When the glycosaminoglycan staining area was compared with Alcian blue staining as an index of chondrocyte differentiation, ATDC5 cells cultured for 21 days under normoxic conditions showed approximately 1.4-fold larger glycosaminoglycan staining area with Pro-Hyp addition compared to the control. The proliferation of chondrocytes was quantified by WST assay on day 1 of culture Pro-Hyp addition did not affect ATDC5 cell proliferation under either normoxic conditions (20% oxygen) or hypoxic conditions (1% oxygen) ( Figure 1A,B). ![]() We analyzed whether Pro-Hyp affects the proliferation and differentiation of ATDC5 cells. Pro-Hyp Regulates Chondrogenic ATDC5 Cells Differentiation in Hypoxic Condition In this study, Pro-Hyp strongly enhanced early chondrogenic differentiation under physiological hypoxic conditions.Ģ.1. Previous studies have reported that Pro-Hyp is involved in the early stages of chondrocyte differentiation, but its effects under physiological hypoxic conditions remain unclear. Therefore, chondrocyte differentiation may be influenced by Pro-Hyp under physiological hypoxic conditions. The physiological oxygen concentration in the cartilage’s deepest layer is about 1%. Cartilage chondrocytes surround a hypoxic environment, unlike other well-vascularized tissues. The transcription of Col2a1 and Aggrecan is strongly induced by sex-determining region Y-box 9 (Sox9), while matrix metalloproteinase 13 (Mmp13) plays essential roles in cartilage degeneration. Ĭhondrocytes are the only cell type existing in cartilage and maintain the equilibrium of ECM and cartilage homeostasis by producing cartilage matrix proteins such as type II collagen α1 (Col2a1) and proteoglycans including Aggrecan (Acan). ![]() One of the final collagen metabolites, prolyl-hydroxyproline (Pro-Hyp), has been shown to increase the number of fibroblasts migrating from the mouse skin and promote the differentiation of osteoblasts, tendon cells, and chondrocytes. Collagen contains at least one glycine (Gly)-X-Y common repeat domain, where X and Y are predominantly proline (Pro) and hydroxyproline (Hyp), respectively, and many bioactive peptides derived from collagen contain Hyp in their sequences. There are different types of collagen molecules: type I collagen is the primary collagen in skin, bone, and tendons type II collagen is the primary collagen in cartilage. ![]() Collagen turnover produces bioactive collagen peptides (CP) from enzymatic degradation. The extracellular matrix (ECM) of tissues is essential for cell adhesion and function, and collagen is a critical component for maintaining ECM homeostasis. Therefore, Pro-Hyp, a bioactive peptide produced during collagen metabolism, may function as a remodeling factor or extracellular matrix remodeling signal that regulates chondrocyte differentiation in hypoxic cartilage. These results demonstrate that Pro-Hyp strongly promotes the early differentiation of chondrocytes under physiological hypoxic conditions. Moreover, Pro-Hyp treatment significantly upregulated the expression of SOX9, Col2a1, Aggrecan, and MMP13 in chondrocytes cultured under hypoxic conditions. The addition of Pro-Hyp resulted in an approximately 18-fold increase in the glycosaminoglycan staining area compared to the control group under hypoxic conditions. This study aimed to investigate whether Pro-Hyp affects the differentiation of ATDC5 chondrogenic cells under hypoxic conditions. However, whether Pro-Hyp can alter chondrocyte differentiation under physiological hypoxic conditions is still unclear. ![]() Prolyl-hydroxyproline (Pro-Hyp), one of the final collagen-derived peptides, has been previously reported to be involved in the early stages of chondrocyte differentiation. Chondrocytes are surrounded by a lower oxygen environment than other well-vascularized tissues with higher oxygenation levels.
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