Bone growth is affected by ambient temperature

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Osteoporosis, a bone disease linked to aging, is characterized by a loss of bone density, micro-architectural deterioration of the bones and an increased risk of fractures.

With one third of postmenopausal women affected, it is a major public health problem.

Through epidemiological analyses, laboratory experiments and state-of-the-art metagenomic and metabolomics tools, a research team from the University of Geneva (UNIGE), in Switzerland, has observed that exposure to warmer ambient temperatures (34 °C) increases bone strength, while preventing the loss of bone density typical of osteoporosis.

Moreover, this phenomenon, linked to a change in the composition of gut microbiota triggered by heat, could be replicated by transplanting the microbiota of mice living in a warm environment to mice suffering from osteoporosis.

Indeed, after the transplant, their bones were stronger and denser. These results, to be discovered in Cell Metabolism, make it possible to imagine effective and innovative interventions for prevention and treatment of osteoporosis.

Many biologists are familiar with Allen’s Rule, from 19th-century naturalist Joel Asaph Allen, according to which animals living in warm areas have a larger surface area in relation to their volume than animals living in colder environment.

Indeed, a larger skin surface allows better evacuation of body heat. “In one experiment, we placed newborn mice at a temperature of 34 °C in order to minimize the heat shock associated with their birth. We found that they had longer and stronger bones, confirming that bone growth is affected by ambient temperature,” explains Mir

ko Trajkovski, Professor at the Department of Cell Physiology and Metabolism and at the Diabetes Centre of the UNIGE Faculty of Medicine, who led the study. But what about adulthood?

Consistent epidemiological data

By placing several groups of adult mice in a warm environment, the scientists observed that while bone size remained unchanged, bone strength and density were largely improved.

They then repeated their experiment with mice after an ovariectomy modeling post-menauposal osteoporosis. “The effect was very interesting,” says Claire Chevalier, then a researcher in Professor Trajkovski’s laboratory and the first author of this work.

“The simple fact of warming the living environment of our mice protected them from the bone loss typical of osteoporosis!”

What about human beings? The research team analyzed global epidemiological data on the incidence of osteoporosis in relation to the average temperature, latitude, calcium consumption and vitamin D levels.

Interestingly, they found that the higher the temperature, the fewer hip fractures—one of the main consequences of osteoporosis – regardless of other factors. “We found a clear correlation between geographical latitude and hip fractures, meaning that in the northern countries the incidence is higher compared to the warmer south,” says Mirko Trajkovski.

“Normalizing the analysis of the known players such as vitamin D or calcium did not modify this correlation. However, when we excluded the temperature as the determinant, the correlation was lost.

This is not to say that calcium or vitamin D do not play a role, either alone or in combination. However, the determining factor is heat – or lack thereof.”

How the microbiota adapts

Specialists in the microbiota, the Geneva scientists wanted to understand its role in these metabolic modifications. To this end, they transplanted the microbiota of mice living in a 34° environment to osteoporotic mice, whose bone quality was rapidly improved.

“These findings may imply an extension to Allen’s rule, suggesting elongation-independent effects of the warmth, which predominantly favors bone density and strength during adulthood through microbiota alterations,” says Mirko Trajkovski.

Thanks to the state-of-the-art metagenomic tools developed in their laboratory, the scientists then succeeded in understanding the role played by microbiota.

When adapts to heat, it leads to a disruption in the synthesis and degradation of polyamines, molecules that are involved in aging, and in particular in bone health.

“With heat, the synthesis of polyamines increases, while their degradation is reduced.

They thus affect the activity of osteoblasts (the cells that build bones) and reduce the number of osteoclasts (the cells that degrade bones).

With age and menopause, the exquisite balance between the osteoclast and osteoblast activity is disrupted,” explains Claire Chevalier. “However, heat, by acting on the polyamines, which we found to be partly regulated by the microbiota, can maintain the balance between these two cell groups.”

These data therefore indicate that exposure to warmth could be a prevention strategy against osteoporosis.

Developing new treatments

The influence of microbiota on metabolism is being better understood. However, in order to be able to use this knowledge to develop therapeutic strategies, scientists must identify precisely the role of particular bacteria in particular diseases.

In the context of their work on osteoporosis, Professor Trajkovski’s team has been able to identify certain important bacteria. “We still need to refine our analyses, but our relatively short-term goal would be to identify candidate bacteria, and develop several ‘bacterial cocktails’ to treat metabolic and bone disorders, such as osteoporosis, but also to improve insulin sensitivity, for example,” the authors conclude.


Bone elongation disorders have multiple underlying causes, ranging from injury and illness to genetic bone disease. Advancing insight into linear growth regulation at the molecular level1 has outpaced development of strategies to offset short stature and/or leg length discrepancy caused by childhood growth failure.

Limb length inequality can lead to disabling health conditions in adulthood, such as scoliosis, chronic back pain, and osteoarthritis.2

Alternatives are needed because existing limb-lengthening procedures involve invasive surgery and/or drug regimens, which are only partially effective.3 A major obstacle to successful bone lengthening by noninvasive means is difficulty in targeting therapeutics to cartilaginous growth plates, which do not have a direct blood supply. Experimental drug delivery approaches include surgically implanted catheters and localized injections into specific growth plates.4,5

Data from our lab and others demonstrate that exposure to warm ambient temperature during growth increases bone blood supply and length in young mice.6,7 While continuous whole body heating does not effectively translate to the clinic, intermittent-targeted heating could be accomplished with a heating pad or temperature cuff.

Localized heat could be an alternative to surgery and a supplement to systemic bone-lengthening drugs to noninvasively achieve limb length equalization.

The objective of this project is to test a unilateral heating model to increase length of specific bones without surgical or drug intervention. We hypothesize that daily heat exposure on one side of the body will unilaterally increase femoral and tibial lengths on the heat-treated side.

Our goal is to develop a low-cost, noninvasive method for lengthening bones that can translate into practical therapy to offset linear growth impediments in children.

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Figure 3.
Extremities are lengthened on the heat-treated side. (A) Error bar plots show > 12% increase in tibial elongation rate on the heat-treated side. (B) Right-left tibial slab sections from the same mouse labeled with OTC. The metaphyseal chondro-osseous junction (COJ) is indicated by the single arrowheads. Double arrowheads show OTC band in metaphyseal bone. Growth rate was calculated by measuring the vertical distance between the arrowheads (gray lines). Yellow segment of the vertical line on the heat-treated side shows the total difference in length measured over 7-days. (C) Ear area and (D) tibial length remain significantly increased on the heat-treated side in skeletally mature adults after only 14 days of juvenile heat exposure (see Fig. 1). Mean ± 1 standard error plotted.

Tissue Collection and Elongation Rate Analyses
Experimental 5-week-old mice (N = 14 females) were euthanized for tissue harvest 1 day after the last heat-treatment. Control mice (N = 6 females) were euthanized at the same endpoint. Experimental 12-week-old mice (N = 6, mixed sex) were euthanized 49 days post-heating to evaluate persistent limb length differences at skeletal maturity (Fig. 1). In addition to limb length, cartilaginous ears were measured to document a treatment effect because ear size increases with ambient temperature.7,18

Tibial elongation rate was measured for all 5-week-old control mice (N = 6) and a subset of the 5-week-old experimental mice (N = 8). Tibiae from the remaining 5-week experimental mice (N = 6) and all 12-week-old mice (N = 6) were kept intact for lengths. Femora and humeri from all mice (N = 26 total) were reserved for length.

The proximal tibial growth plate was selected to measure elongation rate because its relatively flat contour yields a uniform growth rate across the epiphysis.17 The adjacent distal femoral growth plate was not used due to its undulating shape (with varied growth rate) and irregular geometry that changes with age,19 which introduces sampling error and measurement inconsistency. The proximal femoral and distal tibial growth plates were also not used because they contribute least to total limb lengthening,20 with correspondingly reduced growth activity.21

The OTC label was visualized in unfixed slab sections of bisected tibiae. One half of each bone was placed in a specialized holder on a glass slide, and cover-slipped with glycerol in PBS. The other half was reserved for a separate histological study. Fluorescence was visualized using a UV filter on a fluorescence stereomicroscope. Brightfield (to delineate the chondro-osseous junction) and fluorescence images were captured in tandem.

Images were calibrated manually in ImageJ software (version 1.44, National Institutes of Health, USA) from a 2mm stage micrometer, and then two lines were drawn on each image. The first line was drawn across the metaphyseal chondro-osseous junction (COJ), marked by invading vasculature at the lower edge of the growth plate (single arrowheads in Fig. 3B).

The second line was drawn across the leading (proximal) edge of the OTC band in metaphyseal bone (double arrowheads in Fig. 3B). The vertical distance between the lines was measured at 5 equidistant points across the growth plate using ImageJ. Measurements were averaged and divided by the 7-day labeling period to estimate daily elongation rate (μm/day).

DISCUSSION
The goal of this study was to establish a model system using targeted intermittent heat exposure to permanently increase extremity length in mice. Our data support the hypothesis that daily unilateral limb heating increases femoral and tibial lengths on the heat-treated side. Extremity lengthening was correlated with temperature during treatment, particularly in temperature-sensitive cartilaginous ears (Fig. 2B).

The length effect persisted at skeletal maturity after only 14 days of post-weaning treatment. A significant right-left difference was measured without impacting overall body mass, and left-right differences were absent in normal non-treated control mice. Core temperature and respiration were in a physiological range under anesthesia. These results suggest that daily unilateral heating is an effective way to model temperature-enhanced hindlimb elongation in young, rapidly growing mice.

The rationale for the model is to develop methods for increasing bone length with minimally invasive methods that can apply to many different growth-limiting conditions. With these baseline data, our model will allow us to move forward and test mechanisms of heat-enhanced bone lengthening to better tailor future clinical therapies.

For example, using in vivo multiphoton imaging, we have shown that short-term (30-min) hindlimb heating increases molecular uptake in mouse tibial growth plates.24 Heat could potentially be applied on a scheduled regimen with systemic bone lengthening drugs to target their delivery to specific skeletal growth plates.

Routine heat exposure developed in the model here could thus provide a method for augmenting drug-induced limb elongation.

Interestingly, the humerus did not respond to heat-treatment in any of four independent trials conducted for this study (Table 1). Although it is unclear why humeral length did not differ, one potential explanation is the warmer starting temperature of the forelimb when compared to the hindlimb.

The knee joint capsule is normally at least 3–4 °C lower than body core.25,26 Our treatments elevated hindlimb temperature by 10 °C on the heat-treated side (Fig. 2). However, skin temperatures in the humeral region more closely resembled body core, consistent with thermal maps for humans showing that 37 °C core temperature extends into the shoulder region, while extremity temperatures progressively decrease in a proximal-distal gradient.27

Our working hypothesis is that heat-treatments do not impact temperature of the humerus due to the proximity of this joint to the body core, and the disproportionately large volume of warm blood delivered to the shoulder region through the large subscapular artery.28

Since most elongation of the humerus occurs at its proximal growth plate (shoulder), versus in the distal (knee) growth plate of the femur,20,29-31 it is possible that the left-right symmetry in humeral length reflects its relatively constant temperature. This could be tested by decreasing shoulder temperature with cold, which stunts limb elongation in a dose-dependent manner.32

Although heat effects on extremity lengthening have been documented for over a century,6 temperature is still under-recognized for its ability to modulate bone elongation in growth plates. Brookes and May33 demonstrated up to a 20% increase in bone growth rate for every 1 °C increase in incubation temperature in growing chicks.

Doyle and Smart34 used daily application of short-wave diathermy (heat generating treatment) near the growth plate in rats and showed an increase in femoral and tibial lengths. Granberry and Janes35 were not able to replicate these findings using microwave diathermy in dogs; however, their 100 watt treatment produced bone damage that may have prevented heat-related growth acceleration.

Here we found that unilateral exposure of mild, non-damaging 40 °C heat for 40-min per day for only 14 days permanently increased ear area and hindlimb length on heat-treated sides of young mice. No treatment-related damage was observed morphologically or histologically. These results suggest that heat could be a promising strategy for enhancing elongation potential of specific growth plates without affecting the entire skeleton.

One caveat is that the width of the mouse growth plate is only a fraction of that of a human growth plate. It will be important to replicate these results in a larger animal model to ensure that heat can fully penetrate a larger growth plate, so as to avoid potential angular growth deformities.

This should not be problematic, however, since whole body heat-effects on bone length have already been demonstrated in experiments using large animals.18,32 Treatments could be optimized for a more robust effect by using a temperature cuff to target the most active growth plates.

The distal femoral and proximal tibial growth plates contribute most to lower limb lengthening in humans.31 Use of a localized heating device around the knee could be a clinically useful tool to augment existing drug therapies (i.e., growth hormone injections) and to potentially avoid invasive surgical limb length correction.

A practical strategy in children could be to wrap a noninvasive heating device around the knee during the sleep period when growth occurs13 and growth hormone naturally peaks.36

In conclusion, we believe that such heat-based therapies resulting from the model developed here will have advantages over traditional methods that are potentially painful and invasive. Our next step is to establish the effects in a larger animal model, and test them alongside pharmaceutical interventions.

This approach could ultimately lead to development of alternative treatment modalities with better outcomes by reducing costs and side effects of surgery and high-dose systemic pharmaceuticals.

reference link : https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6818498/


More information: Claire Chevalier et al, Warmth Prevents Bone Loss Through the Gut Microbiota, Cell Metabolism (2020). DOI: 10.1016/j.cmet.2020.08.012

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