Collagen-hyaluronic acid hydrogel with embedded chondrocytes as a platform for modeling early stages of endochondral ossification <i>in vitro</i>

Tissue engineering strategies aim to create bone models to enhance our understanding of bone development and improve bone repair. Endochondral ossification (EO), where cartilage is replaced by bone, is difficult to replicate. We developed a collagen-hyaluronic acid (HA) hydrogel system for a three-dimensional (3D) co-cultivation of human chondrocytes, human mesenchymal stem cell (hMSCs), and human umbilical vein endothelial cells (HUVECs) to model the early stages of EO. To our knowledge, this specific tri-culture configuration within such a hydrogel composition has not been previously reported, providing a novel platform for studying EO-related cellular and matrix interactions <i>in vitro.</i> Differentiation and migration of hMSCs and HUVECs were studied in collagen I (Col I) hydrogels, with or without the addition of collagen II (Col II), HA, and chondrocytes. Col II and HA enhanced cell osteogenic differentiation, as documented by an increased <i>COL1A1</i> and <i>ALPL</i> gene expression and enhanced alkaline phosphatase activity. Chondrocytes further promoted osteogenic cell differentiation and temporarily enhanced hypertrophic and angiogenic signaling as evidenced by elevated <i>MMP13</i> and <i>VEGFA</i> expression. Furthermore, elevated <i>SOX9</i> expression and Alcian blue staining indicated a chondrogenic-supportive environment. These findings highlight the synergistic effects of osteogenic and chondrogenic signals, emphasizing the potential of combining Col I and Col II, HA, and essential cell types in hydrogels to optimize bone and cartilage tissue engineering.