Human Enriched Serum Following Hydrolysed Collagen Absorption Modulates Bone Cell Activity: from Bedside to Bench and Vice Versa

Abstract

Collagen proteins are crucial components of the bone matrix. Since collagen-derived products are widely used in the food and supplement industry, one may raise the question whether collagen-enriched diets can provide benefits for the skeleton. In this study, we designed an innovative approach to investigate this question taking into account the metabolites that are formed by the digestive tract and appear in the circulation after ingestion of hydrolysed collagen. Blood samples collected in clinical and pre-clinical trials following ingestion and absorption of hydrolysed collagen were processed and applied on bone-related primary cell cultures. This original ex vivo methodology revealed that hydrolysed collagen-enriched serum had a direct impact on the behaviour of cells from both human and mouse origin that was not observed with controls (bovine serum albumin or hydrolysed casein-enriched serum). These ex vivo findings were fully in line with in vivo results obtained from a mouse model of post-menopausal osteoporosis. A significant reduction of bone loss was observed in mice supplemented with hydrolysed collagen compared to a control protein. Both the modulation of osteoblast and osteoclast activity observed upon incubation with human or mouse serum ex vivo and the attenuation of bone loss in vivo, clearly indicates that the benefits of hydrolysed collagen for osteoporosis prevention go beyond the effect of a simple protein supplementation.

Keywords: absorption; bone; collagen peptides; hydrolysed collagen; metabolites; nutrition; osteoporosis.

Figure 1

Effect of B2000 HC on bone metabolism in vitro and in vivo. (A) MC3T3-E1 proliferation after seven days of culture in 2% FCS (foetal calf serum) in the presence or absence of B2000 (hydrolysed collagen, 0.5 mg/mL) or its isoproteic control (BSA – bovine serum albumin: 0.5 mg/mL); (B) ALP activity in MC3T3-E1 pre-osteoblasts after three or seven days of culture (FCS2%; B2000: 0.5 mg/mL; BSA: 0.5 mg/mL); (C) uterus weight; (D) bone mineral density and (E) RANKL concentration in mouse serum. Values were obtained at the end of the in vivo experiment (eight weeks). Three groups were surgically ovariectomized (OVX), two groups were sham-operated (SH). Mice received a standard diet (modified from the AIN-93M powdered diet) containing 15% or 17.5% of casein or a diet containing 15% of casein + 2.5% of bovine HC (B2000). Groups are as follows: SHAM 15%; SHAM 17.5%; OVX 15%; OVX 17.5% and OVX B2000 (OVX15% casein + 2.5% B2000). Groups with significant differences (p < 0.05) are indicated with different letters (a, b, c) or (*) p < 0.05.

Figure 2

Ex vivo effects of HC in murine bone cells. (A) Hydroxyproline kinetics in mouse serum following B2000 (hydrolysed collagen) gavage; (B) MC3T3-E1 pre-osteoblast proliferation during incubation with FCS (foetal calf serum; 0 to 2%), naïve or enriched mouse serum (7.5% FCS + 2.5% mouse serum); (C) and (D) mineralisation assays and quantification. MC3T3-E1 were cultured as for proliferation assay in combination with β-glycerophosphate (5 mM) and ascorbic acid (25 µg/mL); (E) RAW264.7 pre-osteoclast proliferation at day four after incubation with FCS (2%), naïve or enriched mouse serum (7.5% FCS + 2.5% mouse serum); (F) osteoclast differentiation assays. RAW264.7 pre-osteoclast cells were cultured for four days in the presence of naïve or enriched mouse serum (7.5% FCS + 2.5% mouse serum) in combination with recombinant, murine RANKL (25 ng/mL). Red lines represent osteoclast edges and define osteoclast surface. Groups with significant differences (p < 0.05) are indicated with different letters (a, b, c).

Figure 3

Human serum enrichment and effects on primary cell growth. (A) Human serum protein concentration following HC absorption; (B) human MSC (mesenchymal stem cells) proliferation; (C) human PBMC (peripheral blood mononuclear cells) proliferation. MSCs and PBMCs were cultured in the presence of 10% human enriched serum, optimized for cell culture compatibility. Naïve serum values were used for normalisation. B2000 (bovine HC; mean molecular weight 2kDa); F2000 (fish HC; mean molecular weight 2kDa); P2000 (porcine HC; mean molecular weight 2kDa) and P5000 (porcine HC; mean molecular weight 5kDa). Groups with significant differences (p < 0.05) are indicated with different letters (a and b).

Figure 4

Biological activity screening of HC using human serum enrichment. (A) ALP (alkalin phosphatase) activity in human MSCs subjected to different human enriched sera, (B) TRAP (tartrate resistant acid phosphatase) activity. MSCs and PBMCs were cultured in the presence of 10% human enriched serum, optimized for cell culture compatibility. Naïve serum values were used for normalisation. B2000 (bovine HC; mean molecular weight 2kDa); F2000 (fish HC; mean molecular weight 2kDa); P2000 (porcine HC; mean molecular weight 2kDa) and P5000 (porcine HC; mean molecular weight 5kDa). Groups with significant differences (p < 0.05) are indicated with different letters (a, b, c, d and e).