Inhibiting growth plate ossification without inhibiting growth

Usually, when the ossification stage of endochondral bone growth is inhibited so is growth like when MMP13 is inhibited for example.  However, in the chicken growth plate, ossification can be inhibited without affecting longitudinal bone growth.  If we apply this to humans, we can have eternally open growth plates.

We do know there are instances in humans where ossification is inhibited but tall stature is achieved by means such as aromatase deficiency.  It's possible that the people with aromatase deficiency could have quincidentally had tall stature or they could have had other mutations in addition to aromatase defiency that makes estrogen less essential to longitudinal bone growth.  Perhaps, these mutations are related to making growth plates more like the chicken growth plate.

Pinealectomy in a broiler chicken model impairs endochondral ossification and induces rapid cancellous bone loss

"Adolescent idiopathic scoliosis (AIS) in humans is a lateral curvature of the spine often associated with osteopenia. It has recently been accepted that the development of AIS is closely associated with spinal overgrowth{a more controlled means of inducing spinal overgrowth with a similar pathology to scoliosis may be a possible height increase technique}. Pinealectomy (PNX) in a chicken model consistently induces scoliosis with anatomic features similar to human AIS.
A histomorphometric study was performed to analyze longitudinal bone growth and cancellous bone remodeling before the development of scoliosis. Static and dynamic parameters in cancellous bone and chondro-osseous junction of the 7th thoracic vertebral body at 9 days after hatching were compared between PNX chickens and control chickens with no surgery.
PNX resulted in a rapid and marked loss of cancellous bone volume (7.9±0.9% vs. 14.2±1.8%) and profoundly disrupted trabecular structure with increases in dynamic formative parameters, such as mineralizing surface, mineralization apposition rate, and adjusted appositional rate. In the chondro-osseous junction, activated osteoclasts phagocytized degenerating chondrocytes, leaving a minimal amount of cartilage matrix and activated osteoblasts, losing their scaffolding for bone formation, and directly covering the hypertrophic zone cells. The osteoid surface and thickness in the chondro-osseous junction were significantly increased in PNX chickens (43.1±14.2% vs. 11.6±5.7% and 4.1±0.2μm vs. 2.9±0.4 μm). In the subjacent cartilage regions being protected from further resorption, abundant labeled cartilage remained with higher cellularity.
Fast-growing birds have a unique paradigm of rapid bone elongation with minimal metaphyseal bone production. A bone-forming surface exists at the front of cartilage ossification in the growth plate; therefore, papillae of hypertrophic chondrocytes become included between the trabeculae of metaphyseal bone and the overall thickness of the growth plate increases considerably in addition to distal expansion. Our results indicate that the unique mechanism for rapid bone elongation in chicken is more pronounced after PNX."

"Supplementation with melatonin, the hormone produced by the pineal gland, in PNX chickens produces less severe spinal deformities"<-but does melatonin also reduce the overgrowth?

"Pinealectomy (PNX) alters growth plate morphologies by enlarging the bone-forming surface.  A bone-forming surface exists in front of the cartilage ossification with intermittent penetration of metaphyseal vessels. Papillae of degenerated hypertrophic chondrocytes extend into the metaphysis with osteoblasts on the surface forming primary bone trabeculae in a direction parallel to bone growth. In PNX chickens, osteoblast coverage at the ossification front is enlarged. Some of the osteoblasts align perpendicular to bone growth. Enlargement of the calcein-labeled hypertrophic zone  and higher numbers of proliferative chondrocytes are seen."

PNX increased the hypertrophic zone area.  Longitudinal bone growth was not labeled in this study however.  But "“The unique paradigm of rapid bone growth”  is more pronounced following PNX, suggesting longitudinal over-growth."

"In the growth plates of mammals, the ossification front is essentially straight because of the simultaneous replacement of hypertrophic chondrocytes by bone in a transverse plane and is solely composed of eroded surface. The majority of longitudinal bone growth results from advancing the whole plate by newly synthesized primary spongiosa, ie, cell proliferation and matrix synthesis at the top of the plate is more or less matched by resorption, thus the thickness of the growth plate stays reasonably constant. In the growth plates of chickens, metaphyseal vessels invade the zone of hypertrophy at intervals and resorption does not occur synchronously across the plate and a bone-forming surface exists in the chondro-osseous junction. Growth plate cartilage becomes included within the newly formed bone and the chondro-osseous junction undulates. A bone-forming surface exists and the subjacent region is temporarily protected from further resorption (protected regions). Primary spongiosa is not produced where endochondral bone is produced in direction perpendicular to longitudinal growth. The increase in thickness of the growth plate with reduced metaphyseal bone production considerably contributes to rapid longitudinal bone growth{can we mimic this in humans?}. Following PNX in chickens, activated osteoclasts and osteoblasts enlarge endochondral bone coverage. The subjacent region is protected from further resorption, resulting in the increase in thickness of the growth plates"

"at 6 days following PNX we found histological changes that were seen after activated osteoclasts and/or chondroclasts to a large extent phagocytized degenerating chondrocytes leaving a minimal amount of cartilage matrix. Activated osteoblasts, losing their scaffolding for bone formation, directly cover the hypertrophic cells. The enlarged endochondral bone coverage further minimizes metaphyseal bone production, leading to a rapid and marked loss of cancellous bone volume, and would accelerate bone elongation economically.  Where endochondral bone covers the cartilage, the subjacent region is temporarily protected from further resorption"<-maybe a surgery can be developed to inject endochondral bone into the growth plate preventing growth plate resorption.

Let's look at the genes produced in the chicken growth plate to see if there are any that could be linked to these protected growth plate regions i.e. eternal growth plate formation without fusion.

Screening of differentially expressed genes in the growth plate of broiler chickens with Tibial Dyschondroplasia by microarray analysis.

"Tibial dyschondroplasia (TD) is a common skeletal disorder in broiler chickens. It is characterized by the presence of a non-vascularized and unmineralized cartilage in the growth plate{so we get an even better idea of genes that might protect from resorption}. Previous studies have investigated differential expression of genes related to cartilage development during latter stages of TD. The aim of our study was to identify differentially expressed genes (DEGs) in the growth plate of broiler chickens, which were associated with early stage TD. We induced TD using tetramethylthiuram disulfide (thiram) for 1, 2, and 6 dayss.
We identified 1630 DEGs, with 82, 1385, and 429 exhibiting at least 2.0-fold changes  at days 1, 2, and 6, respectively. These DEGs participate in a variety of biological processes, including cytokine production, oxidation reduction, and cell surface receptor linked signal transduction on day 1; lipid biosynthesis, regulation of growth, cell cycle, positive and negative gene regulation, transcription and transcription regulation, and anti-apoptosis on day 2; and regulation of cell proliferation, transcription, dephosphorylation, catabolism, proteolysis, and immune responses on day 6. The identified DEGs were associated with the following pathways: neuroactive ligand-receptor interaction on day 1; synthesis and degradation of ketone bodies, terpenoid backbone biosynthesis, ether lipid metabolism, JAK-STAT, GnRH signaling pathway, ubiquitin mediated proteolysis, TGF-β signaling, focal adhesion, and Wnt signaling on day 2; and arachidonic acid metabolism, mitogen-activated protein kinase (MAPK) signaling, JAK-STAT, insulin signaling, and glycolysis on day 6."

"thiram-induced TD is not produced through an increase in chondrocyte multiplication in the transition zone, nor by altering the expression of genes causing the arrest of chondrocytes in a pre-hypertrophic state. It acts by creating metabolic dysfunction that leads to the destruction of blood capillaries in the transition zone chondrocytes. Thiram could promote chondrocyte proliferation in the growth plate of chickens, and disturbs the regulation of endochondral calcification and development of normal cartilage. This results in prehypertrophic cell accumulation, angionecrosis, abnormal extracellular matrix synthesis, deferred endochondral calcification, and bone resorption"

" the expression level of secreted frizzled-related protein 4 (sfrp4) was upregulated 2.8-, 73.5-, and 11.6-fold on days 1, 2, and 6, respectively. The expression level of cadherin 1 (cdh1) was upregulated 31.4- and 10.9-fold on days 2 and 6, respectively. Similarly, expression of enolase 2 (eno2) was upregulated 2.3-, 10.5-, and 4.0-fold on days 1, 2, and 6, respectively."

"lysyl oxidase (lox), which was upregulated, participates in crosslinking of extracellular collagen via oxidative deamination of lysine or hydroxylysine"

"Downregulation of prostaglandin E receptor 4 (subtype EP4) (ptger4) and arginine vasopressin receptor 2 (avpr2) on day 1 directly influenced signal transduction and blood vessel elasticity. On day 2, downregulation of a variety of synthases altered the production of lipid compounds that are precursors of hormones, vitamin D, PTGD, and PTGE, all of which likely have an important role in angiogenesis, regulation of transcription and cell proliferation, and bone development."

Detailed comparison of these genes to LSJL genes to be done.  Unfortunately, I could not find a comparison of normal chicken to human growth plates.

There was a weight loading study that weight loading in young chicks increased bone resorption but decreased longitudinal bone growth.  Maybe weight loading destroys the protected areas of the growth plate.  If this were true, then that study would not be relevant to humans as humans do not have the protected zones of the growth plate.

Does Tybial dyschondroplasia cause overgrowth in chickens?

Changes in the tibial growth plates of chickens with thiram-induced dyschondroplasia.

"Tibial dyschondroplasia (TD) is a metabolic cartilage disease of young poultry in which endochondral bone formation is disrupted leading to the retention of a non-calcified, avascular plug of cartilage in the tibial growth plate. Chicks aged 7 days were fed either a control diet or one containing thiram 100 ppm for 48 h to induce TD. Cell multiplication in the growth plate was determined thereafter with bromodeoxyuridine (BrdU) labelling, and metabolic changes by measuring alkaline phosphatase (ALP), tartrate-resistant acid phosphatase (TRAP), and glutathione (GSH) activities. The effect on chondrocyte maturation was examined. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) and DNA fragmentation were used to determine the effects of thiram on cell survival.  Thiram-induced TD was not due to the multiplication of cells in the post-proliferative zones. Thiram did not affect ALP activity, which would have indicated a loss of calcification potential, but it reduced both TRAP and the glutathione concentrations, suggesting that the growth plate metabolism and remodelling functions were adversely affected. Thiram appeared to have no effect on the expression of type X collagen, transglutaminase, RUNX2, or matrix metalloproteinase-2 (MMP) genes suggesting that it did not alter the maturation potential of chondrocytes. On the contrary, the expressions of MMP-13 and vascular endothelial growth factor (VEGF) genes were "up-regulated," suggesting that thiram has pro-angiogenic activity. Thiram induced endothelial cell apoptosis in the capillary vessels of the growth plates, as early as 10 days of age, when TD was not visually evident. The vascular death increased on subsequent days accompanied by massive death of chondrocytes in the transition zone of the growth plate. The induction of apoptosis in the growth plate was also demonstrated by DNA fragmentation.  Thiram induced TD not through an increase in the multiplication of chondrocytes in the transition zone and not by altering the expression of genes causing the arrest of chondrocytes in a prehypertrophic state, but by creating a metabolic dysfunction which led to the destruction of blood capillaries in the transition zone chondrocytes."

Thiriam also depleted Guthione levels.


"Typical morphology of growth plates from control and thiram-fed chickens aged 16 days. (a) Control growth plate. (b) Growth plate with TD severity score of 1 from a thiram-fed chicken. (c) Growth plate with a severity score of 2 from a thiram-fed chicken."<-C looks overgrown but I can't say for sure.  Tibial dyschondroplasia is associated with rapied chicken growth but we can't say in which way is the causal direction.

However, in instances where there are protected regions of the growth plate inhibiting ossification may increase and induce longitudinal bone growth.  This may be the reason for the deformity as only some areas are protected thus only some areas have overgrowth thus resulting in deformity like in C.

Chondrocytes and longitudinal bone growth: the development of tibial dyschondroplasia.

"Chondrocyte proliferation proceeds normally in TD, but markers of the differentiated phenotype, local growth factors, and the vitamin D receptor are abnormally expressed within the prehypertrophic chondrocytes above, and within, the lesion. Tibial dyschondroplasia is associated with a reduced incidence of apoptosis, suggesting that the lesion contains an accumulation of immature cells that have outlived their normal life span. Immunolocalization studies of matrix components suggest an abnormal distribution within the TD growth plate that is consistent with a failure of the chondrocytes to fully hypertrophy. In addition, the collagen matrix of the TD lesion is highly crosslinked, which may make the formed lesion more impervious to vascular invasion and osteoclastic resorption. "

"Abnormal, nonuniform bone growth within the area of the [TD] lesion leads to increased tibial plateau angle and tibial bowing"<-this deformity may be due to overgrowth.

So if we can recreate regions of the growth plate that can undergo longitudinal bone growth without needing the resorption phase like in some parts of the chicken then we can grow taller forever.