Mice growth plates do not go through the fusion stage but they do cease eventually in longitudinal bone growth due to growth plate dysfunction.  If we can reverse growth plate dysfunction in mice than perhaps we can do so in humans and get more height from the growth plate.

This study however does not discuss reversing elderly growth plate dysfunction but rather states that older mice are still capable of undergoing endochondral ossification outside the growth plate.

Fractures in Geriatric Mice Show Decreased Callus Expansion and Bone Volume.

“Using a small animal model of long-bone fracture healing based on chronologic age, we asked how aging affected (1) the amount, density, and proportion of bone formed during healing; (2) the amount of cartilage produced and the progression to bone during healing; (3) the callus structure and timing of the fracture healing; and (4) the behavior of progenitor cells relative to the observed deficiencies of geriatric fracture healing.
Transverse, traumatic tibial diaphyseal fractures were created in 5-month-old (young adult) and 25-month-old (which we defined as geriatric, and are approximately equivalent to 70-85 year-old humans) C57BL/6 mice. Fracture calluses were harvested at seven times from 0 to 40 days postfracture for micro-CT analysis (total volume, bone volume, bone volume fraction, connectivity density, structure model index, trabecular number, trabecular thickness, trabecular spacing, total mineral content, bone mineral content, tissue mineral density, bone mineral density, degree of anisotropy, and polar moment of inertia), histomorphometry (total callus area, cartilage area, percent of cartilage, hypertrophic cartilage area, percent of hypertrophic cartilage area, bone and osteoid area, percent of bone and osteoid area), and gene expression quantification (fold change).
The geriatric mice produced a less robust healing response characterized by a pronounced decrease in callus amount (mean total volume at 20 days postfracture, 30.08 ± 11.53 mm3 versus 43.19 ± 18.39 mm3; p = 0.009), density (mean bone mineral density at 20 days postfracture, 171.14 ± 64.20 mg hydroxyapatite [HA]/cm3 versus 210.79 ± 37.60 mg HA/cm3; p = 0.016), and less total cartilage (mean cartilage area at 10 days postfracture, 101,279 ± 46,755 square pixels versus 302,167 ± 137,806 square pixels; p = 0.013) and bone content (mean bone volume at 20 days postfracture, 11.68 ± 3.18 mm3 versus 22.34 ± 10.59 mm3) compared with the young adult mice. However, the amount of cartilage and bone relative to the total callus size was similar between the adult and geriatric mice (mean bone volume fraction at 25 days postfracture, 0.48 ± 0.10 versus 0.50 ± 0.13), and the relative expression of chondrogenic (mean fold change in SOX9 at 10 days postfracture, 135 + 25 versus 90 ± 52) and osteogenic genes (mean fold change in osterix at 20 days postfracture, 22.2 ± 5.3 versus 18.7 ± 5.2; p = 0.324) was similar{so the deficiencies of older mice to undergo endochondral ossification in response to fracture may be related to things other than gene expression}. Analysis of mesenchymal cell proliferation in the geriatric mice relative to adult mice showed a decrease in proliferation (mean percent of undifferentiated mesenchymal cells staining proliferating cell nuclear antigen [PCNA] positive at 10 days postfracture, 25% ± 6.8% versus 42% ± 14.5%.
the molecular program of fracture healing is intact in geriatric mice, as it is in geriatric humans, but callus expansion is reduced in magnitude.
Our study showed altered healing capacity in a relevant animal model of geriatric fracture healing. The understanding that callus expansion and bone volume are decreased with aging can help guide the development of targeted therapeutics for these difficult to heal fractures.”

So older mice are still capable of chondrogenic fracture healing which is a lot like endochondral ossification.  Therefore, this provides evidence that older humans may be capable of inducing new endochondral ossification.

“At 10 days postfracture, corresponding to peak cartilage, young adult mice produced more total cartilage than geriatric mice (302,167 ± 137,806 versus 101,279 ± 46,755 square pixels”

“No difference was found in the percent cartilage content of the callus at 10 days postfracture (41.17% ± 10.13% versus 28.94% ± 8.74%) and in the percent of total cartilage that was hypertrophic at 15 days postfracture (88.76% ± 14.56% versus 70.21 ± 16.05%; p = 0.078). Osseous tissue formation did not reach high levels until 20 days postfracture with cartilage resorption nearly complete for both groups. Geriatric mice exhibit decreased total cartilage production and delayed resorption”

If you’re deficient in Cyp26b1 then having a Vitamin A deficient diet may be a way to grow taller.

Cyp26b1 Within the Growth Plate Regulates Bone Growth in Juvenile Mice.

“Retinoic acid (RA) is an active metabolite of vitamin A and plays important roles in embryonic development. CYP26 enzymes degrade RA and have specific expression patterns that produce a RA gradient, which regulates the patterning of various structures in the embryo.  Localized RA activities in the diaphyseal portion of the growth plate cartilage were associated with the specific expression of Cyp26b1 in the epiphyseal portion in juvenile mice. To disturb the distribution of RA, we generated mice lacking Cyp26b1 specifically in chondrocytes (Cyp26b1Δchon cKO). These mice showed reduced skeletal growth in the juvenile stage. Additionally, their growth plate cartilage showed decreased proliferation rates of proliferative chondrocytes, which was associated with a reduced height in the zone of proliferative chondrocytes, and closed focally by four weeks of age, while wild-type mouse growth plates never closed. Feeding the Cyp26b1 cKO mice a vitamin A-deficient diet partially reversed these abnormalities of the growth plate cartilage. Cyp26b1 in the growth plate regulates the proliferation rates of chondrocytes and is responsible for the normal function of the growth plate and growing bones in juvenile mice, probably by limiting the RA distribution in the growth plate proliferating zone.”

“Cyp26b1 is expressed in the distal region of developing limb buds, and mice that lack Cyp26b1 show severe limb malformation due to the spreading of the RA signal toward the distal end of the developing limb, causing abnormal patterning of limb skeletal elements”

“In juveniles, focal closures of the growth plate in the distal tibia, the proximal tibia, the distal tibia, elbow, proximal femur and distal femur are caused by the treatment of acne with retinoids or the treatment of hyperkeratinosis with cis-retinoic acid. In guinea pigs, the application of RA caused closure of the growth plates in the proximal tibia”

“Excess intake of vitamin A causes growth impairment and skeletal pain in juveniles”

This is a study that could have massive ramifications on height increase but the the exact mechanism is unknown.  But that cells that have the genetic material of growth plate cells are retained in adult bone is huge news.

Hypertrophic chondrocytes can become osteoblasts and osteocytes in endochondral bone formation.

hypertrophic chondrocytes<-full study

“Chondrocytes and osteoblasts are considered independent lineages derived from a common osteochondroprogenitor. In endochondral bone formation, chondrocytes undergo a series of differentiation steps to form the growth plate, and it generally is accepted that death is the ultimate fate of terminally differentiated hypertrophic chondrocytes (HCs). Osteoblasts, accompanying vascular invasion, lay down endochondral bone to replace cartilage.  [Can] HC become an osteoblast and contribute to the full osteogenic lineage? Here we use a cell-specific tamoxifen-inducible genetic recombination approach to track the fate of murine HCs and show that they can survive the cartilage-to-bone transition and become osteogenic cells in fetal and postnatal endochondral bones and persist into adulthood. This discovery of a chondrocyte-to-osteoblast lineage continuum revises concepts of the ontogeny of osteoblasts, with implications for the control of bone homeostasis and the interpretation of the underlying pathological bases of bone disorders.”

This is huge because it means that the transdifferentiated osteoblasts retain some of the chondrocytic genetic material which means they could possibly dedifferentiate back into chondrocytes!

“The expression of preosteoblastic markers in LHs before the formation of the POC raises the possibility that these cells may transition to an osteoblastic fate”

“HC-Derived Cells Are Present in Fetal, Neonatal, and Adult Bone.”

“The HC-derived cells, morphologically resembling osteoblasts, were found close to the chondro-osseous junction, on the surface of trabeculae, and in the endosteum”<-that means they are in a good position to be involved in neo growth plate formation.

“HC[hypertrophic chondrocytes] Derivatives Transit to the Primary Spongiosa and Become Col1a1-Expressing Cells.”<-the majority of HC derivatives become osteoblasts.

“HC-to-bone transition occurs during postnatal bone growth and that HC-derived cells may be long-lived within the mature bone”

Here’s an image of the HC lineage:

“The reversion of HCs to a prehypertrophic- like state in response to endoplasmic reticulum (ER) stress suggests that hypertrophy is not an irreversible state in vivo”<-Could we revert HC-derived osteoblasts back to hypertrophic chondrocytes back to pre-hypertrophic cells to reform growth plates.

(Michael: That actually makes a lot of sense if one thought about it. It can’t be all dead inorganic bone ECM matter where the physeal cartilage turns into bones (aka Primary Spongiosa). How would bone cells even be able to travel to that middle-region later on if the chondrocytes completely died out and the lucanae was covered by calcium minerals. For the smooth gradual transition from the hypertrophic layer to the vascularization/calcification layer to the mineralization layers to work out, there would be some minority of chondrocytes which would change identities. If all the chondrocytes died out and the layer of the lucanae was completely mineralized, I would not be sure that osteoblasts would be able to reach the primary spongiosa layer.

Here is what I have found years ago. (Source: Chondrogenesis just ain’t what it used to be) Prehypertrophic chondrocytes secrete IHH while the chondrocytes from the perichondrium secrete PTHrP, which effect the PTH/PTHrP receptors which are on the prehypertrophic chondrocytes. In addition, we have to consider the effects of the VEGF. VEGF is secreted by hypertrophic chondrocytes, and that it acts as a major inducer of vascular invasion.

The big thing is the following…

“…demonstrated that Ihh is not only required for chondrocyte hypertrophy, but also for expression of Cbfa1, a transcription factor required for osteoblast differentiation”

If the molecular biologist researchers have been able to identify all of the  major players which causes the chondrocytes to go into apoptosis, and also transdifferentiation, then we at least know which transcription factors causes the changes. I suspect it is Cbfa1.

Refer also the seminal work “Indian hedgehog couples chondrogenesis to osteogenesis in endochondral bone development”

That study proved conclusively that it is IHH that starts everything. IHH stimulates the Cbfa1, which causes the vascularization. I am going to make a guess that somewhere along the way, the two peptides also caused the transdifferentiation.

So how do we actually figure out what type of chemical would lead the osteoblast/osteocytes types to go in reverse and de-differentiate aka some type of reverse transdifferentiation?

We first have to assume that there is some type of external stimuli (mechanical, chemical, electrical) which would be able to even do the de-differentiation.

I propose this idea for Tyler…

Let’s assume that if we change the environment, the cell will start to change its identity. Remember the study which showed that the path which the MSCs will differentiate into can be determined by the shape of space that they are placed into? That suggest that if we change the environment that the osteoblasts/bone cells are in, maybe they will change as well.

Here is my first idea: I propose that we try to remove the calcium crystals/de-mineralize the area. To do that, remember that the PTH and the PTHrP balance from the parathyroid glands controls the level of calcium that is dissolved into the human blood (refer to “Chapter 5 – The parathyroid glands and vitamin D“)

“In bone, within 1 or 2 hours, PTH stimulates a process, known as osteolysis, in which calcium in the minute fluid-filled channels (canaliculi/lacunae) is taken up by syncytial processes of osteocytes and transferred to the external surface of the bone and, thence, into the extracellular fluid. Some hours later, it also stimulates resorption of mineralized bone; a process that releases both Ca2+ and Pi into the extracellular fluid. The Pi is rapidly removed from the circulation because the most dramatic effect of PTH on the kidney is to inhibit reabsorption of Pi in the proximal tubule and markedly increase its excretion“

Let’s increase the level of PTHrP in the local region, which is what I had proposed in a post I had written more than a year ago (The Connection Between Regenerating Deer Antlers and The PTHrP, PTH And IHH pathway for Cartilage Regulation, PTHrP Seems To Be The Answer (Big Breakthrough!)). Decrease the concentration of CA2+ from the ECM, and see what happens to the osteoblasts. Would they de-differentiate into chondrocytes if their environment changes?

It might be that the formation of osteoblasts and the increased levels of mineralization/vascularization/calcification is a positive feed back loop where each part feeds upon itself, which is initialized by VEGF. If we break the positive cycle at the process of mineralization, would the osteoblasts also go in reverse?

Last time it looked as though my right finger which I loaded via LSJL was about 1/4″ longer.  Now it looks like it’s about .375″ inches longer.  I’ve been loading about every day for about a 100 count on each of the three joints of the finger.  I increase the load as fast as possible, I could do more but I worry about injury because the clamp is so much stronger than the finger.  I might work up to more.  Two joints I load side to side but since the hand is in the way for the knuckle I load from top to bottom.  Here’s a post regarding my previous results and some images about how I perform LSJL. Here’s an image of my fingers now: Now it’s an extremely significant increase in finger length that is a result of LSJL.  Now I do have some osteophytes and the finger growth is not the same as normal finger growth.  In some of the other images you can see some finger deformities relative to a normal finger.  But it’s still a strong proof of concept that LSJL works to lengthen long bones. I’d rather prove LSJL sooner rather than later.  Would x-rays help?  I don’t really want to get them if they won’t convince people because it would cost a couple hundred dollar.  A lot of people don’t know exactly what makes you taller.  They can’t connect that long bones make you taller and the finger bones are long bones.  If LSJL can increase the length of finger bones(which are long bones(although they do have some different properties to other long bones)) then LSJL can increase overall height if those long bones are legs. As far as my leg progress though, I find that I can’t get as intense a clamp on my knees as I can on my fingers.  I think part of the reason is that there’s a lot of tissue types you’re clamping when you clamp a synovial joint.  It may take a bit of time before these tissues adapt to the clamping force.  I’ve been clamping for a long time with the C-clamp but there was a lot of slippage so there’s now a lot more force with the Irwin Quick Grip that i’m used to.  So right now I’m clamping with the Irwin Quick Grip to about a count of 130 before the pain in the soft tissues is just too irritating but over time the soft tissues will adapt and I’ll be able to clamp with as much force as I want as I have with bones I’ve been clamping a long time. So I’d recommend not clamping past the point of too much soft tissue pain and just try to increase clamping duration and intensity over time to allow the soft tissues to adapt. Remember, that LSJL is untested so there are guarantees that you won’t get injured or other maladies. Of course, if we could just prove LSJL then more testing can be done.  The question is how can we do it now rather than having to perfect it to increase leg length first?

Michael: The finger seems to be definitely longer, but you said that you clamped in all three joints.

• Does that mean that the clamping was also at metacarpophalangeal joints?
• How did you do that, and how can we not make sure that the MSP Joint did not go into inflammation mode aka swelling?
• There is so much evidence that finger joints can swell up if you hit them on something.

X-Rays seem to be the way to go. We measure the synovial joints of the index finger of the right hand compared to the control of your left hand’s index, which I hoped was never clamped, and see whether the lengthen is from the tissue in the synovial joints thickening as a response. If there is a difference in the distance between the bones in either the PIP and/or MCP joints, then the lengthening was not bone. If the distance in the PIP & MCP joint locations are the same, then we then say that the lengthening was truly bone.

You don’t have to go in for a GP check-up. Look into Urgent Care Centers (Source: Which is Cheaper Out of Pocket: Urgent Care Facility or Hospital ER?). They usually accept Insurance. I’ll even put down $70 for the X-rays if that helps.

This study doesn’t relate directly to height growth but it is by the scientists whose research was the foundation for LSJL.

In vitro and in silico analysis of an inhibitory mechanism of osteoclastogenesis by Salubrinal and Guanabenz

“Synthetic agents such as salubrinal and guanabenz, which attenuate stress to the endoplasmic reticulum, are reported to inhibit development of osteoclasts. However, the mechanism of their inhibitory action on osteoclasts is largely unknown. Using genome-wide expression profiles, we predicted key transcription factors that downregulated nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1), a master transcription factor for osteoclastogenesis. Principal component analysis (PCA) predicted a list of transcription factors that were potentially responsible for reversing receptor activator of nuclear factor kappa-B ligand (RANKL)-driven stimulation of osteoclastogenesis. A partial silencing of NFATc1 allowed a selection of transcription factors that were likely to be located upstream of NFATc1. We validated the predicted transcription factors by focusing on two AP-1 transcription factors (c-Fos and JunB) using RAW264.7 pre-osteoclasts as well as primary bone marrow cells. As predicted, their mRNA and protein levels were elevated by RANKL, and the elevation was suppressed by salubrinal and guanabenz. A partial silencing of c-Fos or JunB by RNA interference decreased salubrinal- and guanabenz-driven reduction of NFATc1 as well as tartrate-resistant acid phosphatase (TRAP) mRNA. Collectively, a systems-biology approach allows the prediction of a RANKL-salubrinal/guanabenz-NFATc1 regulatory axis, and in vitro assays validate an involvement of AP-1 transcription factors in suppression of osteoclastogenesis.”

“Salubrinal and guanabenz are potent chemical agents for the inhibition of protein phosphatase 1 (PP1) that specifically de-phosphorylate eIF2α. Through upregulating the phosphorylated level of eIF2α and reducing translational efficiency of most proteins except for a limited set of proteins, such ATF4, these agents attenuate stress to the endoplasmic reticulum. Gene regulation by salubrinal and guanabenz, however, not only takes place at the level of translation but also at the level of transcription. In osteoclasts, it has been shown that administration of salubrinal and guanabenz suppresses receptor activator of nuclear factor kappa-B ligand (RANKL)-driven activation of nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1) ”

“A partial silencing of c-Fos and JunB decreased the mRNA and protein levels of NFATc1. Furthermore, there was a feedback loop in which a decrease in c-Fos by salubrinal reduced NFATc1 expression, and the reduction in NFATc1 further attenuated the level of c-Fos protein. AP-1 proteins are known to play a critical role in osteoclast differentiation. It is reported that mice lacking c-Fos are osteopetrotic due to abnormal development of osteoclasts”

Unfortunately, no real LSJL insights in this study.