Self-Assembly of Tooth Root Organoid from Postnatal Human Dental Stem Cells.

Challenges remain in simultaneously regenerating the multiple diverse tissues of the tooth root in a spatially organized manner. Previously, our research group has established that scaffold-free tissue engineering approaches enable dental pulp stem/progenitor cells (DPSCs) and periodontal ligament (PDL) stem/progenitor cells (PDLSCs) to self-assemble into dentin-pulp and PDL-cementum organoids, respectively. In this study, we leveraged the innate self-organizing capacity of DPSCs and PDLSCs to now engineer organoids that resemble the full tooth root. Scaffold-free engineered tissues were generated using a heterogeneous mixture of human DPSCs and PDLSCs. Within 2 days of construct formation, PDLSCs and DPSCs became spatially restricted to the periphery and center of the constructs, respectively, emulating their anatomical positions in the tooth root. Histological and microcomputed tomography analyses showed that organoids exhibited a striated mineral pattern with a central unmineralized core, surrounded by a mineralized tissue structure, enclosed within a second peripheral unmineralized tissue, similar to the natural tooth root. Interestingly, DPSCs gave rise to the central unmineralized tissue and the inner portion of the mineralized tissue, and PDLSCs generated the outer portion of the mineralized tissue and the peripheral soft tissue. Quantitative image analysis of immunofluorescent staining revealed increased dentin sialophosphoprotein expression in the region of mineralized tissue associated with DPSCs and increased cementum protein-1 expression in the portion formed by PDLSCs, demonstrating that tooth root organoids comprise two biochemically distinct mineralized tissues characteristic of dentin-like and cementum-like structures, respectively. In addition, PDL-associated protein-1 expression was localized to the peripheral soft tissue, suggesting the formation of a rudimentary PDL-like structure. This study demonstrates that DPSCs and PDLSCs have an inherent ability to orchestrate the formation of a full tooth root-like structure. These organoids present a biomimetic model system to study cellular dynamics driving dental tissue repair or could be utilized therapeutically as biological dental implants.

Differential Effects of Escherichia coli- Versus Porphyromonas gingivalis-derived Lipopolysaccharides on Dental Pulp Stem Cell Differentiation in Scaffold-free Engineered Tissues.

INTRODUCTION: To leverage the therapeutic capabilities of dental pulp stem cells (DPSCs) for regenerative endodontic applications, a better understanding of their innate defense and reparative processes is needed. Lipopolysaccharide (LPS) is a major virulent factor of gram-negative bacteria and contributor to endodontic infections. We have developed 3-dimensional scaffold-free DPSC tissues that self-organize into dentin-pulp organoids comprising a mineralized dentin-like tissue on the periphery and an unmineralized pulp-like core. In this study, scaffold-free DPSC constructs were used as controllable experimental models to study the DPSC response to bacterial challenge. METHODS: Scaffold-free constructs were engineered using DPSCs isolated from human third molars. To simulate bacterial exposure, DPSC constructs were exposed to either Porphyromonas gingivalis-derived LPS or Escherichia coli-derived LPS. The effects of LPS on DPSC differentiation, proliferation, and apoptosis were evaluated. RESULTS: Engineered tissues lacking LPS treatment self-organized into dentin-pulp organoids. LPS treatment did not negatively affect DPSC proliferation or apoptosis in the engineered tissues. Both E. coli LPS and P. gingivalis LPS inhibited the up-regulation of RUNX2 messenger RNA expression and reduced the expression of the odontoblast-associated proteins (P < .05), suggesting that LPS is inhibiting odontoblastic differentiation. However, only E. coli LPS treatment significantly reduced mineral deposition in the DPSC (P < .05) constructs, indicating that E. coli LPS but not P. gingivalis LPS reduced functional differentiation of DPSCs and prevented DPSCs from self-organizing into a dentin-pulp complex-like structure. CONCLUSIONS: This study establishes scaffold-free DPSC constructs as models of oral disease. Furthermore, it emphasizes the diversity of LPS derived from different bacterial species and highlights the necessity of using LPS derived from clinically relevant bacteria in basic science investigations.

Dental Pulp Cell Sheets Enhance Facial Nerve Regeneration via Local Neurotrophic Factor Delivery.

An effective strategy for sustained neurotrophic factor (NTF) delivery to sites of peripheral nerve injury (PNI) would accelerate healing and enhance functional recovery, addressing the major clinical challenges associated with the current standard of care. In this study, scaffold-free cell sheets were generated using human dental pulp stem/progenitor cells, that endogenously express high levels of NTFs, for use as bioactive NTF delivery systems. Additionally, the effect of fibroblast growth factor 2 (FGF2) on NTF expression by dental pulp cell (DPC) sheets was evaluated. In vitro analysis confirmed that DPC sheets express high levels of NTF messenger RNA (mRNA) and proteins, and the addition of FGF2 to DPC sheet culture increased total NTF production by significantly increasing the cellularity of sheets. Furthermore, the DPC sheet secretome stimulated neurite formation and extension in cultured neuronal cells, and these functional effects were further enhanced when DPC sheets were cultured with FGF2. These neuritogenic results were reversed by NTF inhibition substantiating that DPC sheets have a positive effect on neuronal cell activity through the production of NTFs. Further evaluation of DPC sheets in a rat facial nerve crush injury model in vivo established that in comparison with untreated controls, nerves treated with DPC sheets had greater axon regeneration through the injury site and superior functional recovery as quantitatively assessed by compound muscle action potential measurements. This study demonstrates the use of DPC sheets as vehicles for NTF delivery that could augment the current methods for treating PNIs to accelerate regeneration and enhance the functional outcome. Impact statement The major challenges associated with current treatments of peripheral nerve injuries (PNIs) are prolonged repair times and insufficient functional recovery. Dental pulp stem/progenitor cells (DPCs) are known to endogenously express high levels of neurotrophic factors (NTFs), growth factors that enhance axon regeneration. In this study, we demonstrate that scaffold-free DPC sheets can act as effective carrier systems to facilitate the delivery and retention of NTF-producing DPCs to sites of PNIs and improve functional nerve regeneration. DPC sheets have high translational feasibility and could augment the current standard of care to enhance the quality of life for patients dealing with PNIs.