The ability of human deciduous tooth dental pulp cells (HDDPCs) to differentiate into odontoblasts that generate mineralized tissue holds immense potential for therapeutic use in the field of tooth regenerative medicine. Realization of this potential depends on efficient and optimized protocols for the genetic manipulation of HDDPCs. In this study, we demonstrate the use of a PiggyBac (PB)-based gene transfer system as a method for introducing nonviral transposon DNA into HDDPCs and HDDPC-derived inducible pluripotent stem cells. The transfection efficiency of the PB-based system was significantly greater than previously reported for electroporation-based transfection of plasmid DNA. Using the neomycin resistance gene as a selection marker, HDDPCs were stably transfected at a rate nearly 40-fold higher than that achieved using conventional methods. Using this system, it was also possible to introduce two constructs simultaneously into a single cell. The resulting stable transfectants, expressing tdTomato and enhanced green fluorescent protein, exhibited both red and green fluorescence. The established cell line did not lose the acquired phenotype over three months of culture. Based on our results, we concluded that PB is superior to currently available methods for introducing plasmid DNA into HDDPCs. There may be significant challenges in the direct clinical application of this method for human dental tissue engineering due to safety risks and ethical concerns. However, the high level of transfection achieved with PB may have significant advantages in basic scientific research for dental tissue engineering applications, such as functional studies of genes and proteins. Furthermore, it is a useful tool for the isolation of genetically engineered HDDPC-derived stem cells for studies in tooth regenerative medicine.
Cells, Cultured ; DNA Transposable Elements ; Dental Pulp ; cytology ; Humans ; Induced Pluripotent Stem Cells ; cytology ; Nerve Tissue Proteins ; genetics ; Tooth, Deciduous ; cytology ; Transfection

BACKGROUND: Systemic and local factors may lead to disruption of craniofacial growth and development, causing an imbalance between the orofacial skeleton, muscle and soft tissue, dental occlusion, and the dental arch during growth periods. We aimed to reveal whether the prevalence of incompetent lip seal (ILS) varies with age and region, as well as to clarify the factors related to an ILS, in a national, large-scale epidemiological study. METHODS: We surveyed 3399 children, from 3 to 12 years of age, visiting 66 pediatric dental clinics throughout Japan. For this survey, we employed a questionnaire consisting of 44 questions regarding daily health conditions and lifestyle habits. We evaluated the differences in ILS prevalence by age and region (using a Cochran-Armitage test for trend and a Kruskal-Wallis test), and the relationship between ILS and factors investigated in the questionnaire (using Spearman's rank correlation coefficient). RESULTS: We observed that 30.7% of Japanese children exhibited an ILS and that the ILS rate increased with age (p < 0.001). There were no regional differences in the rate of ILS in Japanese children (p = 0.506). We revealed that 12 of 44 survey items exhibited a statistically significant correlation with ILS (p < 0.001), using Spearman's rank correlation coefficient. These items involved orofacial morphology, mouth breathing, and possibly, allergic rhinitis. CONCLUSION The rate of ILS seems to increase with age in children, throughout Japan. Therefore, this disorder may not self-correct during the growth periods in these children. Guidelines are required for pediatric dentists to recognize ILS among children aged 3-12 years.
Child ; Child, Preschool ; Cross-Sectional Studies ; Female ; Humans ; Japan/epidemiology* ; Lip/abnormalities* ; Male ; Prevalence