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

Abstract

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.

Desktop-stereolithography 3D printing of a radially oriented extracellular matrix/mesenchymal stem cell exosome bioink for osteochondral defect regeneration

“…The mechanisms of the immune response towards allografts have not been entirely determined 52. Previously, Vas et al evaluated the immunocompatibility of decellularized cartilage ECM scaffolds in mice and found a predominantly M2 macrophage-mediated immune response towards the decellularized ECM, which is indicative of a favorable long-term outcome and tissue-specific remodeling 53. This is consistent with our results that the 3D printed ECM/GelMA scaffold can effectively induce M2 macrophages.…”

Decellularized Porcine Cartilage Scaffold; Validation of Decellularization and Evaluation of Biomarkers of Chondrogenesis

“…In this study, DNase and RNase treatments were used to remove DNA and RNA. DNase and RNase may require as many as three cycles to accomplish complete depletion of native genetic material [7,19,20,23,[76][77][78][79]. Removal of 99% of genomic material was observed after a six-day wash cycle in our studies [23].…”

“…Our study included the use of freeze-thaw cycles to help increase the porosity by forming more pores Freeze-thaw cycles have been shown to result in the formation of pores after ice crystal formation in addition to contributing to the disruption of resident chondrocytes. These cycles are often conducted in phosphate-buffered saline (PBS) solution to maintain physiological pH and osmolality, which additionally helps remove the residual reagents [7,19,20,23,[76][77][78][79].…”

“…Removal of 99% of genomic material was observed after a six-day wash cycle in our studies [23]. To quantify the DNA present in cartilage samples before and after decellularization, we used a DNA extraction process [21,79]. The DNA content of the sample was also assessed using Hoechst stain [21,23].…”

3D Cancer Models: The Need for a Complex Stroma, Compartmentalization and Stiffness

“…Some of these chemicals can be hard to remove from tissues and they either physically alter the matrix or remain in trace amounts affecting the viability of newly added cells. Recently the vacuum-assisted osmotic shock method has proven to be extremely effective at removal of cellular components whilst maintaining ECM integrity for cartilage and allowing new cell infiltration with a high degree of viability (Vas et al, 2018).…”