Limb Lengthening Surgery

Limb Lengthening Surgery is the premier method for gaining height.  It is the only acknowledged method for height gain(even though physiologically other methods are possible).  There are three steps involved in limb lengthening: fracturing the bone, lengthening the bone(distraction), and healing of the bone(osteogenesis).  The bone is lengthening at a rate of 1mm a day(so about 25 days to grow taller by an inch).  It may even be possible for spinal limb lengthening in the future.  Scientists have also explored the possibility of facial limb lengthening.

Studying limb lengthening is important as it may have implications.  The Periosteum is the primary source of stem cells for the endochondral ossification portion of distraction osteogenesis.  Although there are other types of ossification involved in DO.  DO(Distraction Osteogenesis) may provide us with insight into entirely new height increase methods like hypertrophy of osteoblast mitochondria.

Limb lengthening does not work by causing a macrofracture in the bone.  So we can not apply distraction osteogenesis to microfracutres.  Limb lengthening works by stretching the bony callus that is formed at the fractured ends of the bones.  This stimulates bone growth.  Microfractures may not necessarily form this bony callus.

Another interesting fact about limb lengthening is they don't stretch the fibula.  Maybe they don't do that on purpose to discourage "excessive" height gain.

Here's a study that explains the mechanobiology of distraction osteogenesis.  Let's look at how distraction osteogenesis causes height gain:

Mechanobiology of mandibular distraction osteogenesis: finite element analyses with a rat model.

"Three-dimensional finite element (FE) analyses were performed to characterize the local mechanical environment created within the tissue regenerate during mandibular distraction osteogenesis (DO) in a rat model. Finite element models were created from three-dimensional computed tomography image data of rat hemi-mandibles at four different time points during an optimal distraction osteogenesis protocol (i.e., most successful protocol for bone formation): end latency (post-operative day (POD) 5), distraction day 2 (POD 7), distraction day 5 (POD 10), and distraction day 8 (POD 13). A 0.25 mm distraction was simulated and the resulting hydrostatic stresses and maximum principal tensile strains were determined within the tissue regenerate[Limb Lengthening involves hydrostatic pressure and tensile strain which are two modalities we have been trying to use to induce height gain]. When compared to previous histological findings, finite element analyses showed that tensile strains up to 13% corresponded to regions of new bone formation and regions of periosteal hydrostatic pressure with magnitudes less than 17 kPa corresponded to locations of cartilage formation[So limb lengthening does involve cartilage formation rather than purely intramembranous ossification, 17 kPa is about 127 mmHg which isn't much at all]. Tensile strains within the center of the gap were much higher, leading us to conclude that tissue damage would occur there if the tissue was not compliant enough to withstand such high strains, and that this damage would trigger formation of new mesenchymal tissue. These data were consistent with histological evidence showing mesenchymal tissue present in the center of the gap throughout distraction. Finite element analyses performed at different time points during distraction were instrumental in determining the changes in hydrostatic stress and tensile strain fields throughout distraction, providing a mechanical environment rationale for the different levels of bone formation in end latency, and distraction day 2, 5, and 8 specimens."

A diagram in the study states that compression(like the lateral compression of LSJL) induces more cartilage formation whereas tensile strain induces more bone formation. The type of tension: hydrostatic pressure or tensile strain determined the type of bone formation.  Chondrogenic differentiation from the periosteum with hydrostatic pressure and bone formation from the callus.  So there are essentially two possibilities to make limb lengthening work:  break the bone and then generate hydrostatic pressure in the bony callus or break the bone and then stretch the bony callus.  Both seem to be effective in generating height growth.  Generating a bony callus and then stretching it may be a method of height growth worth considering if we can do it without fracturing the bone of course.

Bone lengthening (distraction osteogenesis): a literature review.‏

"During a DO procedure, tissues are subjected to steady and constant tension and become metabolically activated. New bone formation occurs along the distraction stress line from both extremities of the distracted segment, on the cut ends of the two bony segments[so bone forms at the ends of the bones rather than within]. The proximal and distal parts of the osteotomized bone participate equally in bone regeneration. During this regeneration process, bone formation may show a rate of linear bone formation as high as 200-400 μm/day which is four to eight times faster than physiological physeal growth.

Distraction osteogenesis can be divided into three temporal phases: a latency period of 5 to 10 days, a distraction phase and a consolidation phase. The latency phase allows for the initial trauma response to take place. It starts immediately following the transverse osteotomy and extends until the beginning of distraction. Events taking place during this phase are basically the same as those in the early stages of fracture repair.

During the distraction phase, tensile forces are applied to the callus with a specific rate and rhythm by the distraction device[distraction osteogenesis applies a stretching force to the bony callus at the end of the bone]. 

As the primitive callus is stretched, a central fibrous zone called the fibrous interzone (FIZ) forms. It is rich in chondrocyte-like cells, fibroblasts and oval cells, which are morphologically intermediate between fibroblasts and chondrocytes[so distraction osteogenesis is not really like a growth plate]. The differentiating osteoblasts at the fibrous interzone deposit osteoid along collagen bundles. They subsequently undergo mineral crystallization parallel to the collagen bundles, forming a zone called the microcolumn formation zone (MCF). Microcolumns resemble stalagmite and stalagtites and have been identified as cones of 150-200 μm. Mineralization proceeds both longitudinally along collagen bundles, parallel to the distraction forces, and transversely as more collagen fibers incorporate[so the type II collagen fibers direct the mineralization]. In between the fibrous interzone and the microcolumn formation zone, a zone of highly proliferating cells, called the primary mineralization front (PMF), is observed.

Once the desired bone length is achieved, distraction ceases, marking the beginning of the consolidation phase, where bone and extensive amounts of osteoid undergo mineralization and remodeling.

Bone regeneration during distraction osteogenesis is believed to occur in response to the longitudinal mechanical strain applied to the callus during healing[distraction osteogenesis involves stretching the callus not the bone]. The exact mechanism by which strain stimulates bone formation remains unclear. It has been suggested that living tissues become metabolically activated by slow, steady traction, a phenomenon called "mechano-transduction", characterized by the stimulation of proliferative, secretory and biosynthetic cellular functions. The structural changes in the cells provide the basis for tissue regeneration under mechanical stress. Mitochondria in skeletal muscle hypertropy, showing evidence of increased volume with multiple cristae, and the functional activity of the nuclei was also increased during DO[skeletal muscle mitochondria hypertrophying causes increased volume perhaps an increase in the functional activity of the nuclei in the bone mitochondria can increase height]. Smooth muscle cells in the middle layer of the vessel walls were also activated, their nuclei were hypertrophied, and active euchromatin appeared in the nuclei.

Histological changes occurring in the regenerate under the tensile forces have been widely studied. Three different modes of ossification are identified and implicated in bone formation during DO.

Membranous ossification is the predominant mechanism of ossification during DO, particularly during late stages. Histological observations reveal that cells represent a continuum between fibroblasts, pre-osteoblasts and osteoblasts arranged longitudinally in order of differentiation. The different types of cells are seen along the bone trabeculae oriented along the tension vector within the MCF.

Endochondral ossification occurs during early stages of DO and is characterized by a cartilage tissue transition from fibrous tissue to bone. Ossification occurs through a cartilage intermediate. A hypertrophic cartilaginous callus is progressively invaded by capillaries and new bone will deposit on the surface of eroded cartilage. Enchondral ossification has been identified during distraction and consolidation phases. Enchondral ossification is usually seen at the junction of the FIZ and the newly mineralized membranous bone emanating from the cut ends. This mode of ossification which is characteristic of bone fracture repair has been identified in various experimental models of long bone DO (sheep, dogs, rabbits.) and in mandibular distraction. The ratio of membranous on enchondral ossifications in DO is close to 5/1.

A third mode of ossification called "transchondroid bone formation" has been described as part of DO histological events. During transchondroid ossification, chondroid bone is formed directly by chondrocyte-like cells, with a gradual transition from fibrous tissue to bone (chondroid bone)[so you can grow taller from fibrous tissue]. Transition from fibrous tissue to bone occurs gradually without capillary invasion. Chondrocyte-like cells undergo some kind of an osteogenic differentiation with type I and type II collagen fibres identified in hypertrophic chondrocytes and APL activity present in cartilage matrix in transitional region. Cartilage that forms during DO is usually observed at the level of the periosteum, but not between the cut ends of the cortices within the distraction gap[so stem cells from the periosteum are responsible for any endochondral ossification].

Tissue regeneration within the distraction gap is inevitably consecutive to changes in cellular morphology and function.  There is hyperplasia of cell organelles including mitochondria, endoplasmic reticulum, Golgi complex in skeletal muscle, and blood vessels at the ultrastructural level, under mechanical tension.  Proliferation of osteoblasts is increased by mechanical force. Immunohistochemical analysis showed that proliferating cell nuclear antigen was expressed during the initial period of distraction, indicating the active stimulation of cell proliferation by tension, which was coincident with the appearance of large numbers of fibroblasts in the distraction gap on histological examination . More recently, a very well-documented experimental study analysing the ultrastructural changes occurring within cells under tensile forces in a goat mandibular distraction model clearly showed morphological changes occurring within the cells during the distraction process. In the distraction gap, cells are seen longitudinally oriented along the distraction force 8 days after loading. A week later, at 16 days, cells in the distraction gap begin to differentiate into osteoblasts, showing changes in both protein synthesis and the energy-supplying system. At an ultrastructural level, these cells are hyperplastic in rough endoplasmic reticulum[so hyperplasia of osteoblastic cells can cause height growth?]. Active secretion of collagen fibers in the extracellular matrix is identified. Finally at 32 days, the main ultrastructural character was biosynthesis and secretion of the extracellular matrix. The cells showed numerous rough endoplasmic reticula and abundant mitochondria, smooth membrane vesicles and well-developed Golgi complexes indicating active synthetic and secretory capabilities. Cells were less likely to proliferate and osteoblasts on the surface of newly formed bone secreted collagenous fibres directly on to the matrix surface. In the 48 day group, the bony matrix was more mature and mineralised. Osteoblasts around the bony trabeculae secreted matrix on to the trabeculae, which may help new bone to be modelled.

Recent molecular investigations have also indicated that the molecular signaling cascade plays an important role in the relationship between induced strain and bone regeneration. The molecular signals that drive the regenerative process of DO are similar to those characterizing fracture repairs and include the pro-inflammatory cytokines, the transforming growth factor beta superfamily and angiogenic factors. Various studies have reported that among growth factors, bone morphogenetic proteins (BMPs) may play a central role in the molecular signaling cascade leading to bone renegeration and remodeling in a DO procedure. "

Although hyperplasia of muscle cells causes an increase in volume does it cause an increase in length?  It would seem to be no but it's possible there is an increase in length but there's just no room for more muscle.  Since bone is the limiting factor if hyperplasia of osteoblasts caused an increase in length this wouldn't be an issue. Analyzing hyperplasia of osteoblast mitochondria may be a method of height growth worth studying.

Molecular mechanisms controlling bone formation during fracture healing and distraction osteogenesis.

"

Stage of Fracture Repair	Biological Processes	Expression of Signaling Molecules and their Proposed Functions
Inflammation	Hematoma	IL-1, IL-6, and TNF-α play a role in initiating the repair cascade.
	Inflammation	TGF-β, PDGF, and BMP-2 expression increases to initiate callus formation.
	Recruitment of mesenchymal stem cells	GDF-8 is restricted to day 1, suggesting its role in controlling cellular proliferation.[GDF-8 is myostatin, so myostatin limits how much cellular proliferation you get during healing, so myostatin alters the effectiveness of limb lengthening and many other height growth mechanisms]
Cartilage Formation and Periosteal Response	Chondrogenesis and endochondral ossification begins	TGF-β2, -β3, and GDF-5 peak due to their involvement in chondrogenesis and endochondral bone formation.
	Cell proliferation in intramembranous ossification	BMP-5 and -6 rise.
	Vascular in-growth	Angiopoietins and VEGFs are induced to stimulate vascular in growth from vessels in the periosteum.
	Neo-angiogenesis
Cartilage Resorption and Primary Bone Formation	Phase of most active osteogenesis	TNF-α rises in association with mineralized cartilage resorption. This promotes the recruitment of mesenchymal stem cells and induces apoptosis of hypertrophic chondrocytes.[maybe TNF-alpha should not be inhibited.  Inflammatory cytokines do cause DNA damage so there is likely a better way to induce recruitment of MSCS]
	Bone cell recruitment and woven bone formation	RANKL and MCSF rise in association with mineralized cartilage resorption.
	Chondrocyte apoptosis and matrix proteolysis
	Osteoclast recruitment and cartilage resorption	BMP-3, -4, -7, and -8 rise in association with the resorption of calcified cartilage. They promote recruitment of cells in the osteoblastic lineage.
	Neo-angiogenesis	BMP-5 and -6 remain high during this stage, suggesting a regulatory effect on both intramembranous and endochondral ossification.
		VEGFs are up-regulated to stimulate neo-angiogenesis.
Secondary Bone Formation and Remodeling	Bone remodeling coupled with osteoblast activity	IL-1 and IL-6 rise again in association with bone remodeling, whereas RANKL and MCSF display diminished levels.
	Establishment of marrow	Diminished expression of members of the TGF-β superfam

 "

"Interleukins-1 and -6 (IL-1 and IL-6) and TNF-α have been shown to play a role in initiating the repair cascade. They induce a downstream response to injury by recruiting other inflammatory cells, enhancing extracellular matrix synthesis, and stimulating angiogenesis. They are secreted at the injury site by macrophages, inflammatory cells, and cells of mesenchymal origin."<-this could potentially make anti-oxidents bad by lowering the extracellular matrix synthesis.  However, it may be possible to bypass inflammatory cytokines and go straight to BMP-2 and TGF-Beta1.

"In addition to stimulating osteoclast function, TNF-α promotes the recruitment of mesenchymal stem cells and induces apoptosis of hypertrophic chondrocytes during endochondral bone formation. Its absence delays the resorption of mineralized cartilage and, consequently, prevents the formation of new bone. In situations where TNF-α is over-expressed, such as diabetic healing, there is premature cartilage removal that is associated with deficient bone formation and healing"<-Other studies have shown that TNF-alpha inhibits chondrogenesis.  It's possible you want only a minimal amount of TNF-alpha for maximal height growth.  Just enough for new bone formation to occur.

"Bone regeneration during distraction osteogenesis is believed to occur in response to the longitudinal mechanical strain applied to the callus during healing"<-would the bone increase in length if there was no gap and you just stretched the cells of the callus.

To test if the fracture gap is needed for distraction osteogenesis height growth you would need to cause a fracture in a non-longitudinal direction.  Then apply a tensile strain force to the callus.  If the bone grows longitudinally then stretching the bony callus must provide a signal for the bone to increase in volume on a cellular signaling level(such as increasing osteoblast mitochondrial size).

This study describes the cells of the callus.

Bone remodeling during fracture repair: The cellular picture.

"Here's the inflammatory stage that proceeds callus formation:

The extravasation (bleeding) within the fracture site is contained by the surrounding tissue and develops into a hematoma. Degranulating platelets, macrophages, and other inflammatory cells (granulocytes, lymphocytes, and monocytes) infiltrate the hematoma between the fractured fragments and combat infection, secrete cytokines and growth factors, and advance clotting into a fibrinous thrombus. Over time, capillaries grow into the clot, which is reorganized into granulation tissue. Macrophages, giant cells and other phagocytic cells clear degenerated cells and other debris.

This cellular response is coordinated by and involves the secretion of a range of cytokines and growth factors including transforming growth factor-β (TGF-β), platelet-derived growth factor (PDGF), fibroblast growth factor-2 (FGF-2), vascular endothelial growth factor (VEGF), macrophage colony stimulating factor (M-CSF), interleukins-1 and -6 (IL-1 and -6), bone morphogenetic proteins (BMPs), and tumor necrosis factor-α (TNF-α). This factors facilitate the recruitment of additional inflammatory cells in a positive feedback loop, and also the migration and invasion of multipotent mesenchymal stem cells. Stem cells originating from the periosteum, bone marrow, circulation, and the surrounding soft tissues have been implicated in bone formation and repair."<-So a bony callus begins as a hematoma with inflammatory cytokines and traditional bone forming compounds(FGF-2, TGF-Beta, PDGF, BMPs, and VEGF).

For the soft callus:

"Chondrocytes derived from mesenchymal progenitors proliferate and synthesize cartilaginous matrix until all the fibrinous/granulation tissue is replaced by cartilage. Where cartilage production is deficient, fibroblasts replace the region with generalized fibrous tissue. Discrete cartilaginous regions progressively grow and merge to produce a central fibrocartilaginous plug between the fractured fragments that splints the fracture. In the final stages of soft callus production, the chondrocytes undergo hypertrophy and mineralize the cartilaginous matrix before undergoing apoptosis."<-We can get chondrocytes and fibrous tissue without a fracture based hematoma.  Say within the bone marrow.  If we proceed to stretch this region will it stimulate bone volume(and therefore bone height increase)?

" hard callus can form in the absence of a cartilaginous template in intramembranous bone formation (during conditions of high mechanical stability) or in appositional bone growth, where bone forms directly adjacent to an existing mineralized surface. However, in the majority of orthopaedic instances, some level of endochondral ossification is present."<-There has to be some limiting factor on appositional bone growth because why don't the tips of your fingers constantly grow(if they do then it is incredibly slow)?  Stretching this hard callus must too stimulate height growth.  It's unlikely for appositional bone growth to occur at a fracture gap as then it would be possible for the bones to become two separate bones.

"The initial woven bone matrix contains a combination of proteinaceous and mineralized extracellular matrix tissue. This is synthesized by mature osteoblasts, which differentiate from osteoprogenitors in the presence of osteogenic factors. Members of the BMP family are critical mediators of this process, and have been shown to be sufficient for de novo bone formation"<-If the hard callus is mostly type I collagen then we have tons of type I collagen in the bone to stretch.  Either the loads people have been using to stretch bone have not been sufficient to stretch Type I collagen or stretching Type I Collagen alone does not cause height growth.

What part of stretching the callus makes you taller is unknown as stretching the components of the callus like cartilage or Type I collagen has not been enough to induce height growth(Now of course the stretching force may have never been enough).  The hydrostatic pressure generated by a hematoma may definitely play a role in soft callus fracture healing but hydrostatic pressure is not needed for the hard callus.  There would need to be a control study of stretching type I collagen by 1 mm a day with no fracture.

Here's a study that shows the formation of cartilage islands and bands during distraction osteogenesis.  This is of relevance to LSJL, as cartilage islands and bands are likely to be what's formed when chondrogenic differentiation is achieved.

Bone lengthening osteogenesis, a combination of intramembranous and endochondral ossification: an experimental study in sheep.

"Endochondral ossification from the central fibrous tissue has been shown in the distraction gap in experimental models of distraction osteogenesis in rabbits. On the other hand, intramembranous ossification has been proposed to result when a low distraction rate under stable external fixation is applied"<-Maybe an initial fibrous tissue has to be formed within the bone marrow for chondrogenesis to proceed.

"At the proximal and distal ends of the fibrous tissue, chondrocytes became hypertrophic, and new bone trabeculae were formed through endochondral ossification. The cartilage tissue consisted of hypertrophic chondrocytes invaded by neovessels, and mesenchymal cells, abundant fibrous tissue and new bone gradually replaced the surface of the eroded cartilage. The fibrous tissue showed abundant vessels and mesenchymal cells in both forms of ossification"<-Everything else should be present in the bone marrow except for the abundant fibrous tissue.

Here's a diagram showing cartilage bands and islands within fibrous tissue:

"The cascade of endochondral bone development in association with the role of fibronectin has been described. During mesenchymal cell proliferation, fibronectin is present in a cottony array. During chondrogenesis, it is associated with the pericellular zone of chondrocytes. During chondrolysis, loss of proteoglycans unmasks the fibronectin in the hypertrophic cartilage matrix. This “exposed” fibronectin may then serve as nidus for osteoprogenitor cell attachment and differentiation into osteoblast. In the present study, we observed fibronectin in the cartilage tissue, the central region of the newly formed bone trabeculae, and some of the cells within the bone trabeculae."<-Fibronectin which is in fibrous tissue may be the key to grow taller.


Vascular tissues are a primary source of BMP2 expression during bone formation induced by distraction osteogenesis.

"Bone regeneration during distraction osteogenesis (DO) [is] dependent on vascular tissue development and inhibition of VEGFR signaling [diminishes] the expression of BMP2. Transgenic mice containing a BAC transgene in which β-galactosidase had been inserted into the coding region of BMP2 was used to examine how the spatial temporal expression of the morphogenetic signals that drive skeletal and vascular tissue development is coordinated during DO. BMP2 expression was induced in smooth muscle and vascular endothelial cells of arteries and veins, capillary endothelial cells, hypertrophic chondrocytes and osteocytes. BMP2 was not expressed by lymphatic vessels or macrophages. Separate peaks of BMP2 mRNA expression were induced in the surrounding muscular tissues and the distraction gap and corresponded first with large vessel collateralization and arteriole remodeling followed by periods of angiogenesis in the gap region. Mesenchymal cells, lining cells and chondrocytes, expressed VEGFA, although PlGF{down in LSJL} expression was only seen in mesenchymal cells within the gap region. On the other hand VEGFR2 appeared to be predominantly expressed by vascular endothelial and hematopoietic cells{hematopoietic cells are established by endochondral ossification}. Bone and vascular tissue formation is coordinated via a mutually supporting set of paracrine loops in which blood vessels primarily synthesize the morphogens that promote bone formation while mesenchymal cells primarily synthesize the morphogens that promote vascular tissue formation."

"Cortical bone formation is patterned around the Haversian system, and trabecular bone formation is patterned around the vascular structures that infiltrate the empty lacunae left after chondrocyte apoptosis during endochondral bone formation. Both vascular and skeletal morphogeneses are interdependent on each other: development of vascular tissue precedes bone cell differentiation in BMP2-induced ectopic bone formation"

"studies were performed with male mice at 9–12 weeks of age."

" The osteotomy alone produced a strong angiogenic response in the vasculature within the surrounding musculature, resulting in increased size and number of vessels as compared to the unoperated limb."

"active application of mechanical strain by distraction osteogenesis produced an even greater and profound effect on the existent vasculature. This effect was seen initially as a massive increase in the size of the existent vessels that is most easily observed for the femoral artery during the active distraction period. Formation of smaller vessels was primarily seen during the consolidation period and was observed both within the developing bone and the surrounding muscular space. In contrast, the bones that had undergone osteotomy and no distraction showed an extensive amount of vascular remodeling had occurred by day 31 and actually exhibited a reduction in both the number and size of vessels in the surrounding tissues."

"{For} DO, relative to other processes of bone formation, although extensive numbers of MSCs are recruited into the gap region, they do not undergo terminal differentiation and mineralization until the distraction period is completed. This delay in the osteogenic progression may be evidence of a mechanism that coordinates the processes of vascular morphogenesis and bone morphogenesis, because if the connective tissue were to mineralize prematurely, the blood vessels would be unable to grow into the tissue."

"Two Wnt antagonists, Sost and DKK are induced in each tissue compartment after each peak of BMP2 induction, consistent with emerging indications that they are downstream targets to BMP signaling through the BMPR1A receptor"

"DKK regulates neoangiogenesis within vessels, while Sost serves to control the osteoblast to osteocyte differentiation and mineralization in bone"

Lacunocanalicular fluid flow transduces mechanical tension stress during distraction osteogenesis.

"The mechanotransduction mechanisms linking distraction device activation to new bone formation remain unknown. We hypothesize that the tension stress of activation during distraction osteogenesis is transmitted through lacunocanalicular fluid flow to initiate the osteogenic signaling cascade. Adult Sprague-Dawley rats (N = 24) were subjected to mandibular osteotomy and application of an external distraction device. After a 3-day latency period, half the animals (n = 12) underwent device activation at 0.25 mm twice daily for 6 days (total activation, 3 mm), and the other half (n = 12) had no activation. On day 10, the animals were injected with fluorescent reactive red lacunocanalicular tracer before killing. Mandibles were harvested, embedded, and sectioned, and reactive red epifluorescence lacunocanalicular flow was measured. Protein was harvested for focal adhesion kinase 1 (FAK1), NESPRIN1, SUN1, LAMIN A/C, and SMAD1 Western blotting as well as for bone morphogenetic protein (BMP)-2 enzyme-linked immunosorbent assay and alkaline phosphatase assay. Lacunocanalicular fluid flow was significantly greater in the distracted samples (60.5 ± 14 vs 10.3 ± 4 molecules of equivalent soluble fluorochrome per megapixel). Flow distribution demonstrated the highest lacunocanalicular flow near the center of the distraction gap. Increased lacunocanalicular flow resulted in increased FAK1, NESPRIN1, SUN1, and LAMIN A/C expression. Focal adhesion kinase 1 activation in the presence of BMP-2 protein expression  resulted in increased intranuclear SMAD1 phosphorylation and alkaline phosphatase activity. These findings suggest that activation of the distraction osteogenesis device affects cellular response through changes in lacunocanalicular fluid flow. "

"A common misconception is that mechanosensation (the cellular perception of mechanical force) of distraction device activation occurs through direct cellular stretch. This idea comes from an overly simplistic view of physical force in the intercalary gap and in vitro studies demonstrating osteoblast response to low-amplitude uniaxial microstrain. Unfortunately, in vivo direct cellular stretch could at most enact a displacement on the order of 0.1 nm—far smaller than the threshold of any in vitro study."

" In the tensegrity model, cells are effectively miniature “tents” that are held in a continuous state of tension by filaments and microtubules (the “tent poles”) connected to the extracellular matrix (the “pegs” of the tent). When a force is exerted upon a cell, its “pretension” configuration is altered, and the buckling and bending of the cytoskeleton bring intracellular molecules into proximity enabling the conversion of an external force into a biochemical signal"

"t activation of a distraction device creates hydrodynamic cavitation in the fibrous zone at the center of the intercalary gap. As fluid rushes to fill the cavitation, it draws interstitial fluid including lacunocanalicular fluid from either edge of the osteotomized bone. As the lacunocanalicular fluid flows, we hypothesize that it imparts a sheer stress on the osteoblastic cells near the intercalary gap causing a conformational change that is propagated via integrin-mediated proteins such as focal adhesion kinase 1 (FAK1) through the cytoskeleton and nuclear membrane by sequential activation of a mechanotransductive cascade of proteins (ie, NESPRIN1, SUN1, and LAMIN A/C). The intranuclear transmission of the mechanochemical signaling of the FAK1 pathway ultimately connects with the bone morphogenetic protein (BMP)-SMAD signaling pathway by activating the intranuclear phosphorylated SMAD1, enabling it to bind to its target genes (eg, alkaline phosphatase [ALP] expression) and initiate osteoblastic differentiation."

"distraction device activation creates a low-pressure zone in the intercalary gap that results in ebb of lacunocanalicular fluid toward the center of the distraction zone. As fluid flows from the osteotomized bone edges toward the center of the distraction zone, it imparts a conformational change upon the prestressed configuration of osteoblastic cells in the zone. The fluid flow–induced cell surface conformational changes are propagated through the cytosolic nonreceptor tyrosine kinase protein, FAK1, to the outer nuclear membrane (ie, NESPRIN1) to the inner nuclear membrane (ie, SUN1 and LAMIN A/C). In the nucleus, the FAK1 pathway interests the BMP-SMAD pathway by enabling SMAD1 to bind to its target genes (eg, ALP) and initiate osteoblastic differentiation."<-What about chondrogenic differentiation?

So it appears that the activation of the distraction osteogenesis device produces mechanical forces very similar to LSJL.  However the device necessitates the creation of a gap beforehand.

Fibronectin may be the missing link to growing taller

In the article on distraction osteogenesis, an observation was made that endochondral ossification occurred within the fibrous tissue.  Fibronectin which is produced by fibrous tissue is key to endochondral ossification.  Fibrous tissue is formed in the distraction gap in distraction osteogenesis.  Does LSJL produce fibrous tissue?  Is there anything we can do to encourage the production of fibrous tissue in the bone marrow?

If Fibrous tissue is not currently encouraged by LSJL and fibrous tissue production is key to endochondral ossification related processes and therefore growing taller then finding mechanisms to stimulating fibrous tissue production in the bone marrow may help individuals grow taller.  LSJL alters Fibronectin related proteins.  It upregulates Fank1 and FNDC4.

Fibronectin- and collagen-mimetic ligands regulate bone marrow stromal cell chondrogenesis in three-dimensional hydrogels.

"While adhesion to RGD peptides[cells containing RGD peptides inhibit attachment and induce apoptosis] has been shown to inhibit in vitro chondrogenesis, the effects of extracellular matrix (ECM)-mimetic ligands with complex secondary and tertiary structures are unknown. This study aimed to determine whether collagen- and fibronectin-mimetic ligands would retain biologic functionality in three-dimensional (3D) hydrogels, whether different ECM-mimetic ligands differentially influence in vitro chondrogenesis, and if effects of ligands on differentiation depend on soluble biochemical stimuli. A linear RGD peptide, a recombinant fibronectin fragment containing the seven to ten Type III repeats (FnIII7-10) and a triple helical, collagen mimetic peptide with the GFOGER motif were covalently coupled to agarose gels using the sulfo-SANPAH crosslinker, and bone marrow stromal cells (BMSCs) were cultured within the 3D hydrogels. The ligands retained biologic functionality within the agarose gels and promoted density-dependent BMSC spreading. Interactions with all adhesive ligands inhibited stimulation by chondrogenic factors of collagen Type II and aggrecan mRNA levels and deposition of sulfated glycosaminoglycans. In medium containing fetal bovine serum, interactions with the GFOGER peptide enhanced mRNA expression of the osteogenic gene osteocalcin whereas FnIII7-10 inhibited osteocalcin expression. In conclusion, modification of agarose hydrogels with ECM-mimetic ligands can influence the differentiation of BMSCs in a manner that depends strongly on the presence and nature of soluble biochemical stimuli."

"integrin-mediated adhesion to fibronectin is required for precartilage condensation of limb bud cells"<-Fibronectin may be critical for chondrogenesis.  Demineralized bone matrix binds Fibronectin.  Fibronectin may also be key in the hypertrophic chondrocyte to allow for differentiation into osteoblasts.  If the fibronectin is not exposed in the chondrocyte, then no osteogenic differentiation occurs and thus a chondrogenic phenotype is maintained.  RGE was the most chondrogenic of all the ligands.  RGE is the non-adhesive version of the RGD integrin.  So we want to find ways to make RGD non-adhesive.

"Our data provide additional support for the role of the cytoskeleton and suggest that cell shape, more
so than the specific integrin ligand, is a key regulator of chondrogenesis"<-this is good for LSJL as hydrostatic pressure may induce a more pro-chondrogenic cell shape.

It may be that Fibronectin is key but that it's key for it to be non-adhesive to allow for chondrogenesis.  Perhaps, the adhesive form of Fibronectin is produced post cessation of endochondral ossification and that's what inhibits growth.

TGF-Beta1 may stimulate fibronectin and TGF-Beta1 is very pro-chondrogenic.  We know that Fibronectin is important in the pre-condensation stage of MSCs before chondrogenesis is initiated.

Fibronectin regulates proteoglycan production balance in transforming growth factor-β1-induced chondrogenesis.

"Transforming growth factor (TGF)-β and bone morphogenetic protein (BMP) induce a cartilage-specific extracellular matrix (ECM) gene, aggrecan, in a chondrogenic cell line, ATDC5. The results of our recent study show that TGF-β1, but not BMP-4, strongly induces an ECM gene, fibronectin, during chondrogenesis.  Our results showed that TGF-β1, but not BMP-4, led to versican-dominant proteoglycan production during chondrogenesis of ATDC5 cells. siRNA-mediated reduction of fibronectin and interference in the liaison between fibronectin and integrins by the Arg-Gly-Asp-Ser (RGDS) peptide increased aggrecan expression, and decreased versican expression by TGF-β1 stimulation[so fibronectin affects the aggrecan/versican balance in chondrogenic regions, dependent on whether versican or aggrecan is better for height growth, inhibiting or stimulating fibronectin could be used to grow taller]. These data suggest that fibronectin is a critical mediator for TGF-β-specific production balance of 2 major proteoglycans, aggrecan and versican, during chondrogenesis."

"These data suggest that increased expression of versican in the cartilage correlates with fibrosis and decreases the water-holding capacity, and TGF-βs, which induce versican expression, may be key molecules that increase the degree of fibrosis during chondrogenesis."<-So Aggrecan is likely better than versican for height growth as more water equals a larger growth plate.  Fibrosis usually refers to thickening and scarring of cartilagenous tissue.  So this study indicates that inhibting fibronectin may help grow taller.

Fibroconectin signaling may be modulated by magnetic fields.  PEMFs have been discussed before.

Mechanical integrin stress and magnetic forces induce biological responses in mesenchymal stem cells which depend on environmental factors.

"Because integrin receptors function as mechanotransducers, we applied drag forces to β1 integrins on the apical surface of adherent human MSC. In addition to mechanical forces, the technique we used involved also the exposure of the cells to an inhomogeneous magnetic field. In order to assess the influence of the substrate on cell adhesion, cells were cultured on plain tissue culture polystyrene (TCP) or on coated well plates, which allowed only adhesion to embedded fibronectin or RGD peptides[RGD peptides inhibiting attachment and fibronectin allowing it]. We found that the expression of collagen I, which is involved in osteogenesis, and VEGF, a factor which stimulates angiogenesis, increased as a result of short-term mechanical integrin stress. Whereas, collagen I expression was stimulated by mechanical forces when the cells were cultured on fibronectin and RGD peptides but not on TCP, VEGF expression was enhanced by physical stimulation on TCP. The study further revealed that magnetic forces enhanced Sox 9 expression, a marker of chondrogenesis, and reduced the expression of ALP[magnetic forces help enhance Sox9 which helps you grow taller]. Concerning the intracellular mechanisms involved, we found that the expression of VEGF induced by physical forces depended on Akt activation. Together, the results implicate that biological functions of MSC can be stimulated by integrin-mediated mechanical forces and a magnetic field. However, the responses of cells depend strongly on the substrate to which they adhere and on the cross-talk between integrin-mediated signals and soluble factors."

"Mesenchymal stem cells are also able to sense the elasticity of a substrate, which determines the direction of differentiation or maintains their quiescence"<-We can manipulate this.  Remember, bone becomes more elastic when exposed to acid for instance.  How to do this safely is another question.

"The forces subjected to one bead were adjusted to 2 × 10⁻¹⁰ N. Because of the varying number and location of beads attached to one cell, differential strains across the cell can occur. A cyclic stress of 1 Hz (0.5 s on, 0.5 s off) was applied for 15 min."<-this seems pretty small so it's promising for potential application.

"On TCP, cells can adhere to a mix of extracellular matrix proteins both produced by the cells and contained in the culture medium"  The increase in SOX9 was only observed on Tissue Cultured Polystyrene.    So SOX9 was only induced when cells were free not to bind to Fibronectin and RGD peptides.

DLK1 may inhibit chondrogenic differentiation by interacting with Fibronectin.

Delta-like 1/fetal antigen-1 (Dlk1/FA1) is a novel regulator of chondrogenic cell differentiation via inhibition of the Akt kinase-dependent pathway.

"Delta-like 1 (Dlk1, also known as fetal antigen-1, FA1) is a member of Notch/Delta family that inhibits adipocyte and osteoblast differentiation. We overexpressed Dlk1/FA1 in mouse embryonic ATDC5 cells and tested its effects on chondrogenic differentiation. Dlk1/FA1 inhibited insulin-induced chondrogenic differentiation as evidenced by reduction of cartilage nodule formation and gene expression of aggrecan, collagen Type II and X. Similar effects were obtained either by using Dlk1/FA1-conditioned medium or by addition of a purified, secreted, form of Dlk1 (FA1) directly to the induction medium. The inhibitory effects of Dlk1/FA1 were dose-dependent and occurred irrespective of the chondrogenic differentiation stage: proliferation, differentiation, maturation, or hypertrophic conversion. Overexpression or addition of the Dlk1/FA1 protein to the medium strongly inhibited the activation of Akt, but not the ERK1/2, or p38 MAPK pathways, and the inhibition of Akt by Dlk1/FA1 was mediated through PI3K activation. Interestingly, inhibition of fibronectin expression by siRNA rescued the Dlk1/FA1-mediated inhibition of Akt, suggesting interaction of Dlk1/FA1 and fibronectin in chondrogenic cells. Our results identify Dlk1/FA1 as a novel regulator of chondrogenesis and suggest Dlk1/FA1 acts as an inhibitor of the PI3K/Akt pathways that leads to its inhibitory effects on chondrogenesis."

"suppression of fibronectin expression in ATDC5-Dlk1 cells, rescued the inhibition of insulin-induced Akt activation. Interestingly, these effects were not related to the fibronectin receptor integrin beta 1 (Itgb) or the canonical target of integrin signaling, FAK (focal adhesion kinase) which is one of the upstream regulators of Akt kinases. These results suggest that Dlk1/FA1 may exert its effects through interaction with fibronectin, but that the signaling is not mediated though Fn/Itgb/FAK1"

So Dlk1 may not exert it's height decreasing effects through Fibronectin/integrin binding and you may want to inhibit Fibronectin to grow taller and not just disrupt the binding of fibronectin and integrin.

Fibronectin regulates proteoglycan production balance in transforming growth factor-β1-induced chondrogenesis.

"Transforming growth factor (TGF)-β and bone morphogenetic protein (BMP) induce a cartilage-specific extracellular matrix (ECM) gene, aggrecan, in a chondrogenic cell line, ATDC5. The results of our recent study show that TGF-β1, but not BMP-4, strongly induces an ECM gene, fibronectin, during chondrogenesis. However, the role of fibronectin in chondrogenesis is unclear. In the current study, our results showed that TGF-β1, but not BMP-4, led to versican-dominant proteoglycan production during chondrogenesis of ATDC5 cells. siRNA-mediated reduction of fibronectin and interference in the liaison between fibronectin and integrins by the Arg-Gly-Asp-Ser (RGDS) peptide increased aggrecan expression, and decreased versican expression by TGF-β1 stimulation. These data suggest that fibronectin is a critical mediator for TGF-β-specific production balance of 2 major proteoglycans, aggrecan and versican, during chondrogenesis."

"Aggrecan expression levels increased by both TGF-β1 (7.4-fold) and BMP-4 (7.5-fold) stimulation. However, versican was more strongly induced by stimulation with TGF-β1 (16.2-fold) than by BMP-4 (2.1-fold)"

"Versican is a large chondroitin sulfate proteoglycan expressed by human fibroblasts. The expression levels of versican are much lower than those of aggrecan in human articular cartilage"

"Focal adhesion kinase(FAK) is one of the upstream mediators of MAP kinase, and FAK/Src signaling suppresses early chondrogenesis, including the induction of aggrecan, Sox6, and Ccn2 in mesenchymal cells by connective tissue growth factor gene regulation in micromass culture. On the other hand, versican expression is regulated by activator protein-1 (AP-1) and T cell factor (TCF) transcriptional factors. Moreover, fibronectin induces AP-1 activation through integrin α5β1-dependent Akt, extracellular signal regulated kinase (ERK), and Jun-aminoterminal kinase (JNK) signaling pathways in endothelial cells"

So Fibroconectin may be a key to growing taller but not by stimulating it.  The breaking of the bone may loosen fibrous tissue and decrease the integrin binding to fibroconectin allowing for chondrogenesis.  Acidifying the bone or applying magnetic fields may be possible ways to weaken integrin signaling to allow for chondrogenesis.


Fibronectin and stem cell differentiation - lessons from chondrogenesis.

"The principal integrins for collagen binding are integrin a1b1 and integrin a2b1"

"Integrin receptors that bind laminins include integrins a3b1, a6b1 and a7b1"

"fibronectin binds to integrins a5b1, a4b1, and avb3"

"Tenascin-C binds to fibronectin and modulates cell adhesion"

"During embryogenesis, prechondrogenic mesenchymal cells that reside within the ECM of the limb bud rearrange into condensed cell aggregates"

"Condensed cells are readily detected by staining with labeled peanut agglutinin – which binds to a galactose moiety that is upregulated in cell aggregates during chondrogenic differentiation in vivo and in vitro"

"Condensation is a prerequisite for chondrogenic differentiation and depends on the expression of the cell–cell adhesion proteins N-cadherin and neural cell adhesion molecule (NCAM)."

"SOX9 is not required during condensation."<-But Sox9 enhances condensation.

"Undifferentiated mesenchymal cells and the condensing mesenchyme deposit a fibronectin-rich matrix that also contains versican {upregulated by LSJL}, type I collagen, and hyaluronan"

"Tenascin-C is expressed during condensation and, as condensed cells undergo differentiation, levels of tenascin-C and collagen I decrease while a cartilaginous matrix consisting of collagen types II and IX, and the proteoglycans aggrecan and versican develops during this stage"<-LSJL upregulates Tenascin N or Tnn. This stage is most consistent with LSJL gene expression.

"Increased expression of fibronectin during condensation would result in upregulation of the fibronectin matrix, thus generating additional contact sites between the cells and fibronectin that could positively influence cell proliferation"

"Activation of focal adhesion kinase (FAK) and paxillin occurs during pre-cartilage condensation"

"binding of preadipocyte factor 1 (Pref-1, also known as DLK1, a transmembrane protein that contains EGF-like repeats) to fibronectin [activates ERK]" which upregualtes Sox9.

"reduced sulfation of proteoglycans and downregulation of the cell-surface proteoglycan syndecan-2 substantially reduces fibronectin matrix assembly"<-Syndecan 2 is downregulated by LSJL.

DTDST affects sulfate transport and mutations can decrease height.

"exostosin 1 (EXT1) is involved in heparan sulfate synthesis"  Knockout of EXT1 or EXT2 impairs chondrogenic differentiation.

FGF4

FGF4 may be involved in pre-mesenchymal condensation.

FGF signaling regulates mesenchymal differentiation and skeletal patterning along the limb bud proximodistal axis.

"Fibroblast growth factors (FGFs) are signals from the apical ectodermal ridge (AER) that are essential for limb pattern formation along the proximodistal (PD) axis. We conditionally inactivated fgf receptor 2 (Fgfr2) in the mouse AER to terminate all AER functions; for comparison, we inactivated both Fgfr1 and Fgfr2 in limb mesenchyme to block mesenchymal AER-FGF signaling. imb skeletal phenotypes resulting from loss of AER-FGF signals cannot simply be a consequence of excessive mesenchymal cell death but also must be a consequence of reduced mesenchymal proliferation and a failure of mesenchymal differentiation, which occur following loss of both Fgf4 and Fgf8. chondrogenic primordia formation, marked by initial Sox9 expression in limb mesenchyme, is an essential component of the PD patterning process and that a key role for AER-FGF signaling is to facilitate SOX9 function and to ensure progressive establishment of chondrogenic primordia along the PD axis."

"Of the four FGFs expressed in the AER, Fgf4 and Fgf8 are necessary for limb skeletal formation and when both genes are disrupted prior to limb bud initiation, the entire limb skeleton fails to form"

"when Fgf4 and Fgf8 are disrupted after limb bud initiation, transient AER-FGF signaling allows formation of a severely hypoplastic skeleton"

"stylopod agenesis after loss of Fgf8 is not due to reduced mesenchymal cell numbers, but rather to the failure of Sox9-expressing cells to undergo further chondrogenic differentiation."

"In the absence of both Gli3{up in LSJL} and Plzf, Sox9 is not expressed in early limb mesenchyme, which, as we suggest, results in failure of proximal chondrogenic primordia formation and subsequent failure of proximal skeletal element formation."

Antagonistic effects of FGF4 on BMP induction of apoptosis and chondrogenesis in the chick limb bud.

"We have implanted beads impregnated with [BMPs and FGFs] into chick limb buds between stages 20 and 26. Embryos were sacrificed at the time the bone chondrocyte condensations first appear (stages 27-28). Implantation of beads containing BMPs at the earlier stages (20-22) caused apoptosis to occur, in the most severe cases leading to complete limb degeneration. Application of FGF4, either in the same, or in a different bead, prevented the BMP-induced apoptosis. apoptosis observed on removal of the AER prior to stage 23 of development could be brought about by BMPs. The action of epithelial FGF in preventing BMP-mediated apoptosis in the mesenchyme would define a novel aspect of epithelial-mesenchymal interactions. Implanting the BMP4 beads into the core of the limb bud a day later (stages 25-26) caused intense chondrogenesis rather than apoptosis. FGF4 could again nullify this effect and by itself caused a reduction in bone size. This is the reverse of the functional relationship these growth factors have in mouse tooth specification (where it is BMP4 that inhibits the FGF8 function), and suggests that the balance between the effects of FGFs and BMPs could control the size of the chondrocyte precursor cell pool. In this way members of these two growth factor families could control the size of appendages when they are initially formed."

"Three FGF receptors that affect bone growth have reasonably distinct expression patterns within developing bone, with a major part of the expression of Fgfr-1 being in the periostium, that of Fgfr-2 in the hypertrophic cartilage, and that of Fgfr-3 in the resting cartilage, there is considerable overlap of expression in the chondrocyte condensations of the early limb bud"

Fibroblast growth factor signaling regulates Dach1 expression during skeletal development.

"Dach1 was expressed in the distal mesenchyme of the early embryonic mouse limb bud and subsequently became restricted to the tips of digital cartilages. Dach1 protein was localized to postmitotic, prehypertrophic, and early hypertrophic chondrocytes during the initiation of ossification centers, but Dach1 was not expressed in growth plates that exhibited extensive ossification. Dach1 colocalized with Runx2/Cbfa1 in chondrocytes but not in the forming bone collar or primary spongiosa. Dach1 also colocalized with cyclin-dependent kinase inhibitors p27 (Kip1) and p57 (Kip2) in chondrocytes of the growth plate and in the epiphysis before the formation of the secondary ossification center. Because fibroblast growth factors (FGF), bone morphogenetic proteins (BMP), and hedgehog molecules (Hh) regulate skeletal patterning of the limb bud and chondrocyte maturation in developing endochondral bones, we investigated the regulation of Dach1 by these growth and differentiation factors. Expression of Dach1 in 11 days postcoitus mouse limb buds in organ culture was up-regulated by implanting beads soaked in FGF1, 2, 8, or 9 but not FGF10. BMP4-soaked beads down-regulated Dach1 expression, whereas Shh and bovine serum albumin had no effect. FGF4 or 8 could substitute for the apical ectodermal ridge in maintaining Dach1 expression in the limb buds. FGFR2 and FGFR3 [overlapped] with Dach1 expression during skeletal patterning and chondrocyte maturation. Dach1 is a target gene of FGF signaling during limb skeletal development, and Dach1 may function as an intermediary in the FGF signaling pathway regulating cell proliferation or differentiation."

"Dach2, which overlaps in expression pattern with Dach1, may functionally compensate for the loss of Dach1 during embryonic morphogenesis"

"[Dach1] was expressed in mesenchymal cells before chondrocyte differentiation but was excluded from proliferating chondrocytes. It was re-expressed in postmitotic, prehypertrophic, and early hypertrophic chondrocytes, but not in the terminal stages of hypertrophy. By implanting growth factor–soaked beads into early limb buds in organ culture, we found that Dach1 expression was regulated by FGF signaling, consistent with its coexpression with FGF receptors throughout limb development."

"Dach1 and Mtsh1 may participate in the same developmental or cellular pathways during morphogenesis of the limb and other structures"<-Mtsh1 is upregulated by FGF8 and downregulated by BMP4.

Isolation and characterization of chorionic mesenchymal stromal cells from human full term placenta.

"[We took] mesenchymal stromal cells (MSCs) from the chorion of human full term placenta from 15 donors. Chorionic MSCs revealed homologous fibroblast-like morphology and expressed CD73, CD29, CD105, and CD90. The hematopoietic stem cell markers including HLA DR, CD11b, CD34, CD79a, and CD45 were not expressed. The growth kinetics of their serial passage was steady at the later passages (passage 10). The multilineage capability of chorionic MSCs was demonstrated by successful adipogenic, osteogenic and chondrogenic differentiation and associated gene expression. Chorionic MSCs expressed genes associated with undifferentiated cells (NANOG, OCT4, REX1) and cardiogenic or neurogenic markers such as SOX2, FGF4, NES, MAP2, and NF. TERT was negative in all the samples. chorionic MSCs undifferentiated stem cells [are] less likely to be transformed into cancer cells. A low HLA DR expression suggests that chorionic MSCs may serve as a great source of stem cells for transplantation because of their immune-privileged status and their immunosuppressive effect. chorionic MSCs are pluripotent stem cells that are probably less differentiated than BM-MSCs, and they have considerable potential for use in cell-based therapies."

"SOX2 regulates FGF4 expression"

Synergistic effect of fibroblast growth factor-4 in ectopic bone formation induced by bone morphogenetic protein-2.

"After subcutaneously implanting recombinant human BMP-2 (rhBMP-2) in rats, we examined the expression of FGF-4 and FGF receptors (FGFRs) mRNAs and the effect of exogenous recombinant human FGF-4 (rhFGF-4) on bone formation. Three days after implantation, the pellets containing rhBMP-2 were surrounded by fibroblastic mesenchymal cells; on day 7, cartilage tissue appeared; on day 10, hypertrophic chondrocytes and a small amount of mineralized tissue were observed; and, on day 14, the amount of mineralized tissue increased. FGF-4 expression appeared at early stages (days 3 and 7) and its expression decreased at later stages (days 10, 14, and 21), whereas FGFRs were expressed continuously. on days 3 and 7, FGF-4, and FGFR subtypes 1 and 2 (FGFR-1 and FGFR-2) were expressed in mesenchymal cells and chondrocytes, and in the area of alkaline phosphatase (ALP) expression. On day 10, FGF-4 was not detected, whereas the expression of FGFR-1 and FGFR-2 was detectable in the area of alkaline phosphatase (ALP) expression. Injection of rhFGF-4 on days 2, 3, and 4 enhanced the mineralized tissue formation induced by rhBMP-2; however, neither rhFGF-4 treatment on days 6, 7, and 8 nor rhFGF-4 treatment on days 9, 10, and 11 influenced the amount of rhBMP-2-induced mineralization. Our results indicate that FGF-4 and FGFR signals play important roles during rhBMP-2-induced bone formation.  The combination of rhBMP-2 and rhFGF-4 would be useful for bone augmentation."

"At day 10, although FGF-4 was undetectable, FGFR-1 and FGFR-2 were expressed"

"FGF-4 expression during rhBMP-2-induced ectopic bone formation strongly suggests that the gene implicated in embryonic tissue development is activated during the BMP-2-induced ectopic bone formation process in adult animals"

Does the structure of bone provide insight into growing taller?

Stuart J Warden was a friend of CH Turner who developed part of LSJL.  SJ Warden published a new paper and it provides some insight into bone and possibly insights into height growth.

Specialized Connective Tissue: Bone, the Structural Framework of the Upper Extremity

"Bone is a connective tissue containing cells, fibers, and ground substance. There are many functions in the body in which the bone participates, such as storing minerals[bone is not hydrophillic like cartilage, so if there are high quantities of water it makes sense for stem cells to differentiate into chondrocytes to store water, although water is not a mineral], providing internal support, protecting vital organs, enabling movement, and providing attachment sites for muscles and tendons. Bone is unique because its collagen framework absorbs energy, whereas the mineral encased within the matrix allows bone to resist deformation."<-In biology, structure correlates with function.  Can there be any instances where a longer bone would improve bones function?  Yes, if bone were longer it would serve better at enabling movement in some cases.  But, playing basketball doesn't make people taller.  Neither do bigger muscles or tendons.  Only storing water requires the formation of cartilage which is what enables growing taller via endochondral ossification.

"Cortical bone thins toward the expanded ends (epiphyses) and interposed developing region (metaphysis) of long bones where it plays a lesser, yet clinically significant mechanical role"<-Maybe thicker cortical bone is a blocker of endochondral ossification?

"The periosteum covers external surfaces of most bones and is divided into two distinct layers—an outer fibrous and inner cellular layer. The cellular or “cambium” layer is positioned in direct contact with the bone surface and is of particular interest as it contains mesenchymal stem cells (MSCs), which have the potential to differentiate into osteoblasts and chondrocytes, and differentiated osteogenic progenitor cells. The localization of these cell types has made the cellular layer a target for drug therapies and cell harvesting for tissue engineering purposes."<-Since periosteal stem cells are right there next to the bone, they are a good target for chondrogenic differentiation.  Unfortunately, they are oriented on a longitudinal axis rather than a horizontal(except for the flat bone of the skull).

"The endocortical surface of a bone faces the medullary canal and is lined by the endosteum, a single thin layer of bone lining cells (mature osteoblasts) and osteoblasts, which form a membrane over endocortical and trabecular bone surfaces to enclose the bone marrow. The endosteum contains osteoprogenitor cells, but does not appear to contain either MSCs or hematopoietic stem cells (HSCs). However, a portion of HSCs can be found next to the endosteum suggesting reciprocal communication between cells within the endosteum and multipotent HSCs. The close relationship between the cells forms a so-called stem cell niche, whereby the cells of the endosteum physically support and influence stem cell activity."<-There's no endosteum in the epiphysis of the bone.  Maybe the functions of the endosteum can be mimicked in the epiphysis to help form new cartilagenous growth plates there.

"Lamellae in cortical bone form osteons or bone structural units, which consist of a central canal enveloped in concentric lamellae of bone tissue. Outer lamellae form first along the boundary of the osteon known as the cement line, with each successive lamella being laid concentrically inside the preceding one. In trabecular bone, lamellae are stacked into saucer-shaped bone packets that are separated by cement lines. The first lamellae are formed toward the center of the trabeculae with each successive lamella being stacked in parallel layers toward the bone surface. Uniformly spaced throughout lamellae are lenticular cavities called lacunae from which branching canaliculae radiate in all directions. The canaliculae penetrate the lamellae of the interstitial substance to anastomose with canaliculae of neighboring lacunae to form a continuous network of interconnecting cavities."<-Thus bone has the ability to communicate to other parts of the bone.

"Without the addition of mineral to collagen, bone tissue would have properties similar to a rubber band, whereas without collagen, bone is brittle such as chalk."<-If bone had properties similar to a rubber band it would be much easier to make longer.  Demineralizing the bone is a potential way for height growth to occur.

"Osteoclasts are large, multinucleate cells that exclusively mediate the process of bone resorption. Osteoclastogenesis begins when a HSC is stimulated to generate mononuclear cells, which then become committed preosteoclasts and are introduced into the blood stream[so the bone has a way for stem cells to enter the blood steam we just have to make those cells chondrocytes instead of osteoclasts]. The circulating precursors exit the peripheral circulation at or near the site to be resorbed, and fuse with one another to form a multinucleated immature osteoclast. Mature osteoclasts establish a microenvironment between themselves and the underlying bone by peripherally attaching to the matrix using integrins. The attachment creates a compartment between the ruffled basal border of the osteoclast and the bone surface that is isolated from the general extracellular space. An electrogenic proton pump transports in H⁺ ions to acidify the compartment, which acts to mobilize the mineralized component of bone. This exposes the organic matrix, which is subsequently degraded using proteases. The end result is the removal of bone matrix and the development of characteristic shallow cavities known as Howship’s lacunae."<-So osteoclasts attach to bone matrix using integrins.  Can we use similar integrins to encourage chondrocytes to attach to the site of a new growth plate?  Osteoclasts acidifing the compartment is also useful in making the collagen more elastic.  Elastic bone is much easier to stretch.  However, once bone is degraded by protease there is none to stretch.

"Osteoblasts are bone-forming cells and develop locally after proliferation of MSCs residing in the bone marrow stroma and periosteum. Mature osteoblasts express the matrix proteins type I collagen and osteocalcin, and alkaline phosphatase—a key enzyme in the mineralization process. Rows of active osteoblasts secrete unmineralized matrix (osteoid) before becoming either bone lining cells or incorporated into the bone matrix. Cells that become incorporated into the matrix gradually develop long cytoplasmic processes to remain in communication with surrounding cells and are considered immature osteocytes. As the matrix matures and mineralizes, and the osteoid seam moves further away, the osteocyte becomes entombed in a bony matrix."<-If we could get osteoblasts to secrete matrix on the longitudinal ends of bones then we could become taller.

"Osteocytes are the most numerous bone cells and are dispersed throughout the matrix where they occupy lacunae[although LSJL is about chondrogenic differentiation of mesenchymal stem cells, osteocytes are likely a part of the process]. Lacunae are interconnected by an elaborate network of thin tunnels called canaliculi through which osteocytes pass cytoplasmic or dendritic processes[we can determine which cytoplasmic and dendritic processes help with height growth and mimic them to grow taller]. These processes connect individual osteocytes with neighboring cells via gap junctions to facilitate both the transport of nutrients for osteocyte viability and the conveying of intercellular messages[so osteocytes have the ability to communicate with other cells including possible communicating with mesenchymal stem cells to differentiate into chondrocytes]. Intercellular communication is also facilitated by the osteocytic release of signaling molecules into the extracellular fluid, which flows through the lacuna-canalicular system[LSJL increases this fluid flow thus possibly amplifying these signaling molecules]. Osteocyte function remains unclear; however, their principal role appears to be the sensing of mechanical stimuli. In addition, recent evidence has also found osteocytes have the capacity to regulate mineral metabolism and alter their surrounding matrix."

"Bones of the upper extremity predominantly develop by endochondral ossification wherein condensations of mesenchymal cells differentiate into chondrocytes to form a cartilaginous template (or “anlage”). Exceptions are parts of the clavicles and scapulae, which form via intramembranous ossification that does not involve a cartilaginous precursor. In the anlage, chondrocytes hypertrophy and an ossification center forms by neovascularization of the initially avascular cartilaginous template. Osteoblasts associated with the newly developed vasculature begin secretion and mineralization of a type-I collagen-containing extracellular matrix. As development continues, the ossification center propagates toward the epiphyseal growth plates."<-This mentions the ossification center propagating toward the epiphyseal growth plates this would of course mean that inhibiting estrogen and bone growth would help height growth by giving more time for chondrocytes to grow.  However, this has been found not to be the case and that only very low or high levels of estrogen are detrimental to growth.  If there is an ossification center, the rate of it's growth does not seem to affect height growth.  In fact, it's likely a byproduct of normal endochondral ossification.  Also, if trying to form a new growth plate with LSJL the region should already be vascularized which may prove problematic with forming a new ossification center.

"[Motor paralysis] interferes with skeletal growth and modeling leading to the development of bones with reduced length, mass, and size"<-So exercise definitely affects growth but there may be diminishing returns as exercise does not seem to positively correlate with height past a point.

This study finds a correlation though between muscle size and bone size(although a shared variable like serum levels of myostatin may be involved):

Mechanical loads and cortical bone geometry in healthy children and young adults

 

"Muscle torque was positively associated with tibia length and muscle CSA[cross sectional area], independent of age, sex, and race."<-Increasing tibial length increases muscle torque.

 

"muscle CSA was positively associated with endosteal circumference"<-If the endosteum does help with height growth then this could be a mechanism to how muscle CSA helps with height growth.

"the study had limited power to detect significant differences in muscle–bone relations in the mature vs. growing skeleton"<-the more mature bone is similar to growing bone the more likely that mature bone is able to grow longer.

"changes in estimated bone strength during growth are highly correlated with changes in lean mass and muscle cross-sectional area (CSA) but not fat mass. When the growing bone is not subject to mechanical loading, muscle size and function are reduced and bone lacks the shape necessary for its function. In a study of arm side-to-side differences in growing tennis players, greater muscle size induced by exercise was positively correlated with changes in bone mass, size, and strength. However, the greater muscle size only accounted for 12%–16% of the side-to-side variance in bone outcomes, suggesting that other mechanical factors may contribute to bone adaptation during growth."<-So muscle size may play a role in up to 12% of bone length increasing.

"Tibia length is highly associated with Zp and with muscle CSA."<-Tibial length is correlated to muscle CSA.

"muscle CSA was positively associated with Zp, explaining 85% of the variance in Zp. Conversely, tibia length alone was significantly associated with greater Zp, explaining 84% of the variance in Zp."<-The fact that percentage of variance explained in bone strength is so similar between muscle and bone means that Bone Length and Muscle CSA are likely connected.

Remember, that muscular activity lowers myostatin levels which could trickle into effecting the bone.

So, potential mechanisms to manipulate height growth are altering osteocyte communication(done by LSJL which increases fluid flow), making the bone more acidic so it's more elastic(but how to do this safely?), and integrin binding(would need to involve cellular engineering).  Also, the endosteum is linked to muscle CSA and the endosteum is involved with bone marrow stem cell differentiation so maybe the endosteum is linked to myostatin which is linked to bone muscle and bone marrow stem cells.

Hydrostatic Pressure may not increase height growth for everyone

Hydrostatic pressure is an important basis for the theory of LSJL.  Could some individuals cells be responsive to hydrostatic pressure while others are not?  Note in the following study 10 MPa is used which is far greater than what can be achieved by a clamp.

The effect of cyclic hydrostatic pressure on the functional development of cartilaginous tissues engineered using bone marrow derived mesenchymal stem cells.

"Mechanical signals can play a key role in regulating the chondrogenic differentiation of mesenchymal stem cells (MSCs)[We are trying to use the mechanical signal of LSJL to induce chondrogenic differentiation of mesenchymal stem cells in the epiphyseal bone marrow]. The objective of this study was to determine if the long-term application of cyclic hydrostatic pressure could be used to improve the functional properties of cartilaginous tissues engineered using bone marrow derived MSCs. MSCs were isolated from the femora of two porcine donors[so pig donors], expanded separately under identical conditions, and then suspended in cylindrical agarose hydrogels. Constructs from both donors were maintained in a chemically defined media supplemented with TGF-β3 for 42 days. TGF-β3 was removed from a subset of constructs from day 21 to 42. Loaded groups were subjected to 10 MPa of cyclic hydrostatic pressurisation at 1 Hz for one hour/day, five days/week. Loading consisted either of continuous hydrostatic pressure (CHP) initiated at day 0, or delayed hydrostatic pressure (DHP) initiated at day 21. Free swelling (FS) constructs were cultured in parallel as controls. Constructs were assessed at days 0, 21 and 42. MSCs isolated from both donors were morphologically similar, demonstrated comparable colony forming unit-fibroblast (CFU-F) numbers, and accumulated near identical levels of collagen and GAG following 42 days of free swelling culture[So the MSCs from different donors responded differently to other variables than hydrostatic pressure].  The two donors displayed a differential response to hydrostatic pressure. For one donor the application of CHP resulted in increased collagen and GAG accumulation by day 42, resulting in an increased dynamic modulus compared to FS controls. In contrast, CHP had no effect on matrix accumulation for the other donor[So for one donor the hydrostatic pressure did not work at enhancing chondrogenic differentiation]. The application of DHP had no effect on either matrix accumulation or construct mechanical properties for both donors[So Hydrostatic Pressure works better at initiating chondrogenic differentiation which is what we're working towards with LSJL]. Variability in the response to hydrostatic pressure was also observed for three further donors. Application of long-term hydrostatic pressure can be used to improve the functional properties of cartilaginous tissues engineered using bone marrow derived MSCs by enhancing collagen and GAG accumulation. The response to such loading is donor dependent."

Since the bone marrow was isolated from the bone itself, it cannot be bone properties that altered the resistance of stem cells to hydrostatic pressure.  Some stem cells must be resistant to the stimulus of hydrostatic pressure to induce cartilage growth.


"Cyclic hydrostatic pressure has been shown to enhance chondrogenesis of MSC aggregates, as evidenced by increases in type II collagen and aggrecan mRNA expression and/or proteoglycan and collagen accumulation"<-MSC aggregates means before any signs of chondrogenesis is detected.  Just if there are sufficient number of MSCs aggregated in a certain area.  Hydrostatic Pressure has the ability to initiate chondrogenesis where there is none which is why it's so powerfully potential to induce height growth.

"It has also been demonstrated that the magnitude of hydrostatic pressure (0.1, 1 or 10 MPa) differentially regulates chondrogenesis of MSC aggregates, with greater type II collagen mRNA expression and collagen accumulation at higher pressures"<-Unfortunately, we are limited in how much hydrostatic pressure we can induce but we will still get some Type II collagen mRNA expression and collagen accumulation.

"In contrast, other studies report that hydrostatic pressure has little or no effect on chondrogenic gene expression or matrix accumulation in MSC aggregates, in either the presence or absence of TGF-β1 or BMP-2. Furthermore, hydrostatic pressure has been shown to have no effect on aggrecan and collagen II mRNA expression for MSCs embedded in agarose hydrogels"<-So hydrostatic pressure does not help induce height growth sometimes.

"The response of stem cells to mechanical signals [may be individual] dependent."<-Some stem cells may not send out chondrogenic signals to hydrostatic pressure which would mean that LSJL would not work for those people.

"Our hypothesis was initially motivated by our previous findings that other forms of mechanical stimulation, specifically dynamic compression, can inhibit chondrogenesis of MSCs if applied before chondrogenesis has occurred"<-Weight lifting is dynamic compression so weight lifting could inhibit chondrogenesis.  This does not affect pubertal growth as the MSCs have already engaged in chondrogenesis although there may be some MSCs that have not yet.

"It may be that 3 weeks of delayed hydrostatic pressure was of insufficient duration, as previous studies have also suggested that multiple days of hydrostatic pressure may be required to enhance chondrogenesis of bone marrow derived MSCs"<-So it's possible that some people may need a longer duration of hydrostatic pressure to experience results.

"MSCs from different donors [respond differently] to cytokine induced chondrogenic differentiation. Animal model studies investigating mechanically induced chondrogenesis in vivo often report dramatic donor dependent response to loading"<-MSCs respond differently to various cytokines and other mechanical signals so responding differently to hydrostatic pressure is not out of the question.

So LSJL may not be effective for some individuals based on the responsiveness of their MSCs to hydrostatic pressure.  Now this might not be solely genetic(which can be altered by the environment as well) but environment.  Some MSCs could become conditioned to hydrostatic pressure or some MSCs might not be sufficiently primed for hydrostatic pressure by exposure to compounds like IGF-1 and Hyaluronic Acid.

We really have to know why some MSCs don't respond to hydrostatic pressure to know how to fix it.


Cell-matrix interactions regulate mesenchymal stem cell response to hydrostatic pressure

"Bone marrow derived MSCs were seeded into either agarose or fibrin hydrogels and exposed to 10 MPa of cyclic HP (1 Hz, 4h/day, 5 days/week for 3 weeks) in the presence of either 1 or 10 ng/ml TGF- 3. Agarose hydrogels were found to support a spherical cellular morphology, while MSCs seeded into fibrin hydrogels attached and spread, with clear stress fiber formation. Hydrogel contraction was also observed in MSC-fibrin constructs. While agarose hydrogels better supported chondrogenesis of MSCs, HP only enhanced sulphated glycosaminoglycans (sGAG) accumulation in fibrin hydrogels, which correlated with a reduction in fibrin contraction. HP also reduced alkaline phosphatase activity in the media for both agarose and fibrin constructs, suggesting that this stimulus plays a role in the maintenance of the chondrogenic phenotype. This study demonstrates that a complex relationship exists between cell-matrix interactions and hydrostatic pressure which plays a key role in regulating the chondrogenic differentiation of MSCs."

Which hydrogel is closer to the epiphyseal bone marrow?

"when MSCs are seeded in RGD-modified alginate hydrogels chondrogenic gene expression and matrix accumulation is inhibited relative to arginine-glycine-glutamic acid (RGE)-modified controls to which MSCs cannot adhere"

"The inhibitory effect of RGD can be blocked with the addition of soluble RGD or cytochalasin D (an F-actin cytoskeleton inhibitor), demonstrating a role for cell attachment and actin cytoskeleton formation in suppressing chondrogenic differentiation"

"an intact dynamic actin cytoskeleton under tension has been shown to be necessary for fluid flow-induced changes in Sox-9 gene expression in MSCs"

"chondrocytes in pellet culture respond more favorably to HP relative to cells embedded in alginate hydrogels"

"In the current study, HP increased sGAG synthesis when MSCs were cultured in the presence of 1 ng/ml TGFBeta- 3 but not 10 ng/ml TGFBeta- 3"<-TGFBeta may affect mechanotransduction.

"long term application of HP (6 weeks) can enhance chondrogenesis of MSCs embedded in agarose hydrogels"

Human cells derived from degenerate intervertebral discs respond differently to those derived from non-degenerate intervertebral discs following application of dynamic hydrostatic pressure.

"The intervertebral disc (IVD) is one of the body's most important load-bearing structures with the major mechanical force experienced in the nucleus pulposus (NP) being hydrostatic pressure (HP). Physiological levels of HP have an anabolic effect on IVD matrix metabolism in cells derived from non-degenerate animal and herniated IVD while excessive HP has a catabolic effect. However, no studies have investigated the response of non-degenerate and degenerate human disc cells derived from non-herniated discs to HP. Here we investigate the effect of physiological HP on such cells using a novel loading rig. Human IVD cells (both NP and AF) cultured in alginate were subjected to dynamic HP (0.8-1.7 MPa 0.5 Hz) for 2 h. Cell viability was assessed, RNA extracted and qRT-PCR for 18 s, c-fos, Sox-9, collagen type II, aggrecan and MMP-3 performed. Cell viability was unaffected by the loading regime. In non-degenerate NP cells, HP increased c-fos, aggrecan, Sox-9 and collagen type II[LSJL increases all of these proteins] (significantly so in the case of c-fos and aggrecan), but not MMP-3{up} gene expression. In contrast, application of HP to AF or degenerate NP cells had no effect on target gene expression. Our data shows that cells obtained from the healthy NP respond to dynamic HP by up-regulating genes indicative of healthy matrix homeostasis. However, responses differed in degenerate NP cells suggesting that an altered mechanotransduction pathway may be operational[so HP won't work if the mechanotransductive mechanisms don't work]."

2.5HP has a catbolic effect whereas lower levels especially 0.25 to 1MPa have an anabolic effect.

"Static pressure of 1.7 MPa was applied for 1 h at room temperature "