What genes are upregulated during distraction osteogenesis?

Knowledge of what genes are expressed during distraction osteogenesis may provide insight of what genes to induce without causing fracture.  It also allows us to compare DO to LSJL.

Expression of TGF-beta1, osteonectin, and BMP-4 in mandibular distraction osteogenesis with compression stimulation: reverse transcriptase-polymerase chain reaction study and biomechanical test.

"A total of 42 rabbits were used in this reverse transcriptase-polymerase chain reaction study. In the control group, distraction was performed at 1 mm/day for 8 days. In the experimental group, overdistraction was performed for 10 days, followed by a 3-day latency period and 2 days of compression to achieve the same amount of DO. Three rabbits per subgroup were killed at 0, 5, 13, 20, 27, 34, and 41 days after the initial osteotomy. The levels of TGF-beta1 {TGF-Beta receptor I was downregulated by LSJL but TGF-BetaRII is the better one for height growth}, osteonectin, and BMP-4 in the bone regenerates were measured.

Reverse transcriptase-polymerase chain reaction revealed a greater level of TGF-beta1 in the experimental group immediately after applying the compression force that continued for 2 weeks. The level then decreased to that of the control group at 3 weeks. The greater level of osteonectin in the experimental group after compression than that in the control group continued for 3 weeks. In the experimental group, the level of BMP-4 increased immediately after compression. However, the level in the control group decreased."

"overexpression of TGF-β1 on mesenchymal cells, osteoblasts, neovessels, and collagen in the extracellular matrix near the osteotomy site"

"the level of TGF-β1 expression increased during the latency and distraction periods and decreased 4 weeks after DO"

"positive staining for platelet-derived growth factor, basic fibroblast growth factor, TGF-β, BMP-2, BMP-4, and BMP-7 in the cells and matrix components [occurred 21 days after DO]"

Comparison of gene expression of tissue inhibitor of matrix metalloproteinase-1 between continuous and intermittent distraction osteogenesis: A quantitative study on rabbits.

"[There were] higher mRNA levels of TIMP-1 under continuous distraction than that under intermittent distraction at day-6 and day-10 {upregulated over 6 fold by LSJL}. No significant differences were found at day-14 and day-21."

Expression of osteotropic growth factors and growth hormone receptor in a canine distraction osteogenesis model.

"The aim of the present study was to determine the expression of growth hormone (GH), growth hormone receptor (GHR), insulin-like growth factor I (IGF-I), insulin-like growth factor II (IGF-II), and bone morphogenetic protein 2 (BMP-2) in distraction-induced bone regeneration. Expression of these factors was assessed during the consolidation phase, comparing distraction osteogenesis with osteotomy-induced bone formation.In addition, plasma GH profiles and plasma concentrations of IGF-I, IGF-II, and insulin-like growth factor-binding protein 4 and -6 (IGFBP-4 and -6) were measured to assess their potential systemic role during bone formation. Expression of GHR, IGF-I, and BMP-2 had significantly increased in comparison with the expression of these factors in mature bone. Expression of GHR was significantly higher in distraction-induced bone regenerate than in osteotomy-induced bone {The difference was 78-fold}. No significant differences were found for the expression of IGF-I and BMP-2 between distraction and osteotomy {thus the osteomy may be most important in initiating height growth and not the distraction}. Plasma concentrations of GH, IGF-I, IGF-II, IGFBP-4, and IGFBP-6 did not demonstrate any significant differences between treatment groups and controls. Upregulation of GHR expression in distraction osteogenesis may enhance sensitivity to endogenous systemic GH and thus promote consolidation of the regenerated bone. Changes in the systemic osteotropic growth factors GH{downregulated by LSJL}, IGF-I, IGF-II, IGFBP-4, and IGFBP-6{upregulated by LSJL} do not seem to be of importance during distraction osteogenesis."

"IGFBP-6 preferentially binds and modulates IGF-II"

"The decrease in IGFBP-6 plasma concentrations possibly mediated through TGF-β1 could enhance local availability of IGF-II and thus stimulate bone formation"<-an IGFBP-6 inhibitor may be a way to grow taller?

The role of angiogenesis in a murine tibial model of distraction osteogenesis.

"A simple and reproducible murine model of tibia distraction osteogenesis was developed using a monolateral fixator. Bone formation was assessed histologically over a 35-day time course. The steady state expression of a broad family of angiogenesis-associated genes was assessed by microarray hybridization analyses over the same time course, while the immediate gene response that was induced during each cycle of distraction was assessed at 30 min and 8 h after the first and last rounds of activation of the fixator. Distraction osteogenesis promoted new bone formation primarily through an intramembranous process with maximal osteogenesis during the active distraction period. Histological analysis also showed that dense cortical bone continued to be formed, during the consolidation phase, for 2 weeks after distraction ended. The analysis of steady state mRNA expression levels over the time course of DO showed that VEGF-A and neuropilin, an alternate receptor for VEGF-A, both angiopoietin (Ang) 1 and 2 factors, and the Ang receptor Tie2 were the critical angiogenic factors during DO. A key transcriptional regulator of many of the angiogenic factors, hypoxia-induced factor1alpha (Hif-1a), the FGF binding protein pleiotropin/OSF1{upregulated by LSJL as PTN}, and multiple MMP(s), were also induced during the active distraction period. Examination of the expression of angiogenic factors that were induced after each cycle of activation, demonstrated that Hif-1a, Nrp1, and VEGF-A were all cyclically induced after each increment of distraction. "

"two growth factors pleiotropin/OSF2 and pigment epithelium-derived factor (PEDF) showed the greatest absolute levels of expression during the bone regeneration"

"our results did show chondrocyte-like cells both at the border of trabecular bone adjacent to osteoblast-like cells. While these cells had the appearance of hypertrophic chondrocytes, contrary to fracture healing a distinct endochondral phase was not seen"

"Ang-2 in the presence of VEGF will promote new vessel formation by both inducing remodeling of capillary basal lamina and stimulating endothelial cell sprouting and migration"

Stimulation of Fos- and Jun-related genes during distraction osteogenesis.

" We looked for the expression of early-response genes of the AP-1 transcription factor complex in an in vivo bone regeneration system subjected to mechanical forces because these genes were found to be related to mechanotransduction and important for bone development. Sheep maxillary bone was distracted daily for 15 days. c-Jun and c-Fos were evaluated in biopsy specimens removed at 8 and 15 days and were compared with post-osteotomy but not distracted repair tissue. Elevated levels of c-Jun and c-Fos mRNA were found after 8 days of distraction. Likewise, mesenchyme-like and fibroblast-like cells composing the 8-day distracted regeneration tissue showed increases in the intensity of immunostaining compared to cells in the corresponding non-distracted fracture repair tissue. After 15 days of distraction, when bone trabeculae start to form distally and proximally in the distracted regeneration tissue, mostly preosteoblasts and osteoblasts retained c-Fos and c-Jun immunoreactivity, similar to bone-associated cells in control non-distracted fracture repair tissue."

Jun and Fos genes are upregulated by LSJL.

"The nuclear protooncogenes c-fos, c-jun, fra-2, and jun-D, the expression of which has been followed in the present study, are members of a family of transcription factors that contain a leucine-rich region and a basic region, which are necessary for dimer formation and DNA binding, respectively. Dimerization is a prerequisite for DNA binding to a consensus response element site (5′-TGAG/CTCA-3′) designated activator protein-1 (AP-1) "

It seems that the way distraction osteogenesis initiates height growth is via BMP-2,4, and 7 and TGF-Beta1 and angiogenesis related proteins.  Why can't we just initiate those proteins without the bone breaking?

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.

Limb Lengthening for the Spine!

I primarily focus on a bone deformation and microfracture method but this study may prove useful to those who view limb lengthening as an option.

The Effect of Posterior Distraction on Vertebral Growth in Immature Pigs: An Experimental Simulation of Growing Rod Technique.

This is a Turkish study.  So perhaps there will be a way of limb lengthening in the spine in the future.

"OBJECTIVE.: The aim of this study is to evaluate the vertebral body growth under distraction forces in immature pigs treated with growing rod (GR) technique. SUMMARY OF BACKGROUND DATA.: Distraction forces applied on growth plate of appendicular skeleton stimulate longitudinal growth. However, the effect of distraction forces on axial skeletal growth has not been fully investigated yet. METHODS.: Twelve 10-week-old domestic pigs were used in this experimental model to simulate GR technique. Four of them were lost during postoperative period because of deep wound infection. Cranially T12-L1 and caudally L4-L5 vertebrae were instrumented by pedicle screws bilaterally, while L2 and L3 were skipped. Distraction between pedicle screws was applied at index surgery. The rods were then lengthened twice in a month interval. All subjects were evaluated with anteroposterior and lateral spinal radiograph before surgery, after surgery, and at the final follow-up. The vertebral body heights of distracted segments (HD = L2 and L3) and control segments (HC = T9, T10 and T11) were measured. Average vertebral body heights and the increase percentage in the vertebral body heights were compared among control segments and distracted segments. RESULTS.: The preoperative vertebral body height was similar in 2 groups (preHC: 10.81 mm, n = 19, preHD: 11.27 mm, n = 16, P > 0.05). At the final follow-up, the average vertebral body height in distraction group was significantly higher than the control group (postHC: 17.03 mm, postHD: 18.58 mm, P < 0.05). The increase percentage in vertebral body height was higher in distracted segments, but there was no statistically significant difference between the 2 groups. CONCLUSION.: The vertebral growth continues during GR instrumentation. Distraction forces might stimulate also apophyseal growth of axial skeleton."

So a 1.58mm height increase from spinal limb lengthening and humans are different from pigs(some of them) so the height increase for humans may be greater.