Gelatin-Based Microribbon Hydrogels Guided Mesenchymal Stem Cells to Undergo Endochondral Ossification In Vivo with Bone-Mimicking Mechanical Strength

Most stem cell-based bone tissue engineering strategies to date yield bone through direct bone formation, which mimics intramembranous ossification. However, bone injuries often affect long bones which are formed through endochondral ossification, involving an initial cartilage template formation followed by remodeling to form bones. There remains a critical need to develop scaffolds that enhance stem cell-based bone formation through endochondral ossification with bone-mimicking mechanical strength. Here we evaluated the potential of gelatin-based microribbons (mu RBs) as macroporous scaffolds for enhancing human mesenchymal stem cell (MSC)-based bone formation through endochondral ossification. This material platform was compared with conventional gelatin hydrogels (HGs) as controls. MSCs were encapsulated in mu RB or HG scaffolds, primed in chondrogenic medium in vitro for 2 weeks, and then implanted in a mouse subcutaneous model with no additional factors. mu RB scaffolds supported fast cartilage deposition by MSCs, which was completely remodeled and replaced by mineralized bone. Impressively, the compressive moduli of MSC-seeded mu RB scaffolds increased from 10 to 3224 kPa by week 11, a range that mimics native bone. In comparison, while HG supported endochondral ossification, the speed was much slower, with less matrix deposition and only a modest increase in compressive modulus to 269 kPa. These results validate gelatin mu RBs as a promising scaffold for repairing long bone defects by guiding robust endochondral ossification. Lay Summary Natural bone development and healing occurs through two distinct pathways: intramembranous ossification and endochondral ossification. Most bone injuries affect long bones, which are formed through endochondral ossification, involving an initial cartilage template formation followed by remodeling to form bones. However, scaffolds that can guide stem cell-based bone formation through endochondral ossification with bone-mimicking mechanical strength remain lacking. Here we report that macroporous gelatin-based microribbons (mu RBs) accelerate endochondral ossification by human mesenchymal stem cells (MSCs) in vivo using a mouse subcutaneous model. Impressively, the mechanical properties of MSC-seeded mu RB scaffolds increased over 300-fold over 11 weeks to bone-mimicking range, whereas conventional gelatin hydrogel controls reached less than 10% of the bone modulus. These results validate gelatin mu RBs as a promising novel scaffold for repairing long bone defects by guiding robust endochondral ossification.