Bone regeneration using aged donor cells and visible light-curable hydrogel: An in vitro and in vivo evaluation.

BACKGROUND: Transplantation therapy combining scaffolds and cells can be used for extensive bone regeneration (e.g., to manage severe alveolar bone defects). Gelatin methacryloyl (GelMA) may be a suitable scaffold for cell transplantation. We developed a novel GelMA that polymerizes under visible light, using riboflavin as a photoinitiator (GelMA-RF). Here we investigated the efficacy of bone-regenerative therapy combining GelMA-RF and immature osteoblasts in rats.

METHODS: Rat alveolar bone immature osteoblasts (RAOBs) from 70-week-old rats were encapsulated in GelMA-RF (aged RAOBs + GelMA-RF) and transplanted into palatal (control, RAOBs alone, and RAOBs + GelMA-RF groups; n = 8, respectively) or femoral (control, GelMA-RF alone, RAOBs alone, and RAOBs + GelMA-RF groups; n = 8, respectively) bone defects. Macroscopic assessment, micro-computed tomography, and histological analyses were performed, and mineralization (aged and young RAOBs with or without osteogenic differentiation (OD) groups; n = 8, respectively) and the calcium/phosphorus ratio (aged and young RAOBs in OD+ or OD- groups; n = 8, respectively) for RAOBs under 3D-culture conditions were evaluated. Bone differentiation-related gene expression (aged and young RAOBs in OD+ or OD- groups; n = 8, respectively) was analyzed by quantitative polymerase chain reaction.

RESULTS: Transplantation of RAOBs encapsulated in GelMA-RF into palatal or femoral defects resulted in significantly earlier bone-like tissue formation compared with controls. RAOBs showed sufficient mineralization in a 3D-culture environment. Bone differentiation-related gene expression was significantly increased in 3D cultures.

CONCLUSIONS: Taken together, GelMA-RF, particularly when combined with RAOBs, provides a supportive scaffold for bone differentiation and may represent a novel bone-regeneration therapy for complex/extensive bone defects, even when combined with immature alveolar-bone osteoblasts from aged rats.

PLAIN LANGUAGE SUMMARY: Regenerative medicine combining scaffold materials and cell transplantation shows promise for widespread bone regeneration. This study focused on gelatin methacryloyl (GelMA) as a scaffold material. We developed GelMA (GelMA-RF) that gelated under visible light, avoiding the disadvantage of UV irradiation in conventional methods, and used it as a scaffold for cell transplantation. Furthermore, considering the increasing need for regenerative medicine in elderly patients, this study focused on using immature-osteoblasts derived from aged individuals as transplant cells to verify the bone regenerative capacity. In this study, immature-osteoblasts isolated from the alveolar bone of aged rats were encapsulated in GelMA-RF and transplanted into bone defects, confirming early bone regeneration sufficiently. Furthermore, in vitro analysis confirmed bone formation-related gene expression and calcification capacity, demonstrating that GelMA-RF is a suitable material for bone regeneration. These findings suggest that even alveolar bone immature-osteoblasts derived from aged individuals, when combined with GelMA-RF, hold promise as a novel therapeutic approach for regenerating complex and extensive bone defects.