Osteoporosis International

, Volume 23, Issue 7, pp 1909–1919 | Cite as

Hydrolyzed collagen improves bone status and prevents bone loss in ovariectomized C3H/HeN mice

  • F. Guillerminet
  • V. Fabien-Soulé
  • P. C. Even
  • D. Tomé
  • C.-L. Benhamou
  • C. Roux
  • A. Blais
Original Article

Abstract

Summary

This study evaluates the effect of hydrolyzed collagen (HC) on bone health of ovariectomized mice (OVX) at different ages. Twenty-six weeks after the OVX procedure, HC ingestion was still able to improve significantly bone mineral density (BMD) and some femur biomechanical parameters. Moreover, HC ingestion for 1 month before surgery prevented BMD decrease.

Introduction

HC can play an important role in preserving BMD before osteoporosis appears. The aim of this study was to evaluate the effect of HC on bone health of ovariectomized mice at different ages.

Methods

Female C3H mice were either OVX at 3 or 6 months and fed for 6 months (first experiment) or 3 months (second experiment) with diet including 0, 10, or 25 g/kg of HC. In the second experiment, one group received HC 1 month before surgery, and two groups received the supplementation immediately after surgery, one fed ad libitum and the other by gavage. Mice treated with raloxifene were used as a positive control. BMD, femur intrinsic and extrinsic biomechanical properties, and type I collagen C-terminal telopeptide were measured after 12 and 26 weeks. Food intake and spontaneous physical activity were also recorded.

Results

The OVX procedure increased body weight, while food intake decreased, thus suggesting that resting metabolism was decreased. Ingestion of 25 g/kg of HC for 3 or 6 months reduced bone loss significantly in, respectively, 3- and 6-month-old OVX mice. The lowest HC concentration was less efficient. HC ingestion for 3 months is as efficient as raloxifene to protect 3-month-old OVX mice from bone loss. Our results also demonstrated that HC ingestion before surgery prevented the BMD decreases.

Conclusion

This study confirms that dietary collagen reduces bone loss in OVX mice by increasing the diameter of the cortical areas of femurs and can have a preventive effect.

Keywords

Bone mineral density Hydrolyzed collagen Microarchitecture Ovariectomized mice model 

Introduction

Osteoporosis is a critical disorder characterized by bone loss and an increased risk of fracture. It results from an imbalance between bone formation and bone resorption occurring mainly, although not only, in post-menopausal women as a consequence of estrogen deficiency that increases osteoclast activity [1]. The different factors involved in bone strength include bone mineral density (BMD) and bone crystal characteristics, bone protein matrix and collagen fiber quality, bone microarchitecture, bone geometry and morphology (bone size, shape, microarchitecture), and the intrinsic properties of the bone material [2]. Cortical bone strength, whose main role is to protect bone integrity, is also influenced by mineralization, porosity, orientation of collagen fibers, extent and nature of collagen cross-linking, number and composition of cement lines as well as presence of micro damages [3, 4, 5, 6].

Nutritional factors play a role in bone development during growth and in bone maintenance throughout adulthood [7]. Nutritional components with potential anti-resorptive activity include calcium, cholecalciferol (vitamin D), and proteins that are key nutrients for bone health maintenance [8, 9, 10]. Low protein intake increases the occurrence of osteoporotic fracture which is associated with an imbalance between increased bone formation and bone resorption. This imbalance is partly related to a decrease in plasma levels of insulin-like growth factor-I (IGF-I) [11]. In contrast, despite some controversies related to high protein intake, the majority of the studies show a beneficial effect of an increase in protein intake on bone metabolism and protein repletion after hip fractures in elderly patients. This has been associated with increased plasma levels of IGF-1 and attenuation of proximal femur bone loss [12, 13, 14].

Among different proteins, the intake of hydrolyzed collagen (HC) in animal models has been demonstrated to improve BMD and bone mineral content, and to increase the quantity of type I collagen and proteoglycans in the bone matrix [15, 16, 17, 18, 19]. HC ingestion may modulate bone metabolism through the release of different collagen-derived peptides into the blood including free hydroxyproline (Hyp), Pro-Hyp, and other Hyp-containing peptides such as Ala-Hyp, Leu-Hyp, Ile-Hyp, Phe-Hyp, and Pro-Hyp-Gly [20]. The peptides released from collagen digestion are presumed to mimic those released by osteoclasts during the bone matrix resorption phase. The peptides released have been shown to act on osteoblast activity especially on their mineralization step [21].

The aims of the this study were (1) to evaluate in mice the effect of hydrolyzed collagen intake on bone loss induced by ovariectomy at different ages, (2) to test if the administration route (periodical gavages or ad libitum food intake) can modulate the efficiency of HC ingestion, and (3) to determine whether HC given before the OVX procedure could have a preventive effect on bone loss. Young (3 months) and adult mice (6 months) were ovariectomized. HC was either incorporated into the food or given by gavage. The HC supplementation was started, either 1 month before or immediately after the OVX procedure. Raloxifene was used as a positive control since this selective estrogen receptor modulator is effective in preserving bone OVX-induced changes.

Materials and methods

Hydrolyzed collagens and ovariectomized mouse model

Enzymatic HC was provided by Rousselot SAS, a Vion Company (Puteaux, France), from the Rousselot® Peptan™ range. The HC was of porcine origin (Peptan™ P) with a molecular weight of 5 kDa. The preparation was food grade and was derived from the enzymatic hydrolysis of animal skin which is predominantly type 1 collagen.

One hundred thirty-four 6-week-old C3H/HeN mice (Harlan) were housed in a room controlled for temperature (22 ± 1°C) with a 12:12 light–dark cycle and free access to food and water for a 6-week adaptation period prior to the study. The experiment was carried out according to the guidelines of the French National Committee for Animal Care and the European Convention of Vertebrate Animals Used for Experimentation, under European Council Directive 86/609/EEC dated November, 1986. For surgery, mice were anesthetized with ketamine (100 mg/kg) and xylazine (10 mg/kg). Morphine was used post-operatively as an analgesic.

Experimental design

The design of the animal protocol includes two experiments described in Table 1.
Table 1

Design of the animal studies

 

Number of mice

OVX/SHAM

Age at surgery (months)

Dietary protein source

Diet complementation (g/kg diet)

Collagen intake

Duration of the experiment (weeks)

Denomination

Experiment 1: 64 mice

8

OVX

3

Soy

26

3OVX

8

OVX

3

Soy

Collagen (10)

Ad libitum

26

3OVX10

8

OVX

6

Soy

Collagen (25)

Ad libitum

26

3OVX25

8

OVX

6

Soy

26

6OVX

8

OVX

6

Soy

Collagen (10)

Ad libitum

26

6OVX10

8

OVX

6

Soy

Collagen (25)

Ad libitum

26

6OVX25

8

SHAM

3

Soy

26

3SHAM

8

SHAM

6

Soy

26

6SHAM

Experiment 2: 70 mice

10

OVX

3

Casein

12

OVX

10

OVX

3

Casein

Collagen (25)

Ad libitum

12

OVX25

10

OVX

3

Casein

Collagen (25)

Ad libitum 4 weeks before OVX

12

OVX25-4W

10

OVX

3

Casein

Collagen (25)

Gavage

12

OVX25-G

10

OVX

3

Casein

Raloxifene (0.001)

Ad libitum

12

OVXR

10

SHAM

3

Casein

12

SHAM

10

SHAM

3

Casein

Collagen (25)

Ad libitum

12

SHAM25

The first experiment included eight groups of eight mice and studied the effect of collagen ingestion for 26 weeks. Mice were sham operated (SHAM) or ovariectomized at 3 months (3OVX) or 6 months (6OVX) of age. They were fed ad libitum with a standard AIN-93N diet with 200 g/kg soy protein as a protein source to provide an appropriate level of essential amino acids in the diet. After surgery, the HC-enriched diet in which the total protein content had been adjusted to allow the incorporation of 10 g/kg (3OVX10 and 6OVX10) or 25 g/kg (3OVX25 and 6OVX25) of HC to the diet was used (Table 1). The monitoring of diet consumption showed an intake of about 3.5 g/day. These concentrations correspond to an ingestion of approximately 1 g/kg body weight of HC for OVX10 and 2.5 g/kg for OVX25 for a mouse of 35 g. We used that evaluation to calculate the amount of collagen that should be given by gavage to each animal. Less than 1% of the natural isoflavone content was still present in the soy protein used to prepare the diet.

The second experiment included seven groups of ten mice fed a collagen-supplemented diet for 12 weeks and was designed (1) to evaluate the effect of four additional weeks of collagen supplementation (25 g/kg food) given before the OVX procedure, (2) to study if the way collagen was given (in the food vs. by gavages) modulated its effect, and (3) to compare the effect of collagen to the effect of raloxifene, a drug commonly indicated for osteoporosis. Two groups of mice were sham operated (SHAM), and five groups were ovariectomized at 3 months (OVX) of age. They were fed ad libitum with a standard AIN-93N diet containing 140 g/kg casein as a protein source (previous experiments done in our laboratory showed no difference for any parameter between OVX mice fed with the soy or casein diet). One SHAM and one OVX group received the control diet throughout. The second SHAM group and two groups of OVX mice received a diet in which 25 g/kg of hydrolyzed collagen had been added at the expense of casein (SHAM25, OVX25) (Table 1); one OVX group received the collagen 4 weeks before surgery (OVX25-4W), the other received the collagen only after surgery (OVX25). Of the two remaining OVX groups, one received the hydrolyzed collagen by gavage (OVX25-G) in amounts adjusted to the collagen intake of the OVX25 group, and the last received raloxifene (1 mg/kg) and was used as a positive control (OVXR). Raloxifene was diluted in the soya oil used to prepare the diet. To calculate the amount of raloxifene that should be added to the diet, we take into account the mouse weight and the amount of diet ingested per day. At the end of both experiments, i.e., 26 and 12 weeks after surgery, mice were anesthetized; blood samples were collected by cardiac puncture, immediately aliquoted into EDTA, and centrifuged. Plasma was stored at −80°C until assay. Body composition was determined by dissection. Three white adipose tissue (WAT) pads (retroperitoneal, mesenteric, and total subcutaneous) were removed and weighed. The liver, intestines, uterus, brown adipose tissue, and carcass (muscles and skeleton) were also weighed. Femurs were collected, cleaned of muscle, and stored at −80°C.

Food intake recording

Daily food intake was measured in all mice of experiment 2 by weighing the amount of pellet ingested. In addition, meal pattern and motor activity were measured in SHAM and OVX mice 10 weeks after OVX using metabolic cages designed in the laboratory and equipped with weighed food cups (sensitivity <0.05 g). Spontaneous physical activity was recorded by means of an activity platform placed below the cages (sensitivity <1 g). Data acquisition was performed throughout the day–light cycle and was controlled by a computer running a program developed by the laboratory. Data were recorded at 100 Hz, averaged, and stored in 5-s bins for subsequent analysis.

Bone mineral density evaluation

At weeks 0, 4, 8, 13, and 26 after the surgery for experiment 1 and at weeks 0, 2, 4, 8, and 12 after the surgery for experiment 2, BMD was measured by DXA with a Lumar Piximus densitometer (GE Medical Systems, software version 1.4 X Lumar). The Piximus allows automated, accurate, and precise measurement of bone density for small animals (10–50 g). The bone measurements exhibit excellent correlation with their ash or chemical extraction weights (r = 0.99). The mice were weighed, anesthetized with ketamine (100 mg/kg) and xylazine (10 mg/kg), and placed prone under the X-ray of the Lumar Piximus. BMD was measured for the whole body (excluding head and tail to improve sensitivity as suggested by the manufacturer), lumbar spines, and the right femoral bone of each mouse [22].

Geometric and biomechanical parameters

The mechanical properties of the mouse femurs were assessed by a three-point bending test using a Universal Testing Machine (Instron 4501, Instron, Canton, MA, USA). The distance between supports was 1.1 cm. Each femur was centrally loaded at the mid-diaphysis at a speed of 1 mm/s. The extrinsic biomechanical parameters of stiffness (S, newtons per millimeter) and ultimate strength (FU, newtons) were determined from load–displacement curves. The areas under the curves represent the energies absorbed during elastic and plastic deformation. The intrinsic biomechanical parameters Young's modulus (E, megapascals) and ultimate stress (σU, megapascals) were calculated from the load displacement curves and geometric properties as described previously [23]. The cross-sectional geometry of the diaphysis of each mouse femur was imaged by micro-computed tomography (μCT, Skyscan 1072, Skyscan, Kontich, Belgium). Both medio-lateral and antero-posterior external and internal diameters of the cortical bone were measured at the mid-diaphysis. Cross-sectional cortical area (CSA, square millimeters) and moment of inertia (I, quartic millimeters) in relation to the horizontal axis were calculated as previously described [24].

CTX concentration in plasma

C-terminal telopeptide of type I collagen (CTX) concentration was determined in blood using an ELISA RatLaps™ kit (Osteomedical).

Statistical analysis

Statistical analyses were carried out using SAS version 9.1 with the data expressed as means ± SD. One-way ANOVA was performed using the GLM procedure of SAS with a Duncan multiple comparison test and a Tukey test post hoc. The MIXED procedure was used to perform repeated measures ANOVA.

Results

Body weight, body composition, and food intake

In experiment 1 (Table 2), the OVX procedure reduced uterine weight when it was performed on 3-month-old mice. This phenomenon was not observed when OVX was performed on 6-month-old mice, probably because of the natural uterus atrophy in 6-month-old SHAM-operated mice that is observed when compared with 3-month-old SHAM-operated mice (0.136 ± 0.020 vs. 0.183 ± 0.048 g, respectively, p < 0.05). Significant differences in final body weight are reported between 3OVX and 3SHAM but not between 6OVX and 6SHAM. In contrast, all OVX mice had significantly higher perirenal and subcutaneous adipose tissue pad mass than SHAM mice (Table 2). No difference was observed between SHAM and OVX mice for carcass and kidney weights (data not shown).
Table 2

Body composition of 3- and 6-month-old OVX mice 26 weeks after surgery (experiment 1) and of 3-month-old OVX mice 12 weeks after surgery (experiment 2)

Dietary groups

Initial body weight (g)

Final body weight (g)

Uterus (g)

Mesenteric fat mass (g/g BW)

Perirenal fat mass (g/g BW)

Subcutaneous fat mass (g)

Carcass (g)

Experiment 1

 3SHAM

20.43 ± 0.95

41.90 ± 3.62 a

0.183 ± 0.048 a

0.779 ± 0.109

0.754 ± 0.218 a

3.371 ± 0.631 a

12.09 ± 0.55

 3OVX

21.84 ± 2.30

45.46 ± 2.77 b

0.139 ± 0.039 b

1.019 ± 0.196

1.091 ± 0.163 b

4.047 ± 0.489 b

12.10 ± 0.50

 3OVX10

21.38 ± 2.47

44.67 ± 3.90 ab

0.127 ± 0.040 b

0.923 ± 0.147

0.912 ± 0.201 b

3.984 ± 0.648 b

11.94 ± 0.40

 3OVX25

21.72 ± 1.65

44.05 ± 3.48 ab

0.140 ± 0.054 b

0.939 ± 0.179

0.985 ± 0.246 b

4.087 ± 0.763 b

12.25 ± 0.92

Experiment 1

 6SHAM

28.01 ± 3.04

38.79 ± 4. 89 a

0.136 ± 0.020

0.910 ± 0.233

0.918 ± 0.440 a

3.674 ± 0.764 a

11.54 ± 1.01

 6OVX

30.00 ± 3.31

42.27 ± 4.07 ab

0.124 ± 0.021

1.299 ± 0.380

1.784 ± 0.330 b

4.852 ± 0.788 b

12.46 ± 0.91

 6OVX10

29.81 ± 2.94

46.85 ± 4.94 b

0.121 ± 0.019

1.309 ± 0.344

1.840 ± 0.329 b

5.634 ± 0.737 b

12.92 ± 1.67

 6OVX25

30.31 ± 3.33

47.45 ± 5.87 b

0.122 ± 0.031

1.427 ± 0.281

1.772 ± 0.321 b

5.359 ± 0.317 b

12.15 ± 1.15

Experiment 2

 SHAM

25.14 ± 1.18

36.50 ± 2.34 a

0.253 ± 0.041 a

1.037 ± 0.177

0.780 ± 0.183 a

4.071 ± 0.643 a

11.37 ± 0.59

 SHAM25

24.54 ± 1.18

35.45 ± 2.34 a

0.244 ± 0.039 a

1.004 ± 0.207

0.779 ± 0.109 a

3.781 ± 0.668 a

11.34 ± 0.73

 OVX

24.20 ± 1.43

41.74 ± 2.49 b

0.131 ± 0.029 b

1.421 ± 0.334

1.197 ± 0.223 b

5.158 ± 0.681 b

12.87 ± 1.17

 OVX25

24.75 ± 2.30

39.63 ± 3.65 ab

0.144 ± 0.042 b

1.246 ± 0.177

1.077 ± 0.174 b

5.180 ± 0.731 b

11.60 ± 1.23

 OVX25-4W

25.05 ± 1.90

39.76 ± 3.67 ab

0.140 ± 0.031 b

1.160 ± 0.280

1.045 ± 0.147 b

5.163 ± 0.682 b

11.87 ± 0.82

 OVX25G

25.26 ± 0.97

41.00 ± 2.49 b

0.151 ± 0.038 b

1.324 ± 0.132

1.212 ± 0.214 b

5.461 ± 0.694 b

12.72 ± 1.18

 OVXR

24.32 ± 1.06

34.61 ± 2.87 a

0.127 ± 0.028 b

0.953 ± 0.213

0.751 ± 0.144 a

3.546 ± 0.734 a

11.84 ± 0.72

Values are means ± SD, n = 8 for experiment 1 and n = 10 for experiment 2. Values in a column of the same group with different letters are significantly different (p < 0.05)

In experiment 2 (Table 2), the design was similar to a previous one [19]. The surgery was performed on 3-month-old mice. All the OVX mice, regardless of their treatment, had a significantly lower uterine weight than the SHAM mice. Moreover, the OVX procedure increased body weight and perirenal and subcutaneous fat mass of OVX control mice as compared to SHAM mice. HC ingestion had no effect on any parameter of SHAM mice and did not significantly modify the body weight or the body fat mass increase of OVX mice. However, raloxifene (OVXR) prevented the increase of both body weight and perirenal and subcutaneous fat mass. No differences were observed between groups for carcass and kidney weight (data not shown).

Food intake was decreased in all OVX mice and meal patter analysis performed in control OVX mice showed that they ate less compared with SHAM and were less active (Fig. 1). The food intake was reduced during both day and night whereas the decrease in activity was limited to the night period. Assuming that activity represents 10–15% of total energy expenditure in caged mice, the decrease in spontaneous activity would have reduced energy requirements by only 2–3%, while food intake was reduced by 10–15%. Thus, to explain the increased BW gain and body adiposity of the OVX mice, it is necessary to consider that OVX decreased resting metabolism by at least 15% or that part of the food energy was lost in feces.
Fig. 1

Daily food intake analysis (a) and spontaneous physical activity (b) of SHAM (black) and OVX mice (gray) of the second experiment performed 10 weeks after the OVX procedure. Values are means ± SD, n = 8. Groups with different letters are significantly different (p < 0.05)

Influence of ovariectomy and collagen ingestion on BMD

In experiment 1, surgery was performed either at 3 months when BMD was still increasing or at 6 months when BMD had reached its maximal value.

BMD of 3-month-old OVX mice (Fig. 2a) increased during the first 13 weeks and then remained stable for the next 13 weeks. The OVX procedure reduced BMD throughout the study. HC ingestion did not prevent a transient decrease in BMD (weeks 4 and 8) but allowed a progressive recovery after 13 weeks in OVX25 mice and 26 weeks in OVX10 mice.
Fig. 2

BMD of the whole body 26 weeks after ovariectomy for 3-month-old ovariectomized mice (a) and 6-month-old ovariectomized mice (b) under soy main protein diet. BMD was evaluated for SHAM (hatched), OVX (white), OVX10 (gray), and OVX25 (black) mice. Values are means ± SD, n = 8. Groups with different letters are significantly different (p < 0.05). c Evolution of BMD of the whole body 2, 4, 8, and 12 weeks after ovariectomy for 3-month-old ovariectomized mice under casein main protein diet. BMD was evaluated for SHAM (hatched), OVX (white), OVX25-G (gray), OVX25-4W (black), and OVXR (small squares) mice. Values are means ± SD, n = 10. Groups with different letters are significantly different (p < 0.05)

When the OVX procedure was performed on 6-month-old mice (Fig. 2b), this procedure reduced significantly the BMD until the 13th week after the surgery, but the decrease was no more significant after 26 weeks. In collagen-treated mice, the BMD decrease was less pronounced for both OVX10 and OVX25 mice than for untreated mice. Moreover, BMD values reported for OVX10 and OVX25 mice were never significantly lower than those in SHAM-operated mice. After 26 weeks, the BMD reported for the OVX25 mice was significantly higher than that for OVX mice.

In experiment 2 (Fig. 2c), surgery was performed at 3 months when BMD was still increasing, as in our previous study [19]. Collagen (25 g/kg of diet) did not affect BMD in the SHAM group (SHAM vs. SHAM25). The effect of HC on BMD in OVX mice did not differ according to whether the HC was mixed to the diet or given gavages (OVX25 vs. OVX25-G). Consequently, for the sake of clarity, Fig. 2c includes only the SHAM, OVX25, OVX25-4W, and OVXR groups. As in experiment 1, collagen (25 g/kg) given post-surgery did not prevent a transient decrease in BMD but allowed its recovery after 12 weeks. Interestingly, the protective effect of collagen on BMD was greatly improved by pre-treatment of the mice 4 weeks before OVX (OVX25-4W) in such a way that in these mice, no BMD decrease was observed after 2 weeks. The BMD increase of OVX25-4W mice was delayed at 4 weeks but recovered after 8 and 12 weeks. Treatment with raloxifene (OVXR) prevented the effect of OVX throughout the period.

Bone resorption

In experiment 1 (Fig. 3a), the bone turnover marker CTX was assayed in mouse plasma at the end of the experiment (26 weeks after the OVX procedure). OVX increased plasma CTX whether it was performed at 3 or 6 months. In both groups, ingestion of 10 or 25 g/kg of HC prevented this increase. The OVX procedure realized on 3-month-old mice induced an increase of the CTX level was of 2.01 (from 9.67 to 11.68); however, when the procedure was realized on 6-month-old mice, the increase was of 3.43 (from 8.7 to 12.3) 6 months later. The OVX procedure induces a higher increase of CTX when realized on 6-month-old mice.
Fig. 3

a CTX plasma concentration 26 weeks after ovariectomy. CTX was evaluated for 3-month-old ovariectomized mice (black) and 6-month-old ovariectomized mice (gray) under soy main protein diet. Values are means ± SD, n = 8. Groups with different letters are significantly different (p < 0.05). b CTX plasma concentration 12 weeks after ovariectomy for 3-month-old ovariectomized mice under casein main protein diet. Values are means ± SD, n = 10. Groups with different letters are significantly different (p < 0.05)

In experiment 2 (Fig. 3b), plasma CTX was assayed 12 weeks after surgery. OVX significantly increased plasma CTX, and this increase was prevented by all collagen treatments. This suggests that HC ingestion reduces bone resorption. No significant differences were observed between treatments.

Bone mechanical properties

In experiment 1 (Table 3), an evolution of the biomechanical parameters can be noticed between 3- and 6-month-old SHAM-operated mice. 6SHAM mice had a significantly higher moment of inertia, FU, and CSA than 3SHAM mice. Evaluation of femur mechanical properties for 3SHAM and 3OVX mice showed no significant differences between groups, neither for intrinsic parameters such as ultimate stress, Young's modulus, and stiffness (Table 3) nor for the moment of inertia. However, the stiffness reported for 6OVX mice is decreased compared to 6SHAM mice. CSA and FU were decreased by ovariectomy, and this decrease was prevented by collagen in both 3- and 6-month-old OVX mice. Ingestion of the higher concentration of HC 25 g/kg allowed a better restoration of these parameters. Because the higher HC concentration was the more efficient to restore bone mechanical properties, only the higher HC concentration was used for the second experiment. Table 3 shows, in experiment 2, that 12 weeks after the OVX procedure, for mice ovariectomized at 3 months, values for FU, ultimate stress, CSA, and stiffness are significantly decreased for OVX mice compared with SHAM mice. Twelve weeks after the OVX procedure, raloxifene and collagen ingestion whatever were the conditions of HC administration restored all parameters (FU, ultimate stress, CSA, and stiffness) decreased by the OVX procedure. When mice were ovariectomized at 3 months, we observed an evolution of bone mechanical properties from 3 to 6 months after the OVX procedure for all the parameters except for the Young's modulus.
Table 3

Femur biomechanical parameters of 3- and 6-month-old OVX mice 26 weeks after surgery (experiment 1) and of 3-month-old OVX mice 12 weeks after surgery (experiment 2)

Dietary groups

Moment of inertia I (mm4)

FU (N)

Ultimate stress σU (MPa)

CSA (mm2)

Young's modulus E (MPa)

Stiffness S (N mm−1)

Energy (N mm)

Experiment 1

 3SHAM

0.19 ± 0.03

37.74 ± 3.15 a

269 ± 24

1.47 ± 0.01 a

11,071 ± 1,720

181 ± 24

10.26 ± 1.54

 3OVX

0.17 ± 0.02

32.47 ± 2.48 b

262 ± 20

1.33 ± 0.08 b

10,252 ± 1,362

174 ± 24

9.51 ± 1.66

 3OVX10

0.19 ± 0.02

36.29 ± 2.77 a

259 ± 21

1.44 ± 0.10 ab

9,985 ± 1,576

175 ± 27

10.17 ± 1.47

 3OVX25

0.20 ± 0.03

38.52 ± 2.60 a

263 ± 32

1.46 ± 0.14 a

9,456 ± 1,637

176 ± 19

10.61 ± 2.77

Experiment 1

 6SHAM

0.27 ± 0.04

47.91 ± 3.54 a

254 ± 27

1.74 ± 0.13 a

9,928 ± 1,961

204 ± 26 ab

11.43 ± 2.33

 6OVX

0.24 ± 0.02

42.16 ± 3.74 b

258 ± 21

1.55 ± 0.12 b

9,170 ± 1,305

186 ± 19 b

11.27 ± 2.93

 6OVX10

0.25 ± 0.05

43.86 ± 4.39 b

261 ± 25

1.58 ± 0.17 ab

9,928 ± 1,961

175 ± 27 b

10.17 ± 1.47

 6OVX25

0.27 ± 0.04

46.00 ± 4.76 ab

257 ± 21

1.67 ± 0.17 a

9,170 ± 1,305

227 ± 13 a

12.79 ± 1.87

Experiment 2

 SHAM

0.17 ± 0.03

30.80 ± 1.82 a

245 ± 17 a

1.25 ± 0.06 a

9,278 ± 1,608

155 ± 13 a

8.43 ± 1.59

 SHAM25

0.16 ± 0.02

30.91 ± 1.47 a

248 ± 20 a

1.26 ± 0.09 a

10,781 ± 1,534

157 ± 15 a

8.56 ± 1.32

 OVX

0.16 ± 0.03

27.59 ± 2.17 b

228 ± 25 b

1.08 ± 0.09 b

8,627 ± 1,435

134 ± 15 b

7.61 ± 1.12

 OVX25

0.16 ± 0.03

29.85 ± 2.23 a

248 ± 25 a

1.25 ± 0.07 a

10,340 ± 2,137

151 ± 15 a

8.17 ± 1.31

 OVX25-4 W

0.16 ± 0.03

31.25 ± 1.88 a

256 ± 22 a

1.27 ± 0.08 a

10,971 ± 1,532

167 ± 20 a

9.04 ± 1.25

 OVX25G

0.16 ± 0.03

30.07 ± 1.69 a

250 ± 23 a

1.25 ± 0.07 a

10,921 ± 1,378

165 ± 22 a

8.62 ± 1.18

 OVXR

0.16 ± 0.03

31.65 ± 2.17 a

265 ± 25 a

1.29 ± 0.04 a

11,034 ± 1,906

167 ± 25 a

9.14 ± 1.17

Values are means ± SD, n = 8 for experiment 1 and n = 10 for experiment 2. Values in the same column of the same group with different letters are significantly different (p < 0.05)

Discussion

Two different models were used in this study to document the effects of HC ingestion on bone quality. The OVX procedure was performed either on 3-month-old mice when BMD was still increasing or on 6-month-old mice when BMD had reached its maximal value which may better mimic menopause [25]. This study reports time-dependent changes of bone loss after OVX.

In both models, untreated OVX animals had higher body fat and lower BMD than SHAM mice. We reported a decrease of uterine weight for 3-month-old ovariectomized mice; however, for mice which had undergone surgery at 6 months, the same uterine weight is reported 6 months later for the 6OVX mice compared to 6SHAM mice suggesting that as C3H are getting older, they are less sensitive to estrogen. We thus report a natural uterine atrophy as a function of time for C3H mice.

Analysis of food intake and activity of SHAM and OVX mice showed that the increased body fat observed for OVX mice was not related to a positive energy balance associated to an increase in food intake and/or a decreased activity and thus should result from a decrease of resting energy metabolism. In contrast, OVXR mice did not gain fat compared to SHAM, and since the amount of food ingested was the same as for OVX mice, this result likely indicates that the OVXR mice were more active and/or had a higher resting metabolism.

We evaluated not only total BMD but also femur and vertebral BMD. At the vertebral level, the OVX procedure did not decrease significantly the BMD. Indeed, a previous study has shown that the effect of the OVX procedure varied not only with mouse strain but also with skeletal site [26]. Analysis of skeletal site-specificity changes, such as vertebral trabecular microachitecture, performed to evaluate differences between SHAM and OVX, showed strain dependency, ranging from no difference in C3H to −24% in BALB/c. In contrast, the OVX procedure induced important changes in long bone microachitecture of C3H mice (−39% of the tibia trabecular bone volume) [26]. Consequently, in this study, we examined only femur biomechanical properties.

BMD results showed that 4 weeks after the OVX procedure, the differences between SHAM and OVX were larger for mice ovariectomized at 3 months than for those ovariectomized at 6 months. Moreover, 6 months after the OVX procedure, the difference between OVX and SHAM remained significant for mice ovariectomized at 3 months; however, the difference was no more significant for mice ovariectomized at 6 months. Those results show that, according to the developmental stages, mouse bone cell sensitivity to estrogen deficiency is different, leading to various bone loss patterns. When the ovariectomy was performed at 12 weeks (when BMD was still increasing), we measured a BMD decrease 2 weeks after surgery (Fig. 2c); this result suggests that bone metabolism modifications are more marked immediately after estrogen withdrawal. This observation confirmed our previous results regarding the evolution of bone metabolism by the OVX procedure. We have shown in a previous study using C3H OVX mice that 1 month after the OVX procedure, the CTX plasma level of these mice doubled, and N-terminal propeptide of procollagen (PINP), a marker of bone formation, increased by 30%. However, 2 and 4 months later, only CTX was still significantly increased by the OVX procedure although no significant modulation of PINP was reported [27]. The ovariectomy procedure induced a negative long-term regulation of bone formation leading to a predominance of bone resorption over bone formation as CTX (a specific marker of bone resorption) was still significantly increased after 6 months. Osteogenesis is regulated not only by ovariectomy duration but also by genetic factors [28, 29]. This regulation allows protection against bone loss induced by ovariectomy until exhaustion of the osteogenesis process and may explain why 6 months after surgery, there were no BMD differences between SHAM and OVX mice ovariectomized at 6 months. The early losses in bone mass and strength in OVX mice appeared to be transient and may explain that 6-month-old OVX-C3H mice eventually returned to SHAM levels after 6 months. Our results also showed that, as previously reported, raloxifene was able to maintain BMD and to prevent bone loss due to estrogen deficiency [30, 31, 32]. However, in this study, raloxifene did not prevent uterus atrophy of 3-month-old ovariectomized mice.

Both experiments showed that whenever the OVX procedure was performed (i.e., on rapidly growing (3- month-old) or young adult (6-month-old) mice), HC ingestion reduced the bone deterioration induced by the OVX procedure. The present study indicates that ingestion of HC was able to induce growth of the external diameter of the femur cortical zone without modification of the size of the medullar area in OVX mice whatever mice were ovariectomized at 3 of 6 months. The increase in cortical area of femur induced by HC ingestion was correlated with a significant increase of femur ultimate strength. This increase also suggests a higher level of bone formation for mice eating HC diets. Moreover, no adverse effects were observed when SHAM mice ingested the HC diet. This result is in good agreement with our previous in vitro observations which demonstrate that HC of porcine origin is able to increase osteoblast growth and differentiation. The intrinsic material properties of bone, Young's modulus, and energy were not significantly modulated by the OVX procedure or collagen ingestion, but stiffness was significantly increased by HC for 6OVX25 and OVX25 mice and trend to increase for 3OVX25. The contribution of mineral phase to bone's mechanical properties is well documented, but collagen content or change in collagen cross-linking can also reduce bone strength [30]. In a previous observation, we showed that HC ingestion is able to increase the bone non-mineral content which is almost only type 1 collagen. As HC diets increase the formation of collagen in bone, the increase in bone stiffness can be related to the amount of type 1 collagen as suggested by few studies [33, 34]. Further studies are needed to quantify the new formation of type 1 collagen in mice bones.

We observed a general evolution of bone mechanical properties characterized by bone growth and bone strength improvement as a function of time. These results show that bone structure is still developing even when the mouse growth phase is over. At 6 months, BMD reached its maximal value, and the OVX procedure induced a BMD loss which is related to the estrogen deficiency, but, as previously reported by Li et al. [35], genetic factors also intervene in bone metabolism to restore it after ovariectomy. Indeed, our data suggest that when the OVX procedure is performed on 6-month-old mice, the experimental model may better mimic menopause.

Analysis of CTX plasma concentrations 12 weeks or 6 months after surgery showed that collagen ingestion reduced these concentrations, suggesting that the protective effect of hydrolyzed collagen ingestion on bone is still effective after 6 months. Moreover, the main source of protein in the diet or the way collagen was given to mice, in the food or added by gavages, did not modulate HC efficiency.

One of the most interesting observations was that early supply of collagen 1 month before surgery prevented the decrease in BMD observed 2 weeks after the OVX procedure in 3-month-old ovariectomized mice. This result strongly suggests that this protocol was as efficient as the addition of the selective estrogen receptor modulator raloxifene. HC intake may have a preventive role on bone metabolism before the beginning of bone loss due to menopause when referring to humans. The final CTX plasma level for OVX25-4W mice was significantly lower than in OVX mice, while we observed no significant difference for CTX values between the different groups fed with the 25-g/kg HC diet (OVX25-4W, OVX25, gOVX25-G). Further work is needed to explain the mechanisms of the preventive effect of hydrolyzed collagen given before surgery on BMD loss. Nevertheless, these results are in good agreement with our previous study which reported a direct inhibiting action of collagen on osteoclast differentiation in vitro. Moreover, the effect was found to persist as long as collagen was ingested.

In conclusion, our study shows that HC ingestion reduced bone resorption factors and restored bone mineral density of OVX mice. Moreover, anticipated dietary collagen ingestion can prevent the decrease in BMD as efficiently as raloxifene. Thus, our findings support the potential interest of HC as a nutritional complement to prevent bone loss. Regarding clinical application, further work is needed to evaluate the possible benefits associated with HC supplement in women at high risk for enhanced post-menopausal bone loss.

Notes

Conflicts of interest

None.

References

  1. 1.
    Omi N, Ezawa I (1995) The effect of ovariectomy on bone metabolism in rats. Bone 17:163S–168SPubMedGoogle Scholar
  2. 2.
    Ammann P, Rizzoli R (2003) Bone strength and its determinants. Osteoporos Int 14:S13–S18PubMedCrossRefGoogle Scholar
  3. 3.
    Burr DB (2002) The contribution of the organic matrix to bone's material properties. Bone 3:8–11CrossRefGoogle Scholar
  4. 4.
    Currey JD (2001) Bone strength: what are we trying to measure? Calcif Tissue Int 68:205–210PubMedCrossRefGoogle Scholar
  5. 5.
    Seeman E, Delmas PD (2006) Bone quality—the material and structural basis of bone strength and fragility. N Engl J Med 354:2250–2261PubMedCrossRefGoogle Scholar
  6. 6.
    Turner CH (2006) Bone strength: current concepts. Ann N Y Acad Sci 1068:429–446PubMedCrossRefGoogle Scholar
  7. 7.
    Peters BS, Martini LA (2010) Nutritional aspects of the prevention and treatment of osteoporosis. Arq Bras Endocrinol Metabol 54:179–185PubMedCrossRefGoogle Scholar
  8. 8.
    Bonjour JP (2005) Dietary protein: an essential nutrient for bone health. J Am Coll Nutr 24:526S–536SPubMedGoogle Scholar
  9. 9.
    Kerstetter JE, O'Brien KO, Insogna KL (1998) Dietary protein affects intestinal calcium absorption. Am J Clin Nutr 68:859–865PubMedGoogle Scholar
  10. 10.
    Lips P, Bouillon R, van Schoor NM, Vanderschueren D, Verschueren S, Kuchuk N, Milisen K, Boonen S (2010) Reducing fracture risk with calcium and vitamin D. Clin Endocrinol 73:277–285CrossRefGoogle Scholar
  11. 11.
    Thissen JP, Ketelslegers JM, Underwood LE (1994) Nutritional regulation of the insulin-like growth factors. Endocr Rev 15:80–101PubMedGoogle Scholar
  12. 12.
    Schürch MA, Rizzoli R, Slosman D, Vadas L, Vergnaud P, Bonjour JP (1998) Protein supplements increase serum insulin-like growth factor-I levels and attenuate proximal femur bone loss in patients with recent hip fracture. A randomized, double-blind, placebo-controlled trial. Ann Intern Med 128:801–809PubMedGoogle Scholar
  13. 13.
    Barzel US (1995) The skeleton as an ion exchange system: implications for the role of acid-base imbalance in the genesis of osteoporosis. J Bone Miner Res 10:1431–1436PubMedCrossRefGoogle Scholar
  14. 14.
    Barzel US, Massey LK (1998) Excess dietary protein can adversely affect bone. J Nutr 128:1051–1053PubMedGoogle Scholar
  15. 15.
    Moskowitz RW (2000) Role of collagen hydrolysate in bone and joint disease. Semin Arthritis Rheum 30:87–99PubMedCrossRefGoogle Scholar
  16. 16.
    Koyama Y, Hirota A, Mori H, Takahara H, Kuwaba K, Kusubata M, Matsubara Y, Kasugai S, Itoh M, Irie S (2001) Ingestion of gelatin has differential effect on bone mineral density and body weight in protein undernutrition. J Nutr Sci Vitaminol 47:84–86PubMedCrossRefGoogle Scholar
  17. 17.
    Wu J, Fujioka M, Sugimoto K, Mu G, Ishimi Y (2004) Assessment of effectiveness of oral administration of collagen peptide on bone metabolism in growing and mature rats. J Bone Miner Metab 22:547–553PubMedCrossRefGoogle Scholar
  18. 18.
    Nomura Y, Oohashi K, Watanabe M, Kasugai S (2005) Increase in bone mineral density through oral administration of shark gelatin to ovariectomized rats. Nutrition 21:1120–1126PubMedCrossRefGoogle Scholar
  19. 19.
    Guillerminet F, Beaupied H, Fabien-Soulé V, Tomé D, Benhamou CL, Roux C, Blais A (2010) Hydrolyzed collagen improves bone metabolism and biomechanical parameters in ovariectomized mice: an in vitro and in vivo study. Bone 46:827–834PubMedCrossRefGoogle Scholar
  20. 20.
    Ohara H, Matsumoto H, Ito K, Iwai K, Sato K (2007) Comparison of quantity and structures of hydroxyproline-containing peptides in human blood after oral ingestion of gelatin hydrolysates from different sources. J Agric Food Chem 55:1532–1535PubMedCrossRefGoogle Scholar
  21. 21.
    Reyes CD, Petrie TA, Burns KL, Schwartz Z, García AJ (2007) Biomolecular surface coating to enhance orthopaedic tissue healing and integration. Biomaterials 28:3228–3235PubMedCrossRefGoogle Scholar
  22. 22.
    Nagy TR, Clair AL (2000) Precision and accuracy of dual-energy X-ray absorptiometry for determining in vivo body composition of mice. Obes Res 8:392–398PubMedCrossRefGoogle Scholar
  23. 23.
    Turner CH, Burr DB (1993) Basic biomechanical measurements of bone: a tutorial. Bone 14:595–608PubMedCrossRefGoogle Scholar
  24. 24.
    Di Masso RJ, Font MT, Capozza RF, Detarsio G, Sosa F, Ferretti JL (1997) Long-bone biomechanics in mice selected for body conformation. Bone 20:539–545PubMedCrossRefGoogle Scholar
  25. 25.
    Blais A, Malet A, Mikogami T, Martin-Rouas C, Tome D (2009) Oral bovine lactoferrin improves bone status of ovariectomized mice. Am J Physiol Endocrinol Metab 296:E1281–E1288PubMedCrossRefGoogle Scholar
  26. 26.
    Bouxsein ML, Myers KS, Shultz KL, Donahue LR, Rosen CJ, Beamer WG (2005) Ovariectomy-induced bone loss varies among inbred strains of mice. J Bone Miner Res 20:1085–1092PubMedCrossRefGoogle Scholar
  27. 27.
    Malet A, Bournaud E, Lan A, Mikogami T, Tome D, Blais A (2011) Bovine lactoferrin improves bone status of ovariectomized mice via immune function. Bone 48:1028–1035PubMedCrossRefGoogle Scholar
  28. 28.
    Jochems C, Lagerquist M, Håkansson C, Ohlsson C, Carlsten H (2008) Long-term anti-arthritic and anti-osteoporotic effects of raloxifene in established experimental postmenopausal polyarthritis. Clin Exp Immunol 152:593–597PubMedCrossRefGoogle Scholar
  29. 29.
    Schroeder TM, Jensen ED, Westendorf JJ (2005) Runx2: a master organizer of gene transcription in developing and maturing osteoblasts. Birth Defects Res C Embryo Today 75:213–225PubMedCrossRefGoogle Scholar
  30. 30.
    Cano A, Dapía S, Noguera I, Pineda B, Hermenegildo C, Del Val R, Caeiro JR, García-Pérez MA (2008) Comparative effects of 17β-estradiol, raloxifene and genistein on bone 3D microarchitecture and volumetric bone mineral density in the ovariectomized mice. Osteoporos Int 19:793–800PubMedCrossRefGoogle Scholar
  31. 31.
    Sliwiński L, Folwarczna J, Nowińska B, Cegieła U, Pytlik M, Kaczmarczyk-Sedlak I, Trzeciak H, Trzeciak HI (2009) A comparative study of the effects of genistein, estradiol and raloxifene on the murine skeletal system. Acta Biochim Pol 2:261–270Google Scholar
  32. 32.
    Marie PJ (2008) Transcription factors controlling osteoblastogenesis. Arch Biochem Biophys 473:98–105PubMedCrossRefGoogle Scholar
  33. 33.
    Burr DB (2002) The contribution of the organic matrix to bone's material properties. Bone 31(1):8–11PubMedCrossRefGoogle Scholar
  34. 34.
    Mann V, Hobson EE, Li B, Stewart TL, Grant SF, Robins SP, Aspden RM, Ralston SH (2001) A COL1A1 Sp1 binding site polymorphism predisposes to osteoporotic fracture by affecting bone density and quality. J Clin Invest 107(7):899–907PubMedCrossRefGoogle Scholar
  35. 35.
    Li CY, Schaffler MB, Wolde-Semait HT, Hernandez CJ, Jepsen KJ (2005) Genetic background influences cortical bone response to ovariectomy. J Bone Miner Res 20:2150–2158PubMedCrossRefGoogle Scholar

Copyright information

© International Osteoporosis Foundation and National Osteoporosis Foundation 2011

Authors and Affiliations

  • F. Guillerminet
    • 1
    • 3
  • V. Fabien-Soulé
    • 3
  • P. C. Even
    • 1
  • D. Tomé
    • 1
  • C.-L. Benhamou
    • 2
  • C. Roux
    • 4
  • A. Blais
    • 1
  1. 1.AgroParisTechUMR914 Nutrition Physiology and Ingestive BehaviorParisFrance
  2. 2.Inserm U658Orléans Regional Hospital and University of OrléansOrléansFrance
  3. 3.Rousselot SASPuteaux cedexFrance
  4. 4.Département de Rhumatologie, APHP, Hôpital CochinUniversité Paris DescartesParisFrance

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