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.

Development of a novel direct dental pulp-capping material using 4-META/MMA-TBB resin with nano hydroxyapatite.

In the case of dental pulp exposure, direct pulp capping is often performed to preserve vital dental pulp tissue. Numerous studies regarding the development of direct pulp-capping materials have been conducted, but materials with an appropriate sealing ability, which induce dense reparative dentin formation, have not been developed. Although nano hydroxyapatite (naHAp) is a bone-filling material with bioactivity and biocompatibility, the inductive effects of naHAp on reparative dentin formation remain unclear. In the present study, the effects of dental adhesive material 4-methacryloxyethyl trimellitate anhydride/methyl methacrylate tri-n-butylborane [4-META/MMA-TBB or Super-bond (SB)], which included 10%, 30%, and 50% naHAp (naHAp/SB) on odontoblastic differentiation of dental pulp stem cells (DPSCs) and reparative dentin formation were investigated. Scanning electron microscopy (SEM) and energy dispersive X-ray spectrometer analysis were performed to verify the existence of naHAp particles on the surface of naHAp/SB discs. The tensile adhesive strength of naHAp/SB was measured using a universal testing machine. As a result, 10% naHAp/SB and 30% naHAp/SB showed almost the same tensile adhesive strength as SB but 50% naHAp/SB showed significantly lower than the other experimental group. WST-1 proliferation assay and SEM analysis revealed that naHAp/SB did not affect the proliferation of DPSCs. Calcium release assay, quantitative RT-PCR, and western blotting analysis demonstrated that naHAp/SB did not release calcium ion but 30% naHAp/SB increased the expression of calcium-sensing receptor (CaSR) in DPSCs. Additionally, quantitative RT-PCR, western blotting analysis, Alizarin Red S- and von Kossa staining revealed that 30% naHAp/SB induced odontoblastic differentiation of DPSCs, which was inhibited by a MEK/ERK inhibitor and CaSR antagonist. Furthermore, 30% naHAp/SB promoted dense reparative dentin formation in an experimentally-formed rat dental pulp exposure model. These findings suggest that 30% naHAp/SB can be used as an ideal direct pulp capping material.

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.