Elsevier

Bone

Volume 46, Issue 3, March 2010, Pages 827-834
Bone

Hydrolyzed collagen improves bone metabolism and biomechanical parameters in ovariectomized mice: An in vitro and in vivo study

https://doi.org/10.1016/j.bone.2009.10.035Get rights and content

Abstract

Collagen has an important structural function in several organs of the body, especially in bone and cartilage. The aim of this study was to investigate the effect of hydrolyzed collagen on bone metabolism, especially in the perspective of osteoporosis treatment and understanding of its mechanism of action. An in vivo study was carried out in  12-week-old female C3H/HeN mice. These were either ovariectomized (OVX) or sham-operated (SHAM) and fed for 12 weeks with a diet containing 10 or 25 g/kg of hydrolyzed collagen. We measured bone mineral density (BMD) using dual-energy X-ray absorptiometry (DXA). C-terminal telopeptide of type I collagen (CTX), marker of bone resorption, and alkaline phosphatase (ALP), marker of bone formation, were assayed after 4 and 12 weeks. Femur biomechanical properties were studied by a 3-point bending test and bone architecture by microtomography. The BMD for OVX mice fed the diet including 25 g/kg of hydrolyzed collagen was significantly higher as compared to OVX mice. The blood CTX level significantly decreased when mice were fed with either of the diets containing hydrolyzed collagen. Finally, we have shown a significant increase in bone strength correlated to geometrical changes for the OVX mice fed the 25 g/kg hydrolyzed collagen diet. Primary cultures of murine bone cells were established from the tibia and femur marrow of BALB/c mice. The growth and differentiation of osteoclasts and osteoblasts cultured with different concentrations (from 0.2 to 1.0 mg/mL) of bovine, porcine or fish hydrolyzed collagens (2 or 5 kDa) were measured. Hydrolyzed collagens (2 or 5 kDa) in the tissue culture medium did not have any significant effects on cell growth as compared to controls. However, there was a significant and dose-dependent increase in ALP activity, a well-known marker of osteogenesis, and a decrease in octeoclast activity in primary culture of bone cells cultured with hydrolyzed collagens (2 kDa only) as compared to the control.

It is concluded that dietary hydrolyzed collagen increases osteoblast activity (as measured in primary tissue culture), which acts on bone remodeling and increases the external diameter of cortical areas of the femurs.

Introduction

Osteoporosis is a chronic disorder occurring mainly in postmenopausal women, characterized by reduced BMD and an increased risk of fracture. Bone loss originates from an imbalance between bone formation and bone resorption, especially after the menopause, which induces an increase of bone turnover by excess osteoclast activity. Nutritional components with potential anti-resorptive activity generally include calcium and cholecalciferol, but other components such as hydrolyzed type I collagen are also presumed to have an impact on bone metabolism [1].

Type I collagen is the major structural protein distributed throughout the whole body accounting for 25% of total body protein and for 80% of total conjunctive tissue in humans. It is an important component of bone, being the main extra cellular matrix protein for calcification, which also plays a role in osteoblast differentiation [2], [3]. Some studies suggest that hydrolyzed collagen-enriched diet improves bone collagen metabolism and BMD. Oral administration of hydrolyzed collagen increased bone mass content and density in rats and mice fed a calcium- or protein-deficient diet [4], [5]. Oral administration of hydrolyzed collagen was also demonstrated to increase the quantity of type I collagen and proteoglycans in the bone matrix of ovariectomized rats [1]. Moreover, in patients with osteoporosis, oral intake of hydrolyzed collagen with calcitonin had a stronger inhibitory effect on bone resorption than calcitonin alone [6]. Proteins represent a key nutriment for bone health and thereby in the prevention of osteoporosis [7]. Protein undernutrition contributes to the occurrence of osteoporotic fracture in elderly. This may be associated with an uncoupling between increased bone formation and bone resorption, due to a decreased plasma insulin-like growth factor-I (IGF-I) levels [8]. Previous works showed that protein repletion after hip fracture in elderly patients was associated with increased serum levels of IGF-1 and attenuation of proximal femur bone loss [9].

The mechanisms by which ingestion of hydrolyzed collagen could improve bone formation remain unclear but have been suspected to be associated with the release and absorption of collagen-derived peptides acting on bone metabolism. Since Oesser et al. [10] demonstrated the intestinal absorption and the cartilage accumulation of collagen-derived peptides, it has been generally assumed that collagen-rich diets interact with the bone matrix. Indeed, collagen-derived di- and tripeptides rich in hydroxyproline such as Hyp, Pro-Hyp, Pro-Hyp-Gly or Gly-Pro-Val have been detected in human blood following the ingestion of hydrolyzed collagen [11]. Negligible amounts of the peptide form of hydroxyproline (Hyp) were observed in human blood before collagen ingestion. After its oral ingestion, the peptide form of Hyp significantly increased and reached a maximum level (20−60 nmol/mL of plasma) after 1−2 h and then decreased to half of the maximum level 4 h after ingestion. Moreover, some studies demonstrated the time-dependent degradation of Gly-Pro-Hyp, which is frequently found in collagen sequences, into the free-form Gly and a dipeptide, Pro-Hyp. The PEPT1 proton-dependent transporter assures the transport of Pro-Hyp across the intestinal barrier [12]. Among the collagen-derived peptides, Pro-Hyp-Gly, Pro-Hyp and analogs display chemotactic activity to fibroblasts, peripheral blood neutrophils [13], [14] and monocytes [15], Asp-Gly-Glu-Ala stimulates osteoblast-related gene expression of bone marrow cells [16], Ala-Hyp and Gly-Pro-Val are potential inhibitors of angiotensin-converting enzyme [17], [18], and Gly-Pro-Hyp could be involved in platelet aggregation [19].

In such a context, the aim of this study was to investigate both in vivo and in vitro the effects of hydrolyzed collagen used as a nutritional component on bone metabolism. For such a purpose, we used an ovariectomized C3H/HeN mouse model of postmenopausal osteoporosis and a primary culture of murine bone cells.

Section snippets

Hydrolyzed collagens

Enzymatic hydrolyzed collagens were provided by Rousselot SAS, a Vion Company, (Puteaux, France) from the Rousselot® Peptan™ range. The hydrolyzed collagens were from bovine (Peptan™ B coded RDH), porcine (Peptan™ P coded PCH) and fish (Peptan™ F coded FGH) origins with molecular weights of 2 kDa (RDH-N, PCH-N, FGH-N) and 5 kDa (RDH, PCH, FGH). All preparations were food grade and can be obtained commercially. The hydrolyzed collagens were derived from the enzymatic hydrolysis of animal skins

Body composition of ovariectomized mice fed hydrolyzed collagen supplemented diet

Body weight change as function of time in SHAM, OVX, OVX10 and OVX25 mice is reported on Fig. 1. The OVX procedure induces a higher weight gain as compare to SHAM. However, no difference in body weight could be observed between OVX, OVX10 and OVX25 mice. Analysis of body composition as determined by dissection showed that after 12 weeks, OVX, OVX10 and OVX25 mice had significantly higher subcutaneous, perirenal and periovarian adipose tissue pad masses and a significantly lower uterine weight

Discussion

The present study shows that hydrolyzed collagens are able to increase in vivo and in vitro bone metabolism. To evaluate the efficiency of hydrolyzed collagen to modify bone turnover, we used an ovariectomized mice model. Our results show that feeding a diet enriched with 25 g/kg hydrolyzed collagen is able to significantly increase bone metabolism as well as the biomechanical properties (such as BMD) of OVX animals. Moreover, an assay of the bone turnover marker CTX reinforced the view that a

References (40)

  • W.E. Ward

    Serum equol, bone mineral density and biomechanical bone strength differ among four mouse strains

    J. Nutr. Biochem.

    (2005)
  • M.P. Lynch

    The influence of type I collagen on the development and maintenance of the osteoblast phenotype in primary and passaged rat calvarial osteoblasts: modification of expression of genes supporting cell growth, adhesion, and extracellular matrix mineralization

    Exp. Cell. Res.

    (1995)
  • R.O. Oreffo

    Activation of the bone-derived latent TGF beta complex by isolated osteoclasts

    Biochem. Biophys. Res. Commun.

    (1989)
  • G. Xiao

    Role of the alpha2-integrin in osteoblast-specific gene expression and activation of the Osf2 transcription factor

    J. Biol. Chem.

    (1998)
  • J. Wu

    Assessment of effectiveness of oral administration of collagen peptide on bone metabolism in growing and mature rats

    J. Bone Miner. Metab.

    (2004)
  • Y. Koyama

    Ingestion of gelatin has differential effect on bone mineral density and body weight in protein undernutrition

    J. Nutr. Sci. Vitaminol. (Tokyo)

    (2001)
  • M. Adam

    Postmenopausal osteoporosis. Treatment with calcitonin and a diet rich in collagen proteins

    Cas. Lek. Cesk.

    (1996)
  • BonjourJ.P.

    Dietary protein: an essential nutrient for bone health

    J. Am. Coll. Nutr.

    (2005)
  • J.P. Thissen et al.

    Nutritional regulation of the insulin-like growth factors

    Endocr. Rev.

    (1994)
  • M.A. Schurch

    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.

    (1998)
  • Cited by (0)

    View full text