Michael talked about Ischemia in the past here.  I found a study earlier that suggested that ischemia of cartilage canals may reduce height growth.  Since intense clamping transiently affects the blood vessels to the cartilage it is important to study this.

Chondro-osseous growth abnormalities after meningococcemia. A clinical and histopathological study.

“The cases of nine children who survived the acute stage of meningococcal septicemia and secondary disseminated intravascular coagulation were reviewed. All of the children had major orthopaedic problems as a result of the acute disease. Detailed histological studies were performed on specimens of bone and cartilage, obtained when these patients had either acute amputation for gangrene or subsequent revision for a chondro-osseous deformity. In the specimens that were obtained from the children who had acute gangrene, the histological changes included small-vessel thrombi[blood vessel clot], osteonecrosis, subperiosteal new-bone formation, cortical disruption, cellular disorganization in the physis, and medullary inflammation. These findings were compatible with a combination of inflammation (acute osteomyelitis) and ischemia. In the specimens that were obtained during revision of the amputation, three years or more after the initial infectious or ischemic process, the clinically relevant findings involved the epiphyses and physes. The growth plates showed variable permanent ischemic damage. Bone bridges connecting the epiphysis and metaphysis were observed in various stages of formation, including several early bridges with involvement of only the physis and metaphysis. Endosteal and cortical bone, in contrast, showed complete recovery with no evidence of permanent ischemic damage. We concluded that children who survive meningococcal septicemia are at high risk for complex orthopaedic problems, both acute and chronic. The disseminated intravascular coagulation and focal infections of the acute phase are primarily responsible for the vascular injuries to the growing chondro-osseous tissues. Ischemic changes also selectively involve the physeal circulation, but may take several years to adversely affect longitudinal and transverse growth of bone.”

Image of a growth plate clot:

Growth plate with abnormal “micro” growth plate like structure:

I wrote some about calcium secretions(which relate to ATP oscillations) here.  TGF-Beta forms pre-chondrogenic mesenchymal condensation(which is what creates the growth plate) via ATP oscillations.  ATP oscillations also play a role in FGF and Shh mesenchymal condensations.

Analysis of proteins showing differential changes during ATP oscillations in chondrogenesis.

“Prechondrogenic condensation is a critical step for skeletal pattern formation{ie form growth plates}. ATP oscillations play an essential role in prechondrogenic condensation because they induce oscillatory secretion. We examined how differential changes in proteins are implicated in ATP oscillations during chondrogenesis by using liquid chromatography/mass spectrometry. A number of proteins involved in ATP synthesis/consumption, catabolic/anabolic processes, actin dynamics, cell migration and adhesion were detected at either the peak or the trough of ATP oscillations, which implies that these proteins have oscillatory expression patterns that are coupled to ATP oscillations. On the basis of the results, we suggest that (1) the oscillatory expression of proteins involved in ATP synthesis/consumption and catabolic/anabolic processes can contribute to the generation or maintenance of ATP oscillations and that (2) the oscillatory expression of proteins involved in actin dynamics, cell migration and adhesion plays key roles in prechondrogenic condensation by inducing collective adhesion and migration in cooperation with ATP oscillations.”

So we can compare the proteins altered in ATP oscillations to those in LSJL to help see if LSJL induces similar ATP oscillations as those in growth plate chondrogenesis.

“ATP oscillations depend on Ca2+ dynamics.”

“We used the prechondrogenic ATDC5 cell line”<-It would be more ideal if they used normal mesenchymal stem cells as that’s what we’re trying to use to create new growth plates rather than the ATDC5 pre-chondrogenic cells that are like the pre-cursor cells in the Ring of LaCroix.

Peak versus Trough of ATP oscillations.  Genes up and downregulated in LSJL are mentioned in {}

With categories

Comparison to LSJL genes to be done.

” it was found that many proteins involved in cell adhesion, such as laminin subunit gamma-2, plakophilin-3, neuroplastin, thrombospondin-3 and leukocyte surface antigen CD47, were detected only at the troughs of ATP oscillations whereas many proteins involved in cell migration, such as platelet-activating factor acetylhydrolase IB subunit beta, CD44 antigen, homeobox protein Hox-A5, cell division protein kinase 5, and ephrin-A5, were detected only at the peaks of ATP oscillations”

” cell–cell adhesion and cell movement are stimulated at the troughs and peaks of ATP oscillations, respectively, leading to synchronized oscillations of cellular migration during chondrogenesis”

” A nucleation and growth mechanism in which a critical-size aggregate (nuclei) is required for subsequent growth can be applicable to the cellular aggregation process. Therefore, we propose that prechondrogenic condensation proceeds by a nucleation-growth mechanism. This could explain the reason why the oscillatory expression patterns of proteins that are involved in actin dynamics, cell migration and adhesion are required for prechondrogenic condensation. Thermodynamically, a barrier exists in free energy; this barrier occurs at a certain critical size at the initial stage of condensation. Thus, aggregates smaller than the critical size are unstable owing to energy loss due to surface tension, whereas aggregates larger than the critical size grow irreversibly owing to the energy gain due to cellular adhesions surpassing the energy loss, thereby ultimately forming large cellular aggregates. Therefore, the oscillatory expression patterns of proteins involved in actin dynamics, cell migration and adhesion result in collective migration and adhesion, which aggregates cells collectively within a limited time and then drives more efficient formation of the nuclei than random migration and adhesion. Therefore, coordinated oscillatory expression of the proteins is crucial during the initial step of prechondrogenic condensation.”

“platelets are aggregate into clusters at the site of an injury to the skin or blood vessels.”<-Note that blood is involved in distraction osteogenesis

CCAAT/Enhancer Binding Protein β Regulates the Repression of Type II Collagen Expression during the Differentiation from Proliferative to Hypertrophic Chondrocytes.

“we investigated whether C/EBPβ represses type II collagen (COL2A1) expression and is involved in the regulation of sex-determining region Y-type high mobility group box 9 (SOX9), a crucial factor for transactivation of Col2a1. Endogenous expression of C/EBPβ in the embryonic growth plate and differentiated ATDC5 cells were opposite to those of COL2A1 and SOX9. Overexpression of C/EBPβ by adenovirus vector in ATDC5 cells caused marked repression of Col2a1. The expression of Sox9 mRNA and nuclear protein was also repressed, resulting in decreased binding of SOX9 to the Col2a1 enhancer. Knockdown of C/EBPβ by lentivirus expressing shRNA caused significant stimulation of these genes in ATDC5 cells. Reporter assays demonstrated that C/EBPβ repressed transcriptional activity of Col2a1. Deletion and mutation analysis showed that the C/EBPβ core responsive element was located between +2144 and +2152 bp within the Col2a1 enhancer. EMSA and ChIP assays also revealed that C/EBPβ directly bound to this region. Ex vivo organ cultures of mouse limbs transfected with C/EBPβ showed that the expression of COL2A1 and SOX9 were reduced upon ectopic C/EBPβ expression. Together, these results indicated that C/EBPβ represses the transcriptional activity of Col2a1 both directly, and indirectly through modulation of Sox9 expression. This consequently promotes the phenotypic conversion from proliferative to hypertrophic chondrocytes during chondrocyte differentiation.”

So maybe knockdown of CEBP-Beta could increase height?  It upregulates chondrogenic genes but we’d have to see the effects on the growth plate to be sure.

“The expression of COL10A1, RUNX2, and MMP13 was misexpressed through the tibias that were transfected with C/EBPβ, compared with LacZ control. Forced expression of C/EBPβ may lead the ectopic expression of these genes even in the regions that do not show the morphological hypertrophy because C/EBPβ is reported as a direct regulator of them. Moreover, the expression of SOX9 was also decreased and restricted to a small upper area of the growth plate by overexpression of C/EBPβ, similar to the expression of COL2A1. Together, these results further confirmed that C/EBPβ could be involved in regulation of phenotypic conversion from proliferative to hypertrophic chondrocytes by repressing the genes characteristic of proliferative chondrocytes during chondrocyte differentiation.”

Isopsoralen is also known as Angelicin and is found in Bituminaria bituminosa.  This plant does not seem to be currently available in supplement form.

Isopsoralen Induces Differentiation of Prechondrogenic ATDC5 Cells via Activation of MAP Kinases and BMP-2 Signaling Pathways.

[ATDC5 cells are chondrogenic progenitor cells so it’s much easier to get them to differentiate into chondrocytes than Mesenchymal Stem Cells.  But ATDC5 cells are like prechondrogenic growth plate cells so it may have applications to people with growth plates]

“Endochondral bone formation is the process by which mesenchymal cells condense to become chondrocytes, which ultimately form new bone.  We investigated the possible role of isopsoralen in induction of chondrogenic differentiation in clonal mouse chondrogenic ATDC5 cells. Isopsoralen treatment stimulated the accumulation of cartilage nodules in a dose-dependent manner. Further, ATDC5 cells treated with isopsoralen were stained more intensely with Alcian blue than control cells, suggesting that isopsoralen increases the synthesis of matrix proteoglycans. Similarly, isopsoralen markedly induced the activation of alkaline phosphatase activity compared with control cells. Isopsoralen enhanced the expressions of chondrogenic marker genes such as collagen II, collagen X, OCN, Smad4 and Sox9{all upregulated in LSJL except for Smad4} in a time-dependent manner. Furthermore, isopsoralen induced the activation of extracellular signal-regulated kinase (ERK){stimulated by LSJL} and p38 MAP kinase{LSJL likely upregulates p38}, but not that of c-jun N-terminal kinase (JNK). Isopsoralen significantly enhanced the protein expression of BMP-2 in a time-dependent manner. PD98059 and SB 203580, inhibitors of ERK and p38 MAPK, respectively, decreased the number of stained cells treated with isopsoralen.  Isopsoralen mediates a chondromodulating effect by BMP-2 or MAPK signaling pathways.”

“the upregulation of BMP-2 causes cells to skip cellular condensation stages in early-phase chondrogenic differentiation and also markedly up-regulates the expression of type X collagen mRNA in late-phase differentiation”