Here we present a new model for alveolar jaw bone regeneration, which uses human dental pulp cells (hDPCs) combined with tyrosine-derived polycarbonate polymer scaffolds [E1001(1k)] containing beta-tricalcium phosphate (β-TCP) (E1001(1k)/β-TCP). E1001(1k)/β-TCP scaffolds (5 mm diameter X 1mm thickness) were fabricated to fit a 5 mm rat mandibular ramus critical bone defect. Five experimental groups were examined in this study: 1) E1001(1k)/β-TCP scaffolds seeded with a high density of hDPCs, 5.0 x 105 hDPCs/scaffold (CH); 2) E1001(1k)/β-TCP scaffolds seeded with a lower density of hDPCs, 2.5 x 105 hDPCs/scaffold (CL); 3) acellular E1001(1k)/β-TCP scaffolds (SA); 4) acellular E1001(1k)/β-TCP scaffolds supplemented with 4 µg recombinant human bone morphogenetic protein-2 (BMP); and 5) empty defects (ED). Replicate hDPC-seeded and acellular E1001(1k)/β-TCP scaffolds were cultured in vitro in osteogenic media (OM) for one week prior to implantation for 3 and 6 weeks. Live micro-computed tomography (µCT) imaging at 3 and 6 weeks post-implantation revealed robust bone regeneration in the BMP implant group. CH and CL groups exhibited similar uniformly distributed mineralized tissue coverage throughout the defects, but less than the BMP implants. In contrast, SA treated defects exhibited sparse areas of mineralized tissue regeneration. The ED group exhibited slightly reduced defect size. Histological analyses revealed no indication of an immune response. In addition, robust expression of dentin and bone differentiation marker expression was observed in hDPC-seeded scaffolds, while in contrast BMP and SA implants exhibited only bone and not dentin differentiation marker expression. hDPCs were detected in 3 week but not in 6 week hDPC seeded scaffold groups, indicating their survival for at least 3 weeks. Together, these results show that hDPC seeded E1001(1k)/β-TCP scaffolds support the rapid regeneration of osteo-dentin like mineralized jaw tissue, suggesting a promising new therapy for alveolar jaw bone repair and regeneration.

Here, we describe the characterization of tooth-germ organoids, three-dimensional (3D) constructs cultured in vitro with the potential to develop into living teeth. To date, the methods used to successfully create tooth organoids capable of forming functional teeth have been quite limited. Recently, hydrogel microparticles (HMP) have demonstrated utility in tissue repair and regeneration based on their useful characteristics, including their scaffolding ability, effective cell and drug delivery, their ability to mimic the natural tissue extracellular matrix, and their injectability. These outstanding properties led us to investigate the utility of using HMPs (average diameter: 158 ± 32 µm) derived from methacrylated gelatin (GelMA) (degree of substitution: 100%) to create tooth organoids. The tooth organoids were created by seeding human dental pulp stem cells (hDPSCs) and porcine dental epithelial cells (pDE) onto the HMPs, which provided an extensive surface area for the cells to effectively attach and proliferate. Interestingly, the cell-seeded HMPs cultured on low-attachment tissue culture plates with gentle rocking self-assembled into organoids, within which the cells maintained their viability and morphology throughout the incubation period. The self-assembled organoids reached a volume of ~50 mm³ within two weeks of the in vitro tissue culture. The co-cultured hDPSC-HMP and pDE-HMP structures effectively attached to each other without any externally applied forces. The presence of polarized, differentiated dental cells in these composite tooth-bud organoids demonstrated the potential of self-assembled dental cell HMPs to form tooth-bud organoid-like structures for potential applications in tooth regeneration strategies.

Currently used methods to repair craniomaxillofacial (CMF) bone and tooth defects require a multi-staged surgical approach for bone repair followed by dental implant placement. Our previously published results demonstrated significant bioengineered bone formation using human dental pulp stem cell (hDPSC)-seeded tyrosine-derived polycarbonate scaffolds (E1001(1K)-bTCP). Here, we improved upon this approach using a modified TyroFill (E1001(1K)/dicalcium phosphate dihydrate (DCPD)) scaffold-supported titanium dental implant model for simultaneous bone–dental implant repair. TyroFill scaffolds containing an embedded titanium implant, with (n = 3 each time point) or without (n = 2 each time point) seeded hDPCs and Human Umbilical Vein Endothelial Cells (HUVECs), were cultured in vitro. Each implant was then implanted into a 10 mm full-thickness critical-sized defect prepared on a rabbit mandibulee. After 1 and 3 months, replicate constructs were harvested and analyzed using Micro-CT histological and IHC analyses. Our results showed significant new bone formation surrounding the titanium implants in cell-seeded TyroFill constructs. This study indicates the potential utility of hDPSC/HUVEC-seeded TyroFill scaffolds for coordinated CMF bone–dental implant repair.

Tooth development Each tooth, even though it looks simple, is an organ with complex structure and consists of multiple types of tissues (Ten Cate, 1998). Dentin is the main component, which is a hard and avascular tissue with a small hollow tubular inside. Enamel is the hardest tissue of the whole living body, protecting the dentin underneath by covering the crown dentin. Cementum, including cellular and acellular cementum, is present on the surface of root dentin. Other than those three types of hard tissue, a tooth also contains dental pulp, the highly vascularized soft tissue that acts as nutrition source and biosensor, as well as periodontal ligament (PDL), the tissue with well-organized collagen bundles that anchors the tooth to the surrounding alveolar bone. Among those tissues, only enamel is from the epithelium. The rest of the dental tissues are all from the neural crest-cell-derived mesenchyme (Nanci, 2007). The sequential and reciprocal interactions between the oral epithelium and the cranial neural crest-derived mesenchyme are essential to control tooth formation, including their size, number, and shape (Thesleff and Nieminen, 1996).