Establishing and characterizing human stem cells from the apical papilla immortalized by hTERT gene transfer.

Stem cells from the apical papilla (SCAPs) are promising candidates for regenerative endodontic treatment and tissue regeneration in general. However, harvesting enough cells from the limited apical papilla tissue is difficult, and the cells lose their primary phenotype over many passages. To get over these challenges, we immortalized human SCAPs with lentiviruses overexpressing human telomerase reverse transcriptase (hTERT). Human immortalized SCAPs (hiSCAPs) exhibited long-term proliferative activity without tumorigenic potential. Cells also expressed mesenchymal and progenitor biomarkers and exhibited multiple differentiation potentials. Interestingly, hiSCAPs gained a stronger potential for osteogenic differentiation than the primary cells. To further investigate whether hiSCAPs could become prospective seed cells in bone tissue engineering, in vitro and in vivo studies were performed, and the results indicated that hiSCAPs exhibited strong osteogenic differentiation ability after infection with recombinant adenoviruses expressing BMP9 (AdBMP9). In addition, we revealed that BMP9 could upregulate ALK1 and BMPRII, leading to an increase in phosphorylated Smad1 to induce the osteogenic differentiation of hiSCAPs. These results support the application of hiSCAPs in tissue engineering/regeneration schemes as a stable stem cell source for osteogenic differentiation and biomineralization, which could be further used in stem cell-based clinical therapies.

SATB2: A versatile transcriptional regulator of craniofacial and skeleton development, neurogenesis and tumorigenesis, and its applications in regenerative medicine.

SATB2 (special AT-rich sequence-binding protein 2) is a member of the special AT-rich binding protein family. As a transcription regulator, SATB2 mainly integrates higher-order chromatin organization. SATB2 expression appears to be tissue- and stage-specific, and is governed by several cellular signaling molecules and mediators. Expressed in branchial arches and osteoblast-lineage cells, SATB2 plays a significant role in craniofacial pattern and skeleton development. In addition to regulating osteogenic differentiation, SATB2 also displays versatile functions in neural development and cancer progression. As an osteoinductive factor, SATB2 holds great promise in improving bone regeneration toward bone defect repair. In this review, we have summarized our current understanding of the physiological and pathological functions of SATB2 in craniofacial and skeleton development, neurogenesis, tumorigenesis and regenerative medicine.

Artificial intelligence in digital cariology: a new tool for the diagnosis of deep caries and pulpitis using convolutional neural networks.

BACKGROUND: An accurate diagnosis of deep caries and pulpitis on periapical radiographs is a clinical challenge. METHODS: A total of 844 radiographs were included in this study. Of the 844, 717 (85%) were used for training and 127 (15%) were used for testing the three convolutional neural networks (CNNs) (VGG19, Inception V3, and ResNet18). The performance [accuracy, precision, sensitivity, specificity, and the area under the receiver operating characteristic curve (AUC)] of the CNNs were evaluated and compared. The CNN model with the best performance was further integrated with clinical parameters to see whether multi-modal CNN could provide an enhanced performance. The Gradient-weighted Class Activation Mapping (Grad-CAM) technique illustrates what image feature was the most important for the CNNs. RESULTS: The CNN of ResNet18 demonstrated the best performance [accuracy =0.82, 95% confidence interval (CI): 0.80-0.84; precision =0.81, 95% CI: 0.73-0.89; sensitivity =0.85, 95% CI: 0.79-0.91; specificity =0.82, 95% CI: 0.76-0.88; and AUC =0.89, 95% CI: 0.86-0.92], compared with VGG19 and Inception V3 as well as the comparator dentists. Therefore, ResNet18 was chosen to integrate with clinical parameters to produce the multi-modal CNN of ResNet18 + C, which showed a significantly enhanced performance (accuracy =0.86, 95% CI: 0.84-0.88; precision =0.85, 95% CI: 0.76-0.94; sensitivity =0.89, 95% CI: 0.83-0.95; specificity =0.86, 95% CI: 0.79-0.93; and AUC =0.94, 95% CI: 0.91-0.97). CONCLUSIONS: The CNN of ResNet18 showed good performance (accuracy, precision, sensitivity, specificity, and AUC) for the diagnosis of deep caries and pulpitis. The multi-modal CNN of ResNet18 + C (ResNet18 integrated with clinical parameters) demonstrated a significantly enhanced performance, with promising potential for the diagnosis of deep caries and pulpitis.

Special AT-rich sequence-binding protein 2 (Satb2) synergizes with Bmp9 and is essential for osteo/odontogenic differentiation of mouse incisor mesenchymal stem cells.

OBJECTIVES: Mouse incisor mesenchymal stem cells (MSCs) have self-renewal ability and osteo/odontogenic differentiation potential. However, the mechanism controlling the continuous self-renewal and osteo/odontogenic differentiation of mouse incisor MSCs remains unclear. Special AT-rich sequence-binding protein 2 (SATB2) positively regulates craniofacial patterning, bone development and regeneration, whereas SATB2 deletion or mutation leads to craniomaxillofacial dysplasia and delayed tooth and root development, similar to bone morphogenetic protein (BMP) loss-of-function phenotypes. However, the detailed mechanism underlying the SATB2 role in odontogenic MSCs is poorly understood. The aim of this study was to investigate whether SATB2 can regulate self-renewal and osteo/odontogenic differentiation of odontogenic MSCs. MATERIALS AND METHODS: Satb2 expression was detected in the rapidly renewing mouse incisor mesenchyme by immunofluorescence staining, quantitative RT-PCR and Western blot analysis. Ad-Satb2 and Ad-siSatb2 were constructed to evaluate the effect of Satb2 on odontogenic MSCs self-renewal and osteo/odontogenic differentiation properties and the potential role of Satb2 with the osteogenic factor bone morphogenetic protein 9 (Bmp9) in vitro and in vivo. RESULTS: Satb2 was found to be expressed in mesenchymal cells and pre-odontoblasts/odontoblasts. We further discovered that Satb2 effectively enhances mouse incisor MSCs self-renewal. Satb2 acted synergistically with the potent osteogenic factor Bmp9 in inducing osteo/odontogenic differentiation of mouse incisor MSCs in vitro and in vivo. CONCLUSIONS: Satb2 promotes self-renewal and osteo/odontogenic differentiation of mouse incisor MSCs. Thus, Satb2 can cooperate with Bmp9 as a new efficacious bio-factor for osteogenic regeneration and tooth engineering.

BMP9-initiated osteogenic/odontogenic differentiation of mouse tooth germ mesenchymal cells (TGMCS) requires Wnt/ß-catenin signalling activity.

Teeth arise from the tooth germ through sequential and reciprocal interactions between immature epithelium and mesenchyme during development. However, the detailed mechanism underlying tooth development from tooth germ mesenchymal cells (TGMCs) remains to be fully understood. Here, we investigate the role of Wnt/ß-catenin signalling in BMP9-induced osteogenic/odontogenic differentiation of TGMCs. We first established the reversibly immortalized TGMCs (iTGMCs) derived from young mouse mandibular molar tooth germs using a retroviral vector expressing SV40 T antigen flanked with the FRT sites. We demonstrated that BMP9 effectively induced expression of osteogenic markers alkaline phosphatase, collagen A1 and osteocalcin in iTGMCs, as well as in vitro matrix mineralization, which could be remarkably blunted by knocking down ß-catenin expression. In vivo implantation assay revealed that while BMP9-stimulated iTGMCs induced robust formation of ectopic bone, knocking down ß-catenin expression in iTGMCs remarkably diminished BMP9-initiated osteogenic/odontogenic differentiation potential of these cells. Taken together, these discoveries strongly demonstrate that reversibly immortalized iTGMCs retained osteogenic/odontogenic ability upon BMP9 stimulation, but this process required the participation of canonical Wnt signalling both in vitro and in vivo. Therefore, BMP9 has a potential to be applied as an efficacious bio-factor in osteo/odontogenic regeneration and tooth engineering. Furthermore, the iTGMCs may serve as an important resource for translational studies in tooth tissue engineering.

Role of Special AT-Rich Sequence-Binding Protein 2 in the Osteogenesis of Human Dental Mesenchymal Stem Cells.

Dental mesenchymal stem cells (MSCs) are recognized as a critical factor in repair of defective craniofacial bone owing to the multiple differentiation potential, the ability to regenerate distinct tissues, and the advantage that they can be easily obtained by relatively noninvasive procedures. Special AT-rich sequence-binding protein 2 (SATB2) is a nuclear matrix protein, involved in chromatin remodeling and transcriptional regulation, and has been reported to be as a positive regulator of osteoblast differentiation, bone formation, and bone regeneration in MSCs. In this study, we systematically investigated the capability of SATB2 to promote the osteogenic differentiation of periodontal ligament stem cells (PDLSCs), dental pulp stem cells (DPSCs), and stem cells from human exfoliated deciduous teeth (SHED). RNA-seq analysis and quantitative real-time PCR (RT-PCR) revealed that genes regulating osteogenic differentiation were differentially expressed among three cell types and SATB2 was found to be expressed at a relatively high level. When the three cell types overexpressed SATB2 with AdSATB2 infection, alkaline phosphatase (ALP) staining, ALP activity, Alizarin Red S staining, and quantification tended to increase with an increasing infection rate. It showed opposite results after infection with AdsiSATB2. RNA-seq analysis indicated that the expression of downstream osteogenic genes was affected by AdSATB2 infection and quantitative RT-PCR confirmed that nine osteogenic genes (Spp1, Sema7a, Atf4, Ibsp, Col1a1, Sp7, Igfbp3, Dlx3, and Alpl) were upregulated, to various extents, following SATB2 overexpression. In addition, quantitative PCR results indicated that SATB2 affected the expression of MSC markers. These results suggested an important role of SATB2 in the osteogenesis of PDLSCs, DPSCs, and SHED. Further research is warranted to investigate SATB2-mediated regulation of osteogenic differentiation and to evaluate the therapeutic use of SATB2 for the regeneration of defective craniofacial bone tissue.