Environmental temperature impact on bone and cartilage growth.

“Environmental modulation of tissue temperature can have direct and immediate consequences on cell proliferation, metabolism, matrix production, and mineralization in cartilage. Temperature can also indirectly influence cartilage growth by modulating circulating levels and delivery routes of essential hormones and paracrine regulators.”

The arrows in white are the functional purposes of the bone.  In red are the factors that can affect bone length.  Bone adapts to the demands placed upon but not always in the way we want.  It doesn’t matter if stretching to us makes sense to use to produce longer bones.  It makes what the bones think.  The bone thinks to think:  I’m being stretched I should grow longer.  We need to recreate specific stimulus in the past that produces the desired results.  For example, the growth plate which results in longitudinal bone growth is a result of mesenchymal condensation of progenitor cells.  The goal of LSJL is to create a stimuli of hydrostatic pressure and shear strain that has been shown to induce mesenchymal condensation in adult mesenchymal stem cells and in turn induce chondrogenesis.

“Chondrocytes differentiate from a reserve pool of round quiescent cells into flattened columns of proliferating cells, which then mature into large terminally differentiated hypertrophic cells, and are ultimately replaced with mineral at the chondro-osseous junction where bone-forming osteoblasts invade from the metaphyseal vasculature”

“limb length correlates with temperature and latitude”

“limb length can be modified within a single generation by rearing young littermate animals at warm and cold temperatures during the postnatal growth period”

“Growth plate morphology was unexpectedly similar at cold and warm temperatures.  There were no major appreciable differences in overall size, shape, or organization of the cartilage.”

“variation in adult long bone length between inbred mouse strains was primarily generated by growth rate differences that occurred during a phase of rapid bone elongation between 3 and 5 weeks age.”<-it could be that lack of enough temperature is a limiting factor on height growth only in the most rapid periods of growth.

Usually these temperature studies are capped at one point in temperature so it’s hard to see if there’s an equilibrium temperature.

“normal capillary blood flow in the skin of the tibia (lower leg) was significantly higher than that in the metatarsal (foot) region, indicating a natural proximal-distal gradient in blood supply.”

” at high temperature extremes outside of a tolerable physiological range, heat does negatively impact body growth.”

“Without countercurrent heat exchangers in a cold environment, blood will rapidly lose temperature as it flows to the distal-most parts of the appendages. Core temperature would consequently drop as cool venous blood returns to the body core. One mechanism to combat a whole body cooling effect is to reduce total outflow of blood flow to the appendages by regulating vasomotor tone”

“This initially perplexing finding (no major differences in morphology of the growth plate when bone lengths did differ) may actually be quite informative as to the mechanism of the growth effect. One possibility is that a complex change in gene expression, cell metabolic activity, and/or maturation rate could contribute to the growth rate differences without changing the superficial appearance of the cartilage. The other corresponding possibility is that the growth rate differences were too slight to detect in the static histology “snapshot.” The total difference in tibia bone length between warm and cold was approximately 370 μm”

The study mentions that heat can induce bone marrow expansion.  Equilibrium temperature can increase calcium uptake.  Cold temperature induces endoplasmic reticulum stress.  HSP70 expression is affected by temperature.  Hydrostatic Pressure also increases HSP70.  HMGB1 is negatively correlated with temperature.  HMGB1 seems to encourage the early stages of chondrogenesis.  This would be a good thing in the formation of the growth but could be a bad thing in the later stages as it may effect the degree of endochondral ossification of the growth plate reducing longitudinal bone growth.  But mice that lack HMGB1 have shorter bones.  Heat also can incude TRPV4 activation.  TRPV4 may modulate bone elongation by affecting cell volume in chondrocytes.  However, continuously active TRPV4 mutants have abnormal endochondral ossification and reduced longitudinal bone growth.

” warm temperature could facilitate bone elongation by increasing the number of cells that could become bone forming osteoblasts at the chondro-osseous junction. There is also evidence that cold could limit bone elongation by directly inhibiting osteoblast activity.”<-Which would mean that heat wouldn’t play a role in neo-growth plate formation.

” leptin deficiency produces a contrasting phenotype in the limb bones and spine (short femurs and long spine)”

“Blood flow could be permanently modified by temperature through the route of new vessel formation, or angiogenesis”

“disrupted blood flow markedly impairs limb growth”

“surgical obstruction of the principal nutrient artery within the tibia marrow cavity of rabbits caused blood to be shunted toward the metaphyseal vessels and increased blood flow to the growth plate. Within three months after the surgery, the operated tibia had grown significantly longer compared to the contralateral side that received the normal blood supply”

So blood flow seems to be the most promising way to increase longitudinal bone growth and inducing new growth plates is not a ruled out effect of increasing blood flow so that LSJL likely increases blood flow is a good sign.

The LSJL scientists Yokota and Zhang posted another LSJL related study.  Not a lot can be gathered in relation to LSJL on height growth unless Nfatc1 or ATF4 can be found to be chondrogenic inducers or to increase peak chondrocyte hypertrophy.

Evaluating treatment of osteoporosis using particle swarm on a bone remodelling mathematical model.

“The model formulated a temporal BMD change of a mouse’s whole skeleton in response to ovariectomy, mechanical loading[LSJL] and administration of salubrinal.”<-So even though this study is studying mainly BMD we can extrapolate other LSJL effects via LSJL’s affect on gene expression and cells.

“The best treatment was found to start with mechanical loading followed by salubrinal.”

“Ovariectomy (OVX) was modelled through oestrogen deprivation, whereas salubrinal injection and knee loading were modelled by up-regulating p-eIF2α and inhibiting sclerostin, respectively.”<-In studying eif2a, it was unclear whether it could help increase height.

“To model the observed non-responsiveness to salubrinal at normal oestrogen levels in control mice, the model included a range of p-eIF2α values that does not elicit a change in ATF4 and NFATc1. When oestrogen levels decrease as in OVX mice, this range becomes
smaller, and ATF4 and NFATc1 become more responsive to changes in p-eIF2α.”<-ATF4 has some involvement in height but it doesn’t seem to be a powerful effector like say CNP.

This could connect to cartilage growth via NFATc1 or ATF4.  NFATc’s do affect chondrogenesis.

“Knee loading was applied using a custom piezoelectric loading platform in the lateral-medial direction 3 min/day at 15 Hz, with a peak-to-peak force of 0.5 N”<-LSJL.  Mice were 12 weeks old at time of treatment.

Hydrogel-Based Local Release of Salubrinal Stimulates Healing of Mouse Tibia Fracture

“Salubrinal is a synthetic compound (C21H17Cl3N4OS; 480 Da) which is known to reduce various cellular stresses including stress to the endoplasmic reticulum. It inhibits serine/threonine protein phosphatase 1 alpha (PP1), followed by the elevation of phosphorylated eukaryotic translation initiation factor 2 alpha (eIF2α). Salubrinal is reported to enhance bone formation by stimulating Activating Transcription Factor 4 (ATF4), one of the transcription factors for bone formation, via eIF2α-mediated signaling and stimulating development of bone-forming osteoblasts. It also suppresses nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1), a master transcription factor for osteoclastogenesis, and inhibits development of bone-resorbing osteoclasts. It reduces inflammation and degradation of cartilage tissues{maybe it can delay growth plate cessation?}”

“C57BL/6 female mice (14 weeks, body weight ~20 g”

Here’s how the fracture was induced:

“salubrinal can add calcified mass to osteoporotic bone{maybe salubrinal encourages endochondral ossification}?”

In the stump on the left side the salubrinal group looks taller but less wide.

“salubrinal suppresses the proliferation and maturation of osteoclasts by downregulating AP-1 proteins such as c-Fos and JunB, as well as NFATc1”

Salubrinal improves mechanical properties of the femur in osteogenesis imperfecta mice.

“Salubrinal is an agent that reduces the stress to the endoplasmic reticulum by inhibiting de-phosphorylation of eukaryotic translation initiation factor 2 alpha (eIF2α). We and others have previously shown that the elevated phosphorylation of eIF2α stimulates bone formation and attenuates bone resorption. In this study, we applied salubrinal to a mouse model of osteogenesis imperfecta (Oim), and examined whether it would improve Oim’s mechanical property. We conducted in vitro experiments using RAW264.7 pre-osteoclasts and bone marrow derived cells (BMDCs), and performed in vivo administration of salubrinal to Oim (+/-) mice. The animal study included two control groups (wildtype and Oim placebo). The result revealed that salubrinal decreased expression of nuclear factor of activated T cells cytoplasmic 1 (NFATc1) and suppressed osteoclast maturation, and it stimulated mineralization of mesenchymal stem cells from BMDCs. Furthermore, daily injection of salubrinal at 2 mg/kg for 2 months made stiffness (N/mm) and elastic module (GPa) of the femur undistinguishable to those of the wildtype control. Collectively, this study supported salubrinal’s beneficial role to Oim’s femora. Unlike bisphosphonates, salubrinal stimulates bone formation. For juvenile OI patients who may favor strengthening bone without inactivating bone remodeling, salubrinal may present a novel therapeutic option.”

Salubrinal downregulated Nfatc1 in MSC like cells.  If cells are less likely to become osteoclasts it is more likely more them to become cartilage.

The Influence of Growth Hormone on Bone and Adipose Programming.

“In utero growth hormone exposure is associated with distinct immediate growth responses and long term impacts on adult physiological parameters that include obesity, insulin resistance, and bone function. Growth hormone accelerates cellular proliferation in many tissues but is exemplified by increases in the number of cells within the cartilaginous growth plate of bone{can it increase the number of growth plate progenitor cells?}. In some cases growth hormone also potentiates differentiation as seen in the differentiation of adipocytes that rapidly fill upon withdrawal of growth hormone. Growth hormone provokes these changes either by direct action or through intermediaries such as insulin-like growth factor-I and other downstream effector molecules. The specific mechanism used by growth hormone in programming tissues is not yet fully characterized and likely represents a multipronged approach involving DNA modification, altered adult hormonal milieu, and the development of an augmented stem cell pool capable of future engagement as is seen in adipose accrual.”

“Early therapeutic provision of GH to SGA[small for gestational age] neonates having sufficient GH enhances the velocity of bone growth transiently but only for the duration of GH treatment”

“between birth and 28 days is most influential on bone elongation and adult size. Initiating the elevated GH beyond 28 days of age increases growth but not to the
extent realized with earlier exposure despite the presence of a functional growth plate. At the cellular level, GH accelerates bone growth by hyperplasia[an increase in the number of cells] as opposed to growth plate chondrocyte hypertrophy”<-hyperplasia is more powerful than chondrocyte hyertrophy if it increases the amount of growth plate progenitor cells.  If it only increases chondrocyte proliferation then the effect is transient as chondrocytes have a finite proliferative capacity.