In the oral and maxillofacial region, the treatment of severe bone defects, caused by fractures, cancers, congenital abnormalities, etc., remains a great challenge. In addition, neurological disorders are frequently accompanied by these bone defects or the treatments for them. Therefore, novel bone regenerative techniques and methods to repair nerve injury are eagerly sought. Among them, strategies using dental pulp stem cells (DPSCs) are promising options. Human DPSCs can be collected easily from extracted teeth and are now considered a type of mesenchymal stem cell with higher clonogenic and proliferative potential. DPSCs have been getting attention as a cell source for bone and nerve regeneration. In this article, we reviewed the latest studies on osteogenic or neural differentiation of DPSCs as well as bone or neural regeneration methods using DPSCs and discussed the potential of DPSCs for bone and nerve tissue regeneration.

Mesenchymal stem cells (MSCs), a key cell source for regenerative medicine, are conventionally cultured in human autologous serum (HAS) or fetal bovine serum (FBS). Platelet-rich fibrin-conditioned medium (PRF-CM) has emerged as a potential alternative to FBS, promoting the osteogenic differentiation of dental pulp stem cells (DPSCs) more efficiently than FBS. However, the molecular mechanisms underlying PRF-CM-induced osteogenesis in DPSCs remain unclear. Therefore, the aim of this study was to elucidate these mechanisms. PRF-CM was prepared from peripheral blood samples collected from three healthy donors. DPSCs were derived from the dental pulp extracted from the third molars of three healthy donors. The mRNA expression patterns of DPSCs cultured in FBS or PRF-CM were compared using RNA sequencing (RNA-Seq). Differentially expressed genes (DEGs) were analyzed by Gene Ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses. The mean blood volume collected from the three donors was 51.3 mL, yielding a mean PRF-CM volume of 168 mL. RNA-Seq identified 2258 DEGs, and KEGG pathway and GO analysis revealed that PRF-CM upregulated biological activities associated with transforming growth factor beta (TGF-β) signaling and platelet-derived growth factor (PDGF) binding compared with FBS. Western blotting analysis revealed that pSMAD2/3 and phosphor-Akt (p-Akt) were upregulated in DPSCs treated with PRF-CM, indicating that PRF-CM upregulated TGF-β and PDGF signaling pathways. The addition of SB431542 (a TGF-β inhibitor) or imatinib mesylate (a PDGF inhibitor) to osteogenic differentiation medium with PRF-CM reduced alkaline phosphatase (ALP) activity and osteogenic marker gene expression in DPSCs, respectively. PRF-CM activates TGF-β and PDGF signaling pathways, thereby promoting the osteogenic differentiation of DPSCs. Moreover, the volume of PRF-CM obtained was approximately three times greater than the blood sample volume. These findings suggest that PRF-CM could serve as an effective alternative to HAS or FBS for bone regeneration therapy using MSCs.

Sagittal split ramus osteotomy (SSRO) sometimes induces an irregular split pattern referred to as a bad split. We investigated the risk factors for bad splits in the buccal plate of the ramus during SSRO. Ramus morphology and bad splits in the buccal plate of the ramus were assessed using preoperative and postoperative computed tomography images. Of the 53 rami analyzed, 45 had a successful split, and 8 had a bad split in the buccal plate. Horizontal images at the height of the mandibular foramen showed that there were significant differences in the ratio of the forward thickness to the backward thickness of the ramus between patients with a successful split and those with a bad split. In addition, the distal region of the cortical bone tended to be thicker and the curve of the lateral region of the cortical bone tended to be smaller in the bad split group than in the good split group. These results indicated that a ramus shape in which the width becomes thinner towards the back frequently induces bad splits in the buccal plate of the ramus during SSRO, and more attention should be paid to patients who have rami of these shapes in future surgeries.

Human dental pulp stem cells (DPSCs) exhibit multilineage differentiation capabilities and superior clonogenic and proliferative properties. However, the use of animal-derived components such as FBS raises concerns regarding the clinical application of stem-cell-based therapies. Platelet-rich fibrin (PRF) derived from human blood is rich in fibrin, platelets, and growth factors and acts as a bioactive scaffold for grafting with biomaterials. In this study, we assessed the efficacy of PRF-conditioned medium (CM) in promoting DPSCs proliferation and osteogenic differentiation compared with the standard culture medium supplemented with FBS. A comparison of DPSCs cultured in FBS and PRF-CM revealed no differences in characteristics or morphology. However, cells cultured with PRF-CM exhibited inferior proliferation rates and cell numbers during passage in comparison with those cultured with FBS. In contrast, DPSCs cultured in PRF-CM showed significantly higher levels of calcification, and RT-PCR confirmed that the gene expression levels of markers associated with osteoblast differentiation were significantly increased. The PRF-CM approach offers a convenient, straightforward, and advantageous method for culturing DPSCs, without relying on animal-derived components. In summary, this study introduces a novel application of PRF-CM for enhancing the osteogenesis of DPSCs, which provides an alternative to FBS culture medium and addresses concerns associated with the use of animal-derived components in clinical settings.

Dental pulp stem cells (DPSCs) demonstrate high proliferative and multilineage differentiation potential. As previously reported, the helioxanthin derivative 4-(4-methoxyphenyl)pyrido[40,30:4,5]thieno[2,3-b]pyridine-2-carboxamide (TH) has been demonstrated to induce the osteogenic differentiation of DPSCs. However, the mechanism of osteogenesis induced by TH in DPSCs remains unknown. The objective of this study was to identify functional extracellular vesicle (EV) microRNAs (miRNAs), and the principal genes involved in the TH-induced osteogenesis of DPSCs. DPSCs were derived from dental pulp extracted from the third molars of three healthy subjects, and were cultured with or without TH. miRNAs were extracted from DPSC-derived EVs. The gene expression patterns of mRNA and miRNA were compared using RNA-Seq and miRNA-Seq. To investigate miRNA/mRNA interacting networks, functional analyses were performed by Ingenuity Pathway Analysis. Alkaline phosphatase (ALP) staining demonstrated that treatment with TH resulted in enhanced ALP activity in DPSCs after 7 days. The expression levels of ALP and type 1 collagen alpha 1 were significantly higher in TH-induced DPSCs on day 7. RNA-Seq and miRNA-Seq analyses identified 869 differentially expressed genes (DEGs) and 18 miRNA-DEGs. Gene Ontology analysis of the mRNA-Seq results showed that TH induced several biological activities associated with signal transduction, cell adhesion, and cell differentiation. Integrated miRNA-mRNA analyses showed that these miRNAs contain the targeting information of 277 mRNAs of the DEGs. Among them, 17 target genes known to be involved in the differentiation of osteoblasts, and 24 target genes known to be involved in the differentiation of bone cells were identified. Quantitative real-time PCR showed that WNT5a expression in DPSCs was upregulated by 48 h of TH treatment. Upstream regulator analysis indicated that WNT3a, FOS, and RAC1 may be responsible for gene expression changes in DPSCs after TH treatment. EV miRNA regulatory networks might play crucial roles in TH-induced osteogenic differentiation of DPSCs. Our results presented herein offer valuable insights that will facilitate further research into the mechanism of osteogenesis of DPSCs, which is expected to lead to the clinical application of TH-induced DPSCs for bone regeneration. Furthermore, EVs derived from TH-induced DPSCs might be useful as therapeutic tools for bone defects.