Bone 68 (2014) 85–91

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Original Full Length Article

Peak bone strength is influenced by calcium intake in growing rats S. Viguet-Carrin a,⁎,1, M. Hoppler a,2, F. Membrez Scalfo a, J. Vuichoud a, M. Vigo a, E.A. Offord a, P. Ammann b a b

Centre de Recherche Nestlé, Vers-chez-les-Blanc, 1000 Lausanne 26, Switzerland Service des Maladies Osseuses, Hôpital Universitaire de Genève, Genève, Switzerland

a r t i c l e

i n f o

Article history: Received 25 November 2013 Revised 21 July 2014 Accepted 25 July 2014 Available online 4 August 2014 Edited by: David Fyhrie Keywords: Calcium Bone strength Bone microarchitecture Bone material properties Growing rat Bone turnover

a b s t r a c t In this study we investigated the effect of supplementing the diet of the growing male rat with different levels of calcium (from low to higher than recommended intakes at constant Ca/P ratio), on multiple factors (bone mass, strength, size, geometry, material properties, turnover) influencing bone strength during the bone accrual period. Rats, age 28 days were supplemented for 4 weeks with high Ca (1.2%), adequate Ca (0.5%) or low Ca level (0.2%). Bone metabolism and structural parameters were measured. No changes in body weight or food intake were observed among the groups. As anticipated, compared to the adequate Ca intake, low-Ca intake had a detrimental impact on bone growth (33.63 vs. 33.68 mm), bone strength (−19.7% for failure load), bone architecture (−58% for BV/TV) and peak bone mass accrual (−29% for BMD) due to the hormonal disruption implied in Ca metabolism. In contrast, novel, surprising results were observed in that higher than adequate Ca intake resulted in improved peak bone strength (106 vs. 184 N/mm for the stiffness and 61 vs. 89 N for the failure load) and bone material properties (467 vs. 514 mPa for tissue hardness) but these effects were not accompanied by changes in bone mass, size, microarchitecture or bone turnover. Hormonal factors, IGF-I and bone modeling were also evaluated. Compared to the adequate level of Ca, IGF-I level was significantly lower in the low-Ca intake group and significantly higher in the high-Ca intake group. No detrimental effects of high Ca were observed on bone modeling (assessed by histomorphometry and bone markers), at least in this short-term intervention. In conclusion, the decrease in failure load in the low calcium group can be explained by the change in bone geometry and bone mass parameters. Thus, improvements in mechanical properties can be explained by the improved quality of intrinsic bone tissue as shown by nanoindentation. These results suggest that supplemental Ca may be beneficial for the attainment of peak bone strength and that multiple factors linked to bone mass and strength should be taken into account when setting dietary levels of adequate mineral intake to support optimal peak bone mass acquisition. © 2014 Elsevier Inc. All rights reserved.

Introduction During growth it is important to maximize peak bone mass and strength to reduce the risk of osteoporosis later in life. A low calcium (Ca) intake in children and animals impairs bone mass acquisition whereas the influence of high Ca intake on bone mass remains unclear particularly during periods of bone mineral accrual. Furthermore, although several studies have already investigated the effect of various dietary Ca levels on bone metabolism in growing animals, in most of them, the Ca/P ratio was not kept constant across the different groups which compromised the analysis of the role of calcium intake on bone

⁎ Corresponding author at: CHUV, Rue du Bugnon, 46, 1011 Lausanne, Switzerland. Fax: + 41 21 314 01 37. E-mail address: [email protected] (S. Viguet-Carrin). 1 CHUV, Route du Bugnon, 46, 1011 Lausanne, Switzerland (current address). 2 Roche, Kirchbodenstrasse 64, 8800 Thalwil, Switzerland (current address).

http://dx.doi.org/10.1016/j.bone.2014.07.029 8756-3282/© 2014 Elsevier Inc. All rights reserved.

growth. Indeed, it is important to keep a constant Ca/P ratio to avoid the induction of a secondary hyperparathyroidism. Epidemiological studies have shown an association between a reduced Ca/P intake with a lower BMD [1] and an excess intake of Ca without appropriate phosphorus supplementation with reduced BMD in post-menopausal women [2]. Furthermore, high dietary phosphate intake reduces bone strength and impairs bone growth in growing rats even when Ca intake is sufficient [3,4]. This effect is at least partly mediated by PTH. Thus it can be concluded that adequate Ca/P balance is important to support optimal peak bone mass development. Studies in children and adolescents during periods of bone accrual have shown that supplementation in Ca (1000–1200 mg/day) above the usual dietary intakes (600–800 mg/day depending on studies) enhances their bone mineral content (BMC) and areal bone mineral density (BMD) gain [5–12]. Recently, a meta-analysis of 19 randomized controlled trials including 2859 children aged 3 to 18 years, showed a positive effect of Ca supplementation, with doses ranging between 300 and 1200 mg/day, on total body BMC and upper limb BMD with

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an effect-size of 0.14 standard deviation for both [13]. However, the effect of increasing Ca intake above the usual dietary intakes has been less investigated on bone turnover or bone geometry in children and in adolescents. The effect of high Ca intake on bone formation and bone resorption markers is still controversial: Dibba & Jonhston [6, 7] reported that supplementation in Ca decreased bone formation whereas Rozen et al. [12] showed no change in osteocalcin level but a significant increase in bone-specific alkaline phosphatase concentrations in girls aged 14 years. Furthermore in prepubertal children, high Ca intake had no effect on bone resorption [7,12,14]. More recently, thanks to the development of new imaging techniques such as pQCT, Matkovic et al. [15] found beneficial changes on the geometry of the proximal radius of adolescent Caucasian girls. Girls who were supplemented in Ca had a slightly higher cortical to total area ratio compared to the low Ca intake group. Thus, Ca exerted its action on bone accretion during growth primarily by influencing volumetric bone mineral density and consequently bone strength. Thus, all these data showed that the BMD and also several bone quality parameters respond to Ca intake. Regulation of peak bone mass acquisition is largely an endocrine process. The growth spurt is paralleled by a rapid increase in circulating IGF-I, during the period when peak calcium accretion occurs. IGF-I acts in an autocrine/paracrine fashion to increase bone formation and it is also important for linear and appositional bone growth, bone mineralization and increase in cortical thickness at puberty in human and rodents [16]. Low plasma IGF-I levels are associated with increased fracture risk [17,18]. Maximal strength is determined by bone microarchitecture, geometry, and material level properties. Consequently, any animal model used to analyze the effect of Ca intake on bone strength during growth needs to take these multiple parameters into account. Since calcium is an important contributor to bone strength [19], the purpose of this study was to test the effect of Ca at different doses (low, adequate, higher than recommended dietary intakes but remaining in physiological conditions with a constant Ca/P ratio) on these multiple factors affecting bone strength in young growing male rats and to understand to what extent higher than recommended Ca intakes was beneficial. Moreover, the effect of Ca restriction on these different parameters was also re-evaluated.

in saline-soaked gauze and stored at − 80 °C until testing for physical measurements (bone mineral density, microarchitecture and mechanical testing).

Material and methods

Nanoindentation After sacrifice, cortical diaphysal femur bones were tested using a nanoindentation technique for bone material level properties. The same rat cortical femur bones were used for mechanical testing and the measurements of bone material level properties were performed close to the site of rupture around 5 mm above the condyles in order to preserve bone mechanical properties. Bones were embedded in polymethylmethacrylate and cut. Modulus, tissue hardness and working energy of the bone were determined on hydrated bone tissue samples. A nano-hardness tester (NHT, CSM Instruments, Peseux, Switzerland) was used as follows; force–displacement data of a pyramidal diamond indenter that is pressed into a material are recorded as previously described [21]. Briefly, the mechanical tests included five indentations in the cortical compartment. Indents were set to a 900 nm depth with an approximate speed of 76 mN/min for both loading and unloading. Full rehydration occurs at this distance and is necessary for the nanoindenter of less than 1 μm from the surface of the sample and is stable for up to a period of 60 h. At maximum load, a 5 s holding period was applied. The limit of the maximal allowable thermal drift was set to 0.1 nm/s.

Animal study The study was carried out according to a protocol approved by the Cantonal Veterinary Office of Switzerland. Three groups of 10 weaning 21-day-old male Sprague–Dawley rats (tolerance ± 3 days) were purchased from Charles River (France). The rats were housed individually under ambient conditions of 21 °C with relative humidity of 55% and under a 12 h:12 h light/dark cycle. Animals were allowed to acclimatize to the local environment for 1 week before study initiation. The rats were randomized at day 0 (D0) according to the body weight before allocating the rats in the 3 diet groups. All animals had access to diet (Kliba Nafag, Basel, Switzerland) and deionized sterile water ad libitum throughout the 4 week feeding study. Rats were fed an AIN-93G diet containing either high Ca (HCa: 1.2%), adequate Ca (ACa: 0.5%) or low Ca (LCa: 0.2%) levels. The molar Ca/P ratio remained constant (1.3) between the 3 different AIN-93G diets (Reeves et al. [20]). Food intake and body weight were measured three times weekly. At day 25 and day 28 before sacrifice, calcein (Sigma Aldrich), a fluorescent marker for bone labeling was injected intravenously (10 mg/kg body weight) into the rats. On day 30 (D30), animals were euthanized by isofloran inhalation and body length (nose–anus length) was measured. Blood samples were collected and stored at − 80 °C until biochemical analysis. Femurs were separated from adjacent tissue, cleaned, and wrapped

Bone measurements Bone imaging Bone mineral density (BMD) of the whole femur was assessed by dual-energy X-ray absorptiometry (PIXImus2; GE-Lunar, Madison, WI). Furthermore, bone microarchitecture was analyzed by microcomputed tomography (μCT 20; Scanco Medical AG, Brüttisellen, Switzerland) with a 16 μm resolution. The following variables were measured: cortical thickness (Ct.Th; mm) measured at the diaphysis and the trabecular bone volume fraction (BV/TV, %), trabecular number (Tb.N #), thickness (Tb.Th, μm) and separation (Tb.Sp. μm) were quantified in the distal metaphysis femoral bone. In order to be in the secondary spongiosa, we chose an area of 0.8 mm (or 50 slices) above the growth plate. For the midshaft femur scans were 29 slices (or 0.464 mm) from the middle of the femur (total length divided by 2) and we chose this area as this was comparable to where biomechanical testing in a three point bending test would take place. The cortical area and the total area and the mineralization densities (mg HA/cm3) were also assessed by microcomputed tomography. Femoral length and 3-point bending mechanical testing The length of the femur and the diameter of the femoral diaphysis were measured with a digital caliper. The midline of the shaft was determined and a line was drawn using a marker at this point. Femoral failure load was determined using a three-point bending test using a servocontrolled electro-mechanical system (Instron 1114; Instron Corp., High Wycombe, UK) with the actuator displaced at 2 mm/min. The direction of loading for the femoral 3-point bending tests was done in the posterior–anterior axis. Both displacement and load were recorded. Mechanical properties including femoral maximal failure load (N), stiffness (N/mm), deflection (mm), yield (N) and post yield load (N), failure load (N), post yield deformation (mm), elastic energy (N · mm), plastic energy (N · mm) and total energy (N · mm), were determined from the load–displacement curve. The post yield load is obtained by subtraction of the yield load to the failure load.

Dynamic histomorphometry Histomorphometry was carried out on the tibia. Bones were dehydrated with a sequential change of ethanol and then infiltrated and embedded without decalcification in methylmethacrylate. A longitudinal section of the tibia was cut at a thickness of 8 μm and dynamic

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histomorphometry was performed. The measurements were performed in the metaphysic below the growth plate. The selected quadrants were in the trabecular part and the measurement was done on the endosteal part. The following parameters were performed: the mineral apposition rate (MAR; μm/day) the mineralizing surface (MS/BS; %) and the bone formation rate (BFR/BS; μm3/μm2 per year). All nomenclature and calculations of histomorphometric indices were done to Parfitt et al. [22]. Biomarkers assays C-terminal telopeptide of type I collagen (RatLaps CTX-I; IDS Ltd, UK), insulin-like growth factor I (Octeia Rat/Mouse IGF-I; IDS Ltd, UK) and serum intact PTH (Immutopics, San Clemente, CA) concentrations were measured in duplicate in serum following the kit manufacturer's protocol. The minerals calcium and phosphate were quantified by inductively coupled plasma optical emission spectrometry (ICP-OES Vista-MPX, Varian, Australia). Samples were mineralized with a HNO3/ H2O2 mixture in a microwave digestion system (Mars microwave digestion system, CEM, USA) and quantified by using commercial standards (Titrisol, Merck) for external calibration. Statistical analysis Results are expressed as mean ± standard deviation, unless otherwise stated. For each parameter, Kruskal–Wallis test was first performed. If it indicated a significant difference among groups (p b 0.05), the Mann–Whitney U test was used to determine specific differences. Associations between the two parameters were analyzed by using Spearman correlations. The level of significance was set at p b 0.05 for all statistical tests. All data were analyzed using Statview software. Results Body weight and diets The animals continued growing throughout the experimental period, and the same pattern of body weight evolution was observed in each age group between day 0 (LCa: 103 ± 5 g, ACa: 105 ± 6 g, HCa: 105 ± 4 g) and day 30 (LCa: 293 ± 24 g, ACa: 294 ± 23 g, HCa: 297 ± 18 g). Thus, calcium levels did not influence the body weights of the animals. At baseline (D0), 1-month-old male rats had significantly lower body weight (p b 0.001) than 2-month-old animals (D30). The food intake remained constant between the 3 different groups until the end of the study (p = 0.09). The cumulative food intake was unaffected by the dietary restriction of Ca (LCa: 528 ± 60 g, ACa: 513 ± 64 g, HCa: 539 ± 66 g). Bone size Femur length was similar between the HCa and ACa groups but it slightly decreased at D30 in the Ca-restricted group (p = 0.003; Table 1). Moreover, the diameter of the femur was slightly smaller but not significant in the LCa group compared to the ACa and HCa groups (p = 0.07; Table 1). However, the body length and diameter of the femur were similar within groups (Table 1).

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Bone strength Femoral strength was assessed and reported as femoral failure load. Low Ca intake significantly decreased this parameter (− 15%; p = 0.0001) and also the stiffness (− 20%; p b 0.0001) and plastic energy (−30%; p = 0,003) and total energy (−25%; p = 0,004) at D30 compared to the adequate and high Ca group (Fig. 1). Furthermore, consumption of high Ca improved bone strength compare to the adequate Ca group (failure load +30% p = 0.0009; stiffness +67% p = 0.0002). The yield and post-yield load were increased compared to adequate Ca (yield +66% p = 0.02; post-yield load +33% p = 0.0004) (Table 2). Nanoindentation All parameters measured by nanoindentation were significantly decreased by a low Ca diet when compared to adequate and high Ca diets (Table 3). This included modulus (decreased by N 1.7 GPa; p b 0.0001), tissue hardness (decreased by N103 MPa; p b 0.0001) and the working energy (decreased by N 415 mN nm; p b 0.0001). Interestingly, in the high Ca, the tissue hardness was increased by 35 MPa compared to the adequate Ca (p b 0.0001). BMD and bone microarchitecture The deficiency in Ca affected the BMD and the mineralization densities particularly in the Ca-restricted group (p b 0.0001; Table 4). The mineralization density is lower in the ACa group compared to the HCa group (p = 0.009; Table 4). Moreover, after 4 weeks, the Ca restricted diet induced an alteration of the bone microarchitecture as well as in the cortical bone and in the trabecular bone tissue. The diameters of the femurs were also slightly smaller but not significant in the LCa group compared to the ACa and HCa groups (Table 1). The cortical thickness was significantly reduced in the Ca-restricted group (p b 0.0001) compared to the other groups. Indeed, after 4 weeks of supplementation, the BV/TV (p = 0.0005), Tb.N (p = 0.03), and Tb.Th (p = 0.0003) were significantly lower whereas the Tb.Sp (p = 0.04) was higher in the Carestricted group (Table 4 and Fig. 2) compared to the other groups. In contrast, high Ca intake for 4 weeks during growth did not alter BMD. Furthermore, bone microarchitecture evaluated on cortical and trabecular bone in the high Ca group was not modified compared to the ACa group. Calcium metabolism and bone modeling Plasma concentrations of Ca and phosphate were quantified at D30. We found that the concentration of P remained constant within 3 Ca groups (p = 0.31; Table 5). In contrast, Ca concentrations (p = 0.003) in serum were different between groups (Table 5), being lower in the Ca-restricted group. The level of intact PTH was quantified in serum in order to control for hyperparathyroidism. The level of PTH between the 3 groups of Ca was in the same range after 4 weeks of dietary Ca intake although the rats fed with low Ca showed a trend to increased PTH levels (Table 5). The IGF-I level which is a marker of longitudinal growth, decreased in the adequate Ca and the Ca-restricted group compared to the high calcium group (p = 0.008; Table 5). Furthermore, the

Table 1 Body length, femur length and femur diameter measured on rats fed either low Ca diet (0.2%), adequate Ca (0.5%) or high Ca (1%). Data are presented as mean ± SD.

Body length (cm) Femur length (mm) Femur diameter (mm) a b

Low Ca

Adequate Ca

High Ca

Kruskal–Wallis test

22.75 ± 0.79 32.78 ± 0.52a, b 3.79 ± 0.17

22.65 ± 0.53 33.66 ± 0.60 4.03 ± 0.26

22.30 ± 0.48 33.93 ± 0.74 3.94 ± 0.15

p = 0.31 p = 0.003 p = 0.07

Statistically significant difference compared with high Ca (p b 0.001). Statistically significant difference compared with adequate Ca (p b 0.001).

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A

**

200

Table 3 Effects of Ca intake on cortical bone material level properties (nanoindentation).

Stiffness (n/mm)

175

Low Ca

125

*, **

Modulus (gPa) Tissue hardness (mPa) Working energy (mN ∗ nm)

100 75 50 25 0

Failure Load (N)

B

Total energy (N·mm)

Low Ca

Adequate Ca

High Ca

**

100 80 60

*, **

40 20 0

C

Adequate Ca

High Ca

Kruskal– Wallis test

150

Low Ca

Adequate Ca

High Ca

3153 ± 439a, b

3368 ± 347

3796 ± 591

p b 0.0001

Data are presented as mean ± SD. a Statistically significant difference compared with high Ca (p b 0.03). b Statistically significant difference compared with adequate Ca (p b 0.003).

and mineralization were evaluated by dynamic histomorphometry (Table 6); we found that the MAR of the low calcium group was approximately 13% less than the other diet groups. The BFR of the Ca-restricted group was only 66% that of the ACa and high Ca diet groups. The mineralizing surface (MS/BS) was also significantly lower in the Carestricted group (around − 34%) which may suggest an increase of bone resorption. Nevertheless, rats fed with a high Ca diet had no change on bone resorption and bone formation compared to the animals fed with adequate Ca levels. Discussion Dietary intakes of Ca are set for humans or rodents based on growth curve data. The golden standard measurement for bone health is bone mineral density (BMD). However, many factors contribute to the attainment of maximum peak bone mass and strength during the bone accrual period, which may respond differently to Ca levels. The objective of this study was to evaluate an extensive battery of bone metabolic and structural properties in the growing rat in response to low (0.2%), adequate (0.5%) and high (1.2%) intakes of Ca with the aim of understanding which parameters were most affected by Ca levels and whether higher than adequate levels of Ca were beneficial or not for the achievement of peak bone mass and strength.

50 40

*, ** 30 20 10 0

8.35 ± 1.15a, b 10.06 ± 1.47 10.88 ± 1.54 p b 0.0001 481 ± 93 516 ± 78b p b 0.0001 378 ± 61a, b

Justification of the choice of Ca levels Low Ca

Adequate Ca

High Ca

Fig. 1. Stiffness (A), failure load (B) and total energy (C) were analyzed on femur bones from rats fed with different amounts of calcium (low Ca (0.2%), adequate Ca (0.5%) and high Ca diets) for 4 weeks. Values are mean and SD. ⁎Statistically significant difference compared with high Ca (p b 0.05). ⁎⁎Statistically significant difference compared with adequate Ca (p b 0.05).

high Ca intake for 4 weeks also resulted in a higher IGF-I level compared to the adequate Ca group (increased by 214 ng/ml; p b 0.01). A higher level of CTX-I (LCa: 86.90 ± 11.50 ng/ml, MCa: 53.48 ± 13.72 ng/ml, HCa: 55.60 ± 13.82 ng/ml; p = 0.0006) was seen in the Ca-restricted group compared to the adequate and high Ca, which may suggest an increase of bone resorption (Table 5). Bone formation

Adequate Ca (0.5%) was based on the recommended intake of Ca for growing rats while the deficient Ca diet (0.2%) contained approximately 40% of the required amount, which was previously demonstrated to result in a compromised skeleton. The deficient diet was roughly equivalent to an adolescent consuming a 520 mg Ca/diet, which is within the range expected for children who do not consume supplemental Ca or Ca-fortified foods and sparse dairy products [23]. The high level of dietary calcium representing two-fold of the required amount was selected because it was frequently used in standard commercial rat chow diets (containing 1.2 to 1.5% Ca). In the present study, median body-weight gain by young growing rats and food intake was unaffected by dietary Ca concentration. This finding was in agreement with those of other studies that have examined the effect of dietary Ca on

Table 2 Effects of Ca intake on bone strength (3-point bending test). Low Ca Yield (N) Post yield load (N) Failure load (N) Post yield deformation (mm) Deflection (mm) Plastic energy (N ∗ mm) Elastic energy (N ∗ mm)

33.68 18.10 51.78 0.460 0.91 19.8 7.1

± ± ± ± ± ± ±

9.38a 4.32a 3.01a, b 0.042a, b 0.16 5.0a, b 2.7

Data are presented as mean ± SD. a Statistically significant difference compared with high Ca (p b 0.05). b Statistically significant difference compared with adequate Ca (p b 0.05).

Adequate Ca

High Ca

37.62 23.05 60.67 0.538 0.92 27.2 7.6

55.12 30.63 85.75 0.390 0.84 31 8.2

± ± ± ± ± ± ±

13.51 9.10 2.82 0.050 0.09 6.0 5.0

± ± ± ± ± ± ±

Kruskal–Wallis test 10.16b 6.96b 3.69b 0.027b 0.22 10.0 5.0

p p p p p p p

= = = = = = =

0.002 0.002 0.001 0.001 0.33 0.003 0.91

S. Viguet-Carrin et al. / Bone 68 (2014) 85–91 Table 4 Effect of Ca intake on bone mass, mineralization densities and microarchitecture parameters measured respectively by DEXA and microCT on femur bones. Values are mean ± SD. Low Ca Bone mass BMC (g) BMD (g/cm2)

Adequate Ca

High Ca

Kruskal– Wallis test

0.195 ± 0.017a,b 0.299 ± 0.036 0.303 ± 0.025 p b 0.0001 0.089 ± 0.007a,b 0.126 ± 0.007 0.132 ± 0.006 p b 0.0001

Mineralization densities 1187 ± 42a,b Density (mg HA/cm3)

1250 ± 26

1273 ± 13b

p b 0.0001

Cortical parameters Cortical area 1.649 ± 0.098a,b 2.224 ± 0.201 2.173 ± 0.101 p b 0.0001 Total area 3.902 ± 0.178 4.217 ± 0.458 4.054 ± 0.341 p = 0.10 Ct. Th (mm) 0.28 ± 0.03a,b 0.45 ± 0.03 0.47 ± 0.04 p b 0.0001 Trabecular microarchitecture 14.86 ± 1.09 14.59 ± 2.01 p BV/TV (%) 6.11 ± 1.62a,b 3.37 ± 0.35 3.47 ± 0.49 p Tb.N (#) 2.79 ± 0.44a,b Tb.Th (mm) 0.041 ± 0.004a,b 0.058 ± 0.004 0.056 ± 0.003 p a,b 0.30 ± 0.04 0.30 ± 0.05 p Tb.Sp (mm) 0.38 ± 0.05 a b

= = = =

0.0005 0.03 0.0003 0.04

Statistically significant difference compared with high Ca (p b 0.01). Statistically significant difference compared with adequate Ca (p b 0.05).

weight gain in the rat [24–30]. It is known that either a high Ca/P ratio or a low Ca/P ratio in the diet induces hyperparathyroidism. Previous studies showed that inadequate Ca:P ratios in growing rats had a negative impact on bone growth and also on bone strength [3,4]. Consequently, this study used the standard molar Ca/P ratio from the AIN-G93 diet [20] which is in the appropriate range for bone development in rats. Effect of Ca intake and “peak bone strength” Bone strength is defined by several determinants characterized by bone mass and bone quality. Bone quality is a composite of properties that make bone resist fracture, such as its geometry, its microarchitecture, the quality/quantity of type I collagen and crosslinks, the quality of mineral phase and bone turnover characterizing the bone material level properties. In this study, a low Ca intake led to significant impairment in bone length and bone growth (Table 1). Although bone diameter measured with the caliper did not change (Table 1) the cortical thickness (Table 4) measured by microCT which is a more reliable technique was reduced which may be explained by a decrease of bone modeling and bone formation (Tables 5, 6 and Fig. 2). The outer diameter is the most important determinant with respect to the cortical thickness on bone mechanical properties particularly for the failure load and stiffness. Indeed, the outer diameter of a long bone has been demonstrated to predict up to 55% of the variation in bone strength [31] and individuals with thinner

Low calcium

89

Table 5 Effect of Ca intake on serum biochemical analyses after 4 weeks of feeding. Serum concentrations of calcium, phosphate, PTH, IGF-I, CTX-I and osteocalcin in rats fed either low Ca diet (0.2%), adequate Ca (0.5%) or high Ca (1%). Data are mean ± SD.

Ca (ng/ml) P (ng/ml) PTH (pg/ml) IGF-I (ng/ml) CTX-I (ng/ml) a b

Low Ca

Adequate Ca

High Ca

Kruskal– Wallis test

99.30 ± 2.18a 138.88 ± 9.36 385.43 ± 117.22

102.34 ± 4.56 104.16 ± 2.31 p = 0.003 135.18 ± 11.10 142.92 ± 10.90 p = 0.31 276.58 ± 113.36 278.17 ± 77.14 p = 0.08

1097 ± 190a

1168 ± 194

1382 ± 175b

86.90 ± 11.50a,b

53.48 ± 13.72

55.60 ± 13.82 p = 0.0006

p = 0.008

Statistically significant difference compared with high Ca (p b 0.001). Statistically significant difference compared with adequate Ca (p b 0.01).

cortices are more prone to fracture [32,33]. During growth, the diameter of bones increases, which increases their strength, despite losses of bone mass from the endocortical surface that are greater than the bone deposited on the periosteal surface. In male mice, the wider bone geometry resulted from a net increase in periosteal bone formation during early puberty [34] similar to humans [32,35]. Thus, the cortical bone increases in cross-sectional area with age, increasing the biomechanical strength [36]. Consequently, impairment in bone geometry and the decrease of BMD explain the decrease in bone strength in the Ca-restricted group. Indeed, as shown on Fig. 1, the reduced stiffness, failure load and energy (plastic energy and the total energy to work to fracture) for the bones of Ca-restricted rats imply they are more elastic than those from the adequate and high Ca intake groups. The higher stiffness, failure load, post yield load and the energy and particularly the plastic energy may reflect an increase of the capacity to absorb energy. Moreover, the mineralization densities are lower in the Ca-restricted group and higher in the high Ca intake groups compared to the adequate Ca group. These observations suggest that the Ca intake influenced the mineralization of bone tissue during the skeletal development potentially and at least can partially explain bone tissue material level properties. Interestingly, the bones of the high-Ca fed rats had a greater stiffness, ultimate load, yield, post yield load and mineralization density compared to the adequate Ca intake. In conclusion, the high Ca intake has a favorable impact on peak bone strength acquisition. Effect of Ca intake on bone material level properties The bone material level properties are also an important determinant. Thus, the material properties such as the mineral composition, the shape of crystals and the amount of type I collagen crosslinks may

Adequate calcium

High Calcium

Fig. 2. 3-D microCT reconstructions of images of cortical bone from mid-diaphysis femur and trabecular bone in distal femur. Parameters of mass and microarchitecture of femurs were investigated using microtomographic histomorphometry with high-resolution microCT system (microCT 40; Scanco Switzerland). 3-D images were acquired with a voxel size of 20 μm. Images show rat bones receiving low-Ca (0.2%) (A), adequate Ca (0.5%) (B) and high Ca (C) diets for 4 weeks.

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Table 6 Effect of Ca intake on dynamic histomorphometric parameters of the cortical part of the tibia. The mineral apposition rate (MAR), the mineralizing surface (MS/BS) and the bone formation rate (BFR) were assessed ex vivo at 8 weeks of age. Values are mean ± SD. Low Ca

Adequate Ca

High Ca

Kruskal– Wallis test

1.54 ± 0.12 1.57 ± 0.06 p = 0.0002 MAR (μm/day) 1.34 ± 0.12a,b MS/BS (%) 23.73 ± 4.40a,b 30.82 ± 5.36 30.12 ± 6.14 p = 0.01 3 2 a,b 116 ± 22 174 ± 39 189 ± 29 p = 0.003 BFR (μm /μm /year) a b

Statistically significant difference compared with high Ca (p b 0.01). Statistically significant difference compared with adequate Ca (p b 0.05).

also contribute to bone strength. These bone material level properties may be modulated by Ca intake, which in turn influences bone mechanical properties. Indeed, the result of nanoindentation analyses that reflect the bone material level properties showed that elastic modulus, hardness and working energy were lower in the low-Ca group compared to the adequate Ca group (Table 3). These results suggest that low Ca impacts bone strength not only via the bone structure but also the quality of the bone material. As expected, in the high Ca group, there was an increase in tissue hardness compared to the adequate Ca group. In contrast, there was no macroscopical change in bone geometry (femur length, diameter). Thus, the mineralization densities are higher in the high Ca intake groups compared to the adequate Ca group. These observations suggest that the Ca intake influenced the mineralization of bone tissue and can potentially explain the improvement of bone tissue material level properties. Thus, improvements in mechanical properties in rats fed a high Ca diet may be explained by the improvement of bone material level properties. Effect of Ca intake on bone architecture Mechanical testing was not performed on trabecular bone of the proximal tibia since in growing rats, the growth plate can interfere with the interpretation of mechanical parameter values. However, the microarchitectural properties were evaluated on both cortical and trabecular bones (Table 4 and Fig. 2). Besides bone mass, the different key determinants of mechanical properties were negatively affected by the low Ca diet, especially BV, TV, and Ct.Th on cortical bone and a decrease in Tb.Th and Tb.N and an increase in Tb.Sp. These data confirmed those previously published [24–26,29,30,37]. In contrast, in the animals fed with a high Ca diet, there was no effect on these microarchitectural parameters. Effect of Ca intake on regulatory mediators of bone modeling and metabolism Previous studies in mice have shown that IGF-I plays a critical role in the development of the growing skeleton by establishing both longitudinal and transverse bone accrual [16,38–42]. Courtland et al. [43] described that the depletion of IGF-I during post-natal stage and puberty in mice resulted in a decrease in both trabecular and cortical architecture and also decreased robustness [44]. In the present study, dietary Ca restriction reduced IGF-I level; this hormonal disruption of the Ca metabolism may impact not only bone growth but also bone mass accrual and bone quality as previously described [44]. Moreover, previous studies have shown that IGF-I treatment [45] or IGF-I/IGFBP3 treatment [46] slightly increased bone length and femoral calcium content in rats; however these results are mainly observed during rapid growth. In the present study, although the IGF-I level was slightly increased in the high dietary level of the Ca group compared to the adequate Ca group, bone modeling and mineral apposition rate were unaffected (Tables 5, 6 and Fig. 2). There was no macroscopical change in bone geometry but a slight increase on the polar moment of inertia which positively impact

bone mechanical properties. Consequently, since calcium can regulate IGF-I, it is important to control calcium intake during growth in order to reach the highest peak of bone mass, which is the best protection against age-related bone loss. Dubois-Ferriere et al. [47] showed that by lowering IGF-I level, the synthesis of serum 1,25(OH)2D3 was reduced, and intestinal calcium absorption was reduced and this leads to an increase in PTH levels, regardless of the level of calcium. Similarly, Kasukawa et al. [41] reported that dietary Ca deficiency for 2 weeks in mice during growth caused a significant increase in serum PTH level and decreased serum Ca levels compared with mice fed the normal Ca diet. These effects were enhanced in the IGF-I KO mice compared with wild type mice. We also found a slight but not significant increase in PTH level in the lower Ca group despite the fact that the Ca/P ratio is preserved and also a decrease in plasma Ca concentration but still in normal range compared to the high level of Ca (Table 5). This result indicates that a modest hyperparathyroidism may be possible. Unfortunately we do not have the value of 1,25(OH)2 vitamin D which could help us to understand this mechanism. However the spread of PTH values observed in the 3 groups of Ca intake made it difficult to show significant effects. In the present study, bone formation and bone resorption were evaluated respectively by bone histomorphometry and by bone turnover markers (Tables 5 and 6). Bone modeling was severely altered in the Ca-restricted group as shown by histomorphometric measurement (slower turnover) and by an increase in serum CTX-I level (Table 5) [48]. This finding is in agreement with those of other studies that reported increased bone resorption quantified by the excretion of urinary pyridinium crosslinks in Ca-restricted young rats [27,28,49]. Moreover, MAR, MS/BS and BFR, several histomorphometric parameters characteristics of bone formation (Table 6) were reduced in the Ca-restricted group as described by Peterson et al. [29]. Yoo et al. [50] reported that a decrease in bone formation is correlated with a decrease in circulating IGF-I level. This result may be in agreement with ours. Thus, in the Carestricted group, the lower IGF-I level and the decrease in the modeling process may negatively impact on bone diameter, cortical thickness, BMD and microarchitectural parameters. In contrast, a higher IGF-I level may improve the peak bone strength acquisition.

Conclusion In conclusion, this study confirmed that low dietary calcium negatively impacted bone growth, bone strength, bone architecture and peak bone mass accrual, at least in part due to the hormonal disruption implied in Ca metabolism. In contrast, elevated dietary Ca intake above the recommended level led to a significant increase in bone strength not accompanied by an increase in BMD or changes in bone markers (compared to adequate Ca) in young growing male rats. These findings would suggest that high Ca intake levels, well in excess of requirements, might have a beneficial effect on the attainment of peak bone strength with no evidence of a detrimental effect on bone modeling, at least in the short term. Thus multiple factors linked to bone mass and strength should be taken into account when setting dietary levels of adequate mineral intake to support optimal peak bone mass and strength acquisition.

Conflict of interest The present work was financed by Nestec S.A. Acknowledgments The authors would like to thank M. Marchesini, C. Maubert, and J. Sanchez, for animal management and L. Ameye and I. Badoud for their scientific and technical expertise.

S. Viguet-Carrin et al. / Bone 68 (2014) 85–91

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Peak bone strength is influenced by calcium intake in growing rats.

In this study we investigated the effect of supplementing the diet of the growing male rat with different levels of calcium (from low to higher than r...
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