The dental follicle is a mesenchymal tissue that surrounds the developing tooth germ. During tooth root formation, periodontal components, viz., cementum, periodontal ligament (PDL), and alveolar bone, are created by dental follicle progenitors. Here, we report the presence of PDL progenitors in mouse dental follicle (MDF) cells. MDF cells were obtained from mouse incisor tooth germs and immortalized by the expression of a mutant human papilloma virus type 16 E6 gene lacking the PDZ-domain-binding motif. MDF cells expressing the mutant E6 gene (MDF( E6-EGFP ) cells) had an extended life span, beyond 150 population doublings (PD). In contrast, normal MDF cells failed to proliferate beyond 10 PD. MDF( E6-EGFP ) cells expressed tendon/ligament phenotype-related genes such as Scleraxis (Scx), growth and differentiation factor-5, EphA4, Six-1, and type I collagen. In addition, the expression of periostin was observed. To elucidate the differentiation capacity of MDF( E6-EGFP ) cells in vivo, the cells were transplanted into severe combined immunodeficiency mice. At 4 weeks, MDF( E6-EGFP ) cell transplants had the capacity to generate a PDL-like tissue that expressed periostin, Scx, and type XII collagen and the fibrillar assembly of type I collagen. Our findings suggest that MDF( E6-EGFP ) cells can act as PDL progenitors, and that these cells may be a useful research tool for studying PDL formation and for developing regeneration therapies.

Gelatin methacryloyl (GelMA) is a versatile biomaterial that has been used in various biomedical fields. UV light is commonly used to photocrosslink such materials; however, its use has raised several biosafety concerns. We investigated the mechanical and biological properties of a visible-wavelength (VW)-light-crosslinked gelatin-based hydrogel to evaluate its viability as a scaffold for bone regeneration in bone-destructive disease treatment. Irgacure2959 or riboflavin was added as a photoinitiator to create GelMA solutions. GelMA solutions were poured into a mold and exposed to either UV or VW light. KUSA-A1 cell-laden GelMA hydrogels were crosslinked and then cultured. Mechanical characterization revealed that the stiffness range of GelMA–RF hydrogel was suitable for osteoblast differentiation. KUSA-A1 cells encapsulated in GelMA hydrogels photopolymerized with VW light displayed significantly higher cell viability than cells encapsulated in hydrogels photopolymerized with UV light. We also show that the expression of osteogenesis-related genes at a late stage of osteoblast differentiation in osteoblasts encapsulated in GelMA–RF hydrogel was markedly increased under osteoblast differentiation-inducing conditions. The GelMA–RF hydrogel served as an excellent scaffold for the encapsulation of osteoblasts. GelMA–RF hydrogel-encapsulated osteoblasts have the potential not only to help regenerate bone mass but also to treat complex bone defects associated with bone-destructive diseases such as periodontitis.

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