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Methods: The in vivo effects of CH （collagen hydrolysate） and Pro-Hyp(prolyl-hydroxyproline ) intake on articular cartilage were studied by microscopic examination of sections of dissected articular cartilage from treated C57BL/6J mice. In this study, mice that were fed diets containing excess phosphorus were used as an in vivo model. This mouse line showed loss of chondrocytes and reduced thickness of articular cartilage, with abnormality of the subchondral bone. The in vitro effects of CH, Pro-Hyp, amino acids and other peptides on proliferation, differentiation, glycosaminoglycan content and mineraliza-tion of chondrocytes were determined by MTT activity and staining with alkaline phosphatase, alcian blue and alizarin red. Expression of chon-drogenesis-specific genes in ATDC5 cells was determined by semiquantitative Reverse Transcription Polymerase Chain Reaction (RT-PCR).

Results: In vivo, CH and Pro-Hyp inhibited the loss of chondrocytes and thinning of the articular cartilage layer caused by phosphorus-induced degradation. In vitro study, CH and Pro-Hyp did not affect chondrocyte proliferation but inhibited their differentiation into mineralized chondrocytes. A combination of amino acids such as proline, hydroxyproline and prolyl-hydroxyprolyleglycine did not affect chondrocyte pro-liferation or differentiation. Moreover, CH and Pro-Hyp caused two and threefold increases, respectively, in the staining area of glycosamino-glycan in the extracellular matrix of ATDC5 cells. RT-PCR indicated that Pro-Hyp increased the aggrecan mRNA level approximately twofold and decreased the Runx1 and osteocalcin mRNA levels by two-thirds and one-tenth, respectively.

Conclusion: Pro-Hyp is the first bioactive edible peptide derived from CH to be shown to affect chondrocyte differentiation under pathological conditions.

ª 2009 Published by Elsevier Ltd on behalf of Osteoarthritis Research Society International.