Differential regulation of endochondral bone growth and joint development by FGFR1 and FGFR3 tyrosine kinase domains.

“Fibroblast growth factor receptors (FGFR) 1 and 3 have distinct mitogenic activities in vitro. In several cultured cell lines, FGFR1 transmits a potent mitogenic signal, whereas FGFR3 has little or no mitogenic activity. However, in other in vitro assays the FGFR3 intracellular domain is comparable with that of FGFR1. In vivo, FGFR3 negatively regulates chondrocyte proliferation and differentiation, and activating mutations are the molecular etiology of achondroplasia. By contrast, FGFR1 transmits a proliferative signal in various cell types in vivo. These observations suggest that inhibition of the proliferating chondrocyte could be a unique property of FGFR3 or, alternatively, a unique property of the proliferating chondrocyte. To test this hypothesis, FGFR1 signaling was activated in the growth plate in cells that normally express FGFR3. Comparison of transgenic mice with an activated FGFR1 signaling pathway with an achondroplasia-like mouse that expresses a similarly activated FGFR3 signaling pathway demonstrated that both transgenes result in a similar achondroplasia-like dwarfism. These data demonstrate that suppression of mitogenic activity by FGFR signaling is a property that is unique to growth plate chondrocytes. Surprisingly, we observed that in transgenic mice expressing an activated FGFR, some synovial joints failed to develop and were replaced by cartilage. The defects in the digit joints phenocopied the symphalangism that occurs in Apert syndrome and the number of affected joints was dependent on transgene dose. In contrast to the phenotype in the growth plate, the joint phenotype was more severe in transgenic mice with an activated FGFR1 signaling pathway. The failure of joint development resulted from expanded chondrification in the presumptive joint space, suggesting a crucial role for FGF signaling in regulating the transition of condensed mesenchyme to cartilage and in defining the boundary of skeletal elements.”

“Fgfr1 and Fgfr2 are expressed in the perichondrium”

” in growth plate chondrocytes, activating FGFR1 or FGFR3 signaling pathways had comparable negative proliferative effects. In addition, overactivation of FGFR signaling in developing cartilage caused a failure of joint cavitation as a result of expanded chondrification.”

The chondrogenic response to exercise in the proximal femur of normal and mdx mice.

“Submaximal exercise is used in the management of muscular dystrophy. The effects of mechanical stimulation on skeletal development are well understood, although its effects on cartilage growth have yet to be investigated in the dystrophic condition. The objective of this study was to investigate the chondrogenic response to voluntary exercise in dystrophin-deficient mice.
Control and dystrophin-deficient (mdx) mice were divided into sedentary and exercise-treated groups and tested for chondral histomorphometric differences at the proximal femur.
Control mice ran 7 km/week further than mdx mice on average, but this difference was not statistically significant. However, exercised control mice exhibited significantly enlarged femur head diameter, articular cartilage thickness, articular cartilage tissue area, and area of calcified cartilage relative to sedentary controls and exercised mdx mic{the enlarged femur head is consistent with articular cartilage endohcondral ossification [and] especially considering the area of calcified cartilage increased as well]. No differences were found between other treatment groups.
Mdx mice exhibit a reduced chondrogenic response to increased mechanical stimulation relative to controls. However, no significant reduction in articular dimensions was found, indicating loss of chondral tissue may not be a clinical concern with dystrophinopathy.”

“Exercise treatment consisted of voluntary access to a running wheel that lasted four weeks.”

“it is conceivable that joint forces in mdx mice failed to provide sufficient mechanical pressure to yield significant chondral expansion. Skeletal muscle of mdx mice of similar age to the ones used in this study have been shown to contain extensive fibrosis and degenerative myocytes with diminished muscle force production”<-maybe being able to generate enough force is a similar limiting factor for our purposes and if we could generate more muscular force we could induce longitudinal bone growth.

“a reduction in joint forces resulting from weaker muscle force production and slower running speeds may explain the diminished chondrogenic response to voluntary running exercise in the proximal femurs of mdx mice.”

The response of bone, articular cartilage and tendon to exercise in the horse.

” For bone, alterations in bone mineral content, mineral density and the morphology of the mineralized tissue are the most common end-points. Apparent bone density increases slightly after athletic training in the cortex, but substantially in the major load paths of the epiphyses and cuboidal bones, despite the lower material density of the new bone, which is deposited subperiosteally and on internal surfaces without prior osteoclastic resorption. With training of greater intensity, adaptive change is supervened by patho-anatomical change in the form of microdamage and frank lesions. In tendon, collagen fibril diameter distribution changes significantly during growth, but not after early training. The exact amount and type of protracted training that does cause reduction in mass average diameter (an early sign of progressive microdamage) have not been defined. Training is associated with an increase in the cross-sectional area of some tendons, possibly owing to slightly greater water content of non-collagenous or newly synthesized matrix. Early training may be associated with greater thickness of hyaline but not calcified articular cartilage, at least in some sites. The age at which adaptation of cartilage to biomechanical influences can occur may thus extend beyond very early life. However, cartilage appears to be the most susceptible of the three tissues to pathological alteration. The effect of training exercise on the anatomical or patho-anatomical features of connective tissue structures is affected by the timing, type and amount of natural or imposed exercise during growth and development which precedes the training.”

“Equine joint cartilage has similar appearance and properties to human articular cartilage”

“At both [phalanx] sites, water, DNA and GAG decreased during maturation whereas collagen content, hydroxylysine content and HP cross-links increased. Postnatal adaptation, resulting in biochemical and therefore biomechanical heterogeneity, which is important for future tissue strength and resistance to injury, occurs early and rapidly, possibly because collagen turnover is extremely low at older ages”

“Exercise influenced calcium content and levels of HP and lysylpyridinoline cross-links at the intermittently loaded site but not at the constantly loaded site of the proximal phalangeal bone, levels of lysyl-hydroxylation and lysylpyridinoline cross-linking being lower at the former than the latter site”

“The epiphyseal bone alters dramatically with exercise and is only arbitrarily separable from the subchondral bone.”  the subchondral bone is the bone that is at the end of the articular cartilage and the beginning of epiphyseal bone.

Since blood flow is something we can manipulate, blood flow altering bone growth is something very promising.

Yap/Taz transcriptional activity in endothelial cells promotes intramembranous ossification via the BMP pathway.

“Osteogenesis is categorized into two groups based on developmental histology, intramembranous and endochondral ossification. The role of blood vessels during endochondral ossification is well known, while their role in intramembranous ossification, especially the intertissue pathway, is poorly understood. Here, we demonstrate endothelial Yap/Taz is a novel regulator of intramembranous ossification in zebrafish. Appropriate blood flow is required for Yap/Taz transcriptional activation in endothelial cells and intramembranous ossification. Additionally, Yap/Taz transcriptional activity in endothelial cells specifically promotes intramembranous ossification. BMP expression by Yap/Taz transactivation in endothelial cells is also identified as a bridging factor between blood vessels and intramembranous ossification. Furthermore, the expression of Runx2 in pre-osteoblast cells is a downstream target of Yap/Taz transcriptional activity in endothelial cells. Our results provide novel insight into the relationship between blood flow and ossification by demonstrating intertissue regulation.”

“During endochondral ossification in mammalian systems, the invasion of blood vessels into the cartilage is a well-known process for converting cartilage to bone. Blood vessels in the cartilage provide osteoclast cells, which differentiate from macrophages. Vascular endothelial growth factor (VEGF) secreted by chondrocytes also promotes the replacement of cartilage”

“Yap/Taz localization and transactivation are regulated by substrate stiffness and fluid shear stress, Yap/Taz is also regarded as a mechanosensor.”

“Embryos overexpressing Yap and Taz in endothelial cells showed larger opercle volumes in very early ossification compared to controls”

“Circulation activates Yap/Taz transcriptional activity in endothelial cells and transactivated Yap/Taz promotes Bmp4 expression in endothelial cells, and Runx2 expression in precursor cells of the opercle for its ossification.”

Longitudinal epiphyseal bracket.

“Longitudinal epiphyseal bracket or bracket epiphysis is an uncommon disorder of growth. Alternatively known as a delta phalanx, it is due to an anomalous secondary ossification center that extends longitudinally along the diaphysis{causing anomalous secondary ossification centers may be a way to grow taller}. Although rare, longitudinal epiphyseal bracket most commonly manifests in the hands as clinodactyly and in the feet as hallux varus. ”

“The etiology of longitudinal epiphyseal bracket is not completely understood, but is thought to result from incomplete development of primary ossification centers”<-If this method is the cause then it unfortunately will not likely help us grow taller.

“A typical metatarsal only has a proximal epiphysis, but with a bracket, there is a continuous proximal, medial, and distal epiphysis. The metatarsal cannot elongate normally because of the abnormal epiphysis bracketing the bone.”

Pituitary Adenylate Cyclase Activating Polypeptide (PACAP) Pathway Is Induced by Mechanical Load and Reduces the Activity of Hedgehog Signaling in Chondrogenic Micromass Cell Cultures.

“Pituitary adenylate cyclase activating polypeptide (PACAP) is a neurohormone exerting protective function during various stress conditions either in mature or developing tissues. Previously we proved the presence of PACAP signaling elements in chicken limb bud-derived chondrogenic cells in micromass cell cultures. Since no data can be found if PACAP signaling is playing any role during mechanical stress in any tissues, we aimed to investigate its contribution in mechanotransduction during chondrogenesis. Expressions of the mRNAs of PACAP and its major receptor, PAC1 increased, while that of other receptors, VPAC1, VPAC2 decreased upon mechanical stimulus. Mechanical load enhanced the expression of collagen type X, a marker of hypertrophic differentiation of chondrocytes and PACAP addition attenuated this elevation{maybe if there was a PACAP or PAC1 inhibitor, longitudinal bone growth could be increased}. Moreover, exogenous PACAP also prevented the mechanical load evoked activation of hedgehog signaling: protein levels of Sonic and Indian Hedgehogs and Gli1 transcription factor were lowered while expressions of Gli2 and Gli3 were elevated by PACAP application during mechanical load. Our results suggest that mechanical load activates PACAP signaling and exogenous PACAP acts against the hypertrophy inducing effect of mechanical load.”

“Various cell surface receptors can serve for mechanosensation of chondrocytes, such as
certain purinoreceptors, integrins, stretch activated Ca2+ channels, TRPV4 and NMDA receptors. Downstream targets of these receptors can regulate the morphology, metabolism,
proliferation and Ca2+ homeostasis of chondrocytes. Our group demonstrated a PKA regulated
mechanosensitive activation of Sox9 and PP2A and the involvement of the hedgehog signaling in mechanotransduction”

“PACAP is a short peptide existing in two biological active forms; PACAP 1–38 and PACAP 1–27. Both forms have a very short life span. Three main G-protein coupled receptors of PACAP have been identified; PAC1, VPAC1 and VPAC2, the latter two have lower affinity to the neuropeptide. Activation of these receptors can regulate divers signaling pathways, from which the canonical signaling connection induces the activation of PKA and/or MAPK system”

“exogenous addition of PACAP exerts positive effects both on chondro- and osteogenesis”

“PACAP was proven to prevent apoptosis, ischemic conditions, and oxidative stress”

“PACAP 1–38 at 100 nM was applied on day two of culturing and continuously from day one . MS, mechanical stimulation applied on day two and days 2–3 for 30 min.”

“The enhanced protein levels of collagen type IX and X imply that chondrogenic cells are shifted
toward a prehypertrophic differentiation”

“the mechanical stimulation resulted in a significant elevation of SHH protein expression which was normalized by the application of exogenous PACAP.  The mRNA expression of Indian hedgehog (IHH) was decreased, moreover, the protein expression reduced to an almost undetectable level after PACAP administration. In contrast with this, the mechanical load enhanced the mRNA and protein expression of IHH”

“a simultaneous significant elevation of the amount of type IX collagen[concurrent with an elevation of Type X collagen expression] may reflect on certain matrix reorganization upon mechanical load”

“The presence of collagen type IX determines the architecture of the collagen network in cartilage and its expressional changes have been demonstrated in aging. Addition of PACAP elevated the amount of collagen type II, but reduced the type IX collagen-content in micromass cultures”

“Strong mechanical load is known to induce the apoptotic program of chondrocytes or mesenchymal stem cells resulting in a shift toward the prehypertrophic phenotype. In mature joints, intense mechanical force can result in the reduction of the superficial layer of articulating cartilage and may cause a decrease of the epiphysial growth plate followed by a reduced expression of collagen type II and X”

“Since mechanical load can be regarded as a physical stress to the chondrogenic cells,
one can suppose a mechanoprotective role of PACAP during chondrogenesis.”

“Gli2 has been shown as a primary cilium specific transcription factor influenced by mechanical load and has also been demonstrated to regulate vascularization of cartilage following chondrocyte hypertrophy”

Intermittent applied mechanical loading induces subchondral bone thickening that may be intensified locally by contiguous articular cartilage lesions.

“Right knee joints of CBA mice were loaded: once with 2weeks of habitual use (n = 7), for 2weeks (n = 8) or for 5weeks (n = 5). Both left (contralateral) and right (loaded) knees were micro-CT scanned and the SCB and trabecular bone analysed. Gait analysis was also performed.
These analyses showed a significant increase in SCB[subchondral bone] thickness in the lateral compartments in joints loaded for 5weeks, which was most marked in the lateral femur; the contralateral non-loaded knee also showed transient SCB thickening (loaded once and repetitively). Epiphyseal trabecular bone BV/TV and trabecular thickness were also increased in the lateral compartments after 5 weeks of loading, and in all joint compartments in the contralateral knee. Gait analysis showed that applied loading only affected gait in the contralateral himd-limb in all groups of mice from the second week after the first loading episode.”

However the joints were loaded vertically and not laterally.