Title : In vitro model of endochondral ossification based on collagen-hyaluronic acid hydrogel with embedded chondrocytes
Abstract:
Hydrogels are widely investigated biomaterials due to their potential to mimic the structure and function of the native extracellular matrix. We wanted to prepare a 3D in vitro model of endochondral ossification with focus on the cellular and material composition. To mimic the native cartilage in which ossification occurs, we based our model on a mixed collagen-hyaluronic acid hydrogel containing embedded human chondrocytes. This hydrogel was supported by an underlying poly-ε-caprolactone membrane. We seeded human bone marrow mesenchymal stem cells (hMSCs) and human umbilical vein endothelial cells (HUVECs) onto the membrane, allowing them to migrate into the hydrogel. After a 3-day incubation, we induced hMSCs to differentiate towards osteoblasts, hypothesizing that embedded chondrocytes would enhance cell migration into the hydrogel and promote vascularization and ossification compared to the hydrogel without chondrocytes. Additionally, we exploited the synergistic effect between hMSCs and HUVECs to enhance osteogenic differentiation and induce a capillary-like network formation within the model. To accomplish this, we first optimized the conditions for simultaneous osteogenic differentiation of hMSCs and capillary-like formation by HUVECs by evaluating various cell ratios, seeding densities, media combinations (including signaling molecules), and hydrogel compositions. We then assessed our model by measuring the expression of genes and proteins related to bone and cartilage formation.
Our results indicated that a 1:2 ratio of HUVECs:hMSCs in a 50:50 mixture of endothelial growth and osteogenic media was optimal for osteogenic differentiation and capillary-like network formation. Co-cultured hMSCs exhibited increased alkaline phosphatase activity compared to monoculture, while HUVECs formed capillary-like networks only in the presence of hMSCs. The addition of collagen II to the hydrogels significantly enhanced the production of chondrogenic protein SOX9 by the embedded chondrocytes, compared to pure collagen I hydrogels. However, no significant difference in the production of SOX9 was observed in collagen I hydrogels mixed with varying concentrations of hyaluronic acid. Immunofluorescence staining indicated that the co-culture successfully migrated from the membrane into the hydrogel. The results of real-time PCR showed that the mRNA expression of a chondrogenic marker SOX9 was higher only in the hydrogels with embedded chondrocytes. However, the presence of chondrocytes in hydrogels did not increase the mRNA expression of other osteogenic markers, including collagen I, alkaline phosphatase and osteonectin. Our findings provide important insights for the ongoing construction of our 3D model.
Audience Take Away Notes:
- A 1:2 ratio of HUVECs to hMSCs appears to be optimal to simultaneously support the osteogenic differentiation of hMSCs and the formation of capillary-like networks by HUVECs
- The addition of collagen II to hydrogels significantly enhances the production of chondrogenic protein SOX9 by chondrocytes
- Collagen-hyaluronic acid hydrogels are successfully gelating and supporting the cell migration from the poly-ε-caprolactone membrane and osteogenic activity of hMSCs
- The addition of chondrocytes to the hydrogels does not enhance the mRNA expression of osteogenic markers by hMSCs
