Dental pulp regeneration therapy for the pulpless tooth has attracted recent attention, and clinical trial studies are underway with the tissue engineering approach. However, there remain many concerns, including the extended period for regenerating the dental pulp. In addition, the use of scaffolds increases the risk of inflammation and infection. To establish a basic technology for novel dental pulp regenerative therapy that allows transplant of pulp-like tissue, we attempted to fabricate scaffold-free 3-dimensional (3D) cell constructs composed of dental pulp stem cells (DPSCs). Furthermore, we assessed viability of these 3D DPSC constructs for dental pulp regeneration through in vitro and in vivo studies. For the in vitro study, we obtained 3D DPSC constructs by shaping sheet-like aggregates of DPSCs with a thermoresponsive hydrogel. DPSCs within constructs remained viable even after prolonged culture; furthermore, 3D DPSC constructs possessed a self-organization ability necessary to serve as a transplant tissue. For the in vivo study, we filled the human tooth root canal with DPSC constructs and implanted it subcutaneously into immunodeficient mice. We found that pulp-like tissues with rich blood vessels were formed within the human root canal 6 wk after implantation. Histologic analyses revealed that transplanted DPSCs differentiated into odontoblast-like mineralizing cells at sites in contact with dentin; furthermore, human CD31-positive endothelial cells were found at the center of regenerated tissue. Thus, the self-organizing ability of 3D DPSC constructs was active within the pulpless root canal in vivo. In addition, blood vessel-rich pulp-like tissues can be formed with DPSCs without requiring scaffolds or growth factors. The technology established in this study allows us to prepare DPSC constructs with variable sizes and shapes; therefore, transplantation of DPSC constructs shows promise for regeneration of pulpal tissue in the pulpless tooth.

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Dental pulp regeneration is a promising approach to restore the vitality of necrotic teeth. We have previously reported the fabrication of scaffold-free cell constructs containing only dental pulp stem cells (DPSCs) and their ability to form pulp-like tissue in the pulpless tooth. However, the DPSC construct could not build pulp-like tissue with a full root length because it is difficult to induce blood vessels from a small root canal foramen. Therefore, we hypothesized that vascular structure could be preformed in the DPSC construct by employing endothelial differentiation capability of DPSCs, and vascularized constructs might facilitate dental pulp regeneration in the pulpless tooth. In this study, vascularized DPSC constructs were fabricated by inducing endothelial differentiation, and then we investigated the behavior of differentiated DPSCs, the internal structure of cell constructs, and their pulp regenerative ability in vivo. We observed that DPSCs positive for CD31 and von Willebrand factor were localized at the outer layer of constructs and formed a reticulated lumen structure. The cells constituting the outer layer of the construct expressed endothelial differentiation markers at higher levels than cells in the inner part. These results indicated that DPSCs in the outer layer differentiated into endothelial cells and formed vascular-like structures in the cell construct. Next, a vascularized DPSC construct was transplanted into the human pulpless tooth that was implanted into immunodeficient mice in the subcutaneous space. After 6 wk of implantation, the vascularized construct formed pulp-like tissues with higher density of human CD31-positive blood vessels when compared with specimens implanted with a DPSC construct without prevascularization. These results suggest that the vascular structure formed in the DPSC construct facilitated the blood supply and enhanced pulp regeneration. This study demonstrates that a vascularized DPSC construct is a prospective biomaterial as an implant for novel dental pulp regeneration.

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Freeze‐dry processing improves the operability and stability of cell‐based biomaterials and facilitates sterilization for clinical application. However, there is no established freeze‐drying protocol for engineered tissues. Recently, we reported that biomimetic bone tissues can be fabricated using scaffold‐free three‐dimensional (3D) cell constructs with potential applications as bone graft materials. The purpose of this study was to assess the influence of freeze drying on the morphology and components of 3D cell constructs. Cell constructs freeze dried in phosphate buffered saline (PBS) maintained organic and inorganic components; whereas sodium citrate buffer (SCB)‐treated constructs had significantly lower amounts of calcium and bone‐related proteins. Alkaline phosphatase (ALP) activity in cell constructs was maintained by freeze drying in 10% sucrose‐containing PBS, whereas cell constructs treated with PBS without sucrose or with sucrose‐containing SCB showed significant reductions of ALP activity. In this study, we found that sucrose‐containing phosphate buffer was suitable for freeze drying to maintain minerals and protein functions within 3D cell constructs, whereas citrate buffer was inappropriate. The insights gained by this study may facilitate the development of novel cell‐based biomaterials fabricated by tissue engineering approaches and bone graft biomaterials.