Tuesday, January 1, 2013

A histological analysis of the hyaline cartilage under LSJL

Some of the information below is now found to be incorrect and needs to correct.  But a couple of things, first is that I'm performing LSJL differently and it's more effective.  Rather than loading the tibia and femur separately I'm loading both at once at the center of the knee with the C-clamp, this is closer to the the original LSJL studies.  The ends of the bones are weaker than lower down the epiphysis so this allows you to generate more pressure even if you are loading two bones at once.  I am also loading the tibia differently with the Irwin-Quick Grip F-clamp.  I'm loading at the connection between the calcaneus and the tibia.  I'll update the how to perform LSJL section with this information soon.

I've noticed a couple of new things in the LSJL histology growth plate signs.  The first is that in the bone marrow(the red area)[the trabecular bone is the pink area] the mesenchymal stem cells are much denser.  One of the first steps of chondroinduction is mesenchymal condensation and this observed mesenchymal condensation is further evidence of LSJL chondroinduction.  LSJL increases initial chondrogenic genetic mRNA(Sox9, FGF-2, FGFR1) much more than late stage mRNA growth plate genes(CNP, Beta-Catenin were not observed).

It should also be noted that in slide B the bone marrow in the lower region is much more continuous with no trabecular bone separting it this is evidence that LSJL can degrade bone allowing for no growth plate formtion.

Also, the ossification zone is much developed in B than A although I don't know the significance yet.  If you look under the brackets in slide B you can see that the tissue is bone marrow and not growth plate thus providing evidence that growth plates do not have to be continuous for you to grow taller.  In addition, you can see a zone of bone marrow within the trabecular bone on Slide B on the upper layer.

If anyone can find a growth plate experts to interpret these images it would be a great help for all to grow taller.  I have tried but can't help from experts in the field(general researchers in stem cells don't know).

Also note, that the osteocytes seem less organied in the lacunae in B than A.  The lacunae is pink and the osteocytes are the dots within that lacunae.  I believe that osteoblasts are the dots within the white portion and osteoclasts are the large white circles within the dark red region.

There are several two potential theories as to the Lateral Synovial Joint Loading height increase.  Either it is due to mesenchymal stem cells differentiating into chondrocytes in the epiphysis(the mesenchyaml stem cell theory) or it's due to shear strain on the fibrous capsule releasing periosteal progenitor cells(the periosteal progenitor cell theory).  The fibrous capsule theory is not likely as those cells are tendon derived and they can't really get inside the bone.  Those two could lead to permanent growth which is why they are the most appealing.  It's also a possibility that the height increase is due to an increase in proliferative rate which would not result in new height growth as you need new baby(stem or progenitor) cells to get that new growth.  Which theory is correct perhaps lies in the microscopic analysis of LSJL.

The intensely red represents the bone marrow.  There is more more bone marrow in slide B than A.  The pink color represents the bone.  The bone marrow is much more conjoined in B than A indicating possible osteoclast degradition of the bone which is good with LSJL as it gives room for chondrogenesis.

These slides are from the most important LSJL related study so far "Lengthening of Mouse Hindlimbs with Joint Loading".  Slide A is from the mice who do not undergo LSJL, Slide B is from mice who do.  Slide C is the unloaded group but a special focus on the proliferative and hypertrophic zone of the growth plate.  Slide D is the loaded group.  Now most of the increase in height was due to an increase in the size of the growth plates in the hypertrophic zone.  The bad news about the possibility of permanent height growth is that there was no increase in chondrocyte count in the proliferative zone of the growth plate.  Now if you were getting new periosteal progenitor cells or new stem cells you'd expect the cell count in all layers to be increased.  The chondrocytic cell density in slide B is a lot less than that in slide A.  This doesn't necessarily mean that no new stem cells came in.  If the growth of the hypertrophic cells increased a lot more than the amount of new stem cells coming in then there would be a decrease in chondrocyte density even though overall final growth would be increased.

Or the growth could be so elevated by LSJL that all the chondrocytes mature quickly into the hypertrophic zone.  The fact that the number of chondrocytes increased in the entire growth plate rather than staying constant shows the possibility of a new supply of stem cells or progenitor cells.  No data was collected regarding periosteal shear or microcracks so no way to get information there.  The structure of the bone seems to be different in slide B.  In Slide B you can see a perforation at the top of the bone perhaps where new stem cells came through.  In Slide B, the bone below is a lot more dense than slide A looking more like compact rather than spongy bone.  Well there's only two rats there so it's hard to tell whether any differences are genetically specific to the rats even if such differences are attempted to be eliminated due to using Sprague-Dawley rats.

Also, the ECM matrix in Slide B versus Slide A is a lot whiter.  In Slide A the matrix is the same color as the mesenchymal tissue.  Slide B likely is a whiter ECM as there is likely more of it and less mesenchymal tissue.

Here's a picture where I show evidence of cellular differentiation into chondrocytes(Formation of New Growth Plates):

In the Selection in A you can see no signs of differentiation outside the growth plate.  In Slide B, you see the stem cells condensing which is one of the first steps to forming a growth plate and secreting the white substance(extracellular matrix).  You can also see signs of differentiation in Slide D outside the growth plate in the selection. The area right next to the selection in slide B may also be a premature growth plate although it is hard to tell whether it is a premature growth plate or a bone.

Here's a diagram of histology of stem cells in the bone marrow:

So it's also possible the large white areas are megakaryocytes.  Granulocytes can clearly be seen in B and can be evidence of condensation.

Here's a diagram of osteoclast/osteoblast histology:

Here's a diagram of osteocyte histology:

The large white cells could also be adipose cells:
Here's HSC cell histology:
Histology of Compact Bone:
CL - cement line
End - endosteum
HC - Haversian canal
ICL - internal circumferential lamellae
Os - osteon
VC -Volkmann's canal

Here's the histology of a mesenchymal condenation(in the brackets).  A mesenchymal condensation is a prerequisite to form new growth plates:

"a, b, Histology of wild-type and Plzf-/-;Gli3-/- hindlimb sections at E12.5 (a) and E11.5 (b) stained with haematoxylin and eosin. a, E12.5 Gli3-/-;Plzf-/- limbs show only a single ball of mesenchymal condensation within the proximal region, which lacks the long-bone morphology of wild-type proximal skeletal elements indicated by brackets. b, E11.5 Gli3-/-;Plzf-/- limbs have no discernible chondrogenic mesenchymal condensations."from Plzf and Gli3 are for required for proximal limb condensations.

Info on granulocytes and MSCs:

Dynamic Imaging of Marrow-Resident Granulocytes Interacting with Human Mesenchymal Stem Cells upon Systemic Lipopolysaccharide Challenge

"under inflammatory insults or tissue injury, infused hMSCs accumulate at disease sites"

" multiple hMSCs could be visualized to colocalize in the same general vicinity of the bone marrow with the LysM+ granulocytes, with most hMSCs in close proximity to the granulocytes while few cells were seen to be devoid of contact with the GFP+ cells"

"mouse granulocytes exhibit a highly dynamic “swarming” behavior around hMSCs in response to LPS challenge and that hMSCs remain essentially intact during such an active host innate immune response."


  1. Tyler, are you suggesting that in the rabbit study there is actually a formation of new growth plate due to LSJL?
    If LSJL should theoretically work on adults then why doesnt' any scientist study it?

  2. They are STILL within the range of growth plate. And NOT differentiating but chondrocytes are JUST proliferating as stated in the paper.

  3. The papers say loading has "SMALL" anabolic effects on bone regardless of growth plate. However since LSJL is LATERALLY applied to bone, lengthwise growth seems marginal. So lengthwise pressure works better? (For anonymous#1, Maybe there aren't enough researchers to work on that because academic degree is required to become scientist or something? Not sure)

  4. Annonymous#1: This is a mouse study. What's the rabbit study you are referring to.

    No scientists are studying it because most studies are focused on their own field. They tend to have tunnel vision. Also, there's the unfortunate death of CH Turner. H Yokota seems to be focusing on developing a drug. P Zhang hasn't published a study for a while.

    Science is slow with lots of red tape and you need to get grants and funding.

    Joe: It doesn't seem likely that the chondrocytes would proliferate in that direction. Anyways, several studies have shown that hydrostatic pressure on MSCs can induce chondrogenic differentiation.

    The loads used in the joint loading were fairly low. Higher loads may work better. Lateral loading works partially by generating a pressure gradient that increases pressure throughout the bone. Also, at the site of loading the bone is compressed so there is less room for the blood to travel through which increases hydrostatic pressure further.

    This is why flexing helps to pump more blood through the bone.

    The LSJL studies don't make the link between hydrostatic pressure and the growth plate effects but I believe that is the most likely cause of the height increase.

  5. "I'm performing LSJL differently and it's more effective". SO does this mean that you've grown some more? :D

  6. has there ever been a comparison of LSJL compared to normal walking? walking exerts pressure as well right? I just wonder how much more stronger lsjl is at exerting effect compared to walking, i know weights of dumbbells and clamp pressure is a factor.

  7. I also am wondering if we could figure out a way to do our own distraction ostogenesis, maybe there is a non surgical based way to do so.

  8. Conclusions

    Both VEGFR1 and VEGFR2 are functionally essential in blood vessel and bone formation during DO and are needed to promote osteogenic over chondrogenic lineage progression.

  9. can you take a look at angiotensin and vasopressin (both have to do with blood pressure and water pressure)

    1. Is there any way we can force pump the chondrocytes with water,maximize their water retention? Why exactly is water beneficial for growth is just acting as a pump to push fluid through the limbs? Is water pressure the point? whats the point? is it genes? I see your getting into genes and proteins for height now.

    2. what do you think if we caused microfractures and tried to stretch the bone ourselves to replicate distraction osteogenesis? without incision?

  10. some other terms for searches i thought: (regarding chondrocyte hypertrophy)
    water retention, hydroliphic(Ity), cell volumization

  11. I sent you links on stem cells, (what is your opinion regarding Injecting stem cells for height increase) is it very difficult to obtain stem cells for injection, is the injection process hard to do? How come you never talked about injected stem cells in your Blog?
    Is there a possible way to get them cheaply and grow with injecting them?