Monday, January 28, 2013

Reverse Ossification

This study seems like a game changer but unfortunately it's in persian:

Growth Plate Reappearance after Closure in Ankle Radiography for Trauma

"Bone growth plates or physis are present at the end of long bones and are responsible for longitudinal growth. These plates consist of 4 layers and are lucent in radiography as a line perpendicular to the longitudinal axis, Because of cartilage layer x-ray absorption is less than calcified bone. Gradually increases with age and bone maturity these line will be narrower and as longitudinal bone growth stops, the line disappears. This phenomenon occurs at different ages in different bones of the skeleton but with complete maturity at the age of 19, all growth plates are closed and sclerosed. Re-appearing after closing is uncommon. We introduce two young patients in this study due to trauma have been treated for an ankle cast and the growth plates of tibia and fibula in their control X-ray was re-appeared. Subchondrel Bone Resorbtion is a known phenomenon that will occur after 6 to 8 weeks immobility in any bone. The lucent line caused by imbalance in osteoblast and osteoclast activity and bone absorption. Re-appearing of growth plates can be caused by reversed ossification and bone absorption."

The cited studies may lead to clues on reversed ossification but I could not get access to them:

Effects of tiludronate on bone loss in paraplegic patients

The other links I could find don't seem to have a direct relation to reverse ossification just bone resorption.

Please help out if you know Persian or are an expert at translation documents(Google Translate doesn't like multiple columns).

Some selected translations(manually copying and pasting):

Figure 1: The graph occurs when the ankle and a half before casting

Figure 2: X-ray of the ankle occurred and half occurred after casting
Figure 3: X-ray event occurring half calf 78 days after casting
Figure 6: X-ray of the ankle occurred and half occurred after treatment
Figure 7: The graph and profile ankle occurred 45 days after treatment

Figure 4: X-ray of the ankle occurred before casting
Figure 5: X-ray of the ankle profile

Some notes in my feeble attempts to translate:

"The resurgence of growth plate can be absorbed by tissue"

"Treatment, the growth plates of the tibia and fibula appeared again."

"Patients can reverse the growth plate of the bone at the growth plate cartilage of the past"

Tuesday, January 22, 2013

LSJL Case Study: Felipe

At the point of this x-ray Felipe has been performing LSJL for three months with no reported height gain but has reported an increase in knee thickness and width.

4 months after the x-ray's still no increase in height.  He had been doing MENS routine for 2 years.  MENS is just a fancy name for taking melatonin and high dose niacin..  It is not really a height increase routine.

He reports loading for 3 minutes for very high pressure.  Of note that at this point he was loading at the bumpy part of the bone rather than at the synovial joint intersection.

Here's what open tibial growth plates look like.  A growth plate shows up as a gap in the bone in the xray.
Here's what a normal tibial x-ray looks like.
That's what an enchondroma looks like.

That's what a chondrosarcoma looks like.

What's interesting to note is that while a growth plate just presents itself on a gap in the x-ray. An enchondroma(ectopic endochondral ossification) and a chondrosarcoma(ectopic cartilage formation) are both visible.  However, both sarcomas consist of both cartilage/bone cells and tumor cells so maybe it's the tumor cells that are visible and not the cartilage cells.

Comparing the normal x-ray to felipe's x-ray:

Felipe's x-ray is much whiter on the perimeter of the bone than the normal x-ray(which is more gray) indicating enhanced bone density.  This is despite the fact that the normal x-ray individual has a much more muscular calf than Felipe.

Felipe's x-ray is hollower in the middle of the bone.  Note that in the LSJL study involving drilling, LSJL caused degradation in the middle of the bone.

The normal x-ray no longer has a visible epiphyseal line(scar) whereas Felipe does so the normal person is likely older than Felipe.

After the 3 months of these x-ray's Felipe loaded his knees for four minutes(high intensity) and loaded his ankles for two minutes(also high intensity).

Unfortunately these were the best x-ray's of the normal tibia I could find so if you could provide links to other x-rays that would be helpful.  For example, this person may be fatter than Felipe which may present itself as adipose tissue in the bone marrow which would also explain why Felipe's x-ray is clearer.  X-ray's of athlete tibia's would be helpful.

Since growth plate's present as a gap and it's likely that the visible tissue present in other forms of ectopic cartilage formation are other types of tumor cells.  The hollower state of Felipe's bone indicates that there is room for growth plate formation.  You can see clear through the fibula to the tibia in contrast to the normal xray.  It could be due to x-ray quality but the quality of the muscle and fat seems to be the same on both x-rays.

Also, in Felipe's bone you can see streaks within the bone which may be signs that LSJL induced shear strain and fluid flow.

Even with shifting the load from the bone to more the synovial joints Felipe still didn't gain (visible) height and he reported increasing the load from three to four minutes.

He also stated that he did not use a deconditioning period.

So hypothesis from this case study:
Three minutes(high intensity) of LSJL is enough to degrade bone making room for growth plates.
It is not enough to complete formation of new growth plates.
Thus 5 minutes may be the minimum if not longer.  Human bones are much longer than rat bones so it may take more time for fluid to travel within the bone and build up hydrostatic pressure to induce chondrogenesis.

Friday, January 18, 2013


 Egr1 is upregulated  4.973 fold by LSJL.  Under normal axial loading on 20 week old mice, egr1 was downregulated which could mean that egr1 is a key reason as to why LSJL can increase chondrogenesis but axial loading cannot.  Chondrocytic cells under hydrostatic pressure had egr1 upregulatedEgr1 was also upregulated by dynamic chondrocyte compressionLIPUS can also upregulate EGR1Removing the sciatic nerve increased EGR1 levelsOne study found that Egr1 was upregulated in older rat growth platesIncreases in HMGA2(which is linked to height and upregulated by LSJL) levels also increased EGR1 levelsSurprisingly Egr1 was downregulated in mesodermal progenitor cell differentiation to chondrocytesArachidonic acid can increase Egr1 levels.

The Immediate Early Gene Product EGR1 and Polycomb Group Proteins Interact in Epigenetic Programming during Chondrogenesis.

"Initiation of and progression through chondrogenesis is driven by changes in the cellular microenvironment. At the onset of chondrogenesis, resting mesenchymal stem cells are mobilized in vivo and a complex, step-wise chondrogenic differentiation program is initiated {our goal with LSJL is to induce chondrogenic differentiation of resting mesenchymal stem cells}. Differentiation requires coordinated transcriptomic reprogramming and increased progenitor proliferation; both processes require chromatin remodeling. The nature of early molecular responses that relay differentiation signals to chromatin is poorly understood. Immediate early genes are rapidly and transiently induced in response to differentiation stimuli in vitro {So does EGR1 cause the differentiation or is EGR1 the byproduct?}. Functional ablation of the immediate early factor EGR1 severely deregulates expression of key chondrogenic control genes at the onset of differentiation. In addition, differentiating cells accumulate DNA damage, activate a DNA damage response and undergo a cell cycle arrest and prevent differentiation associated hyper-proliferation. Failed differentiation in the absence of EGR1 affects global acetylation and terminates in overall histone hypermethylation. Polycomb associated histone H3 lysine27 trimethylation (H3K27me3) blocks chromatin access of EGR1. In addition, EGR1 ablation results in abnormal Ezh2 and Bmi1 expression. Consistent with this functional interaction, we identify a number of co-regulated targets genes in a chondrogenic gene network. EGR1 [is involved] in early chondrogenic epigenetic programming to accommodate early gene-environment interactions in chondrogenesis."

ATDC5 cells were used which are technically chondrocyte progenitor cells like those in the growth plate resting zone and not true mesenchymal stem cells.

"Progression through chondrogenesis is in part driven by interaction with a constantly changing microenvironment, which is defined by soluble growth and differentiation factors, hormones, oxygen tension, cell-cell and cell-ECM contacts"

"abnormal skeletogenesis [occurs] in PRC1 LOF[Loss of Function} mice"

"Egr1 mRNA induction in chondrogenesis is transient and precedes transcriptional upregulation of Sox9"

"Sox6 and Agc1-loci do not represent early EGR1-targets for transcriptional activation in chondrogenesis."<-Although aggrecan was upregulated by LSJL.

"Consistent with reduced chondrogenic capacity; shEgr1{silenced-Egr1} significantly reduced Col2a1 expression at the mRNA and protein levels"

"chondrogenic markers Sox9, Agc1, Col2A1 and Col10A1 showed a delayed (5–10 days) differentiation response [after Egr1 is silenced]" All of these genes were up in LSJL.

"Enchondral ossification pathway analysis for predicted EGR1 targets (green) and published PRC1 targets (blue)."

That Runx2 is an EGR1 target could explain how Egr1 could upregulate osteoblast differentiation as well.

There is evidence though that loss Egr1 can be compensated for.

"H3K27me3-decorated chromatin prevents EGR1 from accessing promoters."<-thus an H3K27me3 inhibitor may be a well to allow EGR1 to access chondrogenic promoters again.

Sox9 was not upregulated by Egr1 in H3K27me3 cells.

"EZH2 levels decrease in the context of replicative senescence"

"shEgr1 cultures do not reach super-confluence and do not form chondrogenic nodules"

EGFR ligands drive multipotential stromal cells to produce multiple growth factors and cytokines via early growth response-1. establishes a connection between MSCs and Egr1 but not a relationship between the two and chondrogenic differentiation.

Early growth response genes signaling supports strong paracrine capability of mesenchymal stem cells.

"EGF [facilitates] in vitro expansion of MSCs without altering multipotency. The molecular machinery underlying MSCs' strong paracrine capability lies downstream of EGFR signaling, and we focus on transcription factors EGR1 and EGR2. EGR1 regulates angiogenic and fibrogenic factor production in MSCs{fibrogenic factors are involved in chondrogenesis}, and an EGFR-EGR1-EGFR ligands autocrine loop is one of the underlying mechanisms supporting their strong paracrine machinery through EGR1. EGR2{also up in LSJL} appears to regulate the expression of immunomodulatory molecules."

Egr1 is expressed 3 fold higher in human MSCs than human fibroblasts.

So Egr1 likely plays a role in LSJL but unfortunately this was only established in chondrocyte progenitor cells and not MSCs directly.

Amomum Villosum, can it help you grow upwards?

Amomum villosum is available for sale: Sha Ren(concentrated Extract Powder)(amomum Villosum Fruit)(amomum Fruit / Grains of Paradise) Mayway-5330c.  A bunch of chinese studies where I can't get the full studies.  It may seem like I'm constantly doing products.  I actually do a lot of things but I only put things on the main page that people can take action on.

Amomum villosum induces longitudinal bone growth in adolescent female rats.

"Adolescent female Sprague-Dawley rats were divided into 3 groups and treated for 4 days: control (distilled water, p.o.), recombinant human growth hormone (rhGH; 100 microg/kg, s.c.), and A. villosum (500 mg/kg, p.o.) groups. On day 3, tetracycline (20 g/kg, i.p.) was injected for growth plate identification. On days 2, and 4, 5-bromo-2'-deoxyuridine (BrdU) (50 mg/kg, i.p.) was injected to label proliferating cells. On day 5, tibias were dissected.
The rate of bone growth in the A. villosum and rhGH groups increased to (410 +/- 44) and (389 +/- 46) microm/day, respectively, as compared with the control (330.7 +/- 34.7) microm/day. The thickness of the growth plates also increased to (591 +/- 37) and (598 +/- 32) microm, respectively, as compared with the control (524 +/- 89) microm. The number of [proliferating] cells in the chondrocytes of the A. villosum and rhGH groups was also significantly higher (126 +/- 24) and (143 +/- 18) cells/mm2, respectively) than in the control (109 +/- 25) mm2. Insulin-like growth factor-1 and bone morphogenetic protein-2 in the A. villosum and rhGH groups were highly expressed in the growth plate as compared with the control samples, indicating increased bone formation."

Effects of Amomum villosum on longitudinal bone growth in adolescent rats

"The fruit of Amomum villosum has been used for an improvement of gastrointestinal motility in traditional Korean medicine. [A] herbal mixture containing A. villosum [is] used as medicine for malnutrition associated with growth retardation. This study was aimed to investigate the effect of A. villosum on longitudinal bone growth in adolescent rats. A. villosum was extracted with water for 3h at 100°C in a reflux apparatus. The A. villosum treated group (500mg/kg) and the control group (vehicle) were administered orally twice daily for 4 days. On day 3, tetracycline (20mg/kg) was injected intraperitoneally to form a fluorescent band on the growth plates. On days 2-4, bromodeoxyuridine (BrdU) (50mg/kg) was injected intraperitoneally for labeling proliferating cells. A. villosum caused a significant acceleration of longitudinal bone growth, compared to control group. BrdU-positive cells were increased in the chondrocytes of the A. villosum group. The growth plate width was significantly increased, compared to control group. BMP-2 and IGF-1 were highly expressed in the hypertrophic and proliferative zone, respectively. A. villosum [may] increase longitudinal bone growth by stimulation of the chondrocyte hypertrophy and chondrogenesis, through regulation of IGF-1 and BMP signaling in the growth plate."

[Study on phenolic constituents of Amomum villosum].

"Five compounds were isolated from Amomum villosum. They were identified as: 3-ethoxy-hydroxy benzoic acid (1), vanillic acid-1-beta-D-glucopyranosyl ester (2), isorhamnetin-3-beta-D-glucoside (3), flavanocoumarin (4), isoflavanocoumarin (5)."  Benzoic Acid is in Curculigo Orchiodes and Benzoic Acid may be a BMP-2 stimulator.   Glucosides are derived from glucose.

According to Flavanocoumarins from Guazuma ulmifolia bark and evaluation of their affinity for STAT1., flavanocoumarin can inhibit Stat1.  STAT1 is a catabolic compound that may inhibit height.

isoflavanocoumarin and the glucopyranosyl ester are uncharacterized.

Amomum Villosum may increase height via Benzoic acid upregulation of BMP-2 or flavanocoumarin inhibition of STAT1.  Or, there may be some novel function of the other three compounds.  Also, since we can't access the full study we can't know if the increase in growth rate is due to the nutritional value of Amomum Villosum since calories do increase growth rate.

Wednesday, January 16, 2013

Bite-Jumping Appliance

The mechanism of Class II correction in Herbst appliance treatment. A cephalometric investigation.

"Sagittal skeletal and dental changes contributing to Class II correction in Herbst appliance treatment were evaluated quantitatively on lateral roentgenograms. The material consisted of forty-two Class II. Division 1 malocclusion cases. Twenty-two of these were treated with the Herbst appliance for 6 months. The other twenty cases served as a control group. The results of the investigation revealed the following: (1) Bite jumping with the Herbst appliance resulted in Class 1 occlusal relationships in all treated cases. (2) The improvement in occlusal relationships was about equally a result of skeletal and dental changes. (3) Class II molar correction averaging 6.7 mm. was mainly a result of a 2.2 mm. increase in mandibular length, a 2.8 mm. distal movement of the maxillary molars, and a 1.0 mm. mesial movement of the mandibular molars. (4) Overjet correction averaging 5.2 mm. was mainly a result of a 2.2 mm. increase in mandibular length and a 1.8 mm. mesial movement of the mandibular incisors. (5) Anterior condylar displacement (0.3 mm.), redirection of maxillary growth (0.4 mm.), and distal movement of the maxillary incisors (0.5 mm.) were of minor importance in the improvement in molar and incisor relationships seen. (6) A direct relationship existed between the amount of bite jumping at the start of treatment and the treatment effects on the occlusion and on mandibular growth. For a maximal treatment response, it is suggested that the Herbst appliance be constructed with the mandible jumped anteriorly as much as possible, namely, to an incisal edge-to-edge position. The clinician should be aware of the dental changes occurring during Herbst appliance treatment and make sure that these changes are not incongruous with his over-all treatment goal."

"The aim of the present study was to investigate the temporal pattern of expression of VEGF (Vascular Endothelial Growth Factor) and new bone formation in the condyle during forward mandibular positioning. The importance of vascularization during endochondral ossification was investigated during natural growth of the condyle and compared to that after forward mandibular positioning. The goal was to further our understanding of the cellular responses during functional appliance therapy with a view to extending the experiment into maturity. One hundred and fifty 35 days old Sprague-Dawley rats, 100 fitted with a bite-jumping appliance and 50 untreated, were divided into 10 groups. One group was sacrificed on each of experimental days 3, 7, 14, 21, 30, 33, 37, 44, 51 and 60 respectively. Sagittal sections were cut and stained with VEGF antibodies and Periodic acid and Schiff's reagent (PAS). Each section was quantitatively analyzed with a computer assisted analyzing program and the temporal sequence of expression of VEGF and new bone formation during natural growth and after mandibular forward positioning was compared. There was significant increase in both vascularization and mandibular bone growth upon forward mandibular positioning and the highest amount of both were expressed in the posterior region of the condyle. The highest acceleration of vascularization preceded that of new bone formation. Forward mandibular positioning was found to solicit a sequence of cellular events leading to increased vascularization and subsequently new bone formation resulting in enhanced condylar growth."

"Postnatal partial inhibition of VEGF expression was found to cause stunted body growth, impaired organ development and increased mortality."

"During the late stages of somatic growth, quantitative analysis of the temporal expression of VEGF, in rat condyles past the growth spurt was consistently higher in the posterior region when compared to anterior and middle regions. The pattern of new bone formation in the condyle throughout the same period followed a similar pattern.
After the peak height velocity, both VEGF and bone formation showed a gradual decrease concomitant with the slow down of the growth."

"The posterior region of the condyle showed the highest amount of VEGF expression during natural growth and during advancement. Interestingly, the highest amount of bone formed during natural growth and in response to advancement also occurred in the posterior region of the condyle. Such a close correlation could be explained by the fact that new blood vessels contribute to the population size of non-differentiated mesenchymal cells"

"connective tissues of the invading blood vessels are repository of undifferentiated mesechymal cells"

"VEGF could be the regulator of the process of recruiting new blood vessels into the hypertrophic cartilage matrix of the condyle"

"mechanical strain produced by mandibular advancement [may cause] an increase in the expression of VEGF by hypertrophic chondrocytes and subsequently lead to an increase in vascular invasion into the hypertrophic cartilage layer in the condyle."<-this may not be the only effect though.

"The type of collagenous matrix that forms in the human condyle in situations of repair is known to be type III collagen{up in LSJL}. Type III collagen is the emergency type of collagen that occurs during bone repair as well as bone development. The reason that type III collagen is a good candidate for the repair bone matrix is that the nature of the cross links to stabilize the collagen molecules to form collagen fibrils are weaker than those present in type I collagen matrix. This makes its removal at a later stage and its replacement by the more stable type I collagen matrix an easier process. Type III collagen is then removed at a later stage and replaced with Type I collagen, the more permanent type of collagen matrix and the most stable due to its very strong cross links."

Temporomandibular synovial fluid pressure response to altered mandibular positions.

"Hydrostatic synovial fluid pressure within the superior aspect of the temporomandibular joint space of the growing pig, Sus scrofa domesticus, was examined in response to various acute and chronic alterations of mandibular position. Bilateral measurements of pressure were recorded with chronically implanted wick catheters in three 8-week-old pigs before and at the time of appliance placement and then at 2-day intervals until the animals were 20 weeks old. Besides confirming the observations of a previous study, we noted that forward positioning of the mandible caused an increase in synovial fluid pressure that decayed to baseline levels within 2 hours. Posterior positioning of the mandible effected a larger increase in pressure that partially decayed over 2 hours but did not return to baseline levels over the entire course of the experiment."

This is a very relevant experiment and unfortunately I couldn't get the full study.

VEGF and bone formation in the glenoid fossa during forward mandibular positioning.

"The temporal pattern of vascular endothelial growth factor (VEGF) expression and bone formation in the glenoid fossa during natural growth was identified and compared with that during forward mandibular positioning. We randomly divided 150 female Sprague-Dawley rats, 35 days old, into 10 experimental and 10 control groups. Appliances were fitted to position the mandible forward in the experimental groups. The rats were then killed at different times. Sections were cut and stained with anti-VEGF antibodies to evaluate VEGF expression, and with periodic acid and Schiff's reagent to evaluate new bone formation. Both VEGF expression and newly formed bone were measured by a computer-assisted image analyzing system. during natural growth and forward mandibular positioning, VEGF expression and new bone formation were highest in the posterior region of the glenoid fossa. There were significant increases of VEGF and new bone formation in the experimental groups compared with the controls. The highest amount of VEGF expression occurred before the highest amount of bone formation was reached. Forward mandibular positioning causes significant increases in vascularization and new bone formation in the glenoid fossa. A close correlation exists between vascularization and bone formation."

"an increase of 170% in the VEGF expression [has been attributed to] mandibular advancement"

Skeletal effects of bite jumping therapy on the mandible - removable vs. fixed functional appliances.

"The data for removable and fixed functional appliances were respectively comprehended and analyzed with regard to their attributes in mandibular growth modification. Furthermore, numerous reported findings were assessed by relating them to some important factors influencing the effects of bite jumping, such as treatment timing, treatment duration and post-treatment follow-up, to allow for a more objective and accurate evaluation.
The key differences between removable and fixed appliances are working hours (intermittent vs. continuous), length of treatment time (long vs. short), optimal treatment timing (before puberty growth vs. at or after puberty spurt), and mode of bite-jumping (considerable vertical opening vs. limited vertical opening). These different features lead to different treatment effects on mandibular and TMJ growth, such as the intensity of possibly increased growth (clinically less significant vs. significant), the direction of enhanced growth (vertical vs. horizontal), and the stability of treatment changes (unstable vs. stable). The short-term or long-term post-treatment relapse mainly relates to the rebound of dental position.
The immediate effects of bite jumping functional appliances on the mandibular growth enhancement are convincing during actual treatment. This extra gain of growth might be sustainable during the short-term and long-term post-treatment period."

"In an attempt to elucidate the mechanism by which mesenchymal cells proliferate and differentiate in response to mandibular protrusion, the temporal sequence of cellular changes in posterior aspect of TMJ in rats [was identified]. The mesenchymal cells were found to be stretched and oriented in the direction of the pull, which might trigger the biophysiological path of mesenchymal cells differentiating into bone making cells in TMJ."

"[Some studies have] found no growth stimulation on a short-term basis. A limited number of studies on long-term effects of removable BJFA on mandibular growth have almost invariably reported some relapse after 3–20 years after treatment"

[Expression of BMP/Smads in rabbit condylar cartilage during mandibular forward positioning].

"To identify the relationship between the expression of BMP/Smads in condylar cartilage and condylar growth modifications in rabbits during mandibular forward positioning.
Sixty male rabbits with 8 weeks of age were randomly divided into the experimental group (n=36) and control group (n=24). The mandibles of rabbits in the experimental groups were induced to forward position by a functional appliance. The rabbits in the experimental group and control group were sacrificed after 3 days and 1, 2, 4, 8, 12 weeks, respectively. The expression of BMP-2, Smad1/5, 4 and 6 in condylar cartilage was examined by immunohistochemical staining.
The expression of BMP-2, Smad1/5, 4 and 6 was mainly found in the chondrocytes from the transitional zone and hypertrophic zone, and was also found in the chondrocytes and osteoblasts of the mineralized zone. Compared with those of the age-matched controls, the positive signals for BMP-2, Smad1/5, 4 and 6 in the experimental animals were stronger at early stage, coinciding with the remodeling in condylar cartilage after functional appliance.
The expression of BMP-2, Smad1/5, 4 and 6 is associated with the adaptive remodeling of the condylar cartilage after functional appliance."

Again an important study and I can't find the full version.

"Adaptive remodelling of the condylar cartilage in response to mandibular protrusion constitutes the rationale for bite-jumping appliances to solicit growth modification. By investigating the expression of type X collagen and capillary endothelium, this study was designed to evaluate the osteogenic transition of chondrogenesis during adaptive remodelling of condylar cartilage and compare it with that under natural condylar growth. One hundred female Sprague-Dawley rats, 35 days of age, were divided into five experimental groups (n = 15, fitted with bite-jumping appliances) where condylar adaptation was created by forward repositioning of the mandible, and five control groups (n = 5) where the condyles underwent natural growth. The animals were sacrificed at 3, 7, 14, 21 and 30 days and 7 mum serial sections of the condyles were processed for in situ hybridization and immunohistochemical analyses. The expression of type X collagen in the hypertrophic zone and capillary endothelium in the erosive zone of condylar cartilage were examined to evaluate osteogenic transition, a critical programme leading to endochondral ossification.  (1) The temporal pattern of the expression of type X collagen and capillary endothelium during condylar adaptation coincided with that during natural condylar growth. (2) The amount of the expression of these two factors during condylar adaptation was significantly higher than that during natural growth. It is suggested that condylar adaptation in growing rats triggered by mandibular forward positioning enhances osteogenic transition which eventually results in increased bone formation."

"Using an animal model where the condyle is deviated from the fossa, it has been found that mesenchymal cells in the articular layer are stretched and reorientated towards the pull, leading to an increased mesenchyme population and an enhanced differentiation into chondrocytes, which subsequently results in an adaptive remodelling "<-maybe this can occur within the epiphyseal bone marrow as well.

Forward mandibular positioning up-regulates SOX9 and type II collagen expression in the glenoid fossa.

"the purpose of this study was to investigate the temporal pattern of expression of two key chondrogenesis markers-SOX9 and its target gene, type II collagen-in the glenoid fossa by immunostaining in a 35-day-old Sprague Dawley rat model during both natural growth and forward mandibular positioning. The expression of both factors was up-regulated when the mandible was positioned forward, indicating an enhancement of chondrocyte differentiation and chondroid matrix formation. Our results indicate that chondroid bone formation in the glenoid fossa in response to forward mandibular positioning is regulated by molecular markers indicative of endochondral ossification."

"Expression of SOX9 was found to be localized in both the mesenchymal cells and hypertrophic chondrocytes"

Replicating mesenchymal cells in the condyle and the glenoid fossa during mandibular forward positioning.

"The purpose of this study was to identify and quantify the temporal sequence of replicating mesenchymal cells during natural growth and mandibular advancement in the condyle and the glenoid fossa. One hundred fifty 35-day-old female Sprague-Dawley rats were randomly divided into 10 experimental groups (10 rats each) and 10 control groups (5 rats each). The experimental groups were fitted with appliances that positioned the mandible forward. One hour before the rats were killed, bromodeoxyuridine (BrdU) was intravenously injected into them. Sections were cut and stained with anti-BrdU antibody to evaluate the number of replicating mesenchymal cells. Cellular uptake of BrdU was quantified with the Leica Qwin (Leica Microsystem Imaging Solutions, Cambridge, United Kingdom) system.  the numbers of replicating mesenchymal cells during natural growth were highest in the posterior region of the condyle and the anterior region of the glenoid fossa. In the experimental groups, the posterior region had the highest number of replicating cells for both the condyle and the glenoid fossa, with the condyle having 2 to 3 times more replicating cells than the glenoid fossa. The number of replicating mesenchymal cells, which is genetically controlled, influences the growth potential of the condyle and the glenoid fossa. Mandibular protrusion leads to an increase in the number of replicating cells in the temporomandibular joint. Individual variations in the response to growth modification therapy could be a result of the close correlation between mesenchymal cell numbers and growth."

"an increase in the mitotic activity of mesenchymal cells in the condyle in response to mandibular advancement with an increase in the thickness of the prechondroblastic and chondroblastic zones, and [there was] an increase in DNA synthesis only when the mandible was positioned distally instead {although there have been conflicting reports}."

"In some sections, clusters of mesenchymal cells could be observed that resembled the initial stages of cell condensation"

"The number of mesenchymal cells in anyone is genetically controlled. In a study of genetic and environmental control of variations in neuron cell numbers in mice, it was found that genetic factors are the most important in controlling this variation. Heritable influences were found to contribute
to 76% of the variance, and up to 90% is attributable to genetic factors in a broad sense.  Nongenetic factors were still appreciable and account for a coefficient of variation that averages approximately
3.6%. Similarly, variations in mesenchymal cell numbers would be greatly influenced by genetic factors that might explain the variations in patients’ responses to growth modification."

<-In chinese unfortunately.

"Identical bite-jumping appliances were fixed to upper incisors of animals in experimental groups, causing a continuous mandibular forward positioning. Histological sections were performed through mandibular condyle and were stained with HE under the same condition. Chondrogenesis was quantified by measuring the area of cells in resting, proliferative, hypertrophic and erosive zones in the superioposterior region.
1. comparison among experimental groups revealed a fluctuation in proliferative zone, with 21-day group being the greatest (0.058 +/- 0.004 mm2) and 14-day the least (0.012 +/- 0.001 mm2). A change in erosive zone was also depicted by a peak of 0.112 +/- 0.001 mm2 in 7-day group and a bottom of 0.018 +/- 0.002 mm2 in 14-day group. 2. comparison among the control groups manifested stable zonation, except for a slump of proliferative zone descending from 0.069 +/- 0.005 mm2 in 3-day group to 0.009 +/- 0.001 mm2 in 21-day group. 3. comparison between experimental and control groups demonstrated significant discrepancy in proliferative zone, hypertrophic zone and erosive zone.
Mandibular forward positioning stimulates and accelerates cartilaginous remodeling in mandibular condyle."

The adaptive remodeling of condylar cartilage---a transition from chondrogenesis to osteogenesis.

"Mandibular condylar cartilage is categorized as articular cartilage but markedly distinguishes itself in many biological aspects, such as its embryonic origin, ontogenetic development, post-natal growth mode, and histological structures. The most marked uniqueness of condylar cartilage lies in its capability of adaptive remodeling in response to external stimuli during or after natural growth. The adaptation of condylar cartilage to mandibular forward positioning constitutes the fundamental rationale for orthodontic functional therapy, which partially contributes to the correction of jaw discrepancies by achieving mandibular growth modification. The adaptive remodeling of condylar cartilage proceeds with the biomolecular pathway initiating from chondrogenesis and finalizing with osteogenesis. During condylar adaptation, chondrogenesis is activated when the external stimuli, e.g., condylar repositioning, generate the differentiation of mesenchymal cells in the articular layer of cartilage into chondrocytes, which proliferate and then progressively mature into hypertrophic cells. The expression of regulatory growth factors, which govern and control phenotypic conversions of chondrocytes during chondrogenesis, increases during adaptive remodeling to enhance the transition from chondrogenesis into osteogenesis, a process in which hypertrophic chondrocytes and matrices degrade and are replaced by bone. The transition is also sustained by increased neovascularization, which brings in osteoblasts that finally result in new bone formation beneath the degraded cartilage."

"The repositioning of the mandibular condyle in adult rats led to a reactivation of chondrogenesis in condylar cartilage which otherwise is at resting status, and finally results in increased bone formation"<-so stretching the mandible seems to result in endochondral ossification of the articular cartilage.

"the chondrogenic activity of BMP-2 in vitro involves the action of the cell-cell adhesion protein, N-cadherin, which functionally complexes with beta-catenin"

"the change of condyle position relative to the glenoid fossa constitutes an important trigger for [the endochondral ossification related adaptation of the mandible]. The deviation of the condyle from the glenoid fossa by mandibular forward translation is the basis for orthodontic functional therapy, which aims to enhance condylar growth and therefore to eliminate the discrepancy between upper and lower jaws."

"a decrease in compressive loading enhances condylar growth, whereas an increase in loading inhibits growth"

Changes in condylar cartilage after anterior mandibular displacement in juvenile pigs.

"Twenty juvenile pigs were randomly divided into two experimental groups, where the treatment group was fitted with mandibular advancement splints, and the control group was not. Changes in the mRNA content of condylar cartilage tissue was then were measured after 4weeks of treatment.
The temporal pattern of the expression of Col1 and MMP13 during condylar adaptation coincided with that during natural condylar growth. The amount of the expression of Col10 during condylar adaptation was significantly lower, whereas the expression of Col2, MMP8 and VEGF was significantly higher compared to natural growth.
It is suggested that condylar adaptation in growing pigs triggered by mandibular forward positioning results not only from passive adaptation of cartilage, but also involves growth affected processes. Mechanical strain produced by mandibular advancement induced remodelling and revascularization in the posteriocranial mandibular condyle."

"The mandibular condylar cartilage serves as both an articular condyle, and as a growth centre in the juvenile mandible."

Dentoskeletal effects and facial profile changes in young adults treated with the Herbst appliance.

"Early adolescent subjects in the permanent dentition who had been treated with the Herbst appliance were used for comparison. Lateral headfilms from before and after an average treatment period of 8.5 months for the young adults and 7.1 months for the adolescents were evaluated. All adult and adolescent subjects were treated to either Class I or overcorrected Class I occlusal relationships. In both groups the improvement in sagittal incisor and molar relationships was achieved more by dental changes than by skeletal ones. The amount of skeletal change contributing to overjet and molar correction was smaller in the young adult group (22% and 25%, respectively) than in the early adolescent group (39% and 41%, respectively). Skeletal and soft tissue facial profile convexity was reduced in adults and adolescents. Facial profile improvement did not differ between the two groups."

Young adults were defined as individuals who had a fused radius(arm) bone.  Young adults ranged from 13.6-19.8.

Adolescents increased in mandibular length by +2.5mm more than young adults.  young adults still increased in mandibular length by 1.5mm.

Zones of undifferentiated mesenchyme and undifferentiated growth cartilage are seen in the adult condyle.

Tuesday, January 15, 2013

Ectopic endochondral ossification genes versus LSJL

A gene expression profile for endochondral bone formation: oligonucleotide microarrays establish novel connections between known genes and BMP-2-induced bone formation in mouse quadriceps.

"This study evaluated several aspects of the osteogenic effect of hBMP-2 protein injected into quadriceps of female C57B1/6J SCID mice. Mice were euthanized 1, 2, 3, 4, 7, and 14 days postinjection and muscles were collected for several methods of analysis. Hematoxylin and eosin-stained sections of muscles injected with formulation buffer showed no evidence of osteogenesis. In contrast, sections of muscles injected with hBMP-2 showed evidence of endochondral bone formation that progressed to mineralized bone by day 14. In addition, radiographs of mice injected with hBMP-2 showed that much of the quadriceps muscle had undergone mineralization by day 14. Labeled mRNA solutions were prepared and hybridized to oligonucleotide arrays designed to monitor approximately 1300 murine, full-length genes. Changes in gene expression associated with hBMP-2 were determined from time-matched comparisons between buffer and hBMP-2 samples. A gene expression profile was created for 215 genes that showed greater than 4-fold changes at one or more of the indicated time points. One hundred twenty-two of these genes have previously been associated with bone or cartilage metabolism and showed significant increases in expression, e.g., aggrecan (Agc1){up}, runt related transcription factor 2 (Runx2), bone Gla protein 1 (Bglap1), and procollagens type II (Col2a1){up} and X (Col10a1){up}. In addition, there were 93 genes that have not been explicitly associated with bone or cartilage metabolism. Two of these genes, cytokine receptor-like factor-1 (Crlf1){up} and matrix metalloproteinase 23 (Mmp23), showed peak changes in gene expression of 15- and 40-fold on days 4 and 7, respectively. In situ hybridizations of muscle sections showed that Mmp23 and Crlf1 mRNAs were expressed in chondrocytes and osteoblasts, suggesting a role for both proteins in some aspect of cartilage or bone formation"

"BMP-2-induced changes in the expression of gene"

Genes upregulated on Day2-3 versus LSJL:
Col6a1(no change on day 1) {up}

No Change Day1-3 with some transient upregulation(noted if occurred) versus LSJL:
Sdc2 {down}
Vcam1 (transient on day 2) {down}
Col6a2 {up and down}
Lum {up}
Bsp {up}
Col2a1 (transient decrease on day 3) {down}
Wisp2 {up}
Agc1 {up}
Cd9 {down}
Ptpra {down}
Anxa8 {up}
Socs3 (transient increase on day 2) {up}
Ptpn12 {down}

Generally Downregulated:
Col10a1 {up}
Col9a1 {up}
Col15a1 {up}
Eln {up}
Col9a3 {up}

Full-size image (305 K)"Ectopic cartilage induced: A histological time course of the effects of a single intramuscular injection of buffer or hBMP-2 protein into quadriceps muscles of C57B1/6J SCID females. A, C, E, G, and I correspond to muscles removed after 1, 3, 4, 7, and 14 days, respectively, following an injection of formulation buffer. B, D, F, H, and J correspond to muscles removed after 1, 3, 4, 7, and 14 days, respectively, following an injection of 50 μg of hBMP-2 protein. Muscles were fixed and stained with H&E as described under Methods. Abbreviations: M, normal skeletal muscle fiber; I, inflammatory cells; DM, degenerated skeletal muscle fiber; F, fibroplasia; Cb, chondroblast; C, chondrocyte; Ob, osteoblast; Oc, osteoclast; B, bone; BM, bone marrow. The bar in each panel = 0.1 mm."

"The expression profile for cytokine receptor-like factor 1 (Crlf1) is qualitatively similar to that of Cyr61 and peaks on day 4"<-Crlf1 and Cyr61 may be key components of the chondroinduction by LSJL.

This study has a lot of useful information which will neccessitate returning to it in the future.

Monday, January 14, 2013

AKG for height augmentation?

According to Wikipedia AKG contains 2-oxyglutaric acid: Olympian Labs - A-Akg, 90 g powder.  2-oxyglutaric acid is a synonym for α-ketoglutarate.

The effect of dietary administration of 2-oxoglutaric acid on the cartilage and bone of growing rats.

"2-Oxoglutaric acid (2-Ox), a precursor to hydroxyproline, [proline, glutamate, arginine and asparagine], exerts protective effects on bone development during different stages of organism development. [What's] the influence of dietary 2-Ox supplementation on the growth plate, articular cartilage and bone of growing rats? A total of twelve male Sprague-Dawley rats were used in the study. Half of the rats received 2-oxoglutarate at a dose of 0·75 g/kg body weight per d in their drinking-water.  Rats receiving 2-Ox had an increased body mass and absolute liver weight. Femoral length and bone mineral density, overall thickness of growth plate and the thickness of femoral articular cartilage{so 2-oxoglutaric acid could also cause a miniscule increase in height in adults} were also increased. Dietary supplementation with 2-Ox to growing rats exerts its effects mainly on cartilage tissue, having only a slight influence on bone."

"2-Ox, together with Fe2+, have been proposed to be active participants in the conversion of proline to hydroxyproline, the main amino acid of bone collagen. Moreover, 2-Ox acts as a cofactor for Fe2+ absorption from the intestine"<-Iron decreases FGF23, which affects height.

" resting and calcification zones of the femoral growth plate and calcification zone of the tibial growth plate were significantly thicker in the [supplement] group"

A stands for supplement group, C stands for control.  Above is femoral growth plate.
Above is tibial growth plate.
Above is the increase in articular cartilage thickness and the difference is pretty big(28%).  Unfortunately, articular cartilage thickness only makes a small difference in height.  Perhaps if it affects the intervertebral discs as well.

"2-Ox is a N scavenger and a source and precursor of glutamine, synthesised in human skeletal muscles, which improves and stimulates protein synthesis and inhibits protein degradation in skeletal muscles"<-Thus another connection between muscle and height growth.

" The question that arises is whether 2-Ox is the cause of the cartilage thickening, in particular the articular cartilage, or whether the bone loading (by a heavier body mass) is the key factor stimulating better nutrient utilisation facilitated by 2-Ox abundance?"<-So the heavier bone mass is a confounding variable.  Would anything that makes heavier bones stimulate GP's and AC's in the same way?  It should be noted that LSJL makes bones heavier too but other effects unrelated to bone weight have been established due to LSJL such as fluid flow and an increase in pressure within the bone.

"Within the femur articular cartilage, the most loaded point of the knee joint, thickening was the predominant reaction to additional 2-Ox."

"Unlike articular cartilage, growth plate cartilage is a tissue which is sensitive to load and overload and may slow down the growth, which is mostly related to the hypertrophy zone of this particular tissue. And yet we observed an increase in growth plate thickness, in the present study, in the rats treated with 2-Ox. "

The question is also would byproducts of 2-Ox like arginine have similar effects and thus there would be no need for both.  Since AKG is so promising in bodybuilding perhaps there are answers.

But so far, based on the results in this study it seems possible that AKG supplementation may increase height by miniscule amounts via articular cartilage thickness it may also help people grow taller via their growth plates.

Thursday, January 10, 2013

Gene Expression Patterns of Bone under normal mechanical load versus LSJL

Each day I go through studies to put into old posts and most of them are not that helpful but I found one that's full of one and insight's into the legendary microfracture theory sprouted by Sky and others.  Search for (*NEW*) to find it.  The study has a lot of great diagrams and the full study is available.  It's a rich informative study so it would be beneficial for other people to look into it.  Ultimately, you can see that the load required to induce a microfracture may be too much and if kicking with ankle weights induced the microfractures such as in this study you would notice them due to the massive appositional bone formation(thus if you did grow taller with such a method you'd have lots of bumps on your bones--chicks dig scars but do they dig microfractures?).  However, it may be possible for tapping with a sufficient load enough times may be enough to induce such a fracture.  I have no idea what that load is though.

CH Turner was one of the leading researchers behind Lateral Synovial Joint Loading before he passed away and he was the researcher who was most directly interested in bone(Hiroki Yokota for example was more interested in mechanotransduction).

CH Turner's studies are still coming out.  Here's one about the gene expression of bone on mechanical loading.  It will be interested to compare that to gene expression under LSJL.<-Read that study, it's important!

Gene expression patterns in bone following mechanical loading.

"The primary goal of this study was to determine the time sequence for gene expression in a bone subjected to mechanical loading during key periods of the bone-formation process, including expression of matrix-related genes, the appearance of active osteoblasts, and bone desensitization. A standard model for bone loading was employed in which the right forelimb was loaded axially for 3 minutes per day{so axial loading rather than our lateral loading, note that the epiphysis still gets loading under axial loading as there's no way to avoid it}, whereas the left forearm served as a nonloaded contralateral control. We evaluated loading-induced gene expression over a time course of 4 hours to 32 days after the first loading session. Six distinct time-dependent patterns of gene expression were identified over the time course and were categorized into three primary clusters: genes upregulated early in the time course, genes upregulated during matrix formation, and genes downregulated during matrix formation. Genes then were grouped based on function and/or signaling pathways. Many gene groups known to be important in loading-induced bone formation were identified within the clusters, including AP-1[The C-Fos and c-Jun complex]-related genes in the early-response cluster, matrix-related genes in the upregulated gene clusters, and Wnt/β-catenin signaling pathway inhibitors in the downregulated gene clusters. Chemokine-related genes, were upregulated early but downregulated later in the time course; solute carrier genes{these help with chondrocyte hypertrophy}, were both upregulated and downregulated; and muscle-related genes,  were primarily downregulated{This is interesting, as many anabolic pathways are shared between muscle and bone; this gives weight to the theory that muscle and bone compete for resources}."

The Peak Load used was 13N. "Compressive load was applied as an oscillating Haversine waveform for 360 cycles at a frequency of 2 Hz"  24 hours between loading.  Genes upregulated in supplementary material were taken from all time points from 4 hours to 32 days.  Mice were 20 weeks old.

"Bone responds in an anabolic manner to physiologic dynamic loading. For example, the midshaft humerus in the throwing arm of baseball pitchers and catchers showed enhanced bone mass, structure, estimated strength, and resistance to torsion compared with the nonthrowing control arm.{bone length was measured but the comparative length was not presented, something to ask Stuart J. Warden} In contrast, bone mineral density (BMD) in astronauts decreased 1.0% and 1.5% in the spine and hip, respectively, per month of spaceflight{This is not that much considering astronauts are reported to gain 3 inches in space, this shows you the potential of your intervertebral discs in terms of height gain}"

"Mechanical loading uses pathways currently being investigated for new drug development, such as low density lipoprotein receptor–related protein 5 (LRP5) and sclerostin"<-supplements to look into

"New osteoblasts appear on the bone surface 24 to 48 hours after initiating mechanical loading, and bone formation is observed within 96 hours of loading. Bone formation increases between 5 and 12 days after starting loading, but after 6 weeks of loading, bone formation returns to baseline levels."

This is for osteoblasts not chondrocytes which is what we are mainly looking for with LSJL but  it may take 12 days after starting LSJL to notice new osteoblast bone deposition(never mind that LSJL requires a chondrocyte phase beforehand).  A month is not enough time to measure results.

The shaft of the bone was used so stem cell genes should be detected.  So if any mesenchymal chondrogenesis occurred it should show up.

Looking at what was upregulated in axial Loading and Lateral Loading share many of the same pathways like TGF-Beta and WNT/B-Catenin in addition to many ECM related proteins.

Some genes involved in LSJL that were not involved in axial loading hyaluronan synthase(involved in hyaluronic acid).  No MMP3 in axial loading in contrast to lateral loading(MMP-3 is stimulatory to chondrocytes). 

And of course no induction of chondrogenic differentiation of stem cells in axial loading(that's more due to hydrostatic pressure though than genes).  Though it should be represented by genes which I think it is given the upregulating of ECM genes.  Chondrogenic differentiation produces ECM but ECM doesn't always indicate chondrogenic differentiation.

BMP-2 and TGF-Beta were produced by axial loading.  Both of which can induce chondrogenic differentiation.  Axial Loading + LIPUS may be enough to gain height.

Interleukin 1 receptor-like 1 was expressed by both axial and LSJL.  Stat3 was expressed which induces Lin28B expression.  It also downregulated FGF23 which may be involved in growth plate reactivation.

Upregulated Genes of Note(see supplementary material):
Acan(upregulated in LSJL)
Cntn1(up in LSJL)
Col2A1 alpha1(up in LSJL)
Col3A1 alpha1(up in LSJL)
GPR180(down in LSJL)
Hapln1(up in LSJL)
Hnf4a(down in LSJL)
Id2(down in LSJL)
IRS-1(down in LSJL)
Junb(also upregulated in LSJL)
MMP2(up in LSJL)
MMP14(up in LSJL)
PTGS2(up in LSJL)
PTN(up in LSJL)
SOCS2(Anti-height gene)
SOCS3(upregulated in LSJL)
Syndecan 4(also upregulated in LSJL)
TIMP1(up in LSJL)
Vcan(up in LSJL)
Zfp36(upregulated in LSJL)

Axial loading upregulated a few chondrogenic genes like Acan and COL2A1 but nowhere near the amount of Collagens upregulated by LSJL which also upregulated Col9.  Also key, is that Sox9 is not upregulated in axial loading whereas it is in LSJL.

Downregulated genes of note:
Anxa3(down in LSJL)
BMPR1B(up in LSJL)
ccnb1(down in LSJL)
Egr1(up in LSJL)
IGFBP6(up in LSJL)
Leptin Receptor
Prkg2(up in LSJL)
Rsad2(down as Pcaf)
Vcam1(down in LSJL)

The differential expression of Egr and BMPR1B between Axial Loading and Lateral Loading could be key to LSJL's ability to induce chondrogenesis.

The gene expression data for LSJL was taken 1 hour after the last loading and in this study genes were taken 4 hours after the first loading.  So we can compare these early response genes to see how they compare to LSJL.

Junb(up in LSJL)
Syndecan4 (up)

Regulatory mechanisms in bone following mechanical loading.

"The right forelimb [of rodents] was loaded axially for three minutes per day, while the left forearm served as a non-loaded, contralateral control. Animals were subjected to loading sessions every day, with 24 hours between sessions. Ulnas were sampled at 11 time points, from 4 hours to 32 days after beginning loading."

Mice were 20 weeks old.

"The peak load achieved during loading was 13 N"

Stat5B was upregulated 4 hours following loading. Stat5b is downregulated in LSJL

The expression of COL1 differs greatly with LSJL.  At 14 days there was almost no COL1 expression whereas at 14 days there was still extremely high COL1 levels with Axial Loading.

"The CREB-related transcription factors are important for bone formation, specifically ATF4, which is required for collagen synthesis by mature osteoblasts. The CREB motif was predicted to be positive at 2d, 4d, 6d, and 8d. The transcription factors that bind to the CREB motif include cAMP responsive element binding protein 1 (CREB1), cAMP responsive element modulator (CREM), activating transcription factor 1 (ATF1), ATF2, ATF3, ATF4, and ATF7. The CREB motif was present in the promoter of an important matrix gene, fibronectin 1 (Fn1), and in genes that promote collagen construction and cross-linking, including Lox, prolyl 4- hydroxylase beta polypeptide (P4hb), and procollagen C-endopeptidase enhancer (Pcolce)"

"at 32d, the system was less responsive to loading and had shifted from bone forming to baseline bone maintenance"<-maybe every 32 days take a break from LSJL?

Collagen 1 alpha 1 did not begin to rise until two days after loading.  The LSJL study took gene expression at 49 days after first loading.  In the axial loading Col1A was upregulated 3-fold seven days after loading whereas with LSJL it was upregulated only 2 fold.

Alternative Splicing in Bone Following Mechanical Loading

Alternative splicing means that gene expression was altered in mRNA.  The whole bone was ground.

"Compressive load was applied as an oscillating Haversine waveform for 360 cycles at a frequency of 2 Hz using a Bose ElectroForce 3200 Series electromechanical actuator"<-Peak Load was 13N.  Axial loading was used.

"Rats were subjected to loading sessions every day, with 24 hours between sessions."  Rats were 20 weeks old.  Gene expression data was taken up to 4 hours to 32 days.

The greatest alteration of gene expression occurred at 16 days or about 2 weeks.  Maybe this is when conditioning effect starts to inhibit gene expression?

According to this Study Sox9 mRNA was not altered at any time point.  Col2a1and Acan mRNA were altered.  Tgfbeta1 and Tgfbeta2 expression was altered.  The key stature genes HMGA2 and Lin28b were altered in LSJL but not here.

Altered genes of note:
BMPr1b{up in LSJL}
BMP2{up in LSJL}
Esr2{up in LSJL}
FGF2{up in LSJL}
GH1{down in LSJL}
Smad7(inhibits BMP signaling, Smad6 which inhibits TGF-Beta signaling is not altered)

Here's the Partek GSEA Analysis to compare to LSJL for chondrogenic related genes, Bold means the p-value < 0.05, no fold changes were given and no fold cutoff is used:

Chondroblast Differentiation(6.77) 100%:

Chondrocyte Differentiation(5.16) 45%:

Cartilage Condensation(8.32) 58.32%:

Chondrocyte Development(1.80) 33.33%<-this could be a key between LSJL and axial loading which has an enrichment score of 3.80.

Cartilage Development(12.70) 44.2%

Cartilge Development Involved in Endochondral Bone Morphogenesis(2.10) 42.8%

Growth Plate Cartilage Development(0.27) 14.29%

Endochondral Ossification(1.66) 29.41%

Systemic effects of ulna loading in male rats during functional adaptation.

"The aim of this study was to determine the effects of loading of a single bone on adaptation of other appendicular long bones and whether these responses were neuronally regulated. Young male Sprague-Dawley rats were used. The right ulna was loaded to induce a modeling response. In other rats, a second regimen was used to induce bone fatigue with a mixed modeling/remodeling response; a proportion of rats from each group received brachial plexus anesthesia to induce temporary neuronal blocking during bone loading. Sham groups were included. Left and right long bones (ulna, humerus, tibia, and femur) from each rat were examined histologically 10 days after loading. In fatigue- and sham-loaded animals, blood plasma concentrations of TNF-α, RANKL, OPG, and TRAP5b were determined. Loading the right ulna induced an increase in bone formation in distant long bones that were not loaded and that this effect was neuronally regulated{LSJL increased length in bones not loaded}. Distant effects were most evident in the rats that received loading without bone fatigue. In the fatigue-loaded animals, neuronal blocking induced a significant decrease in plasma TRAP5b at 10 days. Histologically, bone resorption was increased in both loaded and contralateral ulnas in fatigue-loaded rats and was not significantly blocked by brachial plexus anesthesia. In young, growing male rats we conclude that ulna loading induced increased bone formation in multiple bones. "

"The periosteum is the skeletal tissue with the greatest density of sensory nerve fibers, which are arranged in a dense netlike meshwork that is optimized for detection of mechanical distortion. Nerve branches or single neurons enter the bone cortex, often in association with the microvasculature, and connect individual bone cells to the central nervous system via unmyelinated sensory neurons."

"In the load and block + load groups, loading was performed for 1500 cycles at 4 Hz, with an initial peak strain of −3,750 µɛ (−18 N entered into materials testing machine, −16.8 N applied to ulna). In the fatigue and block + fatigue groups, cyclic loading was performed at 4 Hz. Loading was initiated at −16 N, and the load applied to the ulna was increased incrementally until fatigue was initiated. Loading then was terminated when 40% loss of stiffness was attained."

"TRAP5b is expressed on both immature and mature osteoclasts; plasma TRAP5b concentrations are proportional to osteoclast number."


Healing of non-displaced fractures produced by fatigue loading of the mouse ulna.

"Using adult (5 month) C57Bl/6 mice, we first determined that cyclic compression of the forelimb under load-control leads to increasing applied displacement and, eventually, complete fracture. We then subjected the right forelimbs of 80 mice to cyclic loading (2 Hz; peak force approximately 4N) and limited the displacement increase to 0.75 mm (60% of the average displacement increase at complete fracture). This fatigue protocol created a partial, non-displaced fracture through the medial cortex near the ulnar mid-shaft, and reduced ulnar strength and stiffness by >50%. Within 1 day, there was significant upregulation of genes related to hypoxia (Hif1a) and osteogenesis (Bmp2, Bsp) in loaded ulnae compared to non-loaded, contralateral controls. The gene expression response peaked in magnitude near day 7 (e.g., Osx upregulated 8-fold), and included upregulation of FGF-family genes (e.g., Fgfr3 up 6-fold). Histologically, a localized periosteal response was seen at the site of the fracture; by day 7 there was abundant periosteal woven bone surrounding a region of cartilage. From days 7 to 14, the woven bone became denser but did not increase in area. By day 14, the woven-bone response resulted in complete recovery of ulnar strength and stiffness, restoring mechanical properties to normal levels. In the future, the fatigue loading approach can be used create non-displaced bone fractures in transgenic and knockout mice to study the mechanisms by which the skeleton rapidly repairs damage."

Monotonic loading: "Both forelimbs of five mice were loaded by a displacement ramp (0.5 mm/sec) to complete, displaced fracture in order to determine monotonic mechanical properties. Mice were euthanized immediately after loading. Ultimate force (mean ± SD) was 4.32 ± 0.21 N, and stiffness was 3.98 ± 0.26 N/mm."

Fatigue loading:  "Both forelimbs of 14 mice were cyclically loaded at peak compressive forces (F) ranging from 2.1 to 3.5 N (50 to 80% of average ultimate force) until complete fracture. "

Force loading, to partial non-displaced fracture: "Right forelimbs of 80 mice were cyclically loaded at peak forces ranging from 3.75 to 4.10 N (70–75% of ultimate force) while displacement was monitored. Loading was terminated when peak displacement increased by 0.75 mm relative to the peak displacement at cycle 10."

The key here is to see if any chondrogenic genes were upregulated in partial, non -displaced fractures(so like a microcrack).  Col2a1 was highly upregulated at day 7.  BMP2[1.1-1.9 fold] and FGF2[1.1-1.8 fold] were moderately upregulated.  FGF2 was more highly upregulated in LSJL than here whereas BMP2 was more highly upregulated here than LSJL.  Hif1a the chondrogenically related transcription factor was more significantly upregulated peaking at 3.0 at day 3 and increasing before and decreasing after.

It should be noted that LSJL gene expression was done by microarray whereas this study was done with RT-PCR with the exception of BMP2 which was also done by PCR.

Here's what a bone microfracture-microcrack looks like:

M stands for marrow. CB stands for cortical bone.  WB stands for woven bone.

"Longitudinal sections of fatigue-loaded ulnae (H&E) show that the fracture occurred as a non-displaced, oblique crack through the medial cortex (arrows). On day 1 after loading, a clot is seen on both ends of the crack. On day 3, the periosteum is expanded and filled with cellular, fibrovascular tissue; nascent woven bone is seen sub-periosteally. On days 7 and 11 there is abundant woven bone on the medial periosteum. In approximately one-half of specimens the callus contained no cartilage (not shown), but in the others there was cartilage (*) in the center of the woven bone."

"Cyclic loading of the rat forelimb (~18 N peak force) to 85% of fracture displacement resulted in a non-displaced fracture localized to the medial cortex of the ulna and an associated loss of ulnar strength and stiffness of 55 and 80%, respectively. Because the fracture in the rat ulna was partial and non-displaced, and because the repair process involved negligible cartilage formation, we referred to this as a “stress fracture”, consistent with descriptions by others"<-now we've considered that that 0.5N in LSJL is equivalent to 100N on a 200lbs human which is already a challenge.  Imagine the challenge of generating 18N peak force.  It's possible that less force may be needed if the force is applied cyclically over a long period of time as long as that force causes residual damage in the bone.

"Cartilage was often observed at 7 and 11 day timepoints and appeared only on the medial surface, corresponding to the periosteal fracture location. By comparison, in studies of complete fracture in mice cartilage is seen on both sides of the bone as well as between the fractured ends"

The fracture occurred on the medial side and although the majority of the activity is on the medial side there is some enhanced activity on the lateral side giving weight to the possibility of gradually lengthening the bone through microfracture(as the unfractured side does seem to adapt).  Although the force required to induce a sufficient microfracture may be too large to be induced under normal physiological circumstances(and you would notice a bone adaptation as large as that depicted).  That does not preclude the possibility that rapid loading that is large enough to induce residual damage to bone is enough to induce such a microfracture as well.  Something like tapping.

Tibial loading increases osteogenic gene expression and cortical bone volume in mature and middle-aged mice.

"We examined this question in female BALB/c mice of different ages, ranging from young to middle-aged (2, 4, 7, 12 months). We first assessed markers of bone turnover in control (non-loaded) mice. Serum osteocalcin and CTX declined significantly from 2 to 4 months. There were similar age-related declines in tibial mRNA expression of osteoblast- and osteoclast-related genes, most notably in late osteoblast/matrix genes. For example, Col1a1 expression declined 90% from 2 to 7 months. We then assessed tibial responses to mechanical loading using age-specific forces to produce similar peak strains (-1300 µε endocortical; -2350 µε periosteal). Axial tibial compression was applied to the right leg for 60 cycles/day on alternate days for 1 or 6 weeks. qPCR after 1 week revealed no effect of loading in young (2-month) mice, but significant increases in osteoblast/matrix genes in older mice. For example, in 12-month old mice Col1a1 was increased 6-fold in loaded tibias vs. controls. In vivo microCT after 6 weeks revealed that loaded tibias in each age group had greater cortical bone volume (BV) than contralateral control tibias, due to relative periosteal expansion. The loading-induced increase in cortical BV was greatest in 4-month old mice (+13%). Non-loaded female BALB/c mice exhibit an age-related decline in measures related to bone formation. Yet when subjected to tibial compression, mice from 2-12 months have an increase in cortical bone volume. Older mice respond with an upregulation of osteoblast/matrix genes, which increase to levels comparable to young mice."

Unfortunately, no chondrogenic genes were studied.

Global gene expression analysis in the bones reveals involvement of several novel genes and pathways in mediating an anabolic response of mechanical loading in mice

"We applied mechanical loads[4-point bending] to the right tibias of the B6 mice at 9 N, 2 Hz for 36 cycles per day, with the left tibias used as unloaded controls"

"4 days of loading"

"Twenty-four hours after last stimulation"<-whereas LSJL was 1 hour after last stimulation.

"Ten-week-old C57BL/6J female mice"

Complete list of supplementary gene comparison to LSJL to be done.  Gene comparison of just genes on main paper and spot comparisons done below.
Genes upregulated in bone to four point bending also upregulated by LSJL:


Monday, January 7, 2013

Grow Taller with Folinic Acid

For the first time, there is evidence that Folinic Acid treatment may increase length in developing individuals see (*NEW*).  Considering that Folinic Acid should not be harmful to length development, if an individual has active growth plates it's definitely a supplement one should add to their regime.

Hypermethylation of the growth plate chondrocytes may be a way to induce supranatural height growth.  DNA Methyltransferase stopping the addition of methyl groups to stem cell chondrocytes in the resting zone may be an indication to cease height growth.  High levels of HGH may also cause hypermethylation explaining gigantism(but why Gigantism is not inducible in all with HGH is unclear).  Folinic Acid is available as a supplement:Folinic Acid 800 mcg.

Are there any other ways of inducing DNA Hypermethylation(Bare in mind that DNA Hypermethylation increases the spread of cancer as DNA Methylation is one of the negative feedback mechanisms on cell growth)?  And since hypermethylation may not be possible to induce in growth plates general methods of increasing DNA synthesis like Folinic Acid may have similar effects.

Damaging effects of chronic low-dose methotrexate usage on primary bone formation in young rats and potential protective effects of folinic acid supplementary treatment.

"Methotrexate (MTX) is a most commonly used anti-metabolite in cancer treatment and as an anti-rheumatic drug. MTX chemotherapy at a high dose is known to cause bone growth defects in growing bones. We examined effects on bone growth of long-term MTX chemotherapy at a low dose in young rats, and potential protective effects of supplementary treatment with antidote folinic acid (given ip at 1 mg/kg 6 h after MTX). After two cycles of 5 once-daily MTX injections (at 0.75 mg/kg, 5 days on/9 days off/5 days on), MTX at this dose caused significant reduction in heights of growth plate and primary spongiosa bone on day 22 compared to controls. In contrast, a similar dosing regimen but at a lower dose (0.4 mg/kg) caused only slight or no reduction in heights of both regions. However, after the induction phase at this 0.4 mg/kg dosing, continued use of MTX at a low dose (once weekly at 0.2 mg/kg) caused a reduction in primary spongiosa height and bone volume on weeks 9 and 14, which was associated with an increased osteoclast formation and their bone surface density as well as a decreased osteoblast bone surface density in the primary spongiosa. Folinic acid supplementation was shown able to prevent the MTX effects in the primary spongiosa. Acute use of MTX can damage growth plate and primary bone at a high dose, but not at a low dose. However, long-term use of MTX at a low dose can reduce primary bone formation probably due to decreased osteoblastic function but increased osteoclastic formation and function, and supplementary treatment with folinic acid may be potentially useful in protecting bone growth during long-term low-dose MTX chemotherapy."

Folinic Acid(leucovorin) prevents damage to the bone marrow.  It may help by reducing the number of micronuclei.

"Low-dose MTX is able to inhibit other folate-dependent enzymes such as thymidylate synthase, which further blocks the de novo purine synthesis by directly inhibiting the activity of 5-aminooimidazole-4-carboxamide ribonucleotide transformylase, causing an increase in both intracellular and extracellular adenosine (an potent anti-inflammatory mediator), and an increase in cAMP"

No data was taken on Folinic Acid treatment alone.

A comparison of vitamin A and leucovorin for the prevention of methotrexate-induced micronuclei production in rat bone marrow.

"Methotrexate [is] a folate antagonist. In rats, methotrexate is known to induce micronuclei formation, leading to genetic damage, while vitamin A is known to protect against such methotrexate-induced genetic damage. Leucovorin (folinic acid) is generally administered with methotrexate to decrease methotrexate-induced toxicity.
We aimed to determine whether vitamin A and leucovorin differed in their capacity to prevent formation of methotrexate-induced micronuclei in rat bone marrow erythrocytes. The present study also aimed to evaluate the effect of combined treatment with vitamin A and leucovorin on the formation of methotrexate-induced micronuclei.
Male and female Wistar rats were injected with 20 mg/kg methotrexate (single i.p. dose). The control group received an equal volume of distilled water. The third and fourth groups of rats received vitamin A (5000 IU daily dose for 4 successive days) and leucovorin (0.5 mg/kg i.p. dose for 4 successive days), respectively. The fifth and sixth groups of rats received a combination of vitamin A and a single dose of methotrexate and a combination of leucovorin and methotrexate, respectively. The last group of rats received a combination of leucovorin, vitamin A and single dose of methotrexate. Samples were collected at 24 hours after the last dose of the treatment into 5% bovine albumin. Smears were obtained and stained with May-Grunwald and Giemsa. One thousand polychromatic erythrocytes were counted per animal for the presence of micronuclei and the percentage of polychromatic erythrocyte was determined.
Comparison of methotrexate-treated rats with the control group showed a significant increase in the percentage of cells with micronuclei and a significant decrease polychromatic erythrocyte percentage. Combined methotrexate and vitamin A therapy and combined methotrexate and leucovorin therapy led to significant decreases in the micronuclei percentage and an increase in polychromatic erythrocyte percentage when compared to rats treated with methotrexate alone. Leucovorin was found to be more effective than vitamin A against the formation of methotrexate-induced micronuclei."

"rats treated with a combination of methotrexate and vitamin A had a significantly reduced frequency of micronuclei formation when compared to methotrexate (20mg/kg)-treated rats"

Leucovorin helps to prevent genetic damage.  Does it do it in cases not caused by Methotrexate?

Inhibitory effect of folinic acid on radiation-induced micronuclei and chromosomal aberrations in V79 cells.

"Folinic acid (FA), clinically called leucovorin, has been widely used as a nutrient supplement in dietary intake and is capable of inhibiting cytotoxicity and chromosomal damage induced by chemicals. However, data on its antigenotoxic effect on radiation-induced chromosomal damage are limited. The present study was, therefore, performed to investigate the effect of FA on radiation-induced (X-rays and UV radiation) micronuclei (MN) and structural chromosomal aberrations (SCA) concurrently in V79 Chinese hamster lung cells. Exponentially growing cells were exposed to five doses of X-rays (1-12 Gy) and UV radiation (50-800 microJ x 10(2)/cm2) and post-treated with 5 or 50 micrograms FA/ml of culture medium for 16 h. The slides were analyzed for the presence of MN and SCA using standard procedures. X-ray treatment alone produced dose-related cytotoxicity. X-rays produced a clear dose-related clastogenicity as measured by percent of micronucleated binucleated cells (MNBN) (5-79%) and percent of aberrant cells (11-92%). FA at 5 micrograms/ml slightly decreased X-ray induced chromosomal damage in both assays; however, the inhibition was significant (12-46% of MNBN, 14-48% in aberrant cells) only when X-ray-treated cultures were post-treated with 50 micrograms FA/ml. Post-treatment of FA had no effect on X-ray induced cytotoxicity as measured by NDI and MI. A similar a dose-related increase in % MNBN (0.5-10.3%) and percent aberrant cells (6-35%) was produced by UV radiation treatment alone. There were significant percentages of MNBN and aberrant cell inhibitions at both 5 and 50 micrograms/ml in both assays. As in the case of X-ray-treated cells, there was a clear dose-related cytotoxicity in UV-treated cells alone. No reduction in NDI or MI was found when UV-exposed cells were post-treated with 5 or 50 micrograms of FA. FA [decreases] radiation-induced chromosomal damage."

"X-rays produce DNA double-strand breaks (DSBS), DNA single-strand breaks(SSBs), base damages and DNA-protein cross-links and that DSBs are the main lesions responsible for chromosome aberrations"

Folinic Acid protects against many sources of DNA damage(and DNA damage could include damage to DNA Methyltransferase explaining it's growth stunting properties).

Low expression of gamma-glutamyl hydrolase mRNA in primary colorectal cancer with the CpG island methylator phenotype.

"The CpG island methylator phenotype (CIMP+) in colorectal cancer (CRC) is defined as concomitant and frequent hypermethylation of CpG islands within gene promoter regions. We previously demonstrated that CIMP+ was associated with elevated concentrations of folate intermediates in tumour tissues[elevated folate levels may cause hypermethylation]. In the present study, we investigated whether CIMP+ was associated with a specific mRNA expression pattern for folate- and nucleotide-metabolising enzymes. An exploratory study was conducted on 114 CRC samples from Australia. mRNA levels for 17 genes involved in folate and nucleotide metabolism were measured by real-time RT-PCR. CIMP+ was determined by real-time methylation-specific PCR and compared to mRNA expression. Candidate genes showing association with CIMP+ were further investigated in a replication cohort of 150 CRC samples from Japan. In the exploratory study, low expression of gamma-glutamyl hydrolase (GGH) was strongly associated with CIMP+ and CIMP+-related clinicopathological and molecular features. Trends for inverse association between GGH expression and the concentration of folate intermediates were also observed. Analysis of the replication cohort confirmed that GGH expression was significantly lower in CIMP+ CRC. Promoter hypermethylation of GGH was observed in only 5.6% (1 out of 18) CIMP+ tumours and could not account for the low expression level of this gene. CIMP+ CRC is associated with low expression of GGH, suggesting involvement of the folate pathway in the development and/or progression of this phenotype. Further studies of folate metabolism in CIMP+ CRC may help to elucidate the aetiology of these tumours and to predict their response to anti-folates and 5-fluorouracil/leucovorin."

Folate is necessary for hypermethylation as Folate is a vitamin essential for DNA synthesis.  Although, excess levels of Folate will likely just be urinated out.

Folate and cancer: how DNA damage, repair and methylation impact on colon carcinogenesis.

"Inappropriate diet may contribute to one third of cancer deaths. Folates, a group of water-soluble B vitamins present in high concentrations in green, leafy vegetables, maintain DNA stability through their ability to donate one-carbon units for cellular metabolism. Folate deficiency has been implicated in the development of several cancers, including cancer of the colorectum, breast, ovary, pancreas, brain, lung and cervix. Generally, data from the majority of human studies suggest that people who habitually consume the highest level of folate, or with the highest blood folate concentrations, have a significantly reduced risk of developing colon polyps or cancer. However, an entirely protective role for folate against carcinogenesis has been questioned, and recent data indicate that an excessive intake of synthetic folic acid (from high-dose supplements or fortified foods) may increase human cancers by accelerating growth of precancerous lesions. Nonetheless, on balance, evidence from the majority of human studies indicates that dietary folate is genoprotective against colon cancer. Suboptimal folate status in humans is widespread. Folate maintains genomic stability by regulating DNA biosynthesis, repair and methylation. Folate deficiency induces and accelerates carcinogenesis by perturbing each of these processes."

Folic Acid (Nature Made Folic Acid Supplement, 400 mcg, 250-Count Tablets (Pack of 3)) as well as Folinic Acid can maximize height growth by protecting against DNA damage.  But excess folic or folinic acid are only necessary but not sufficient conditions for DNA Methylation to occur.  Every child undergoing growth should ensure that they have appropriate quantities of folic and folinic acid to maximize height growth.

DNA Hypermethylation needs something else to occur and one suggested mechanism is S-Adenosyl methionine.


Effect of methotrexate and folinic acid on skeletal growth in mice.

"Four equal groups of Balb/c young male mice (6 animals in each group; mean body weight 11.9 +/- 0.25 g, in their rapid growth phase[3 weeks]) were subjected to the following drug treatment for a period of 3 wk. Group 1 was given intraperitoneal MTX (3.5 mg kg(-1) body weight) every second day. Group 2 received folinic acid (7.0 mg kg(-1) body weight) intraperitoneally every second day. Group 3 was given both drugs (MTX every second day and folinic acid 8 h post-MTX injection). Group 4 was injected with physiological saline every other day to serve as a control group.
Mean lengths of both the tibia and femur of animals were compared in the four treatment groups. A significant decrease in the mean lengths was observed in the group receiving MTX alone. Similarly, there was a significant decrease in the height of the femoral and tibial growth plate in this group when compared with the other groups. The main effect of MTX seemed to be on the hypertrophic proliferative zone of chondrocytes in the growth plate. Furthermore, animals in this MTX-treated group also showed increased levels of MTX in plasma and low levels of erythrocyte folate{thus folate deficiency may play a role in the length decrease}.
Chronic administration of MTX induces suppression of skeletal growth in mice, possibly through the inhibition of the pathway of de novo DNA synthesis{thus other compounds that inhibit new DNA synthesis may also decrease height}. Folinic acid treatment following MTX administration appears to reverse this growth inhibition."

Folinic Acid alone non-significantly increased both femur and tibia length.

A is saline, B is folinic acid, C is MTX, D is MTX + folinic acid.  The growth plate quality seems higher in group B.

Same groups as above.  Again, GP with highest quality is B.  What's interesting to note is that in D the hypertrophic zone seems to be disorganized but the proliferative zone seems expanded.  Therefore, Folinic Acid may revert MTX growth inhibition by more an expansion of the proliferative zone rather than reverting all the effects of MTX.

Growth plate height was non-significantly higher in Folinic Acid group than in the other groups by almost 10% for the femur.  I think why it was shown as non-significant is that the p value chosen was very low p <0.001.

Folinic Acid increased folate levels in erythrocytes by about 40%.

"chronic folinic acid supplementation can prevent methotrexate-induced chondrocyte apoptosis and preserve chondrocyte columnar arrangement and number in the growth plate. In the metaphysis, folinic acid supplementation can preserve primary spongiosa heights and secondary spongiosa trabecular volume by preventing osteoblasts from undergoing apoptosis and suppressing methotrexate-induced marrow adiposity and osteoclast formation. Systemically, plasma of folinic acid supplemented rats, in comparison to plasma from rats treated with MTX alone, contained a significantly lower level of IL-1β and suppressed osteoclast formation in vitro in normal bone marrow cells. The importance of IL-1β in supporting plasma-induced osteoclast formation was confirmed as the presence of an anti-IL-1β neutralizing antibody attenuated the ability of the plasma (from MTX-treated rats) in inducing osteoclast formation."

This study did not study Folinic Acid supplementation without MTX.