoskeleton is essential for diverse cellular processes such as the driving cell shape changes, cellular motility, adhesion, cytokinesis, and endocytosis"<-Processes which are all involved in chondroinduction.

"Cytochalasin D, a blocker of actin polymerization and elongation of actin, has been extensively used for the study of chondrogenesis. It has been known to induce chondrogenesis of limb mesenchymal cultures"

"Staurosporine, a broad spectrum protein kinase inhibitor, disrupts the actin stress fibers and restores the differentiated functions of dedifferentiated chondrocytes"

"Actin binding proteins regulate disassembly and assembly of actin filaments by sequestering G-actin and by depolymerizing actin. Formation of actin stress fibers is induced by myosin light chain (MLC) phosphorylation which regulates the activity of non-muscle myosin type II. Cofilin binds to both monomeric and filamentous actin and increase actin dynamics by depolymerizing filaments form their pointed ends. The activity of cofilin is regulated by phosphorylation of Serine 3. Phosphorylation of cofilin abolishes the ability of cofilin to bind to F-actin leading to loss of its ability to depolymerize F-actin"

"Staurosporine treatment [on undifferentiated cells] for 2 days induced the expression of type II collagen. Cytochalasin D treatment also resulted in disintegration of the stress fibers but F-actin exhibited an aggregated pattern. Cytochalasin D-treated cells also expressed collagen type II as in the case of staurosporine. "

"Cells which still [had] stress fibers, even thin and small in number, were not stained for collagen type II."

"the relative amount of filamentous (F)-actin, globular (G)-actin, and relative abundance of F-actin compared with G-actin [is the] F/G-actin ratio"<-So more G-actin is pro-chondrogenic.

"F-actin which was dissolved by staurosporine or cytochalasin D, showed a diffused or spotted distribution, respectively. The relative amount of G-actin was decreased with staurosporine treatment and increased with cytochalasin D treatment, which were reflected in the F/G actin ratio. Both of staurosporine and cytochalasin d reduced F-actin/G-actin ratio with more effect by cytochalasin D"

"Cytochalasin D decreases the phosphorylation of cofilin. Cytochalasin B itself has a weak severing effect. Hcytochalasin D reduces phosphorylation of ADF/cofilin which would activate cofilin and thereby depolymerize F-actin. cytochalasin D promotes F-actin depolymerization by reduction of cofilin phosphorylation in concert with the prevention of F-actin polymerization by its immediate effect on the organization of cytoskeletal networks."

"Inhibition of protein kinase C, protein kinase A, and Ca2+/calmodulin-dependent protein kinase with specific inhibitors did not induce the chondrogenesis of mesenchymal cells"

"ROCK controls actin dynamics by regulating MLC phosphorylation directly or indirectly through MLC phosphatase or by regulating cofilin phosphorylation through LIM kinases"

"Staurosporine reduced the activity of RhoA.  RhoA regulates ROCK activity but inhibition of ROCK activity neither affects MLC phosphorylation nor induces chondrogenesis.  RhoA uses other pathway than ROCK for the chondrogenic differentiation"

"cytochalasin D reduced phosphorylation of cofilin"

Cellular accommodation and the response of bone to mechanical loading.

"The ulnae of Sprague-Dawley rats were loaded in axial compression. The animals received loading for 15 weeks with progressively decreasing loads, increasing loads, or a constant load. The results showed the largest increases in geometry in the decreasing load group, followed by the constant load group. Bone formation rates (BFRs) were significantly greater in the decreasing load group during the first 2 weeks of the study as compared to all other groups (P<0.05)[the decreasing load group had the highest load first]. After the first few weeks of mechanical loading, the BFR in the loaded ulnae returned to the values of the nonloaded ulnaeAfter the initial weeks of loading, bone stopped responding so the degree of adaptation was proportional to the initial peak load magnitude."

Stem cells ability to differentiate into chondrocytes may stop responding as well especially if osteocytes release chemical signals that alter this mechanism.  This means that you probably don't want to gradually work up but start with the maximum planned LSJL load right away followed by a deconditioning period.

"Bone cells must process loading information locally because bone tissue is poorly innervated and, unlike many mechanoreceptor cells, cannot rely on the central nervous system to integrate and distribute information about mechanical signals"

"the MES[minimum effective strain] may well be location dependent within bones"

"the groups that had the largest force applied at the start of the study saw the largest overall changes. After the initial weeks of the loading, bone stopped responding to mechanical loading"

"10 weeks of loading desensitized bone cells so no further adaptation response was possible in the last 5 weeks"

"significant trabecular bone formation occurring in response to loading within the first week of loading, with significantly less in weeks 2–4 of loading"

This next study will help answer our question of whether loading produces adaptations in marrow cells that would reduce their responsiveness to chondroinduction by LSJL.  By studying unloading we can see if there's any indication of the sensitivity of genes increasing.

"Hind limb unloading (HU) [was performed on] the tibiae of young C57BL/6J male mice. We focused on the effects of HU in chondrogenic, osteogenic, and marrow mesenchymal cells.
We analyzed for expression of genes and proteins at two time points after HU (7 and 14 days), and at 14 days after recovery from HU.  We studied genes involved in osteogenesis (alkaline phosphatase (AP), osteocalcin (OC){up in LSJL}, bonesialoprotein (BSP){up in LSJL}, membrane type1 matrix metalloproteinase (MT1-MMP)), in extracellular matrix (ECM) formation (procollagenases (BMP1), procollagenase enhancer proteins (PCOLCE)) and remodeling (metalloproteinase-9 (MMP9), RECK), and in bone homeostasis (Stro-1, CXCL12, CXCR4, CD146).
We report the following patterns and timing of changes in gene expression induced by HU: 1) transient or stable down modulations of differentiation-associated genes (AP, OC), genes of matrix formation, maturation and remodelling, (BMP1, PCOLCEs MMP9) in osteogenic, chondrogenic and bone marrow cells; 2) up modulation of MT1-MMP in these same cells, and uncoupling of its expression from that of AP; 3) transient down modulation of the osteoblast specific expression of BSP; 4) for genes involved in bone homeostasis, up modulation in bone marrow cells at distal epiphysis for CXCR4{CXCR4 plays a role in stem cell migration}, down modulation of CXCL12, and transient increases in osteoblasts and marrow cells for Stro1{Stro1 positive stem cells decline with age}. 14 days after limb reloading expression returned to control levels for most genes and proteins in most cell types, except AP in all cells, and CXCL12, only in bone marrow."

So temporary unloading periods can result in permanent decrease in CXCL12 positive cells.  Unloading also decreased expression of procollagens in bone marrow that was not recovered by load.

"RECK is an endogenous membrane inhibitor of MMPs"

"CXCR4 is the receptor for Stromal cell-derived factor-1 (SDF-1 or CXCL12), which is constitutively secreted by osteoblasts and bone marrow stromal cells. "

"Dynamic levels of CXCL12 and CXCR4 expression play a key role in the homing of hematopoietic cells to the bone marrow"

"Activation of the CXCR4/CXCL12 pathway induces proliferation of hematopoietic and mesenchymal progenitors. CXCL12 also stimulates mononucleate cell fusion and TRAP activity and is a key factor in the normal homeostatic regulation of bone development and remodeling"

"Stro1 [is] a stage- and/or lineage-specific stromal antigen"

Do calcium fluxes within cortical bone affect osteocyte mechanosensitivity?

"Osteocytes directly participate in calcium homeostasis by regulating dissolution and deposition of calcium in the perilacuno-pericanalicular space. In the bulk of the canalicular space, the fluid flow due to chemical gradient generated by deposition or dissolution of calcium is negligible compared to the fluid flow due to hydraulic pressure. However, at the osteocyte proximity, the presence of calcium gradient generated sufficient fluid flow to induce significant changes in the shear stress on the osteocyte membrane. Calcium deposition and dissolution on the canalicular wall resulted in increased or decreased shear stress on the osteocyte membrane respectively. Strong calcium fluxes due to whole body calcium homeostasis may affect mechanical forces experienced by osteocytes."

"The pore space is filled by a pericellular matrix, which slows down fluid flow. The presence of chemical gradients (such as directional calcium fluxes between the bone matrix and the interstitial fluid) can result in osmotic fluid flow. In addition, the collagen fibers and the phospholipids present at the canaliculus and osteocyte walls, respectively, produce a negative surface charge density at these interfaces. As a consequence, these surfaces attract the cations present in the interstitial fluid, forming an electrical double layer composed of a layer of cations adsorbed by the charged surface (Stern layer) and a layer of mobile cations close to the interface (diffuse or Gouy–Chapmann layer), which may induce electro-osmotic fluid flow. Electrical phenomena in bone canaliculi and other nanoscale porous media have been shown to noticeably affect both ionic diffusion and fluid transport."

"Under the action of a chemical gradient, the chemical species move to compensate this difference in concentration, dragging with them some interstitial fluid through a viscous force."

"The interstitial fluid flows from the entrance to the exit in response to the hydraulic and electrical gradients (e.g., generated by blood pressure). In this situation, a calcium/phosphate deposition onto the canalicular wall (loss of chemical species in the fluid) corresponds to a higher concentration of calcium at the entrance of the canaliculus comparing to its exit, i.e. to a negative calcium gradient. As a consequence, the osmotic velocity is positive\ and concomitant to the hydraulic and electro-osmotic velocities"

"in the presence of a calcium/phosphate dissolution from the canalicular wall (gain of chemical species in the fluid), the amount of calcium within the canaliculus is lower at the entrance than at the exit. Thus, the calcium gradient is positive and the osmotic velocity is negative and opposite to the hydraulic and electro-osmotic velocities "

"If the hydraulic shear stress is too low to activate osteocyte's response, a concomitant calcium flux could increase the total shear stress up to values activating osteocyte's response. Conversely, if the hydraulic shear stress is such as to activate osteocyte's response, opposite calcium fluxes can decrease the total shear stress to values that osteocytes are not sensitive to."