Rousselot

Light‐based volumetric bioprinting enables fabrication of cubic centimeter‐sized living materials with micrometer resolution in minutes. Xolography is a light sheet‐based volumetric printing technology that offers unprecedented volumetric generation rates and print resolutions for hard plastics. However, the limited solubility and reactivity of current dual‐color photoinitiators (DCPIs) in aqueous media have hindered their application for high‐resolution bioprinting of living matter. Here, we present a novel three‐component formulation that drastically improves photoreactivity and thereby enables high‐resolution, rapid, and cytocompatible Xolographic biofabrication of intricately architected yet mechanically robust living materials. To achieve this, various relevant additives are systematically explored, which revealed that diphenyliodonium chloride and N‐vinylpyrrolidone strongly enhance D‐mediated photoreactivity, as confirmed by dual‐color photo‐rheology. This enables Xolographic bioprinting of gelatin methacryloyl‐based bioresins, producing >1 cm³ constructs at ≈20 µm positive and 125 µm negative resolution within minutes. Multimaterial printing, molecular patterning, and grayscale‐mediated mechanical patterning are explored to programmably create intricate, biomimetic, and concentration‐controlled architectures. We demonstrate the Bioxolographic printing of various cell types, showing excellent cell viability, compatibility with long‐term culture, and ability for nascent protein deposition. These results position Bioxolography as a transformative platform for rapid, scalable, high‐resolution fabrication of functional living materials with encoded chemical and mechanical properties.

Volumetric Bioprinting (VBP), enables to rapidly build complex, cell‐laden hydrogel constructs for tissue engineering and regenerative medicine. Light‐based tomographic manufacturing enables spatial‐selective polymerization of a bioresin, resulting in higher throughput and resolution than what is achieved using traditional techniques. However, methods for multi‐material printing are needed for broad VBP adoption and applicability. Although converging VBP with extrusion bioprinting in support baths offers a novel, promising solution, further knowledge on the engineering of hydrogels as light‐responsive, volumetrically printable baths is needed. Therefore, this study investigates the tuning of gelatin macromers, in particular leveraging the effect of molecular weight and degree of modification, to overcome these challenges, creating a library of materials for VBP and Embedded extrusion Volumetric Printing (EmVP). Bioresins with tunable printability and mechanical properties are produced, and a novel subset of gelatins and GelMA exhibiting stable shear‐yielding behavior offers a new, single‐component, ready‐to‐use suspension medium for in‐bath printing, which is stable over multiple hours without needing temperature control. As a proof‐of‐concept biological application, bioprinted gels are tested with insulin‐producing pancreatic cell lines for 21 days of culture. Leveraging a multi‐color printer, complex multi‐material and multi‐cellular geometries are produced, enhancing the accessibility of volumetric printing for advanced tissue models.

Osteoarthritis (OA) is a common disease in dogs with severe impact on their welfare. The multimodal management of OA includes feeding therapeutic diets and nutraceuticals to slow down OA progression. Collagen hydrolysates (CH) are a nutritional supplement that may exert anabolic effects on osteoarthritic joint cartilage as well as disease-modifying effects. After oral intake, CH is absorbed, mainly as amino acids, di- and tripeptides that are transported amongst others to the joint. In addition to reducing cartilage degradation, CH metabolites may reduce synovial inflammation and subchondral bone sclerosis during OA. Preliminary evidence in dogs suffering from the consequences of OA support the clinical efficacy of CH with reported reductions in lameness. However, effects on biomarker level of cartilage metabolism and inflammation are inconclusive. Additionally, current studies show a lack of standardised dosing regimens and the use of not validated outcomes. Future work should therefore elucidate further on the bioavailability of CH in dogs in order to establish adequate dosing recommendations. Furthermore, high-quality placebo-controlled randomised controlled trials are essential to dstudies have evaluated the cetermine the clinical efficacy of CH to reduce lameness, prevent OA progression and thereby improve the level of evidence.

In response to nutrients, intestinal L‐ and K‐cells naturally secrete glucagon‐like peptide 1 (GLP‐1). GLP‐1 regulates postprandial blood glucose by increasing insulin secretion, slowing down gastric emptying and inducing satiety. A selection of specifically developed collagen hydrolysates was screened for their ability to enhance natural GLP‐1 production in vitro. The best performing hydrolysate, H80 (Nextida GC), was orally administered at different doses to lean, normoglycemic mice and overweight, prediabetic mice. Lean mice were acutely challenged 45 min before an oral glucose load. While daily supplemented for 6 weeks, prediabetic mice were acutely challenged at day 21 and 34. Oral glucose tolerance, plasma insulin and GLP‐1 levels were assessed, and a gastric emptying assay performed in prediabetic mice. H80 significantly lowered the blood glucose response in lean and prediabetic mice, at a 4 g/kg dose (−25% and −36%, respectively), compared to vehicle. In chronically supplemented, prediabetic mice, acute H80 administration slowed down gastric emptying (−60%) after 21 days and increased plasma insulin (+166%) after 35 days of supplementation. H80 increased plasma active GLP‐1 in lean (+217%) and prediabetic (+860%) mice. Overall, the data indicate that the specific collagen hydrolysate, H80, has significant GLP‐1‐mediated effects on oral glucose tolerance in lean and prediabetic mice. Furthermore, effects on postprandial glucose tolerance were evaluated in a small, human, proof of concept study. H80 reduced the postprandial glucose response at a 5 g dose in healthy, normoglycemic and prediabetic participants. Oral supplementation with H80 might thus be a promising strategy to maintain normal glucose tolerance.

Background A healthy skin provides protection against intrinsic and extrinsic factors. Skin aging is characterized by structural and morphological alterations affecting skin health, integrity, and functionality, resulting in visible aging signs. Aim The primary objective of this study was to assess the effect of a collagen peptide dietary supplement on skin aging in the East Asian population. Methods Eighty‐five healthy women, aged from 43 to 65 years old, were randomly assigned to the collagen supplement (CP, 5 g) or placebo (maltodextrin, 5 g) group. To standardize daily skin care, the volunteers in both groups used a specific face cream for 28 days prior to and throughout the supplementation period, creating an equal baseline for the assessment of the efficacy of CP on several skin parameters. At baseline, day 28 and day 84, the following hallmarks of skin and nail aging were assessed: dermis density, skin moisture and elasticity, wrinkle visibility, beauty perception, and nail color. Results After 84 days, a significant improvement of dermis density and skin moisture was observed in the collagen peptides group compared to the placebo group. Positive effects on skin elasticity, wrinkle visibility, nail color, and overall beauty perception were already observed within 28 days of supplementation in the CP group, while the same effects in the placebo group were only observed after 84 days. Conclusion Taken together, these results show that, in addition to a standardized skin care, daily supplementation with 5 g of collagen peptides positively affects visible signs of skin and nail aging in the East Asian population.

Background Collagen hydrolysates (CH) in functional foods and supplements are dietary sources of amino acids (AAs) and di-and tripeptides linked to various health benefits. This study aimed to investigate the single-dose bioavailability of skin- and hide-derived CH from fish, porcine and bovine origin with different molecular weights (bovine 2,000 and 5,000 Da). Methods A randomized, double-blind crossover clinical study was performed with healthy volunteers assessing the plasma concentration of free and peptide-bound hydroxyproline (Hyp) as well as selected peptides reported to be abundantly present in collagen. Results The pharmacokinetic endpoints demonstrated comparable uptake of free Hyp from all CH. A higher amount of total compared to free Hyp indicated the uptake of substantial amounts of Hyp-containing di- or tripeptides. Conclusion Independently of source and molecular weight, all CH yielded relevant plasma concentrations of the investigated metabolites. Larger studies are needed to estimate an ideal level of selected circulating metabolites needed to trigger distinct physiological reactions in target tissues.

Gelatin nanoparticles (GNPs) have been widely studied for a plethora of biomedical applications, but their formation mechanism remains poorly understood, which precludes precise control over their physicochemical properties. This leads to time-consuming parameter adjustments without a fundamental grasp of the underlying mechanism. Here, we analyze and visualize in a time-resolved manner the mechanism by which GNPs are formed during desolvation of gelatin as a function of gelatin molecular weight and type of desolvating agent. Through various analytical and imaging techniques, we unveil a multistage process that is initiated by the formation of primary particles that are ∼18 nm in diameter (wet state). These primary particles subsequently assemble into colloidally stable GNPs with a raspberry-like structure and a hydrodynamic diameter of ∼300 nm. Our results create a basic understanding of the formation mechanism of gelatin nanoparticles, which opens new opportunities for precisely tuning their physicochemical and biofunctional properties.

Purpose The primary aim of this study was to examine whether a glycine-rich collagen peptides (CP) supplement could enhance sleep quality in physically active men with self-reported sleep complaints. Methods In a randomized, crossover design, 13 athletic males (age: 24 ± 4 years; training volume; 7 ± 3 h·wk ¹ ) with sleep complaints (Athens Insomnia Scale, 9 ± 2) consumed CP (15 g·day ¹ ) or a placebo control (CON) 1 h before bedtime for 7 nights. Sleep quality was measured with subjective sleep diaries and actigraphy for 7 nights; polysomnographic sleep and core temperature were recorded on night 7. Cognition, inflammation, and endocrine function were measured on night 7 and the following morning. Subjective sleepiness and fatigue were measured on all 7 nights. The intervention trials were separated by ≥ 7 days and preceded by a 7-night familiarisation trial. Results Polysomnography showed less awakenings with CP than CON (21.3 ± 9.7 vs. 29.3 ± 13.8 counts, respectively; P = 0.028). The 7-day average for subjective awakenings were less with CP vs. CON (1.3 ± 1.5 vs. 1.9 ± 0.6 counts, respectively; P = 0.023). The proportion of correct responses on the baseline Stroop cognitive test were higher with CP than CON (1.00 ± 0.00 vs. 0.97 ± 0.05 AU, respectively; P = 0.009) the morning after night 7. There were no trial differences in core temperature, endocrine function, inflammation, subjective sleepiness, fatigue and sleep quality, or other measures of cognitive function or sleep (P > 0.05). Conclusion CP supplementation did not influence sleep quantity, latency, or efficiency, but reduced awakenings and improved cognitive function in physically active males with sleep complaints.

Background: Joint discomfort is a widespread and growing problem in active adults. The rising interest in preventative nutrition has increased the demand for supplements reducing joint discomfort. Protocols assessing the effect of a nutritional intervention on health commonly involve a series of face-to-face meetings between participants and study staff that can weigh on resources, participant availabilities and even increase drop-out rates. Digital tools are increasingly added to protocols to facilitate study conduct but fully digitally run studies are still scarce. With the increasing interest in real-life studies, the development of health applications for mobile devices to monitor study outcomes could be of great importance. Objective: The purpose of the current real-life study was to develop a specific mobile application, Ingredients for LifeTM, to conduct a 100% digital study testing the effectiveness of a hydrolyzed cartilage matrix (HCM) supplement on joint discomfort in a heterogeneous group of healthy, active consumers. Methods: The 'Ingredients for LifeTM ' mobile app using Visual Analog Scale (VAS) was specifically developed to monitor the variation in joint pain after exercise by the study participants. A total of 201 healthy and physically active, adult women and men (18 to 72 years old) with joint pain completed the study over a period of 16 weeks. Participants were randomly allocated to the study groups and did not receive any dietary or lifestyle advice. Each participant indicated one area of joint pain and logged the type and duration of their weekly activities. They received blinded study supplements and took a daily regimen of 1 g of hydrolyzed cartilage matrix (HCM-G) or 1g of maltodextrin (placebo group; P-G) for 12 weeks while weekly logging joint pain scores in the app. This was followed by a 4-week wash out period during which participants continued reporting their joint pain scores (until the end of week 16). Results: Joint pain was reduced within 3 weeks of taking a low dosage of HCM (1g/day), regardless of gender, age group and activity intensity when compared to the placebo-group. After stopping supplementation, joint pain scores gradually increased but still remained significantly lower than placebo after 4 weeks of washout. The low dropout rate (< 6% of participants, mainly in the P-G) demonstrates the digital study was well received by the study population. Conclusions: The digital tool allowed to measure a heterogeneous group of active adults in a real-life setting (without any lifestyle intervention), thus promoting inclusivity and diversity. With low dropout rates, it demonstrates that mobile applications can generate qualitative, quantifiable, real-world data showcasing supplement effectiveness. The study confirmed that the oral intake of a low dose (1g/day) of HCM led to a significant reduction of joint pain from 3 weeks after starting supplementation.

Purpose We examined the effects of collagen peptides (CP) supplementation on exercise-induced gastrointestinal (GI) stress. Methods In a randomized, crossover design, 20 volunteers (16 males: $\dot{V}$ V ˙ O 2max , 53.4 ± 5.9 ml·kg ⁻¹ ) completed 3 trials: a non-exercise rest trial, with no supplement (REST) and then an exercise trial with CP (10 g·day ⁻¹ ) or placebo control (CON) supplements, which were consumed for 7 days prior to, and 45 min before, a 70 min run at 70–90% of $\dot{V}$ V ˙ O 2max . Outcome measures included urinary lactulose and rhamnose (L/R), intestinal fatty acid binding protein (I-FABP), lipopolysaccharide (LPS), anti-LPS antibody, monocyte-chemoattractant protein-1 (MCP-1), interleukin (IL) 6 and 8, cortisol, alkaline phosphatase (ALP) (measured pre, 10 min post and 2 h post) and subjective GI symptoms. Results There were no differences in heart rate, perceived exertion, thermal comfort, or core temperature during exercise in the CP and CON trials (all P > 0.05). I-FABP was higher in CP (2538 ± 1221 pg/ml) and CON (2541 ± 766 pg/ml) vs. REST 2 h post (1893 ± 1941 pg/ml) (both P < 0.05). LPS increased in CON vs. REST 2 h post (+ 71.8 pg/ml; P < 0.05). Anti-LPS antibody decreased in CON and CP vs. REST at post (both P < 0.05). There were no differences in MCP-1, IL-6, and IL-8 between the CP and CON trials (all P > 0.05), and no differences in L/R or GI symptoms between CON and CP (all P > 0.05). Conclusion Collagen peptides did not modify exercise-induced changes in inflammation, GI integrity or subjective GI symptoms but LPS was higher in CON 2 h post-exercise and thus future studies may be warranted.

In the medical device sector, bloom index and residual endotoxins should be controlled, as they are crucial regulators of the device’s physicochemical and biological properties. It is also imperative to identify a suitable crosslinking method to increase mechanical integrity, without jeopardising cellular functions of gelatin-based devices. Herein, gelatin preparations with variable bloom index and endotoxin levels were used to fabricate non-crosslinked and polyethylene glycol succinimidyl glutarate crosslinked gelatin scaffolds, the physicochemical and biological properties of which were subsequently assessed. Gelatin preparations with low bloom index resulted in hydrogels with significantly (p < 0.05) lower compression stress, elastic modulus and resistance to enzymatic degradation, and significantly higher (p < 0.05) free amine content than gelatin preparations with high bloom index. Gelatin preparations with high endotoxin levels resulted in films that induced significantly (p < 0.05) higher macrophage clusters than gelatin preparations with low endotoxin level. Our data suggest that the bloom index modulates the physicochemical properties, and the endotoxin content regulates the biological response of gelatin biomaterials. Although polyethylene glycol succinimidyl glutarate crosslinking significantly (p < 0.05) increased compression stress, elastic modulus and resistance to enzymatic degradation, and significantly (p < 0.05) decreased free amine content, at the concentration used, it did not provide sufficient structural integrity to support cell culture. Therefore, the quest for the optimal gelatin crosslinker continues.

Rationale It is important to investigate the behavior of protein hydrolysate components in both in vitro and in vivo studies, to support the elucidation of their biological functions. As protein hydrolysates and biological matrices are highly complex mixtures, it is essential to apply fully reliable and flexible analytical approaches. Methods A novel and generic Liquid Chromatography/Mass Spectrometry methodology was developed to analyze short peptides. A stable‐isotope‐labeled labeling agent 6‐aminoquinolyl‐N‐hydroxysuccinimidyl carbamate (¹³C3) was synthesized and used to prepare internal standards from non‐labeled analyte peptides. The amino acid and peptides p, pG, Pp, GPp and PpG (where p stands for hydroxyproline) were used for proof of principle. Results The method showed acceptable performance in solvent, in simulated gastrointestinal fluid and in serum. The (linear) dynamic range expanded to over four orders of magnitude, which is very useful when multiple analytes are analyzed in a biological matrix, due to the large differences in concentrations observed for endogenous and protein hydrolysate components. The method provides absolute‐quantitative results and is fully accountable on the single‐sample and single‐component level. Conclusions The methodology can be applied to reliably quantify protein hydrolysate nutraceutical components at various stages during their in vivo processing. Internal standards can also be synthesized for other short peptides whenever they are expected to have biological relevance and require quantification. Overall this provides an excellent analytical tool to support the elucidation of the biological functions of protein hydrolysate components.

This study examined whether consuming collagen peptides (CP) before and after strenuous exercise alters markers of muscle damage, inflammation and bone turnover. Using a double-blind, independent group’s design, 24 recreationally active males consumed either 20 g day⁻¹ of CP or a placebo control (CON) for 7 days before and 2 days after performing 150 drop jumps. Maximal isometric voluntary contractions, countermovement jumps (CMJ), muscle soreness (200 mm visual analogue scale), pressure pain threshold, Brief Assessment of Mood Adapted (BAM +) and a range of blood markers associated with muscle damage, inflammation and bone turnover C-terminal telopeptide of type 1 collagen (β-CTX) and N-terminal propeptides of type 1 pro-collagen (P1NP) were measured before supplementation (baseline; BL), pre, post, 1.5, 24 and 48 h post-exercise. Muscle soreness was not significantly different in CP and CON (P = 0.071) but a large effect size was evident at 48 h post-exercise, indicative of lower soreness in the CP group (90.42 ± 45.33 mm vs. CON 125.67 ± 36.50 mm; ES = 2.64). CMJ height recovered quicker with CP than CON at 48 h (P = 0.050; CP 89.96 ± 12.85 vs. CON 78.67 ± 14.41% of baseline values; ES = 0.55). There were no statistically significant effects for the other dependent variables (P > 0.05). β-CTX and P1NP were unaffected by CP supplementation (P > 0.05). In conclusion, CP had moderate benefits for the recovery of CMJ and muscle soreness but had no influence on inflammation and bone collagen synthesis.

Osteoarthritis (OA) is a degenerative joint disease for which there are no disease modifying therapies. Thus, strategies that offer chondroprotective or regenerative capability represent a critical unmet need. Recently, oral consumption of a hydrolyzed type 1 collagen (hCol1) preparation has been reported to reduce pain in human OA and support a positive influence on chondrocyte function. To evaluate the tissue and cellular basis for these effects, we examined the impact of orally administered hCol1 in a model of posttraumatic OA (PTOA). In addition to standard chow, male C57BL/6J mice were provided a daily oral dietary supplement of hCol1 and a meniscal-ligamentous injury was induced on the right knee. At various time points post-injury, hydroxyproline (hProline) assays were performed on blood samples to confirm hCol1 delivery, and joints were harvested for tissue and molecular analyses were performed, including histomorphometry, OARSI and synovial scoring, immunohistochemistry and mRNA expression studies. Confirming ingestion of the supplements, serum hProline levels were elevated in experimental mice administered hCol1. In the hCol1 supplemented mice, chondroprotective effects were observed in injured knee joints, with dose-dependent increases in cartilage area, chondrocyte number and proteoglycan matrix at 3 and 12 weeks post-injury. Preservation of cartilage and increased chondrocyte numbers correlated with reductions in MMP13 protein levels and apoptosis, respectively. Supplemented mice also displayed reduced synovial hyperplasia that paralleled a reduction in Tnf mRNA, suggesting an anti-inflammatory effect. These findings establish that in the context of murine knee PTOA, daily oral consumption of hCol1 is chondroprotective, anti-apoptotic in articular chondrocytes, and anti-inflammatory. While the underlying mechanism driving these effects is yet to be determined, these findings provide the first tissue and cellular level information explaining the already published evidence of symptom relief supported by hCol1 in human knee OA. These results suggest that oral consumption of hCol1 is disease modifying in the context of PTOA.

The article contains sections titled: 1.Introduction2.Structure and Properties2.1.Structure and Function of Collagens2.2.Collagen Types of Importance for the Gelatin Industry2.3.Collagen Type I Chemistry2.4.Chemical Properties of Gelatin2.5.Physical Properties of Gelatin2.6.Quality Specifications2.6.1.Worldwide Gelatin Associations2.6.2.Traceability2.6.3.Microbes, Residues, and Chemicals3.Raw Materials and Production3.1.Raw Materials3.2.Production4.Uses4.1.General Aspects4.2.Food Industry4.3.Pharmaceutical and Surgical Applications4.4.Cosmetics4.5.Photography4.6.Technical Gelatin and Glue4.6.1.Matches4.6.2.Coated Abrasives4.6.3.Paper Sizing4.6.4.Adhesives4.6.5.Gelatin Fibers4.6.6.Other Uses4.7.Collagen Peptides' Nutraceutical Value5.Economic Aspects

Background: Skin dryness and an accelerated fragmentation of the collagen network in the dermis are hallmarks of skin aging. Nutrition is a key factor influencing skin health and consequently its appearance. A wide range of dietary supplements is offered to improve skin health. Collagen peptides are used as a bioactive ingredient in nutricosmetic products and have been shown in preclinical studies to improve skin barrier function, to induce the synthesis of collagen and hyaluronic acid, and to promote fibroblast growth and migration. Our aim was to investigate the effect of oral supplementation with specific collagen peptides on skin hydration and the dermal collagen network in a clinical setting. Methods: Two placebo-controlled clinical trials were run to assess the effect of a daily oral supplementation with collagen peptides on skin hydration by corneometry, on collagen density by high-resolution ultrasound and on collagen fragmentation by reflectance confocal microscopy. Human skin explants were used to study extracellular matrix components in the presence of collagen peptides ex vivo. Results: Oral collagen peptide supplementation significantly increased skin hydration after 8 weeks of intake. The collagen density in the dermis significantly increased and the fragmentation of the dermal collagen network significantly decreased already after 4 weeks of supplementation. Both effects persisted after 12 weeks. Ex vivo experiments demonstrated that collagen peptides induce collagen as well as glycosaminoglycan production, offering a mechanistic explanation for the observed clinical effects. Conclusion: The oral supplementation with collagen peptides is efficacious to improve hallmarks of skin aging.

The theory of inventive problem solving (TRIZ)was developed to solve inventive problems in different industrial fields. In recent decades, modern innovation theories and methods proposed several different knowledge sources. These knowledge sources are all built independently of the specific application and their different levels of abstraction make it quite difficult to use them without extensive knowledge about different engineering domains. Considering that all the TRIZ knowledge sources are described in short-text, the missing links among the TRIZ knowledge sources are defined based on short-text semantic similarity, which also makes it possible to ease the use of TRIZ. Meanwhile, the ontology reasoning mechanism deployed on Protégé and JESS, is used to provide heuristic solutions dynamically for TRIZ users. Firstly, TRIZ users start solving the inventive problem with the TRIZ knowledge source of their choice. Then other similar knowledge sources are obtained according to a calculation of semantic relatedness. Finally, with the help of the heuristic abstract solutions and pointers to physical-chemical-geometrical effects, specific solutions are obtained through ontology reasoning. A particular case of a “Diving Fin” is studied to show the heuristic processes of searching abstract solutions and pointers to physical-chemical-geometrical effects in detail.

Even if TRIZ is developing increasingly both in research and education, new users always encounter difficulties in their first attempts to practice it. In such situation, Altshuller's original contradiction matrix often appears as an "easy-to-begin-with" tool. However, while not being representative of what TRIZ really is, it continues to seduce new users, teachers and trainers. This article presents an innovative method for facilitating the use of the contradiction matrix, using a semantic similarity approach and case-based reasoning.

Health claims for food products in Europe are permitted if the nutrient has been shown to have a beneficial nutritional or physiological effect. This paper defines health claims related to bone health and provides guidelines for the design and the methodology of clinical studies to support claims. Regulation (EC) no. 1924/2006 on nutrition and health claims targeting food products was introduced in Europe stating that health claims shall only be permitted if the substance in respect of which the claim is made has been shown to have a beneficial nutritional or physiological effect. The objective of this paper is to define health claims related to bone health and to provide guidelines for the design and the methodology of clinical studies which need to be adopted to assert such health claims. Literature review followed by a consensus discussion during two 1-day meetings organized by the Group for the Respect of Ethics and Excellence in Science (GREES). The GREES identified six acceptable health claims related to bone health based on the potential of food products to show an effect on either the bioavailability of calcium or osteoclast regulatory proteins or bone turnover markers or bone mineral density or bone structure or fracture incidence. The GREES considers that well-designed human randomized controlled trial on a relevant outcome is the best design to assess health claims. The substantiation of health claim could also be supported by animal studies showing either an improvement in bone strength with the food product or showing the relationship between changes induced by the food product on a surrogate marker and changes in bone strength. The consensus reached is that the level of health claim may differ according to the surrogate endpoint used and on additional animal studies provided to support the claim.