Me: I have been having this issue for the longest time with any method or technique that does not involve at least comes kind of  distraction in the bones we want to stretch and lengthening because I have been having the hardest time wrapping my head around how such methods could possibly work. I have talked to my coworker about this and Tyler but the answer was always “chondrogenesis” which I couldn’t understand. Finally I decided to send Tyler an email so he can clarify what that means. Also, I wanted to ask him about this specific issue when applied to his method, the LSJL, and why it works.

Questions

Inbox…

Natural Height Growth, naturalheightgrowth@gmail.com,  to Tyler,  Sep 30 (2 days ago)

My coworker and i are having a discussion on a main point. how is lsjl able to break apart and stretch out the hard inorganic calcium phosphate bone matrix of the cortical bone even if chondrocytes are being created at such a high level. it should not do anything since you are pushing against something that is harder than concrete.

Tyler Davis to me, Oct 1 (1 day ago)

LSJL doesn’t try to push apart the bones.  LSJL tries to induce chondrogenesis in the epiphysis of the bone.  The chondroinductive properties of LSJL have been validated by gene expression results and histology diagrams.  The ability for hydrostatic pressure, fluid flow, and LIPUS to induce chondrogenesis of adult human MSC’s has also been validated in vivo.  LSJL induces similar stimuli as HP, FF, and LIPUS

Natural Height Growth naturalheightgrowth@gmail.com, to Tyler,  2:12 AM (19 hours ago)

i still don’t get how creating more cartilage cells in the epiphysis will lead to longitudinal growth. even if this type of external stimuli is inducing the right type of genes to produce and release the right type of protein, how can they get around the hard bone? the epiphysis may not be as hard as the diaphysis but it is still very strong.

People keep telling me that the answer is in the chondrogenesis like her but I can’t figure it out. Here is my thinking…

1. So you produce a lot of chondrocytes from MSC differentiation in the cancellous external cavity in the epiphysis ends….

2. The loading (assume it is lsjl method) causes certain genes in the MSC and other HSC in the bone marrow to up regulate and release growth hormones and increase rate of changing into the right type of

3. Some BMPs, FGF, and maybe some other growth factors which are proteins and hormones increases in formation and output or gets turned on

How does the epiphysis increase in length? I realize now that my focus or at least what is easier for me to understand is the orthopedics part, less of the endocrine stuff, and least of all the genetics stuff. So I am focusing on the orthopedics right now.

You say that you are trying to induce the generation and proliferation of chondrocytes in the inner cavity of the epiphysis. How does the chondrocytes then make the overall bone longer? The epiphysis still has a very hard surface so the cell should not be able to pass through the outer bone but then it has the peristeum to deal with. If you remember, the growth plate passes completely through the long bone for it to separate the two bone parts further from each other.

The only explanation I can think of is that you are assuming that the constant rate of osteoblast and osteoclast formation and resorption on the outside and inside might get the inner produced chondrocytes to be pushed out of the inner cavity and once it reaches close to the surface, it causes a bone lengthening process similar tot eh growth plate. However, growth plates have chondrocytes stacked neatly on top of each other. Please explain the process because I am stuck over and over again. the science from a engineering and physics point of view makes no sense.

this is the same kind of problem i am having with fully understanding the ilizarov method. I am still confused whether the fracture is made completely through the bone, thus opening up to the bone marrow cavity or is it just the outer dense bone that is cracked open.

maybe i am being really stupid here so call me “billy bob” but this one part makes no sense.

Tyler David to me, 2:43 AM (19 hours ago)

The principal is that cartilage is capable of growth from within whereas bone is not.  Growth plates have to be capable of pushing incredible amounts of weight like an elephant for example.  Bone can only grow on the outside whereas cartilage is capable of interestitial growth.  Therefore the growth plate doesn’t have to be completely from one end to the bone to the other.  The periosteum is capable of lengthening as well.

If you look at this picture of a finger fracture: http://www.heightquest.com/2010/02/empirical-evidence-of-possibility-of.html.  You can see that my finger increased in length(diagonally) without the whole finger being broken.

And LSJL increases matrix degradation via MMP3, MMP2, and MMP14.  Thus addressing the cortical bone problem.

Growth plate chondrocytes are often not stacked neatly on top of each other.  Also the epiphysis is porous with trabecular bone.  It has a lot of empty spaces filled with marrow.  Yellow/Red doesn’t matter because adiposal stem cells are capable of chondrogenic differentiation too.

Cartilagenous growth plates are capable of interstitial growth.  Growth plates always work against force as it is trapped between two bone ends.  Thus, growth plates should be able to generate growth from within a bone.  One issue is that static compression decreases growth plates and as you say bones are heavy.  So after inducing chondrogenesis with say LSJL it may be best to use microgravity afterwards say inversion or just giving your legs a break for an hour.  Inversion is hard to do for long periods of time though.

In summary: Growth plates can beat forces by definition as they are always trapped between a rock and a hard place(epiphysis and diaphysis).  The sides of the bone are just another force.

Feel free to include this conversation as a “guest post” just include the backlink.

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Natural Height Growth, naturalheightgrowth@gmail.com, to Tyler, 1:30 PM (8 hours ago)

Okay. Now I am finally starting to reach some kind of understanding on what you mean. However, there is still two small points that I haven’t figured out yet.

1. So you increase the chondrocytes. The chondrocytes release the collagen type ii and proteoglycan which is supposed to form a cartilage matrix.

Am i supposed to assume that cartilage is being formed in the epiphysis cavity from doing these loadings?

If you say yes, then the problem is that I have never heard of an incidence where cartilage is actually being formed inside the marrow cavity.

I have always thought that it requires chondrocytes and cartilage matrix to push apart bone, not just chondrocytes. don’t you need cartilage matrix to push the bones.?

in principle and theory, your idea does make sense.

From a physics point of view, if you can get the chondrocytes to multiply, and multiply some more , they will push. The forces produced in the epiphysis will defintiely increase the hydrostatic pressure.

However the force/area which is pressure on the surface area of the saivty will be divided and distributed across the entire thing.

We both know that long bones have high compressive force and tensile strength so force exerted on the axis directions will have to be large. You are pressing laterally from the inside and they say the pressure needed at least from the outside inwards direction is only 50 MPa.

I am visualizing a picture of the femur in my mind as a cylinder with two spheres on the end, like a dumbbell shape. the cortical bone will be strong so the forces there won’t do much. Nature wants to take the path of least resistance so the hydrostatic pressure and chondrocytes will try to put a stretch on the area which is the weakest, which should than be in the epiphysis, but in the lateral direction.

With my logic, this means that LSJL should make the epiphysis ends wide, but only a little bit longer in on the axis which we want, which is longitudinally. Your theory makes sense but the affects will be so small like an exact few millimeters in long bone lengthening.

2. The other main concern which I see is that the original growth plates were not encapsulated like the new chondrocytes. I agree that the original growth plates did have a lot of pressure exerted on them. growth plates are only a few millimeters and they have to support and hold up a 200 lb person so there is a lot of force/ sqr inch. they are pushing up and down. but the sides are not clamped shut.

I will say this again, the chondrocytes produced are pushing in a closed system which is surrounding by upwards of 1 cm thick of bone that is concrete in strength. and the way the pressure will force upon the inner ways will be distributed in a way that goes towards the weaker epiphysis sides.

I stated before that the tensile strength of long bone is 150 MPa. this means the chondrocytes needed to push up to at least 70% of this amount to cause some deformation. They also have to push in the right direction, along the axis. they are trapped in 1 cm thick physically mature adult femur bones.

If I can figure out or calculate how much force/area the original growth plates had to deal with, and it comes out to be anything close to 50-100 MPa then LSJL will definitely work to increase long bones longitudinally. if the values are not close, then I would guess most of the bone changes would be towards increased epiphysis thickness, and very little for real lengthening.  You will get some bone lengthening but little.

I just need to do more research to see what the biomechanical values are of the bones and growth plates. can you explain away my two main concerns?

Tyler Davis to me, 2:01 AM (11 hours ago)

Unfortunately there haven’t been a lot of studies on forces required to lengthen bone or I haven’t come across them.  There’s a study that I mention here http://www.heightquest.com/2011/05/why-does-hydrostatic-pressure-induce.html about mitotic cell rounding and

The idea is that differentiating chondrocytes will automatically secrete matrix.

Unfortunately, I can’t explain away your concerns now.  Ideally we’d need to the growth plates in action live to see what forces they generate.

I have done research myself on the biomechanical forces of the bone and growth plates and couldn’t find very much.

So unfortunately I can’t explain away your concerns at this time.

Natural Height Growth, naturalheightgrowth@gmail.com, to Tyler, Oct 3 (2 days ago)

Okay. I’ll take this conversation and turn it into another post with the back link. It won’t be posted until I get through the protein pathway and endocrinology posts which could take up to 2 weeks. Im just getting around to doing all the reading and research.

Woohae Cho for The International Herald Tribune

Results

Distinct ability to induce an osteogenic marker, alkaline phosphatase (ALP), by 14 BMPs in the osteoblast progenitor C2C12 cells in vitro

In order to comprehensively analyze the distinct osteogenic activity of BMPs, we have recently constructed a panel of recombinant adenoviral vectors that express the 14 types of human BMPs, designated as AdBMPs.⁴⁵ As shown in Figure 1a, the level of transgene expression of the AdBMPs were in general comparable (ie, <2-fold difference among different BMPs), as demonstrated by RT-PCR analysis. These PCR products should be specifically derived from the adenoviral vector-mediated expression (rather than from the endogenous genes), as the 3′-end primer was derived from the SV40 poly-A cassette. Using these adenoviral vectors, we have recently demonstrated that BMPs displayed a distinct ability to induce osteoblast differentiation of mesenchymal progenitor cells in vitro.⁴⁵ In this study, we sought to determine the relative in vivo osteogenic activity of the 14 types of BMPs. We first tested the ability of individual AdBMPs to induce the earlier osteogenic marker alkaline phosphatase in the C2C12 line, which is myoblastic and can be trans-differentiated into osteoblast progenitors upon BMP stimulation. As shown in Figure 1b, ALP activity was remarkably induced by five of the 14 BMPs at four days after AdBMP infections. Among the five osteogenic BMPs, BMP-2, BMP-6, and BMP-9 induced the ALP activity to a much greater extent (approx. 169-, 215-, and 273-fold over the GFP control, respectively), while BMP-4 and BMP-7 increased ALP activity by 44- and 73-fold, respectively. These findings are consistent with our previous studies.⁴⁵ It should be pointed out that several BMPs (eg, BMP-10, and BMP-13) also induced a 2–3-fold increase of ALP activity over the basal level under the same assay conditions.

Orthotopic bone formation effectively induced by several but not all BMPs in athymic mice

We next sought to test the in vivo osteogenic effect of the 14 BMPs. In order to effectively assess the osteogenic ability of the BMPs, we used an orthotopic ossification animal model, in which C2C12 cells were first transduced with AdBMPs and subsequently implanted into the quadriceps muscles of athymic nude mice by intramuscular injection. Orthotopic ossification was assessed by X-ray radiography and histological evaluation at 3 and 5 weeks after injections. As illustrated in Figure 2, ossification was readily detected on X-ray radiographies from the animals injected with AdBMP-2, 6, 7, and 9-transduced C2C12 cells at 3 weeks (Figure 2a) and 5 weeks (Figure 2b). For BMP-6 and BMP-9, histologic examination at both time points (Figure 3a and b) revealed robust and highly mineralized woven bone with scattered osteoblast-rimming and occasional osteoclasts. Osteoid was also present. In addition, BMP-6 showed bone marrow elements with a range of hematopoietic cells. At the 3-week time point, BMP-2 was characterized by well-calcified foci without bone formation; however, at the 5-week time point, these foci had developed into mature bone. BMP-7, on the other hand, was shown to induce much weaker ossification. Interestingly, we failed to detect any signs of ossification in the animals injected with AdBMP-4-transduced C2C12 cells, which is surprising because we have demonstrated that BMP-4 is capable of inducing ALP activity in vitro (Figure 1b).⁴⁵ These results were reproducible in two additional rounds of animal studies using different batches of AdBMP-4 preparations, which were consistently shown to induce ALP activity in C2C12 cells in vitro (data not shown). Further, our RT-PCR analysis demonstrated that the expression level of BMP-4 was comparable with that of other BMPs, especially BMP-2, BMP-6, and BMP-9 under the same assay condition (Figure 1a). Currently, we are still searching for any satisfactory explanations for this discrepancy between BMP-4’s in vitro versus in vivo osteogenic activity. Nevertheless, all of the above findings were reproducible in three batches of independent experiments. The remaining samples had no evidence of ossification at 5 weeks. The injection site in these sections demonstrated exuberant granulation tissue and reparative changes.

Histology of BMP-induced bone formation

We next examined the histology of the recovered injection sites. Overall, the histology correlated well with the findings from X-ray radiography. At the 3-week time point, BMP-2, BMP-6, BMP-7, and BMP-9 demonstrated varying degrees of ossification. BMP-2 and BMP-7 were the least developed with small foci of woven bone (Figure 3a). Both BMP-6 and BMP-9, however, had multiple foci of immature woven trabecular bone. In addition, BMP-9 demonstrated focal cartilaginous differentiation. The bone in BMP-6 and BMP-9-treated animals formed a shell-like rim around a proliferating mass of spindle-shaped C2C12 cells. The 5-week samples demonstrated increased maturation with more mature osteoid matrix and trabecular bone-like structures with accentuation of the shell-like rim, especially in BMP-6 and BMP-9-treated animals. There was less extensive ossification in the BMP-2 sections. Bone marrow elements were present in the BMP-6 sections and chondrocytes and cartilaginous matrix were increased in the BMP-9 sections (Figure 3b). Interestingly, the injected C2C12 cells formed a desmoid-like cell mass in nonosteogenic BMP injections and the GFP control. Even in the animals injected with BMP-2, BMP-6, BMP-7, and BMP-9-transduced C2C12 cells, such cell mass was still visible, and multiple ossification centers were observed at the periphery of the cell mass. BMP-2-, BMP-6-, BMP-7-, and BMP-9-induced osteogenesis was further confirmed by Masson’s Trichrome staining (Figure 3c).

Antagonistic effect of BMP-3 on BMP-induced bone formation

We sought to investigate how the osteogenic BMPs were affected by BMP-3, a known negative regulator of bone formation, as BMP-3 knockout animals exhibited an increase in bone density.⁵² When C2C12 cells transduced by AdBMP-3 and one of the four osteogenic AdBMPs were coinjected intramuscularly for 3 weeks, BMP-2 and BMP-6-induced ossification was completely blocked by BMP-3, and most of the BMP-7-induced ossification was inhibited by BMP-3 (Figure 4a). However, BMP-3 coinjection did not exert any effect on BMP-9-induced calcification (Figure 4a), strongly suggesting that BMP-9 may exert its osteogenic activity via a distinct signaling mechanism. Similar results were obtained for the 5-week groups (data not shown). The histological findings were consistent with those from X-ray radiographic results (Figure 4b). The three samples without ossification demonstrated C2C12 cell proliferation with entrapped skeletal muscle while the BMP-3+BMP-9 sections had multiple foci of woven trabecular bone similar to BMP-9 injection alone.

Bone formation induced by direct intramuscular injection of AdBMPs

Recent studies suggest that skeletal muscles may harbor pluripotent mesenchymal stem cells, including osteoblast progenitors.^34, 44, 53 We next tested the osteoinductive activity of the 14 BMPs via direct intramuscular injection of AdBMPs. At the 3 and 5-week time points, we did not observe apparent ossification on X-ray radiography (data not shown). However, when the 5-week injection sites were examined histologically, various degrees of cartilaginous and/or osteoid matrix formation were observed in BMP-2, BMP-6, BMP-7, and BMP-9-injected animals (Figure 5). Samples derived from BMP-2 and, to a lesser extent, BMP-7 injection sites contained more cartilage-like chondrocyte-containing structure, while osteoid matrix and mature lamellar bone were present with evidence of bone marrow colonization and remodeling in BMP-6 and BMP-9-injected animals. Unlike in the experiments with C2C12 injections, direct intramuscular injections with AdBMPs (ie, the above-mentioned four osteogenic BMPs) induced more diffuse ossification. This may also explain why the calcification (ie, by BMP-9) was not readily detected by X-ray radiography. These findings also suggest that orthotopic osteogenesis induced by direct intramuscular injection with osteogenic AdBMPs may be less efficient than that induced by introduction of AdBMP-transduced osteoblast progenitor cells, implying that osteoblast progenitor cell-based gene therapy may be a more efficacious approach to bone regeneration, although it is possible that the reduced bone formation was resulted from the potentially less-efficient gene transfer associated with direct intramuscular injections than that with AdBMP-transduced C2C12 cells.

Successful bone regeneration mediated by biofactors could revolutionize the clinical management of musculoskeletal disorders, including fracture healing and spinal fusion.⁵⁴ Several biological factors, such as TGF, BMP, FGF, PDGF, IGF, and LMP-1, have been investigated for their potential use in bone regeneration and skeletal repair.^{55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69} BMPs have been shown to be the most promising, and clinical trials with recombinant BMP-2 and -7 are ongoing.^{54, 70, 71, 72, 73} These BMPs have shown varying degrees of success in the clinical setting and further study on their mechanisms of action and optimal formulations is required to optimize effectiveness of this strategy for promoting osteogenesis.

To the best of our knowledge, this reported study represents the first of its kind to evaluate the in vivo osteogenic activity of BMPs in a comprehensive fashion. The observation that BMP-2 exhibited osteogenic activity in our study is consistent with early data from human clinical trials.^25, 27 While BMP-7 exhibited apparent osteogenic activity, its ability to induce ossification was significantly less robust than that of BMP-2, BMP-6, and BMP-9. These findings mirror the moderate success of the rhBMP-7 (ie, OP-1) in a recent clinical trial.²⁶

It is intriguing, however, that BMP-6 and BMP-9 emerged as the most potent inducers of orthotopic ossification in vivo. Although considerable genetic and developmental studies have been conducted to elucidate the biological functions of BMP-6, its osteogenic activity has not been investigated to any significant degree in animal studies or clinical trials. BMP-6-deficient mice are largely unremarkable, with the exception of a defect in the sternum.⁷⁴ Its expression during embryogenesis is closely coupled with BMP-2, and the lack of noticeable defects in BMP-6-deficient mice may be due to functional compensation by BMP-2.⁷⁴ Nevertheless, our findings corroborate well with a recent study in which BMP-6 was shown to induce the most rapid tissue calcification when compared with BMP-2 or BMP-4 in an athymic nude rat model,⁷⁵ although for reasons to be determined AdBMP-4 reproducibly failed to induce orthotopic bone formation in this study.

BMP-9 is one of the least studied members of the BMP family. Originally identified from fetal mouse liver cDNA libraries, BMP-9 (a.k.a., GDF-2) is highly expressed in the developing mouse liver, and recombinant human BMP-9 (rhBMP-9) stimulates hepatocyte proliferation.⁷⁶ BMP-9 has also been shown to be a potent synergistic factor for hematopoietic progenitor cell generation and colony formation,⁷⁷ and may also play a role in the induction and maintenance of the neuronal cholinergic phenotype in the central nervous system.⁷⁸ In addition, it has recently been shown that BMP-9 exhibits an apparent osteoinductive effect in rat models.^21, 23, 79 However, the mechanisms underlying BMP-9-mediated osteogenic signaling remain to be defined. It is a very intriguing finding that BMP-9-mediated bone formation was not inhibited by BMP-3 in our studies. This result strongly suggests that BMP-9 may transduce a distinct osteogenic signaling pathway that is significantly different from that of BMP-2, BMP-6, and BMP-7. Through an expression profiling analysis, we have recently identified a group of downstream targets that may play an important role in the osteogenic BMP signaling pathway mediated by BMP-2, BMP-6, and BMP-9.⁸⁰ Interestingly, while BMP-2, BMP-6, and BMP-9 induced a very similar overall gene expression pattern, the clustering analysis revealed that BMP-2 and BMP-9 exhibited a more similarly related expression pattern.⁸⁰

In conclusion,

we have demonstrated the relative osteogenic ability of 14 BMPs and identified BMP-6 and BMP-9 (in addition to the currently used BMP-2 and BMP-7) as the most potent BMPs to induce orthotopic bone formation in vivo. Our results also suggest that the stem/progenitor cell-based ex vivo gene therapy may represent a more effective approach to bone regeneration. Future studies will focus on elucidating the major signaling differences among BMPs so that maximal synergy in bone formation can be achieved by combining BMPs that act through overlapping or converging signaling pathways. This line of investigation would help to elucidate the molecular mechanisms underlying bone formation and lead to the development of more efficacious approaches towards bone regeneration.

Recombinant adenoviral vectors expressing BMPs

The cDNA clones for human BMP-2, -3 (a.k.a., osteogenin), -4, -5, -6, -8 (a.k.a., OP-2), -9 (a.k.a., GDF-2), -10, -12 (a.k.a., GDF-7 or CDMP-3), and -13 (a.k.a., GDF6 or CDMP2) were kindly provided by the Genetics Institute (Cambridge, MA, USA). The coding sequences for BMP-7 (a.k.a., OP-1), -11 (a.k.a., GDF-11), -14 (a.k.a., GDF-5 or CDMP-1), and -15 (a.k.a., GDF-9) were PCR amplified from a human osteosarcoma cDNA library. The coding regions of the above BMPs were subcloned into pAdTrack-CMV, resulting in pAdTrack-BMPs; recombinant adenoviruses expressing BMPs (ie, AdBMPs) were subsequently generated as previously described. Continue reading →