Hyaluronic Acid Induction Promotes the Differentiation of Human Neural Crest-like Cells into Periodontal Ligament Stem-like Cells.

Periodontal ligament (PDL) stem-like cells (PDLSCs) are promising for regeneration of the periodontium because they demonstrate multipotency, high proliferative capacity, and the potential to regenerate bone, cementum, and PDL tissue. However, the transplantation of autologous PDLSCs is restricted by limited availability. Since PDLSCs are derived from neural crest cells (NCs) and NCs persist in adult PDL tissue, we devised to promote the regeneration of the periodontium by activating NCs to differentiate into PDLSCs. SK-N-SH cells, a neuroblastoma cell line that reportedly has NC-like features, seeded on the extracellular matrix of PDL cells for 2 weeks, resulted in the significant upregulation of PDL marker expression. SK-N-SH cell-derived PDLSCs (SK-PDLSCs) presented phenotypic characteristics comparable to induced pluripotent stem cell (iPSC)-derived PDLSCs (iPDLSCs). The expression levels of various hyaluronic acid (HA)-related genes were upregulated in iPDLSCs and SK-PDLSCs compared with iPSC-derived NCs and SK-N-SH cells, respectively. The knockdown of CD44 in SK-N-SH cells significantly inhibited their ability to differentiate into SK-PDLSCs, while low-molecular HA (LMWHA) induction enhanced SK-PDLSC differentiation. Our findings suggest that SK-N-SH cells could be applied as a new model to induce the differentiation of NCs into PDLSCs and that the LMWHA-CD44 relationship is important for the differentiation of NCs into PDLSCs.

Characterization of a clonal human periodontal ligament stem cell line exposed to methacrylate resin-, bioactive glass-, or silicon-based root canal sealers.

The aim of this study was to characterize a clonal human periodontal ligament (PDL) stem cell line (line 2-23 cells) cultured with root canal sealers based on methacrylate resin (SuperBond sealer; SB), bioactive glass (Nishika Canal Sealer BG; BG), or silicon (GuttaFlow 2; GF). The sealers were set in rubber molds to form sealer discs. Line 2-23 cells were cultured with or without the discs for 3 days. The cell viability was evaluated by direct cell counting and MTT assay. Inflammation-, PDL-, collagen-, and cell cycle-related gene expression was investigated by real-time RT-PCR. Collagen production was analyzed by Picro Sirius Red staining. Calcium ion concentration in the culture was measured by a QuantiChrom calcium assay kit. Line 2-23 cells survived when cultured with GF discs, but decreased cell viability was observed with SB and BG discs. The expression of inflammation-related genes was higher in cells cultured with SB discs, and expression of PDL-related genes was lower in cells exposed to SB and BG discs. These discs also down-regulated collagen production in line 2-23 cells. BG discs increased calcium ion concentration in the culture medium. Cells exposed to GF discs exhibited the same inflammation-, PDL-, collagen-, and cell cycle-related gene expression and collagen production as untreated cells. These results suggested that the characteristics of line 2-23 cells cultured with GF discs was highly resemble to untreated cells throughout the 3 days of the culture model.

Mineral trioxide aggregate immersed in sodium hypochlorite reduce the osteoblastic differentiation of human periodontal ligament stem cells.

White mineral trioxide aggregate (WMTA) is a root canal treatment material, which is known to exhibit a dark brown color when in contact with sodium hypochlorite solution (NaOCl). This study aimed to investigate the effects of NaOCl on the surface properties of WMTA discs and WMTA-induced osteoblastic differentiation of periodontal ligament stem cells (PDLSCs). Mixed WMTA (ProRoot MTA) was filled into the molds to form WMTA discs. These discs were immersed in distilled water (D-WMTA) or 5% NaOCl (Na-WMTA). Their surface structures and Ca2+ release level was investigated. Moreover, they were cultured with a clonal human PDLSC line (line 1-17 cells). The main crystal structures of Na-WMTA were identical to the structures of D-WMTA. Globular aggregates with polygonal and needle-like crystals were found on D-WMTA and Na-WMTA, which included Ca, Si, Al, C and O. However, many amorphous structures were also identified on Na-WMTA. These structures consisted of Na and Cl, but did not include Ca. NaOCl immersion also reduced Ca2+ release level from whole WMTA discs. Line 1-17 cells cultured with D-WMTA formed many mineralized nodules and exhibited high expression levels of osteoblast-related genes. However, cells incubated with Na-WMTA generated a small number of nodules and showed low expression levels of osteoblast-related genes. These results indicated that NaOCl reduced Ca2+ release from WMTA by generating amorphous structures and changing its elemental distribution. NaOCl may also partially abolish the ability of WMTA to stimulate osteoblastic differentiation of PDLSCs.

Secreted Frizzled-Related Protein 1 Promotes Odontoblastic Differentiation and Reparative Dentin Formation in Dental Pulp Cells.

Direct pulp capping is an effective treatment for preserving dental pulp against carious or traumatic pulp exposure via the formation of protective reparative dentin by odontoblast-like cells. Reparative dentin formation can be stimulated by several signaling molecules; therefore, we investigated the effects of secreted frizzled-related protein (SFRP) 1 that was reported to be strongly expressed in odontoblasts of newborn molar tooth germs on odontoblastic differentiation and reparative dentin formation. In developing rat incisors, cells in the dental pulp, cervical loop, and inner enamel epithelium, as well as ameloblasts and preodontoblasts, weakly expressed Sfrp1; however, Sfrp1 was strongly expressed in mature odontoblasts. Human dental pulp cells (hDPCs) showed stronger expression of SFRP1 compared with periodontal ligament cells and gingival cells. SFRP1 knockdown in hDPCs abolished calcium chloride-induced mineralized nodule formation and odontoblast-related gene expression and decreased BMP-2 gene expression. Conversely, SFRP1 stimulation enhanced nodule formation and expression of BMP-2. Direct pulp capping treatment with SFRP1 induced the formation of a considerable amount of reparative dentin that has a structure similar to primary dentin. Our results indicate that SFRP1 is crucial for dentinogenesis and is important in promoting reparative dentin formation in response to injury.

Generation of biohybrid implants using a multipotent human periodontal ligament cell line and bioactive core materials.

We aimed to generate periodontal ligament (PDL) tissue-like structures from a multipotent human PDL cell line using three-dimensional (3D) bioprinting technology and to incorporate these structures with bioactive core materials to develop a new biohybrid implant system. After 3D bioprinting, single-cell spheroids were able to form 3D tubular structures (3DTBs). We established three types of complexes using 3DTBs and different core materials: 3DTB-titanium core (TIC), 3DTB-hydroxyapatite core (HAC), and 3DTB without a core material (WOC). The expressions of PDL-, angiogenesis-, cementum-, and bone-related genes were significantly increased in the three complexes compared with monolayer-cultured cells. Abundant collagen fibers and cells positive for the above markers were confirmed in the three complexes. However, more positive cells were detected in HAC than in WOC or TIC. The present results suggest that 3D-bioprinted structures and hydroxyapatite core materials can function similarly to the PDL and may be useful for the development of a new biohybrid implant system.

Possible function of GDNF and Schwann cells in wound healing of periodontal tissue.

OBJECTIVE: The purpose of this study was to evaluate the function of Schwann cells in wound healing of periodontal tissue. BACKGROUND: In our previous study, glial cell line-derived neurotrophic factor (GDNF) promoted the migration of human periodontal ligament (PDL) cells and that GDNF expression increased in wounded periodontal tissue. GDNF reportedly induces the migration of Schwann cell precursors. Schwann cells play a crucial role in the regeneration of peripheral tissues, including bone tissue. However, the role of Schwann cells on periodontal tissue regeneration remains unclear. METHODS: A transwell assay and a WST-1 (water-soluble tetrazolium compound-1) proliferation assay were used to determine whether GDNF promotes the migration and proliferation of Schwann cells, respectively. Quantitative RT-PCR and Alizarin Red S staining were performed to examine the effect of these cells on the differentiation of human preosteoblast (Saos2 cells) using conditioned medium from YST-1 (YST-1-CM). Western blotting analysis was performed to determine whether YST-1-CM activates ERK signaling pathway in Saos2 cells. The expression of Schwann cell markers, S100 calcium-binding protein B (S100-B) and growth associated protein 43 (GAP-43), was determined in normal and wounded periodontal tissue by immunofluorescent staining. RESULTS: Glial cell line-derived neurotrophic factor promoted the migration of YST-1 cells but did not affect the proliferation of YST-1 cells. Saos2 cells cultured with YST-1-CM increased the expression of osteoblastic markers and mineralization. YST-1-CM also induced phosphorylation of ERK1/2 in Saos2 cells. The number of S100-B-immunoreactive cells which also expressed GAP-43 was increased in rat wounded periodontal tissue during healing process. CONCLUSION: The accumulation of Schwann cells in wounded periodontal tissue suggests that they play a significant role in wound healing of this tissue, especially alveolar bone tissue.

Expression and function of dopamine in odontoblasts.

Dopamine (DA) is produced from tyrosine by tyrosine hydroxylase (TH). A recent study has reported that DA promotes the mineralization of murine preosteoblasts. However, the role of DA in odontoblasts has not been examined. Therefore, in this investigation, we researched the expression of TH and DA in odontoblasts and the effects of DA on the differentiation of preodontoblasts (KN-3 cells). Immunostaining showed that TH and DA were intensely expressed in odontoblasts and preodontoblasts of rat incisors and molars. KN-3 cells expressed D1-like and D2-like receptors for DA. Furthermore, DA promoted odontoblastic differentiation of KN-3 cells, whereas an antagonist of D1-like receptors and a PKA signaling blocker, inhibited such differentiation. However, antagonists of D2-like receptors promoted differentiation. These results suggested that DA in preodontoblasts and odontoblasts might promote odontoblastic differentiation through D1-like receptors, but not D2-like receptors, and PKA signaling in an autocrine or paracrine manner and plays roles in dentinogenesis.