For media contact information. Skip to main content. Toggle menu Go to search page. Search Field. Bone Health In Brief. Nutrition Research Macronutrients Protein-energy Essential Fatty Acids Micronutrients Calcium Fluoride Magnesium Phosphorus Potassium Sodium Vitamin A B Vitamins Vitamin C Vitamin D Vitamin K Lifestyle Factors Alcoholic Beverages Coffee Physical Activity Smoking.

Indeed, in response to blood calcium lowering and when calcium from food is limited, PTH and vitamin D can prompt bone degradation, which releases calcium and phosphorus into the circulation, thereby threatening bone integrity. It is thus critical to obtain enough calcium from food to limit bone loss in response to fluctuating blood calcium concentrations.

It is the nutrient intake value that is estimated to meet the requirement of nearly all healthy people of a particular gender and age group in a population. It is a target value for an individual. This type of study tests the efficacy of an active treatment on a specific outcome compared to a control.

Placebo - a substance without therapeutic effect. It contributes to regulate blood volume through "colloid osmotic pressure" and transports various molecules fatty acids, metals, ions, hormones, etc. in the circulation Low-albumin protein-energy malnutrition PEM - laboratory diagnosis of marginal PEM usually includes measures of serum albumin.

Stunting of stature due to malnutrition is linked to stunting of mental development. Cell-signaling pathways play important roles in regulating numerous cellular functions in response to changes in a cell's environment.

Also refers to as cell transduction pathways or signaling cascades Peak bone mass - for an individual, it refers to the maximum amount of bone acquired by the time a stable skeletal state has been attained and depends on both genetic and environmental lifestyle factors Primary prevention - it refers to a range of activities undertaken to prevent or reduce the risk of an injury or disease before it occurs.

In particular, the substitution of ultra-processed foods and soft drinks for unprocessed meals, milk, and other calcium-rich food is likely to damage bone health on the long run.

Also refers to as cell-transduction pathways or signaling cascades Hydroxyapatite crystals - calcium phosphate salts forming crystals Ca 10 PO 4 6 OH 2 that constitutes the main mineral in teeth and bones Mineralization - the process by which calcium and phosphorus are laid on the organic tooth and bone matrices Nucleic acids - refers to DNA deoxyribonucleic acid and RNA ribonucleic acid Osteomalacia - sometimes called "adult rickets"; it is characterized by a softening of the bones in adults caused by a lack of matrix mineralization Rickets - a condition characterized by soft and deformed bones and due to an impaired incorporation of calcium and phosphorus in growing bones EAR - estimated average requirement.

It is also the best estimate of an individual's requirement and thus may be used to assess the adequacy of an individual's usual intake of the nutrient RDA - recommended dietary allowance. It is a target value for an individual UL - tolerable upper intake level.

Set by the Institute of Medicine, the UL of a specific nutrient is the highest level of daily intake likely to pose no risk of adverse effects in almost all individuals of a specified age.

HIGHLIGHT: Vitamin A supplements should be reserved for undernourished populations and those with evidence of vitamin A deficiency. Set by the Institute of Medicine, the RDA is the average daily dietary intake level of a nutrient sufficient to meet the requirements of nearly all healthy individuals in a specific life stage and gender group.

Genetic variations, skin color, aging, magnesium deficiency, obesity, and specific diseases e. HIGHLIGHT: VITAMIN D IN FOOD Few foods contain vitamin D: Some fatty fish mackerel, salmon, sardines , eggs from vitamin D-fed hens, and mushrooms grown under UV light.

Dairy products, cereal, bread, and fruit juices may be fortified with vitamin D. HIGHLIGHT: Vitamin K deficiency is uncommon in healthy adults.

Thereafter, bone regenerative applications such as bone cements, scaffolds, implants, and coating techniques using calcium phosphates have emerged, and some have been commercialized [ 16 , 17 , 18 ]. Similar to these, the characteristics of calcium phosphates have been studied for bone regenerative applications.

Hierarchical structure of bone ranging from macroscale skeleton to nanoscale collagen and HAP [ ]. Every implantable material must be biocompatible, meaning that inflammation or foreign body response should not occur in the living system and tissue.

Calcium phosphates were discovered to be biocompatible because they can be dissolved in body fluids and are present in large amounts in solid forms [ 19 ]. The properties of calcium phosphates affect bioactivity, such as adhesion, proliferation, and new bone formation in osteoblasts. To exhibit these bioactive features, degradation and ion release in calcium phosphates are important [ 19 ].

These phenomena increase the local concentration of calcium and phosphate ions and stimulate the formation of bone minerals on the surface of calcium phosphates. They also affect the expression of osteoblastic differentiation markers such as COL1, ALP, BMPs, OPN, OCN, BSP, ON, and RunX2 [ 20 , 21 , 22 , 23 , 24 ].

Calcium phosphates play important roles in cell adhesion and tissue formation by affecting the adsorption of extracellular matrix proteins on the surface [ 25 , 26 ]. Their properties also influence bone regeneration by affecting newly formed bone minerals [ 27 ]. First, calcium ions affect cells and living systems in several ways.

Calcium is one of the ions that form the bone matrix, and it exists mostly in the form of calcium phosphates in bone tissues [ 28 ]. These calcium ions cause bone formation and maturation through calcification. In addition, calcium ions affect bone regeneration through cellular signaling.

Calcium stimulates mature bone cells through the formation of nitric oxide and induces bone growth precursor cells for bone tissue regeneration [ 29 , 30 ]. Furthermore, calcium ions regulate the formation and the resorptive functions of osteoclasts [ 33 , 34 ].

Phosphorus ions are present in the human body in large amounts. They are involved in a variety of substances such as proteins, nucleic acid, and adenosine triphosphate, and they affect physiological processes [ 35 , 36 ]. In addition, phosphate has a negative feedback interaction between the RANK-ligand and its receptor signaling and regulates the ratio of RANK-ligand:OPG to inhibit osteoclast differentiation and bone resorption [ 39 , 40 ].

The osteoinductive and osteoconductive features of calcium phosphates are also important for bone regeneration. Osteoinduction is the ability to induce progenitor cells to differentiate into osteoblastic lineages [ 41 , 42 ], whereas osteoconduction is the ability of bone growth on the surface of materials [ 43 ].

Osteoinduction and osteoconduction support cell adhesion and proliferation [ 41 , 42 , 43 ]. Cell adhesion is strongly influenced by the ability to adsorb extracellular matrix proteins. It is influenced by the surface characteristics of calcium phosphates, such as surface roughness, crystallinity, solubility, phase content, porosity, and surface energy [ 42 ].

Osteoconduction and osteoinduction depend on several factors. Some studies suggested that calcium phosphates are osteoinductive even in the absence of supplements [ 42 ]. For example, surface chemistry and surface charge affect protein adsorption, and osteoblastic differentiation occurs via the interaction between cells and the extracellular matrix.

Surface morphology can also exert these effects [ 42 ]. The role of the surface roughness of calcium phosphate is determined by the grain size and particle size of the calcium phosphate crystal structure. The roughness affects protein adhesion on the calcium phosphate surface.

Surface roughness also has an effect on cell adhesion [ 46 ]. The porosity of calcium phosphate also has an effect on bioactivity. The increase in porosity improves contact with body fluids on the surface area.

Thus, dissolution rate is enhanced [ 19 ] and the presence of pores on the surface affects protein adsorption. This effect was also observed with an increase in the number of pores. Additional, pore size impacts bone ingrowth and angiogenesis [ 50 , 51 ]. Because of the existence of pores, calcium phosphate exhibits mechanical properties such as high brittleness, low impact resistance, and low tensile stress [ 41 ].

However, its compressive strength is better than that of natural human bone, and it is used in non-load bearing implants, defect filling, and coating methods. Hydrophilicity is a critical factor in osteogenesis regulation.

Hydrophilic surfaces are essential for cell adsorption and increases fibroblastic cell response [ 55 ]. They increase the maturation and differentiation of bone cells as well as osteointegration, and they also affect cellular reactions [ 56 , 57 ].

Moreover, surface hydrophilicity increases the adhesion and proliferation of osteoblasts [ 58 , 59 ]. The dissolution process of calcium phosphates is affected by surface area per unit volume, fluid convection, acidity, and temperature [ 19 , 41 ].

Stable and low-solubility calcium phosphates show low ion exchange with their surroundings and slow recrystallization rate on the surface, thus determining protein concentration and conformation by electrostatic interaction at the charged site.

On the other hand, calcium phosphates with high solubility easily change the local pH and ion concentration so that protein adhesion is affected.

Protein adhesion causes cell adhesion and determines the effectiveness of bone regeneration [ 60 , 61 , 62 ]. Therefore, it is important to control these characteristics and choose the calcium phosphates with properties that are appropriate for specific applications.

Calcium phosphates with bioactive features in many crystalline phases have been studied Fig. Schematic illustration of the crystal structure of a HAP [ ], b α-TCP, c β-TCP [ ], and d WH [ ]. Copyright American Chemical Society.

TEM and SEM images of e HAP [ ], f α-TCP, g β-TCP [ ], and h WH [ ]. XRD data of i HAP [ ], j α-TCP and β-TCP [ ], and k WH [ ].

Hydroxyapatite HAP has been widely used in bone regeneration. It is a naturally occurring form of calcium phosphate that constitutes the largest amount of inorganic components in human bones [ 63 ].

HAP is naturally formed and can be collected, but various ions and vacancies form defective structures. Therefore, HAP used in actual research or clinical applications is obtained by synthesis in aqueous solution systems [ 65 ].

Stoichiometric structures can have both monoclinic and hexagonal phases, but in biological environments, they take on a hexagonal phase, which is more stable structure [ 66 , 67 ]. HAP is the most stable calcium phosphate with low solubility in physiological environments defined by temperature, pH, body fluids, etc.

In addition, HAP does not cause inflammatory reactions when applied clinically [ 71 ]. HAP is known to be osteoconductive but not osteoinductive [ 42 , 72 ]. Therefore, ions such as fluoride, chloride, and carbonate ions are substituted as needed [ 73 ]. For example, the use of fluoride as an anionic substitution increased the stability and the use of magnesium as a cationic substitution increased the biological effect [ 42 ].

Studies have been conducted to utilize the biocompatible characteristics of HAP, showing that in vivo bone regeneration was improved with enhancing the differentiation or promoting the proliferation of mesenchymal stem cells by increased adhesion of osteoblasts [ 74 , 75 ].

Research on the clinical applications of HAP in bone regeneration began in the mids. It has been used in implant coatings [ 76 , 77 ] and graft materials [ 78 , 79 ], and synthetic HAP has been studied in bone regenerative applications such as granules, cements, and pastes [ 80 , 81 ].

Though HAP has been investigated for clinical applications, it has not been used in cases where high load is applied because of its unique hard and brittle properties, and it has been used mainly as coatings [ 66 , 82 ]. For example, coatings on the surface of metallic implants have been prepared to improve osteoblast activity [ 83 ] or to increase the contact area of bone implants [ 84 ].

In this way, HAP coatings improved the biological fixation, biocompatibility, and bioactivity of implants [ 85 ]. In addition, deposition methods such as spraying, sputtering, pulsed laser deposition, and sol-gel techniques have been attempted, and several reports have been published whereby bone formation was promoted by increasing cellular response [ 86 , 87 , 88 ].

Furthermore, studies on bone regenerative applications have been carried out by mixing HAP with soft materials such as polymers to complement the drawbacks. Studies are underway to control the porosity, mechanical strength, bioactivity, and ease of use, mainly using synthetic scaffolds [ 89 , 90 , 91 ].

α-TCP has the crystal structure of a monoclinic space group and β-TCP has the crystal structure of a rhombohedral space group [ 92 , 93 ]. β-TCP has a more stable structure and higher biodegradation rate than those of α-TCP. Therefore, β-TCP is generally used in bone regeneration [ 95 ].

β-TCP is less stable than HAP but has a faster degradation rate and higher solubility. In addition, it has a high resorption rate and is widely used to increase biocompatibility [ 95 , 96 ]. β-TCP promotes the proliferation of osteoprecursor cells such as osteoblasts and bone marrow stromal cells [ 97 , 98 ].

These properties are due to the excellent biomineralization and cell adhesion by the nanoporous structure of β-TCP [ 99 ].

The characteristics of β-TCP have been actively studied for bone regeneration purposes, and β-TCP has been widely used in bone cements and bone substitution [ , ]. In order to simultaneously utilize the characteristics of TCP and HAP, biphasic materials have been developed.

Biphasic or multiphasic calcium phosphates exist in a form that is not separated because each component is homogeneously and intimately mixed at the submicron level [ ]. The biphasic form of calcium phosphates was first prepared in as a mixture of HAP and β-TCP [ ].

These biphasic calcium phosphates generally combine two more incompatible calcium phosphates, such as the more stable HAP and the more soluble TCP, and they have bene evaluated mainly in terms of bioactivity, bioresorbability, and osteoinductivity [ , ].

Biphasic calcium phosphates have been used and studied as bone grafts, bone substitute materials, and dental materials [ , ]. The mixture of HAP and β-TCP to stimulate the osteogenic differentiation of mesenchymal stem cells, increase cell adhesion, attach growth factors, and enhance mechanical properties has been actively carried out [ , , ].

Ramay et al. The biphasic calcium phosphate scaffolds were found to have microporous structures that influenced cell growth and vascularization. Whitlockite WH is a calcium phosphate-based ceramic that contains a magnesium ion and has the chemical formula Ca 9 Mg HPO 4 PO 4 6 [ , ].

Compared to HAP, WH showed mechanically higher compressive strength [ ]. Its solubility was higher in physiological condition and higher amount of ions could be released continuously [ ].

WH has been difficult to synthesize and thus, research on WH has not progressed well. However, as a result of recent advances, it has been possible to synthesize WH easily in low-temperature conditions.

It has been reported that WH is formed when Mg ions are present in acidic solutions containing calcium phosphate [ ]. In addition, in vivo formation of WH occurs under acidic conditions via the release of acidic molecules when osteoclasts resorb old bone [ , ].

Jang et al. WH induced higher expression of osteogenic genes than did HAP and β-TCP [ ]. Moreover, in vivo bone regeneration of a rat calvarial defect model with composite hydrogel showed that WH promoted growth and osteogenic activity better than HAP did [ ]. These results suggested that the continuous release of magnesium and phosphate ions promoted bone growth by controlling osteogenic differentiation.

Especially, magnesium ions seemed to increase bone formation because they play a role in decreasing the activity of osteoclasts [ ]. It has recently been shown that osteogenic activity was increased when WH and HAP coexisted at a ratio of approximately , a similar ratio to that in native human bone [ ].

These results suggested that the roles and formation mechanisms of WH in native bone need to be studied. The high osteogenic activity of WH and its role in native bone are expected to contribute to future research on calcium phosphate materials.

In addition, octacalcium phosphate OCP , which is present in human teeth [ , ], has a triclinic crystal structure [ ] and is considered to play a role in the initial phase of HAP formation in bone mineral formation [ , ]. OCP plays a role as a precursor of bone mineralization [ ] and showed high biocompatibility [ , ].

Thus, it has been extensively studied in bone implantation and coating [ , ]. The amorphous form of calcium phosphate [ ] has been utilized in clinical applications where certain functions are performed through ion substitution and the use of various impurities [ , ]. Similarly, several types of calcium phosphate-based materials have been studied and utilized.

Although the bioactive properties of calcium phosphate have been studied and used for bone regeneration, there are some drawbacks such as mechanical disadvantages in clinical applications.

Therefore, research has been carried out to utilize calcium phosphate as composite materials with other materials.

Although calcium phosphate has been widely used for bone treatment as a raw material itself, many studies have been made using processed calcium phosphate applications for better utilization. It is used as coating materials for improving bioactivity of bone implants.

And also, it is used as composites with biomaterials to alter mechanical properties, control biodegradability, and encapsulate drugs Fig.

Calcium phosphate based applications. a WH incorporated hydrogel scaffold [ , ]. b Cranial segment made of tetracalcium phosphate and β-TCP [ ]. c The injectable paste included calcium phosphate nanoparticles [ ]. d Mixed zirconia calcium phosphate deposited on dental implant [ ].

e 3D printed calcium-deficient HAP scaffolds [ ]. f 3D printed calcium phosphate cement [ ]. Calcium phosphate coatings can be applied to various materials to enhance bioactivity. Coating of calcium phosphate is mainly performed using sol-gel and electrodeposition methods [ , ].

Research on calcium phosphate coatings is mainly conducted for metal implant applications, aiming to prevent implant corrosion and increase bioactivity [ , ].

Xu et al. This coating technology increased bioactivity, cytocompatibility, osteoconductivity, and osteogenesis. In vivo studies were conducted to compare this surface to that of conventional magnesium alloys.

Experimental results showed that calcium phosphate-coated Mg alloy had significantly improved surface bioactivity. In the osteogenesis process, statistical differences in the expression of bone growth factor BMP-2 and TGF-β1 were observed compared to that on uncoated Mg alloys, resulting in more compact and uniform osteoid tissues.

In addition, studies on calcium phosphate coatings have resulted in improved surface reactivity and enhanced cell adhesion [ , ]. Nguyen et al. On top of this, a thin HAP surface was formed using a sol-gel coating technique to improve post-implantation bone ingrowth and osteoconductivity.

HAP was coated on the porous surface of cylindrical implants. Using this alloy, in vivo testing of rabbit bone was carried out, and osteoconductivity was enhanced by increasing preferential protein adsorption. Many studies have been conducted to encapsulate anti-bacterial agents and growth factors to enhance their effectiveness [ , ].

To reduce infection and improve cell-material interaction and antimicrobial activity, AgNO 3 and TCP were coated using the laser-engineered net shaping method on the surface of Ti metal by Roy et al.

The optimally controlled Ag-TCP-coated Ti showed a significant decrease in bacterial colonies. Calcium phosphate cements are used to fill and heal bone defects. Cements are mainly incorporated with polymers such as alginate, chitin, chitosan, cellulose, gelatin, collagen, and synthetic polymers such as polyethylene glycol PEG , poly lactic-co-glycolic acid PLGA , polycaprolactone PCL , and poly L-lactic acid PLLA [ ].

As a composite of these polymers, calcium phosphate cements were able to control properties such as injectability, porosity, mechanical properties, and degradation rate [ ]. Hesaraki et al. β-TCP pastes were mixed with hyaluronic acid or PEG to make calcium phosphate cement.

The enhanced viscosity and thixotropy of the calcium phosphate cement were investigated and the effect on injectability was reported. There are some problems of calcium phosphate cements such as the difference between bone regeneration rate and degradation rate, limit of ingrowth due to pore size, lack of mechanical strength, and inflammatory reaction of synthetic polymers.

Efforts are continuously being made to overcome these problems [ , ]. Much effort has been devoted to control pore size and improve mechanical strength [ ], improve degradation rate by adjusting contact with body fluid [ ], add materials to improve mechanical strength [ ], and minimize foreign body response by using natural polymers [ , ].

Studies are also conducted to increase the effectiveness of cements by encapsulating drugs and growth factors [ , ]. PLGA and calcium phosphate complex compound cements prepared for sustained delivery of recombinant human bone morphogenetic protein-2 rhBMP-2 were investigated by Ruhe et al.

Ohura et al. prepared a mixed cement of monocalcium phosphate monohydrate MCPM and β-TCP as another effective carrier of rhBMP rhBMPtransplanted β-TCP-MCPM showed good effect on bone regeneration as a carrier of rhBMP-2 with suitably controlled concentration.

Calcium phosphate has been used in combination with scaffolds. Calcium phosphate scaffolds provide stable properties and allow the control of porosity and biocompatibility. The pore size of the scaffold improves revascularization and bone remodeling, enabling the ingrowth of cells and proteins and enhancing biocompatibility, making them suitable for implant use [ 89 , , ].

A variety of materials such as collagen, gelatin, PCL, PLGA, and PLLA can be used as scaffolding materials [ 89 , , , ]. Studies have been actively conducted to improve the bioactivity based on the characteristics and functions of various substances by enhancing the mechanical properties [ , ], cell proliferation, and osteogenic differentiation [ , ].

Zhao et al. Calcium phosphates consisting of tetracalcium phosphate and dicalcium phosphate anhydrate were combined with alginate hydrogel microbeads encapsulating human umbilical cord mesenchymal stem cells to compensate for the lack of mechanical strength in the hydrogel for load-bearing.

This combination could solve the difficulty in seeding cells deep within the scaffold and the inability of injection in minimally invasive surgeries.

This alginate hydrogel scaffold was injectable and showed increased mechanical properties than those of conventional hydrogels. Drugs and growth factors have been encapsulated within scaffolds [ , ]. Koempel et al. Scaffolds were implanted in rabbit calvarial defect models and after four weeks, the degree of bone formation was observed.

rhBMPloaded implants showed more effective bone formation. In addition, rhBMP-2 was shown to enhance osteointegration, allowing HAP scaffolds to be held in place. Therefore, it was confirmed that BMP loaded on macroporous calcium phosphate scaffolds promoted new bone formation, prevented displacement, minimized host bone resorption, and decreased the incidence of infection and extrusion.

In summary, osteoconductive and osteoinductive features of calcium phosphate affect cell adhesion, proliferation, and new bone formation. Bioactivity can be altered and controlled by ion release and physical property of calcium phosphate on it.

The ion release affects osteogenic cells, tissues, physiological processes and pathways. Bioactive characteristics are different depending on the type of calcium phosphate such as HAP, TCP, and WH. As mentioned above, calcium phosphates are often used with other biomaterials to control and improve their properties.

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There were no significant main effects of diet on tissue-level mechanical properties. There was a significant main effect of diet on yield force, and non-exercised mice on the supplemented diet and mice exercised at low speed on the supplemented diet had significantly greater yield force than baseline.

Exercise had a significant main effect on ultimate stress, decreasing bone strength. Yield stress and ultimate stress were significantly greater than baseline in non-exercised mice fed the supplemented diet. There was a significant main effect of exercise on serum Ca only on day For mice on the supplemented diet, there were no significant effects of exercise on serum Ca.

There were no significant effects of diet or exercise on serum P on day 9. On day 30, there was a significant diet and exercise interaction on serum P. Both high- and low-speed exercised groups on the supplemented diet had significantly lower day 30 serum P than all other groups.

Error bars removed for clarity. The supplemented diet significantly increased serum Ca on days 9 and Exercise only significantly increased serum Ca on day Diet and exercise had a significant interactive effect on serum P on day There were no significant main effects of diet, suggesting exercise was more impactful on bone metabolism than diet on day 9.

There were significant main effects of exercise on serum CTX and PINP on day 30, and there was a significant main effect of diet on serum CTX on day After thirty days of interventions, diet had a greater impact on bone mass and strength than exercise Figs. The diet and exercise regimen in this study 4 weeks, 20 min exercise per day resulted in similar effects on tibial bone mass and strength as previous studies that involved longer durations of exercise 8 weeks, 30 min exercise per day 2 , 5.

Direct tibial loading in rodents has diminished effectiveness when applied for a large number of load cycles per day, but the number of loading cycles used here was an order of magnitude greater Thus, longer durations of treadmill exercise are not likely to be more beneficial to bone health.

The diet and exercise interventions were most impactful on tibial trabecular bone. This study showed, for the first time, that this supplemented diet increases trabecular bone volume in the proximal tibia Fig.

The response of bone to mechanical and other stimuli can be site specific Changes in trabecular, but not cortical bone after a short intervention could occur because trabecular bone metabolic rate can be higher than in cortical bone.

Longer-term exercise for 8 weeks increases cortical area 2 , and the high-speed exercise program used here may also have increased cortical area if the exercise program duration was increased.

Since exercise appeared to have no effect on mice on the supplemented diet while providing some benefits to mice on the control diet, the effects of exercise may be dependent on dietary mineral supply. Exercise appears to be most impactful when dietary mineral supply is insufficient.

High-speed exercise significantly decreased body weight by the end of the study Fig. Mice in the high-speed exercise groups did not have lower tibial bone mass or mechanical strength, as might be expected with decreased body weight 28 , Normalizing bone mass and strength by body weight did not reveal any additional insights, as no body-weight normalized properties were significantly affected by exercise data not shown.

Thus, the high-speed exercise program allowed mice to reach the same bone mass and strength at a lower body weight compared to non-exercised mice.

The supplemented diet caused significantly greater serum Ca than the control diet on day 9 and day 30 Fig. Mice exercised at both high and low speeds while fed the supplemented diet had significantly lower day 30 serum phosphorus than non-exercised mice and mice exercised while fed the control diet Fig.

Rodents subjected to treadmill exercise can have increased demand for phosphorus as well as calcium Although the supplemented diet has twice the phosphorus as the control diet, this concentration may still not be sufficient for the increased mineral demands from exercise.

Exercise did not decrease serum phosphorus for mice on the control diet, possibly due to the lower Ca:P ratio in the control diet. The elevated Ca:P ratio in the supplemented diet may be beneficial towards serum calcium, but detrimental towards serum phosphorus.

Mice were not assigned to groups of equivalent baseline serum phosphorus. Initial differences in serum phosphorous may have been a factor in the significant group differences seen on day 30, though only the exercised mice on the supplemented diet had a decrease in serum phosphorus from day 9 to day This change in bone metabolism would be expected to lead to decreased bone formation.

This decrease in growth is transient, as there are no significant differences in cortical area and trabecular bone volume in exercised mice after thirty days Figs. Mice exercised while on the supplemented diet may be reaching peak bone mass at a faster rate than non-exercised mice on the same diet.

There was no significant effect of exercise speed on any morphological or mechanical measurements of either cortical or trabecular bone. Increasing treadmill speed may not be impactful on bone if it does not lead to greater peak loads. High-speed exercise did lead to greater average loading frequency, but may not have been a great enough change to have an effect.

In the direct tibial loading model, increasing loading frequency is associated with increased bone formation rate There were some limitations to this study.

We were unable to measure the magnitude of peak strain applied to the bones during exercise. Thus, we could not determine whether increasing treadmill speed led to increased strain magnitude on the bones.

However, we measured increased mouse step rate at the higher speed of exercise, indicating an increase in strain rate was likely being applied to the bones. Future work could examine the effects of these interventions in different populations of mice at different durations of diet and exercise.

Additionally, food consumption and mouse cage activity levels were not measured, so it is not known whether the loss of body weight is due to the increase in activity or decrease in food consumption. Mice running at speeds similar to our high-speed treadmill exercise can have decreased food consumption 33 , However, all bone mass and strength measurements indicated bone mass and strength were increased and maintained, respectively.

Thus, it appears that all high-speed exercised mice received sufficient nutrient intake for bone health. This study was done in mice that run using four legs.

The results may vary in humans which run on two legs and may have different distribution of loading on the bones during running. After thirty days of high-speed treadmill exercise, mice had lower body weight, regardless of dietary mineral supply. These changes in weight did not come at the expense of tibial cortical bone mass, trabecular bone volume, or mechanical properties.

Together, these changes in bone properties and body weight suggest combining a high-speed exercise program with a mineral-supplemented diet could be best for maximizing bone mass and strength to prevent bone fractures in humans, along with additional health benefits of weight loss.

These results could lead to the design of diet and exercise interventions for weight loss without detrimental effects to bone health.

All animal procedures were done with the approval of the University of Michigan University Committee on Use and Care of Animals protocol and complied with the relevant guidelines and regulations. This study is reported in accordance with ARRIVE guidelines.

The mice were single housed, and they were fed the control diet for 2 weeks of acclimation. On day 1, at 15 weeks of age, mice were assigned to one of 7 groups— 1 a baseline group sacrificed on day 1, 2 a non-exercise group fed the control diet, 3 a non-exercise group fed the supplemented diet, 4 a low-speed exercise group fed the control diet, 5 a low-speed exercise group fed the supplemented diet, 6 a high-speed exercise group fed the control diet, and 7 a high-speed exercise group fed the supplemented diet.

Mice were divided into groups of equal mean body weight and baseline serum Ca concentration. Baseline serum Ca was measured 5 days before experiment day 1 day After 30 days of treatment s , mice from all of the experimental groups were sacrificed at 19 weeks of age, and left tibiae were harvested for analysis.

Diet formulations were the same as we have used previously 2 , 5. The control diet was an AING diet TestDiet®, Richmond, IN that was modified by adding dicalcium phosphate to contain 0. Dietary mineral amounts and Ca:P ratio were all designed to increase serum Ca by increasing passive absorption of Ca in the intestines 2 , 5 , 35 , 36 , For the control diet, the food had 3.

All other nutrients were equivalent between the diets. All exercise was performed by treadmill running during light hours Columbus Instruments, Model M, Columbus, OH. The treadmill lanes were covered such that the moving lanes were in the dark, and if mice fell off the treadmill they would be in the light.

This provided motivation to stay on the treadmill. This speed was the maximum speed mice could sustain running for 20 min without falling off the treadmill at this incline and is similar to the maximum speed mice can run at Exercise compliance was maintained by tapping the tails of mice that fell off the treadmill during exercise.

Frame-by-frame video analysis of mice running gave an estimated average frequency of 3. Cortical and trabecular bone micro-CT measurements were obtained as we have detailed 5. Whole tibiae were embedded in agarose, and scanned using a micro-CT specimen scanner µCT Scanco Medical, Bassersdorf, Switzerland.

The scan settings were voxel size of 12 μm, 70 kVp, µA, 0. Scans were analyzed using algorithms in the Scanco IPL software. For measurement of cortical geometry metrics—cortical area fraction Ct.

Ar , total cross-sectional area, cortical thickness Ct. Th , and volumetric tissue mineral density TMD , a μm thick transverse section from a standard site located The cortical bone section is located approximately at the center of the four-point bending mechanical testing region. A separate μm thick transverse section at the fracture site was analyzed for cortical geometry measurements used in calculations of tissue-level mechanical properties moment of inertia, distance from neutral axis.

Tibial scans were also analyzed for trabecular bone properties. Proximal tibial metaphyseal sections μm thick from immediately below the growth plate were analyzed using freehand traced volumes of interest. N , trabecular thickness Tb. Th , trabecular separation Tb. Sp , and TMD.

Structural- and tissue-level mechanical properties were measured in all groups as we have described previously 2 , 5. Structural-level properties force, deformation, stiffness, work were measured from a 4-point bending to failure test 3-mm inner and 9-mm outer spans.

Tibiae were loaded to failure with the medial side of the mid-diaphysis in tension under displacement control at 0. Tissue-level mechanical properties stress, strain, modulus, toughness were estimated using beam bending theory with geometric measurements moment of inertia about anterior—posterior axis, distance from centroid to medial side of the bone from micro-CT data at the fracture site Fasting blood samples taken before daily exercise were collected by submandibular vein bleeding as we have described 2.

Mice fasted at the start of light hours, and blood was collected six hours later. Blood samples were collected on day -4 baseline , day 9 after the first day of full-speed running for all exercise groups , and on day 30 the final day of exercise.

Our previous work showed significant changes in serum bone metabolism markers occur after one day of exercise, but are not maintained long-term 2. Thus, serum was examined early in the study, just after high-speed exercised mice began running at full speed for the entire exercise session.

Serum was isolated by centrifuge. Calcium and phosphorous concentrations were measured at all time points using the Calcium CPC LiquiColor test kit Stanbio Laboratory, Boerne, TX and the Phosphorus Liqui-UV kit Stanbio Laboratory. ELISAs were used to measure markers of bone formation and resorption—pro-collagen type I amino-terminal peptide PINP and carboxy-terminal collagen crosslinks CTX Immunodiagnostic Systems, Inc.

All statistical analysis was performed using Graphpad Prism. Data was checked for normality using the Shapiro—Wilk test. Outliers were removed using the GraphPad ROUT function for detecting outliers using nonlinear regression Bouxsein, M.

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McNerny, E.

In the late s, it was proposed that crystalline HA forms via the step-wise assembly of Posner's clusters.

Advances in analytical methods have led to the proposition that hydrated Posner's clusters with the formula Ca 9 PO 4 6 H 2 O 30 are the structural building blocks of ACP.

This is consistent with the fact that protonated orthophosphate species are present at the superficial surface of bone that is in contact with physiological solution, whereas unprotonated Pi species are prevalent in deep bone tissue. Several potential sites of mineral association with collagen fibrils have been identified.

As bone formation progresses, the mineral content undergoes a gradual transformation from amorphous to crystalline, resulting in HA nanocrystals. Any remaining amorphous phase may contribute toward maintaining ionic homeostasis.

This transformation has previously been spatially resolved by mapping the transformation of ACP to mature mineral in zebrafish fin rays. Acidic calcium orthophosphates, including dibasic calcium phosphate dihydrate CaHPO 4 ·2H 2 O, DCPD, also known as brushite and octacalcium phosphate Ca 8 HPO 4 2 PO 4 4 ·5H 2 O, OCP , have long been implicated as transient phases that evolve from ACP prior to the formation of HA.

Whilst intermediate phases remain difficult to detect during bone biomineralisation, several recent studies have discovered the presence of beta-tricalcium phosphate β-Ca 3 PO 4 2 , β-TCP , DCPD-like and OCP phases in the early stages of in vivo bone formation, providing some of the strongest evidence to date as to the crucial roles of transient orthophosphate phases that are part of the biologically mediated transformation of ACP to HA.

Under correctly regulated conditions, biomineralisation nearly always results in crystals that exhibit specific size, shape and molecular organisation. Biological apatite crystals are typically formed on the nanoscale.

HA nanocrystals formed in bone are described as platelets and are often assigned approximate dimensions of 50 nm × 25 nm × 3 nm that align on the c -axis parallel to the direction of collagen 72 Fig. It has been proposed that OCP, which has a triclinic crystal structure considered similar to that of bone mineral HA, may act as a template during crystal growth.

Bone has both high strength and toughness, while its organic components are soft and ductile and inorganic mineral is considerably stiff and brittle. Biomacromolecules are believed to play crucial roles in enacting spatial and temporal control over the formation of mineral at the molecular scale.

The precipitation of calcium and orthophosphate ions from supersaturated solution in the absence of biomacromolecules generally results in crystals of considerable size.

In the physiological environment, however, nanocrystals may well be more kinetically stable whilst also being of ideal size to form interactions with protein structures and other physiological molecules. Bone possesses a sufficient concentration of NCPs, many of which are phosphoproteins, which may act to both control crystal growth and facilitate interfacial interaction between collagen and HA.

Taken together, these different NCPs have been implicated in playing a highly orchestrated role in biomineralisation by means of promoting, inhibiting and regulating crystal growth. Expectedly, elemental phosphorus is a prominent component of each of these mineralised human tissues Whilst not a substituting component of the HA lattice, normal mineralised tissue also contains a small proportion of PPi 0.

In the context of calcium oxalate deposition, pyrophosphate is known to act as a nucleation and crystal growth inhibitor. It is likely both pathways are important and pyrophosphate species certainly have a significant and critical role to play in the mineralisation of hard tissues nonetheless.

Alkaline phosphatase ALP is an enzyme responsible for dephosphorylating biomolecules. It is found in a wide range of organisms with the same general function but in different structural forms to suit specific environments.

TNAP plays a crucial role promoting mineralisation of the extracellular matrix by restricting the concentration of PPi. Mutations in the gene encoding TNAP cause hypophosphatasia, an inheritable form of rickets and osteomalacia. Thus, PPi can act as both a local inhibitor and source of orthophosphate to drive mineralisation.

As well as acting via inorganic pathways, PPi has been demonstrated to influence biomolecular mineralisation pathways. For example, PPi has been shown to upregulate the NCP osteopontin OPN , found mainly in the bone matrix.

OPN is also present in other cell types, such as hypertrophic chondrocytes, odontoblasts, cementoblasts, macrophages, as well as endothelial, smooth muscle and epithelial cells.

At the age of 4—6 months, OPN deficient mice had twice the volume of trabecular bone and three times the number of osteoclasts compared to wild type mice. Additionally, OPN deficient mice were more resistant to ovariectomy-induced bone resorption.

Studies have shown that TNAP activity increases in response to PPi. Dense polyP granules are found in many cell types, particularly within the mitochondria. This includes osteoblasts, where polyP is found in high concentrations 0. Biological reactions involving polyP can be grouped into two categories: chain-lengthening and chain-shortening.

Chain-lengthening reactions are catalysed by the polyP kinase family of enzymes. These enzymes can polymerise orthophosphate to form polyP, or take phosphate residues from other molecules and add them to the polyP chain, for example from ATP. Indeed, polyP has been shown to increase extracellular ATP and ADP generation in SaOS-2 osteoblast-like cells.

In addition to its role as a substrate, polyP has also been shown to have a direct influence on biomineralisation pathways by inducing the expression of bone formation markers OPN, OC and ALP, leading to the mineralisation of pre-osteoblast MC3T3 cells.

As well as a role in bone formation, polyP has been shown to increase cartilage production, but for a short time before it is degraded by the polyphosphatases released by chondrocytes.

An integral role of polyP in bone remodelling has been proposed. Enzymes in the osteoclast then polymerise the orthophosphate into polyP. The calcium—polyP complex is left behind by the osteoclast in the bone resorption pit, or transferred through the extracellular space either freely or in vesicle compartments to a previous resorption pit.

The calcium—polyP complex can then be used as a substrate by osteoblasts to produce bone mineral. This general idea outlines the importance of polyP as both a substrate to be broken down, as well as a molecule capable of preventing mineral formation, but in doing so facilitates high concentrations of calcium and phosphate to coexist.

However, this mechanism is somewhat speculative. The enzymes associated with osteoblasts, such as ALP, used to produce orthophosphate are well researched, , as are the enzymes and processes used by osteoclasts to break down apatite and resorb bone.

One of these enzymes has also been found in Dictyostelium discoideum , a unicellular eukaryote. Nevertheless, the enzyme has not been found in mammalian cells to date, therefore the assumption that such an enzyme exists in mammalian osteoclasts remains unconfirmed.

This mechanism also fits with several current theories of bone formation. Recently, in addition to ACP, amorphous calcium carbonate ACC has been proposed as another pre-curser to bone mineral. Furthermore, calcium—polyP may lead to the formation of ACP when enzymatically degraded, which over time may form HA.

Another theory suggests that the calcium—polyP complex could directly infiltrate the collagen matrix by capillary action before being be broken down in situ by osteoblast-secreted enzymes to form HA. Alternatively, the polyP can be stored in and utilised by either osteoblasts or vesicles.

Condensed linear phosphates are becoming increasingly recognised as multifunctional species with important physiological roles. Most notable are the ability of phosphate polymers to stimulate osteogenic markers, regulate the localised concentrations of other phosphate species in a given biological microenvironment, increase the levels of orthophosphate when broken down and antagonise biological crystallisation processes Fig.

View PDF Version Previous Article Next Article. DOI: B , , 7 , Received 16th September , Accepted 29th October Abstract Humans utilise biomineralisation in the formation of bone and teeth.

Physiological serum concentrations and p K a values of orthophosphate in vivo. The mono- and di-protonated forms are by far the most common at physiological pH, temperature and salt concentrations, consistent with a p K a value of 7.

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Engineering Fellowship. I would also like to thank Erin McNerny and Joe Gardinier for their guidance and assistance. The University of Michigan, N University Ave.

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Calcium and phosphorus supplemented diet increases bone volume after thirty days of high speed treadmill exercise in adult mice. Sci Rep 12 , Download citation. Received : 10 March Accepted : 23 August Published : 26 August Anyone you share the following link with will be able to read this content:.

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nature scientific reports articles article. Download PDF. Subjects Biomedical engineering Bone Preclinical research. Abstract Weight-bearing exercise increases bone mass and strength. Figure 1. Full size image. Figure 2. Figure 3. Figure 4. Figure 5.

Figure 6. Figure 7. Discussion After thirty days of interventions, diet had a greater impact on bone mass and strength than exercise Figs. Methods Animals and treatments All animal procedures were done with the approval of the University of Michigan University Committee on Use and Care of Animals protocol and complied with the relevant guidelines and regulations.

Diets and exercise program Diet formulations were the same as we have used previously 2 , 5. Cortical geometry and trabecular architecture measurements Cortical and trabecular bone micro-CT measurements were obtained as we have detailed 5. Mechanical testing Structural- and tissue-level mechanical properties were measured in all groups as we have described previously 2 , 5.

Serum analysis Fasting blood samples taken before daily exercise were collected by submandibular vein bleeding as we have described 2. Statistical analysis All statistical analysis was performed using Graphpad Prism.

Data availability All data is posted in the manuscript figures and supplemental files. References Bouxsein, M. Article PubMed Google Scholar Friedman, M. Article CAS PubMed PubMed Central Google Scholar Gardinier, J. Article CAS PubMed Google Scholar McNerny, E. Article CAS PubMed PubMed Central Google Scholar Friedman, M.

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Acknowledgements This work was supported by the NIH award RAR and the National GEM Consortium Ph. Author information Authors and Affiliations The University of Michigan, N University Ave.

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