Decellularized Cartilage Directs Chondrogenic Differentiation: Creation of a Fracture Callus Mimetic

Complications that arise from impaired fracture healing have considerable socioeconomic implications. Current research in the field of bone tissue engineering predominantly aims to mimic the mature bone tissue microenvironment. This approach, however, may produce implants that are intrinsically unresponsive to the cues present during the initiation of fracture repair. As such, this study describes the development of decellularized xenogeneic hyaline cartilage matrix in an attempt to mimic the initial reparative phase of fracture repair. Three approaches based on vacuum-assisted osmotic shock (Vac-OS), Triton X-100 (Vac-STx), and sodium dodecyl sulfate (Vac-SDS) were investigated. The Vac-OS methodology reduced DNA content below 50 ng/mg of tissue, while retaining 85% of the sulfate glycosaminoglycan content, and as such was selected as the optimal methodology for decellularization. The resultant Vac-OS scaffolds (decellularized extracellular matrix [dcECM]) were also devoid of the immunogenic alpha-Gal epitope. Furthermore, minimal disruption to the structural integrity of the dcECM was demonstrated using differential scanning calorimetry and fluorescence lifetime imaging microscopy. The biological integrity of the dcECM was confirmed by its ability to drive the chondrogenic commitment and differentiation of human chondrocytes and periosteum-derived cells, respectively. Furthermore, histological examination of dcECM constructs implanted in immunocompetent mice revealed a predominantly M2 macrophage-driven regenerative response both at 2 and 8 weeks postimplantation. These findings contrasted with the implanted native costal cartilage that elicited a predominantly M1 macrophage-mediated inflammatory response. This study highlights the capacity of dcECM from the Vac-OS methodology to direct the key biological processes of endochondral ossification, thus potentially recapitulating the callus phase of fracture repair.