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

## TL;DR

A new hydrogel system with chondrocytes, stem cells, and blood vessel cells models early bone development in the lab.

## Contribution

A novel tri-culture hydrogel system combining collagen I and II, hyaluronic acid, and specific cell types to model endochondral ossification.

## Key findings

- Collagen II and hyaluronic acid enhanced osteogenic differentiation of cells in the hydrogel.
- Chondrocytes promoted osteogenic differentiation and increased hypertrophic and angiogenic signaling.
- The system supported chondrogenesis, as shown by elevated SOX9 and Alcian blue staining.

## Abstract

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 in vitro. 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 COL1A1 and ALPL gene expression and enhanced alkaline phosphatase activity. Chondrocytes further promoted osteogenic cell differentiation and temporarily enhanced hypertrophic and angiogenic signaling as evidenced by elevated MMP13 and VEGFA expression. Furthermore, elevated SOX9 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.

Graphical Abstract