Increase Height by inhibiting Chondrocyte DeDifferentiation

Lateral Synovial Joint Loading involves inducing mesenchymal stem cell differentiation into chondrocytes by a combination of TGF-Beta1 release by osteoblasts and hydrostatic pressure on the stem cells.  Once this differentiation has occurred then these chondrocytes can undergo endochondral ossification.  Chondrocytes seem to function relatively independently until the columnar stage of growth.

However, in cartilage tissue engineering the dedifferentiation of chondrocytes is a problem.   This dedifferentiation can be prevented by housing the chondrocytes in an extracellular matrix.  Chondrocytes themselves can produce this matrix.  And even after growth ceases, some extracellular matrix remains until the bone is fully remodeled and vascularized.  LSJL may induce chondrogenic differentiation but this dedifferentiation that follows must be prevented.

How do we prevent chondrocyte dedifferentiation following LSJL?  So we can successfully grow taller with the LSJL height increase program?

Relation of low-intensity pulsed ultrasound to the cell density of scaffold-free cartilage in a high-density static semi-open culture system.

"We attempted to prevent dedifferentiation and reverse the phenotypic modulations by adjusting the cell density. We investigated whether low-intensity pulsed ultrasound (LIPUS) enhances matrix synthesis of the scaffold-free cartilage construct.
Rat articular chondrocytes multiplied in monolayers were seeded onto the synthetic porous membrane at stepwise cell densities (i.e., 1.0, 2.0, and 4.0 × 10(7) cells/cm(2)) to allow formation of a scaffold-free cartilage construct via cell-cell interaction. The cartilage constructs were then stimulated by LIPUS for 20 min/day. To investigate the effect of LIPUS stimulation on matrix synthesis, expression of mRNA for cartilage matrix molecules was quantified by a real-time reverse transcription-polymerase chain reaction. Synthesis of type II collagen, type I collagen, and proteoglycan was also assessed histologically.
Only the chondrocytes cultured at high cell densities in the 2.0 × 10(7)cells/cm(2) group became concentrated and formed a plate-like construct similar to native articular cartilage by macroscopic and histological assessments[It may be necessary for a certain threshold of chondrocytes to be differentiated into for LSJL to be effective]. Statistical analysis on the matrix gene expression demonstrated that the levels of type II collagen and aggrecan mRNA of the 2.0 × 10(7)cells/cm(2) group were significantly higher than with the other two cell-density groups. Interestingly, the LIPUS application led to a statistically significant enhancement of aggrecan gene expression only in the 2.0 × 10(7) cells/cm(2) group.
The current study presents a semi-open static culture system that facilitates production of the scaffold-free constructs from monolayer-cultured chondrocytes. It suggests that the LIPUS application enhances matrix production in the construct, and its combination with the scaffold-free construct might become a feasible tool for production of implantable constructs of better quality."

Now LIPUS may not be necessary.  Tapping may work as well and also longer durations of LSJL may be needed to result in chondrocytes secreting ECM before they dedifferntiate.

Endoglin differentially regulates TGF-β-induced Smad2/3 and Smad1/5 signalling and its expression correlates with extracellular matrix production and cellular differentiation state in human chondrocytes.

"Transforming growth factor-β (TGF-β) plays a critical role in cartilage homeostasis and deregulation of its signalling is implicated in osteoarthritis (OA). TGF-β isoforms signal through a pair of transmembrane serine/threonine kinases known as the type I and type II TGF-β receptors. Endoglin is a TGF-β co-receptor that binds TGF-β with high affinity in the presence of the type II TGF-β receptor. We have previously shown that endoglin is expressed in human chondrocytes and that it forms a complex with the TGF-β signalling receptors. Our objective was to determine whether endoglin regulates TGF-β/Smad signalling and extracellular matrix (ECM) production in human chondrocytes and whether its expression varies with chondrocyte differentiation state.
Endoglin function was determined by overexpression or antisense morpholino/siRNA knockdown of endoglin in human chondrocytes and measuring TGF-β-induced Smad phosphorylation, transcriptional activity and ECM production. Alterations in endoglin expression levels were determined during subculture-induced dedifferentiation of human chondrocytes and in normal vs OA cartilage samples.
Endoglin enhances TGF-β1-induced Smad1/5 phosphorylation and inhibits TGF-β1-induced Smad2 phosphorylation, Smad3-driven transcriptional activity and ECM production in human chondrocytes[Smad 1/3/5 phosphorylation is bad but Smad 2/3 is good so Endoglin has negative effects]. In addition, the enhancing effect of endoglin siRNA knockdown on TGF-β1-induced Smad3-driven transcription is reversed by ALK1 overexpression. Furthermore, endoglin levels are increased in chondrocytes following subculture-induced dedifferentiation and in OA cartilage as compared to normal cartilage.
 Together, our results suggest that endoglin regulates the balance between TGF-β/ALK1/Smad1/5 and ALK5/Smad2/3 signalling and ECM production in human chondrocytes and that endoglin may represent a marker for chondrocyte phenotype."

Inhibiting Endoglin levels is a way to encourage height growth and discourage chondrocyte dedifferentiation by increasing ECM production.

Inducing Hypoxia may be another way to prevent chondrocyte dedifferentiation.

Lack of oxygen in articular cartilage: consequences for chondrocyte biology.

"Controlling the chondrocytes phenotype remains a major issue for cartilage repair strategies. These cells are crucial for the biomechanical properties and cartilage integrity because they are responsible of the secretion of a specific matrix. But chondrocyte dedifferentiation is frequently observed in cartilage pathology[chondrocyte dedifferentiation occurs normally in the body outside of tissue engineering] as well as in tissue culture, making their study more difficult. Given that normal articular cartilage is hypoxic, chondrocytes have a specific and adapted response to low oxygen environment."

"hypoxia is a strong promoter of matrix deposition by the chondrocytes "<-Hypoxia encourages matrix deposition which helps prevent dedifferentiation.

"Cells can sense surrounding oxygen level through Hypoxia–Inducible transcription Factors (initially named HIF1-) whose expression and function are mainly post-translationally regulated by hydroxylation reactions. Under high oxygen environment, these proteins have a very short half-life (<5 min)[Maybe encouraging matrix deposition by freshly differentiated chondrocytes is possible within this five minutes?]. This is due to specific hydroxylated proline that are recognized by the Von Hippel-Lindau protein (pVHL-containing E3 ubiquitin ligase complex) and that targets them to degradation via the proteasome. Conversely, when oxygen level is low (5–1%), hydroxylation decreases and HIF-1 is prevented from a rapid degradation. Then HIF1- heterodimerizes with the constitutively expressed HIF-1β (also called Aryl hydrocarbon Nuclear Translocator ARNT), translocates into the nucleus, and binds specific consensus sequences (-RCGTG-) on gene promoters "<-Usage of a PHD inhibitor which degrades HIF1 may be another way to enhance HIF1 levels without lowering bone oxygen content by say performing LSJL for a longer duration.

"deletion of pVHL in chondrocytes (which results in HIF-1 and -2 overexpression) increases matrix deposition during growth-plate development"

"As PHDs are the true oxygen sensors of hypoxia, these enzymes may be critical in controlling the chondrocyte phenotype.  All the PHDs are expressed in maturing zone of the mouse growth plate "<-So it's not so much increasing HIF1 but inhibiting PHD expression in the bone.

So increasing cell density, inhibiting of endoglin, and induction of hypoxia are ways to prevent chondrogenic dedifferentiation.  The inhibition of PHD strongly suggests the usage of longer applications of LSJL as PHD levels may be returned to normal after clamp placement is no longer impeding oxygen delivery to the epiphyseal bone marrow.