New study related to lateral synovial joint loading:

Knee Loading Enhances the Migration of Adipose‑Derived Stem Cells to the Osteoarthritic Sites Through the SDF‑1/CXCR4 Regulatory Axis

“Osteoarthritis (OA) is a whole joint disorder that is characterized by cartilage damage and abnormal remodeling of subchondral bone. Injecting adipose-derived stem cells (ASCs) into the knee joint cavity can assist in repairing osteoarthritic joints, but their ability to migrate to the damaged site is limited. Our previous studies have shown that knee loading can improve the symptoms of OA, but the effect and mechanism of knee loading on the migration of ASCs in OA remain unclear. We employed a mouse model of OA in the knee and applied knee loading (1 N at 5 Hz for 6 min/day for 2 weeks) after the intraarticular injection of ASCs. The cartilage and subchondral bone repair were assessed by histopathological analysis. Immunofuorescence assays were also used to analyze the migration of ASCs. Using cell cultures, we evaluated the migration of ASCs using the transwell migration and wound healing assays. In vivo experiments showed that knee loading promoted the migration of ASCs, increased the local SDF-1 level, and accelerated the repair of the OA-damaged sites. Mechanistically, the observed effects were blocked by the SDF-1/CXCR4[SDF-1/CXCR4 promotes neovascularization which is great] inhibitor. The in vitro results further revealed that knee loading promoted the migration of ASCs and the inhibition of SDF-1/CXCR4 significantly suppressed the beneficial loading effect. The results herein suggested that the migration of ASCs was enhanced by knee loading through the SDF-1/CXCR4 regulatory axis, and mechanical loading promoted the joint-protective effect of ASCs”

Now we don’t know how much more lateral knee loading stimulates neo-vascularization etc. than regular exercise.  Nor do we know if knee loading promotes subchondral bone repair independently of adipose derived stem cells.  But neovascularization and new cartilage formation are very important findings that could potentially be linked to what’s needed to grow taller.

“A variety of physical rehabilitation regimens can afect the remodeling process of articular cartilage and subchondral bone and alter the symptoms of OA, such as whole-body vibration, axial loading, and bending loading. The knee loading modality, developed by our laboratory, is a mild non-invasive physical therapy, which applies transverse loading to a synovial joint, such as the elbow, knee, and ankle. Knee loading has been shown as an effective loading option for improving OA symptoms. Previous studies showed that knee loading reduced MMP13 activity and prevented OA-induced cartilage degeneration through cross-talk of the cartilage with subchondral bone. Mechanical loading also mitigated OA symptoms by regulating the stress to the endoplasmic reticulum and autophagy. Further, knee loading facilitated repairing osteoporotic OA by relieving the abnormal remodeling of the subchondral bone via Wnt/β-catenin signaling. It contributed to promoting the diferentiation of MSCs into osteogenic and chondrogenic fates”

“Stromal cell-derived factor 1 (SDF-1), a chemokine of a CXC family, and its receptor CXC chemokine receptor 4 (CXCR4) exist widely in the body, and they regulate a variety of physiological processes, including cell proliferation, migration, adhesion, differentiation, and wound healing ”

“A total of 108 female C57BL/6 mice (14 weeks old, Animal Center of Academy of Military Medical Sciences, China) were used.”<-not quite as old as we would like.

“After the induction of OA, mice were injected with GFP-ASCs, followed by daily knee loading for 2 weeks. The injected ASCs in the knee joints of all groups were identified by immunofuorescence staining with antibodies against GFP. In vivo studies showed that GFP-positive cells were found in the OA cartilage 2 weeks after their injections”

” The number of chondrocytes and vacuolar cells was counted. The number of chondrocytes in the OA group was significantly decreased compared to the sham-treated control group, but the number was restored by knee loading or the injection of ASCs”<-it’s very promising that the chondrocyte number was restored be knee loading independently of stem cell injection but again these mice are not as old as we would like.

” IHC analysis showed that collagen II-positive cells in the OA group were significantly decreased. However, the numbers in the OAL group and OA+ASCs group were increased.”<-again very promising that collagen II positive cells were increasing independently of ASC injection

In the top images you can see that the mice still have their growth plate.  It’d be hard to distinguish between changes in the bone caused by Osteoarthritis and those caused by loading.  But if you look at the loaded groups(Loading with and without ASC) you can see in red cartilage in places that did not have cartilage in either the control or the osteoarthritis group at the longitudinal ends of the bone.  And you don’t really see that in the OA+ASC group.  I could be reading it wrong though.

Again here you see cartilage at the sides of the longtiduinal ends of the bone that you don’t really see in the non-loaded groups.

New LSJL study was published that has some good stuff.  Maybe the paper will finally give the insight on how to use mechanical loading to gain height.

Loading-driven PI3K/Akt signaling and erythropoiesis enhanced angiogenesis and osteogenesis in a postmenopausal osteoporosis mouse model

“Bone vasculature influences osteogenesis[any type of new bone formation and haematopoiesis[production of blood cells in the bone marrow] in the bone microenviroment. Mechanical loading has been shown to stimulate the formation of osteogenesis-related type H vessels in an ovariectomy (OVX)- induced osteoporosis mouse model. To determine the loading-driven mechanism of angiogenesis and the formation of type H vessels in bone, we evaluated the roles of PI3K/Akt signaling and erythropoiesis in the bone marrow. The daily application of mechanical loading (1 N at 5 Hz for 6 min/day) for 2 weeks on OVX mice inhibited osteoclast activity{although osteoclast activity is good for our purposes as degradation of bone is needed to theoretically create a new growth plate}, associated with an increase in the number of osteoblasts and trabecular volume ratio. Mechanical loading enhanced bone vasculature and vessel formation, as well as PI3K/Akt phosphorylation[Akt phosphorylation indicates general anabolism] and erythropoiesis[production of red bood clells] in the bone marrow. Notably, LY294002, an inhibitor of PI3K signaling, blocked the tube formation by endothelial progenitor cells, as well as their migration and wound healing. The conditioned medium, derived from erythroblasts, also promoted the function of HUVECs with elevated levels of VEGF, CD31, and Emcn. Collectively, this study demonstrates that mechanical loading prevents osteoporotic bone loss by promoting angiogenesis and type H vessel formation. This load-driven preventing effect is in part mediated by PI3K/Akt signaling and erythropoiesis in the bone marrow”

“A specific vessel subtype called a type H vessel, which is positive for CD31 and endomucin (CD31hiEmcnhi), has been reported to link angiogenesis with osteogenesis”

“Vascular-forming endothelial cells provide a framework of bone homeostasis and metabolism, acting as a cellular highway for blood cells, leukocytes, and other types of cells throughout the body ”

“Erythropoiesis is characterized by the movement of lineage-committed cells with progenitors, precursors, and mature red blood cell (RBC) compartments, which are located in the fetal liver and the postnatal bone marrow”

“Molecular signals by mechanosensing and transduction, leading to a series of cellular responses. The phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway has been shown to play a critical role in the regulation of diverse cellular functions and is also involved in angiogenesis and erythropoiesis. joint loading increases the expression of Akt, which may promote bone formation and homeostasis. The effect of PI3K signaling on endothelial progenitor cells (EPCs) in the bone marrow of OVX mice, however, has not yet been demonstrated in response to joint loading. ”

“we hypothesized that the reduction in type H vessels and the hematopoietic function in OVX mice is suppressed by mechanical loading through PI3K/Akt signaling, which stimulates erythroid differentiation of hematopoietic stem cells (HSC) and promotes vessel formation”<-so joint loading may not suppress osteoclast activity in normal bone as the OVX environment is different.

“Ninety female C57BL/6 mice (~16 weeks of age, Animal Center of Academy of Military Medical Sciences, China) were used. Mice were randomly divided into three groups: the sham control group (Sham, n = 30), the ovariectomized group (OVX, n = 30), and the loading-treated ovariectomized group (OVXL, n = 30).”<-unfortunately no loaded control group.

Above is histological slides.  Can’t really see anything abnormal.  There may be something atypical going on in F where the added arrow is pointing.

According to figure 2G PI3K, AKT, and VEGF activity was greatly enhanced by joint loading even over controls.  But according to 2H, I, J it did not increase the ratios of phosphorylated PI3K and AKT to total PI3K and AKT and VEGF relative to Beta-Actin.

“The formation of type H vessels was correlated to the stimulation of osteogenesis, and the result suggests that the promotion of type H formation and angiogenesis are important in response to load-driven suppression of OVX-induced bone loss. ”

“knee loading stimulated PI3K/Akt phosphorylation in the bone, and this stimulation can be linked to loading-driven bone metabolism”<-although this alone is definitely not sufficient to induce new longitudinal bone growth.

“Knee loading enhanced cell migration and wound healing”

“in OVX mice, load-driven angiogenesis via erythropoiesis contributed to suppressing the loss of osteoporotic bone. “<-erythropoiesis is the creation of new red blood cells.  Erythropoiesis is thought to not typically occur in adult bones so if joint loading can induce new erythropoiesis that is a big deal.

The mice here were subjected to 1n loading at 5Hz for 6 min/day.  This is actually higher than that performed on the original bone lengthening LSJL study 5-min bouts at 0.5 N[at 5Hz).

Here’s a graphical representation in the study at how mechanical loading might stimulate bone:

The estimated increase in type H vessels is dramatic.  Osteoclast activity as depicted is way done too.

And here’s the description of type H vessels “Type H vessels are noted most abundantly in young, growing animals, adjacent to the growth plate. The elongation of long bones by the process of endochondral ossification occurs at the growth plate and involves continuous replacement of cartilage matrix by calcified bone to acquire their typical shapes through bone modeling.”

Could type H vessels be the key for new longitudinal bone growth here’s a study explaining type H vessels.

Type H blood vessels in bone modeling and remodeling

“In the mammalian skeletal system, osteogenesis and angiogenesis are intimately linked during bone growth and regeneration in bone modeling and during bone homeostasis in bone remodeling. Recent studies have expanded our knowledge about the molecular and cellular mechanisms responsible for coupling angiogenesis and bone formation. Type H vessels, termed such because of high expression of Endomucin (Emcn) and CD31, have recently been identified and have the ability to induce bone formation. Factors including platelet-derived growth factor type BB (PDGF-BB), slit guidance ligand 3 (SLIT3), hypoxia-inducible factor 1-alpha (HIF-1α), Notch, and vascular endothelial growth factor (VEGF) are involved in the coupling of angiogenesis and osteogenesis.”

“in bone modeling associated with endochondral ossification, hypertrophic chondrocytes express high levels of vascular endothelial growth factor (VEGF), which promotes the vascular invasion of cartilage, recruits chondroclasts to resorb hypertrophic cartilage and osteoblasts to build the bone matrix”

“In bone modeling in response to mechanical loading, macrophage/non-resorbing osteoclast lineage cells secrete platelet-derived growth factor type BB (PDGF-BB) to recruit both endothelial and osteoblast precursor cells to couple angiogenesis with osteogenesis”

“activation of mechanistic target of rapamycin complex (mTORC) in articular chondrocytes promoted VEGF secretion into subchondral bone and stimulated type H vessel formation.”

“type H vessels were reported to digest the cartilage matrix, which may be another mechanism of cartilage degeneration in OA.”

“In fracture healing, type H vessels appear during the stage of fibrocartilaginous callus formation. Type H vessels form in the fracture callus and induce bone formation. Factors, such as SLIT3, can promote fracture healing by augmentation of type H vessel induced osteogenesis.”<-Limb lengthening basically if LSJL can induce type H vessels and if type H vessels are important in neocartilage formation than that would be a big find.  But I can only find that Type H vessels are important in absorbing the cartilage matrix not in creating the cartilage matrix.  Although increased Type H Vessels as shown in this study could be indicative of enhanced and maybe even new growth plate activity would would be massive.  IF the mice got new Type H blood vessels past skeletal maturity that would be a huge find 16 week of the type used in this study are pretty far along skeletal wise so new type H vessels is still a big deal but they are still not quite old enough.

Here’s the supplementary data attached

This for the sham and the joint loaded control no osteoarthritis.  The bones look significantly different too me with the loaded bone significantly bigger and and maybe slightly longer(hard to tell). I’d say the width difference is striking and huge. The growth plate is clearly open.  Bone parameters are bigger in the loaded group despite not being “statistically significant”.

4-Phenylbutyric acid (4-PBA, Benzenebutyric acid) is a histone deacetylase (HDAC) inhibitor and a key epigenetic inducer of anti-HCV hepatic hepcidin.  Here’s some stuff on histones.   MOre on histones.  “HDAC4, which is expressed in prehypertrophic chondrocytes, regulates chondrocyte hypertrophy and endochondral bone formation by interacting with and inhibiting the activity of Runx2”  It’s possible since histone inhibition does enhance differentiation it may increase adult height.  But it also make differentiation happen sooner thus reducing adult height.  So there’d need to be experimentation done to see whether this makes children grow faster but reduces overall growth plate growth or whether it it makes children faster and increases overall growth plate based growth.

A compound called tubacin could also increase height by histone HDAC6 inhibition.  There’s still no over the counter tubacin.

4PBA reduces growth deficiency in osteogenesis imperfecta by enhancing transition of hypertrophic chondrocytes to osteoblasts

“Short stature is a major skeletal phenotype in osteogenesis imperfecta (OI), a genetic disorder mainly caused by mutations in genes encoding type I collagen. However, the underlying mechanism is poorly understood and no effective treatment is available. In OI mice that carry a G610C mutation in COL1A2, we previously found that mature hypertrophic chondrocytes (HCs) are exposed to cell stress due to accumulation of misfolded mutant type I procollagen in the endoplasmic reticulum (ER). By fate mapping analysis of HCs in G610C OI mice, we found that HCs stagnate in the growth plate, inhibiting translocation of HC descendants to the trabecular area and their differentiation to osteoblasts. Treatment with 4-phenylbutyric acid (4PBA), a chemical chaperone, restored HC ER structure and rescued this inhibition, resulting in enhanced longitudinal bone growth in G610C OI mice{so we don’t know if this will help in normal people}. Interestingly, the effects of 4PBA on ER dilation were limited in osteoblasts and the bone fragility was not ameliorated. These results highlight the importance of targeting HCs to treat growth deficiency in OI. Our findings demonstrate that HC dysfunction induced by ER disruption plays a critical role in the pathogenesis of OI growth deficiency, which lays the foundation for developing new therapies for OI.”

This would have to be tested on normal mice to see if it works.

Evolution and future of growth plate therapeutics

“Longitudinal bone growth is regulated by multiple endocrine signals (e.g. growth hormone, insulinlike growth factor I, estrogen, androgen) and local factors (e.g. fibroblast growth factors and their receptors and the C-natriuretic peptide/NPR-2 pathway).”

“The resting zone chondrocytes, farthest from the primary ossification center, replicate at a slow rate and act as stem-like cells that replenish the pool of proliferative chondrocytes{if we can reintroduce these resting zone chondrocytes we may be able to reopen the growth plate}. Resting zone chondrocytes produce a ‘growth plate-orienting factor’ that instructs the proper spatial orientation of adjacent proliferative chondrocytes. As cells within the resting zone divide, the proliferative zone is formed in which chondrocytes replicate at a high rate, become arranged into columns, and contribute to bone elongation. Hypertrophic chondrocytes generated from terminal differentiation of proliferating zone chondrocytes enlarge in columns parallel to the axis of elongation. Cell swelling during chondrocyte hypertrophy enables chondrocytes within the growth plate to enlarge rapidly. This phase of endochondral ossification, during which chondrocytes increase their height about 6- to 10-fold, serves as the major factor regulating the growth rate amongst endochondral bones. Hypertrophic chondrocytes calcify surrounding extracellular matrix and produce factors that attract bone cell precursors, bone cells and blood vessel growth, and undergo apoptosis shortly before the blood vessels invade the chondrocyte lacuna. The overall effect of this process of chondrocyte proliferation, hypertrophy, and extracellular matrix secretion is elongation of bones and progressive creation of new bone tissue at the bottom of the growth plate. With age, the rate of longitudinal bone growth declines, caused primarily by a decrease in chondrocyte proliferation associated with other hormone-independent structural, functional, and molecular changes termed growth plate senescence. Evidence suggests that growth plate senescence occurs because the progenitor chondrocytes in the resting zone have a limited replicative capacity – gradually exhausted with increasing cell division{things like estrogen and FGFR3 may contribute to reducing the replicative capacity}.  When the proliferative capacity of stem-like cells in the resting zone is exhausted, and in the presence of sex hormones, growth plate cartilage becomes completely replaced by bone, an event termed epiphyseal fusion”

” According to the dual-effector hypothesis, GH promotes recruitment of resting chondrocytes into a proliferative state and stimulates local production of IGF-I, which then acts in a paracrine/autocrine fashion to increase chondrogenesis. The IGF-I signaling pathway plays an important role in promoting complete hypertrophic chondrocyte formation. These effects are predominantly due to growth plate-generated IGF-I, and IGF-I deficiency results in pre- and post-natal growth retardation marked in the growth plate by disorganized columnar chondrocytes, decreased cell proliferation and cell hypertrophy, increased apoptosis, and delayed vascular invasion. ”

“Estrogen influences longitudinal growth primarily and indirectly by augmenting GH secretion during puberty, but also through both growth-enhancing and -attenuating direct actions on the growth plate. A direct effect of estrogen is to advance growth plate senescence causing proliferative exhaustion, and thus epiphyseal fusion. An important mediator of this growth plate closure process is vascular endothelial growth factor (VEGF), the production of which is stimulated by estrogen in both males and females. Androgens stimulate linear growth partly due to aromatization to estrogens within growth plate cartilage, but also by direct interaction with androgen receptors on growth plate chondrocytes – explaining the GH- and IGF-I-independent growth stimulating effects of non-aromatizable compounds such as dihydrotestosterone and oxandrolone. Linear growth is slowed by deficiency of and accelerated by excess thyroid hormone. Hypothyroidism indirectly impedes linear growth by diminishing GH secretion and IGF-I, but also by decreasing chondrocyte proliferation and hypertrophy, slowing of vascular/bone cell invasion, and disruption of column organization. Glucocorticoid (GC) excess slows longitudinal bone growth by inhibiting chondrocyte proliferation, hypertrophy, and cartilage matrix synthesis. Diminished GH secretion and/or altered IGF-I bioavailability have been described in some GCtreated patients. Slowing of growth plate senescence due to GC appears to explain the phenomenon of catch-up growth following transient GC exposure and hypothyroidism.”

“Members of the FGF family of receptors (FGFRs) and their ligands are required for proper chondrocyte function, endochondral ossification and overall skeletal development. Proliferative chondrocytes express FGFR3 and prehypertrophic/hypertrophic chondrocytes express FGFR1. These pathways inhibit the proliferation of chondrocytes, thereby limiting the longitudinal growth of bones. Thus, activating mutations in FGFRs impede linear growth and cause skeletal phenotypes such as achondroplasia and hypochondroplasia. Accelerated linear growth and epiphyseal growth plate maturation in obese children, even in the setting of decreased GH production may be due to effects of increased insulin concentrations and activation of the insulin receptor in the growth plate. Leptin, increased in obese children, has direct effects on skeletal growth centers, enhancing chondrocyte proliferation and subsequent cell differentiation. Leptin also increases growth plate aromatase activity{this may explain why some obese children are taller than normal and others are shorter based on differential responses to estrogen} which along with estrogen produced through adipose tissue aromatization, accelerates skeletal maturation. Parathyroid hormone-related protein (PTHrP) supports chondrocytes and maintains the width of the growth plate. Mutations affecting PTHrP action (e.g. Gs-alpha) can result in a shortening of the proliferating zone, accelerated differentiation of hypertrophic chondrocytes, premature closure of the growth plate, and short stature. Vitamin D facilitates normal linear growth indirectly by increasing intestinal calcium and phosphate absorption, but vitamin D metabolites produced locally in the growth plate also decrease the proliferation of chondrocytes through the PTHrP pathway. Thus, the full effect of vitamin D on the growth plate physiology is incompletely understood.”

The paper some options for treatment for increasing growth plate based growth.  I recommend reading the whole paper.  I highlighted some stuff here because it does a great job explaining the mechanics of growth plate based height increase.