JSPS Postdoctoral Fellow - Division of Anatomy and Cell Biology of the Hard Tissue
Dental Pulp Biology
Henry F. Duncan, Yoshifumi Kobayashi, Yukako Yamauchi, Angela Quispe-Salcedo, Zhi Chao Feng, Jia Huang, Nicola C. Partridge, Teruyo Nakatani, Jeanine D’Armiento, and Emi Shimizu
Frontiers in Cell and Developmental Biology, eISSN: 2296634X, Published: 21 April 2022 Frontiers Media SA
Matrix-metalloproteinase-13 (MMP13) is important for bone formation and remodeling; however, its role in tooth development remains unknown. To investigate this, MMP13-knockout (Mmp13−/−) mice were used to analyze phenotypic changes in the dentin–pulp complex, mineralization-associated marker-expression, and mechanistic interactions. Immunohistochemistry demonstrated high MMP13-expression in pulp-tissue, ameloblasts, odontoblasts, and dentin in developing WT-molars, which reduced in adults, with human-DPC cultures demonstrating a &gt;2000-fold increase in Mmp13-expression during mineralization. Morphologically, Mmp13−/− molars displayed critical alterations in the dentin-phenotype, affecting dentin-tubule regularity, the odontoblast-palisade and predentin-definition with significantly reduced dentin volume (∼30% incisor; 13% molar), and enamel and dentin mineral-density. Reactionary-tertiary-dentin in response to injury was reduced at Mmp13−/− molar cusp-tips but with significantly more dystrophic pulpal mineralization in MMP13-null samples. Odontoblast differentiation-markers, nestin and DSP, reduced in expression after MMP13-loss in vivo, with reduced calcium deposition in MMP13-null DPC cultures. RNA-sequencing analysis of WT and Mmp13−/− pulp highlighted 5,020 transcripts to have significantly &gt;2.0-fold change, with pathway-analysis indicating downregulation of the Wnt-signaling pathway, supported by reduced in vivo expression of the Wnt-responsive gene Axin2. Mmp13 interaction with Axin2 could be partly responsible for the loss of odontoblastic activity and alteration to the tooth phenotype and volume which is evident in this study. Overall, our novel findings indicate MMP13 as critical for tooth development and mineralization processes, highlighting mechanistic interaction with the Wnt-signaling pathway.
Chihiro Imai, Hiroto Sano, Angela Quispe-Salcedo, Kotaro Saito, Mitsushiro Nakatomi, Hiroko Ida-Yonemochi, Hideyuki Okano, and Hayato Ohshima
Journal of Oral Biosciences, ISSN: 13490079, eISSN: 18803865, Pages: 77-84, Published: March 2022 Elsevier BV
Kuniko Nakakura‐Ohshima, Angela Quispe‐Salcedo, Hiroto Sano, Haruaki Hayasaki, and Hayato Ohshima
Dental Traumatology, ISSN: 16004469, eISSN: 16009657, Pages: 677-690, Published: October 2021 Wiley
BACKGROUND/AIM Root length is a critical factor for dental pulp regeneration following tooth replantation. The aim of this study was to analyze the effects of reducing the root length by apicoectomy on the pulp healing process using a model for tooth replantation. MATERIAL AND METHODS After extraction of the upper first molars (M1) of 3-week-old mice, the roots from the experimental group (EG) were shortened to half to two-thirds of their length before replantation, whereas in the control group (CG) the extracted teeth were immediately repositioned into their alveolar sockets. To determine the effects of root resection on the survival of inherent pulp cells, this study included tooth transplantation with root resection using wild-type (WT) and green fluorescent protein (GFP) transgenic mice. The M1 of GFP transgenic mice were transplanted into the alveolar socket of the M1 of WT mice. The roots of the right M1 were shortened (EG), whereas the left M1 remained untreated (CG). RESULTS Apoptotic cells in the EG significantly decreased in number compared with the CG at day 3. Cell proliferative activity in the EG was significantly higher than that in the CG in the root pulp during days 3-5, and nestin-positive odontoblast-like cells began to arrange themselves along the pulp-dentin border in the cusp area at day 5 in the EG but not in the CG. At week 2, tertiary dentin had formed throughout the pulp in the EG, whereas the combined tissue of dentin and bone occupied the pulp space in 60% of the CG. Root resection also positively affected the survival of inherent pulp cells to differentiate into odontoblast-like cells as demonstrated by transplantation using GFP transgenic mice. CONCLUSIONS Reducing the root length accelerated pulp regeneration following tooth replantation due to the better environment for revascularization.
Yoshifumi Kobayashi, Angela Quispe-Salcedo, Sanika Bodas, Satoko Matsumura, Erhao Li, Richard Johnson, Marwa Choudhury, Daniel H. Fine, Siva Nadimpalli, Henry F. Duncan, Amel Dudakovic, Andre J. van Wijnen, and Emi Shimizu
Biochemical and Biophysical Research Communications, ISSN: 0006291X, eISSN: 10902104, Volume: 567, Pages: 72-78, Published: 27 August 2021 Elsevier BV
Enhancer of zeste homolog 2 (EZH2) is the catalytic core of polycomb repressive complex 2 (PRC2), which primarily methylates lysine 27 on histone H3 (H2K27me3), generating transcriptionally suppressed heterochromatin. Since EZH2 suppresses expression of genes involved in dentin formation, we examined the role of EZH2 in tooth development. Intriguingly, microCT analysis of teeth from mice with conditional Ezh2 knockout in uncommitted mesenchymal cells showed hyper-mineralization of enamel, which is produced by the epithelial-lineage cells, ameloblasts. Scanning electron microscopy analysis and nano-indentation of the incisor enamel from knockout mice revealed smaller inter-rod spaces and higher hardness compared to wild type enamel, respectively. Interestingly, expression of the calcium channel subunit gene, Orai2, was decreased compared to its competitor, Orai1, both in knockout mouse incisors and the ex vivo culture of ameloblasts with the surrounding tissues under EZH2 inhibition. Moreover, histological analysis of incisor from knockout mice showed decreased ameloblastin and expedited KLK4 expression in the ameloblasts. These observations suggest that EZH2 depletion in dental mesenchymal cells reduces enamel matrix formation and increases enamel protease activity from ameloblasts, resulting in enamel hyper-mineralization. This study demonstrates the significant role of the suppressive H3K27me3 mark for heterochromatin on enamel formation.
Angela Quispe-Salcedo and Hayato Ohshima
Journal of Clinical Medicine, eISSN: 20770383, Published: 1 August 2021 MDPI AG
The dental pulp is a soft connective tissue of ectomesenchymal origin that harbors distinct cell populations, capable of interacting with each other to maintain the vitality of the tooth. After tooth injuries, a sequence of complex biological events takes place in the pulpal tissue to restore its homeostasis. The pulpal response begins with establishing an inflammatory reaction that leads to the formation of a matrix of reactionary or reparative dentin, according to the nature of the exogenous stimuli. Using several in vivo designs, antigen-presenting cells, including macrophages and dendritic cells (DCs), are identified in the pulpal tissue before tertiary dentin deposition under the afflicted area. However, the precise nature of this phenomenon and its relationship to inherent pulp cells are not yet clarified. This literature review aims to discuss the role of pulpal DCs and their relationship to progenitor/stem cells, odontoblasts or odontoblast-like cells, and other immunocompetent cells during physiological and pathological dentinogenesis. The concept of “dentin-pulp immunology” is proposed for understanding the crosstalk among these cell types after tooth injuries, and the possibility of immune-based therapies is introduced to accelerate pulpal healing after exogenous stimuli.
Angela Quispe-Salcedo, Takuichi Sato, Junko Matsuyama, Hiroko Ida-Yonemochi, and Hayato Ohshima
Regenerative Therapy, eISSN: 23523204, Pages: 216-225, Published: December 2020 Elsevier BV
Introduction Responses of oral-microflora-exposed dental pulp to a triple antibiotic paste (TAP), a mixture of ciprofloxacin, metronidazole, and minocycline in ointment with macrogol and propylene glycol, remain to be fully clarified at the cellular level. This study aimed to elucidate responses of oral-microflora-exposed dental pulp to capping with TAP in mouse molars. Methods A cavity was prepared on the first molars of 6-week-old mice to expose the dental pulp for 24 h. The exposed pulp was capped with TAP (TAP group) or calcium hydroxide cement (CH group), in addition to the combination of macrogol (M) and propylene glycol (P) (MP, control group), followed by a glass ionomer cement filling. The samples were collected at intervals of 1, 2, and 3 weeks, and immunohistochemistry for nestin and Ki-67 and deoxyuride-5′-triphosphate biotin nick end labeling (TUNEL) assay were performed in addition to quantitative real-time polymerase chain reaction (qRT-PCR) analyses. Results The highest occurrence rate of pulp necrosis was found in the control group followed by the CH group at Weeks 2 and 3, whereas the highest occurrence rate of healed areas in the dental pulp was observed in the TAP group at each time point. Tertiary dentin formation was first observed in the dental pulp of the TAP group at Week 2. In contrast, bone-like and/or fibrous tissues were frequently observed in the CH group. qRT-PCR analyses clarified that TAP activated the stem and dendritic cells at Weeks 1 and 2, respectively. Conclusions The use of TAP as a pulp-capping agent improved the healing process of oral-microflora-exposed dental pulp in mouse molars.
Vilma Chuquihuaccha-Granda, , Angela Quispe-Salcedo, , , and
Journal of Oral Research, ISSN: 07192460, eISSN: 07192479, Pages: 446-448, Published: November-December 2020 Facultad de Odontologia, Universidad de Concepcion
Mitsushiro Nakatomi, Angela Quispe-Salcedo, Masaka Sakaguchi, Hiroko Ida-Yonemochi, Hideyuki Okano, and Hayato Ohshima
Histochemistry and Cell Biology, ISSN: 09486143, eISSN: 1432119X, Volume: 149, Pages: 383-391, Published: 1 April 2018 Springer Science and Business Media LLC
The Nestin gene encodes type VI intermediate filament and is known to be expressed in undifferentiated cells during neurogenesis and myogenesis. To regulate Nestin expression, the first or second intron enhancer is activated in a tissue-dependent manner, for example, the former in mesodermal cells and the latter in neural stem cells. Although Nestin has also been used as a differentiation marker for odontoblasts during tooth development, how Nestin expression is regulated in odontoblasts remains unclear. Therefore, this study aimed to compare the expression patterns of Nestin-GFP (green fluorescent protein) with that of endogenous Nestin in developing teeth of Nestin-EGFP (enhanced GFP) transgenic mice, in which the second intron enhancer is connected with the EGFP domain, at postnatal 7d, 3w, and 8w. Immunohistochemical and in situ hybridization analyses revealed that endogenous Nestin protein and Nestin mRNA were intensely expressed in differentiated odontoblasts, while GFP immunoreactivity, which reflects the activity of Nestin second intron enhancer-mediated transcription, was mainly observed in the subodontoblastic layer. These results indicate that the first intron enhancer may be activated in differentiated odontoblasts. Intriguingly, Nestin-GFP expression in the subodontoblastic layer was found to be restricted to the coronal pulp of molars, which is susceptible to tooth injuries. Because the subodontoblastic layer serves as a reservoir of newly differentiated odontoblast-like cells upon exogenous stimuli to dentin, our findings suggest that the original odontoblasts and regenerated odontoblast-like cells may differently regulate Nestin expression.
Walid D. Fakhouri, Kareem Metwalli, Ali Naji, Sarah Bakhiet, Angela Quispe-Salcedo, Larissa Nitschke, Youssef A. Kousa, and Brian C. Schutte
Scientific Reports, eISSN: 20452322, Published: 1 December 2017 Springer Science and Business Media LLC
Interferon Regulatory Factor 6 (IRF6) and TWIST1 are transcription factors necessary for craniofacial development. Human genetic studies showed that mutations in IRF6 lead to cleft lip and palate and mandibular abnormalities. In the mouse, we found that loss of Irf6 causes craniosynostosis and mandibular hypoplasia. Similarly, mutations in TWIST1 cause craniosynostosis, mandibular hypoplasia and cleft palate. Based on this phenotypic overlap, we asked if Irf6 and Twist1 interact genetically during craniofacial formation. While single heterozygous mice are normal, double heterozygous embryos (Irf6+/−; Twist1+/−) can have severe mandibular hypoplasia that leads to agnathia and cleft palate at birth. Analysis of spatiotemporal expression showed that Irf6 and Twist1 are found in different cell types. Consistent with the intercellular interaction, we found reduced expression of Endothelin1 (EDN1) in mandible and transcription factors that are critical for mandibular patterning including DLX5, DLX6 and HAND2, were also reduced in mesenchymal cells. Treatment of mandibular explants with exogenous EDN1 peptides partially rescued abnormalities in Meckel’s cartilage. In addition, partial rescue was observed when double heterozygous embryos also carried a null allele of p53. Considering that variants in IRF6 and TWIST1 contribute to human craniofacial defects, this gene-gene interaction may have implications on craniofacial disorders.
S. Matsumura, A. Quispe-Salcedo, C.M. Schiller, J.S. Shin, B.M. Locke, S. Yakar, and E. Shimizu
Journal of Dental Research, ISSN: 00220345, eISSN: 15440591, Pages: 1153-1161, Published: 1 September 2017 SAGE Publications
Eph receptors belong to a subfamily of receptor tyrosine kinases that are activated by membrane-spanning ligands called ephrins. Previously, we demonstrated that the ephrinB1-EphB2 interaction regulates odontogenic/osteogenic differentiation from dental pulp cells (DPCs) in vitro. The goal of this study was to identify the molecular mechanisms regulated by the EphB2/ephrinB1 system that govern tertiary dentin formation in vitro and in vivo. During tooth development, ephrinB1, and EphB2 were expressed in preodontoblast and odontoblasts at postnatal day 4. EphrinB1 was continuously expressed in odontoblasts and odontoblastic processes until the completion of tooth eruption. In addition, ephrinB1 was expressed in odontoblastic processes 2 wk following tooth injury without pulp exposure, whereas EphB2 was expressed in the center of pulp niches but not odontoblasts. In a model of tooth injury with pulp exposure, ephrinB1 was strongly expressed in odontoblasts 4 wk postinjury. In vitro studies with human and mouse DPCs treated with calcium hydroxide (CH) or mineral trioxide aggregate (MTA) showed an increased expression of insulin-like growth factor 1 (IGF-1). Experiments using several inhibitors of IGF-1 receptor signaling revealed that inhibiting the Ras/Raf-1/MAPK pathway inhibited EphB2 expression, and inhibiting the PI3K/Akt/mTOR pathway specifically inhibited ephrinB1 gene expression. Tooth injury in mice with odontoblast-specific IGF-1 receptor ablation exhibited a reduced tertiary dentin volume, mineral density, and ephrinB1 expression 4 wk following injury. We conclude that the IGF-1/ephrinB1 axis plays significant roles in the early stages of tooth injury. Further research is needed to fully understand the potential of targeting ephrinB1 as a regenerative pulp therapy.
Angela Quispe-Salcedo, Hiroko Ida-Yonemochi, and Hayato Ohshima
Journal of Oral Biosciences, ISSN: 13490079, eISSN: 18803865, Pages: 124-130, Published: 1 May 2015 Elsevier BV
Abstract Objectives Glucose uptake plays a crucial role in early tooth morphogenesis and size determination. Recently, enzymatically synthesized glycogen (ESG), with the characteristics of natural glycogen (a major storage form of glucose), has been developed. This study aimed to elucidate the effectiveness of ESG on the pulpal healing process following intentionally delayed tooth replantation in mice. Methods The upper first molar was extracted, immersed in phosphate buffered saline (PBS) or ESG (5000kDa) solution (1mg/mL) for 60min, and then replanted. Immunohistochemistry (for nestin, osteopontin, and Ki-67), TUNEL assay, and reverse transcription-polymerase chain reaction were performed at different time points. Results Increased apoptosis occurred in the dental pulp of mice from both treatment groups at Day 7, followed by active cell proliferation at Day 14 and tertiary dentin and/or bone-like tissue deposition at Day 21, in the PBS group. In contrast, active cell proliferation and coronal immunoreaction for nestin occurred around Day 10, and hard tissue deposition were observed at Day 14, in the ESG group. The mRNA expression of genes encoding dentin sialophosphoprotein and nestin first reappeared in the ESG group at Day 5, while expression levels of alkaline phosphatase and osteopontin , as well as Cd11c , tended to increase from Day 3 in both groups, and that of the stem cell marker, octamer-binding transcription factor Oct3 / 4 , greatly enhanced at Day 1, particularly in the ESG group. Conclusions ESG improved the pulpal healing process of extracted teeth following intentionally delayed replantation, although both ESG and PBS may induce the formation of bone-like tissue.
Angela Quispe-Salcedo, Hiroko Ida-Yonemochi, and Hayato Ohshima
Journal of Endodontics, ISSN: 00992399, Pages: 1566-1572, Published: 1 October 2014 Elsevier BV
INTRODUCTION This study analyzed the detailed biological events underlying pulpal dynamics evoked by 3Mix (the mixture of ciprofloxacin, metronidazole, and minocycline) solution after intentionally delayed tooth replantation because 3Mix improves pulpal healing after tooth injuries. METHODS The maxillary first molars of 3-week-old mice were extracted and immersed in 3Mix solution for 30 minutes in comparison with phosphate buffered saline (PBS) alone. Cell proliferation, apoptosis, and differentiation were assessed in extracted/replanted teeth during days 0-14 using immunohistochemistry, apoptosis assay, and reverse-transcriptase polymerase chain reaction. RESULTS 3Mix solution accelerated odontoblast differentiation in the coronal pulp on day 7 and tertiary dentin formation on day 14, whereas the regenerative process was delayed in the PBS group. Cell proliferation and apoptosis occurred in the pulp of the 3Mix group during days 5-7 and subsequently decreased from days 7-14. On day 5, dentin sialophosphoprotein and nestin were first recovered in the 3Mix group, whereas expression levels for alkaline phosphatase, osteopontin, and osteocalcin increased in the PBS group. The expression levels for octamer-binding factor 3/4A and 3/4B reached the maximum level on day 1 and were sharply decreased on day 3 in both groups. High expression levels of Cd11c were first observed in the 3Mix group on day 1 and later at days 5 and 7. CONCLUSIONS The results suggest that the application of 3Mix may suppress osteoblast differentiation by the migration of dendritic cells to the injury site and via the activation of stem/progenitor cells, resulting in the acceleration of odontoblastlike cell differentiation.
Angela Quispe-Salcedo, Hiroko Ida-Yonemochi, and Hayato Ohshima
Journal of Oral Biosciences, ISSN: 13490079, Pages: 91-100, Published: May 2013 Elsevier BV
Abstract Objective A mixture of ciprofloxacin, metronidazole, and minocycline (3Mix) has been reported to be effective against oral bacteria from carious and endodontic lesions in vitro and in vivo . The objective of this study was to establish an animal model using mice for the application of 3Mix following intentionally delayed tooth replantation and to investigate the effects of 3Mix on the healing process of dental pulp and periodontal tissues. Methods Upper first molars of ICR mice were extracted, immersed in 3Mix solution at different concentrations for 5–60min with or without the use of a transfer solution (phosphate buffer solution (PBS)), in addition to transfer solution alone, and subsequently repositioned in the sockets. Immunohistochemistry for nestin and Ki-67, histochemistry for TRAP, and TUNEL assay were performed to assess pulpal healing during days 7–21. Results Increased apoptosis was observed in the PBS group at week 1, followed by cell proliferation at week 2, and tertiary dentin and/or bone-like tissue formation at week 3. In contrast, nestin-positive, newly differentiated, odontoblast-like cells began to align along the pulp–dentin border following the appearance of Ki-67- and TUNEL-positive cells during weeks 1–2 in the 3Mix groups, suggesting that pulpal healing was accelerated. Severe root ankylosis was observed exclusively in the 3Mix groups. Rinsing with PBS before replantation partially rescued the viability of the periodontal ligament, but pulpal healing was delayed. Conclusions The application of 3Mix promotes pulpal regeneration of intentionally delayed replanted teeth; however, its use may induce severe damage to periodontal tissues.
Angela Quispe-Salcedo, Hiroko Ida-Yonemochi, Mitsushiro Nakatomi, and Hayato Ohshima
Biomedical Research, ISSN: 03886107, eISSN: 1880313X, Pages: 119-132, Published: April 2012 Biomedical Research Press
Differentiated odontoblasts could not be identified by one unique phenotypic marker, but the combination of expression of dentin phosphoprotein (Dpp), dentin sialoprotein (Dsp), dentin matrix protein 1 (Dmp1), and nestin may be valuable for the assessment of these cells. However, the findings using these proteins remain controversial. This study aimed to compare two odontoblast differentiation markers: nestin and Dsp in the process of dentinogenesis in mice. We performed immunohistochemistry and/or in situ hybridization technique for nestin and Dsp using 3-week-old incisors as well as postnatal 1-day- to 8-week-old molars. Preodontoblasts began to express nestin and Dsp proteins and Dsp mRNA, which increased in their intensity according to the progress of odontoblast differentiation in both incisors and developing molars. Nestin was consistently expressed in the differentiated odontoblasts even after the completion of dentin matrix deposition. The expression of Dsp mRNA coincided with the odontoblast secretory activity for dentin matrix deposition. In contrast, other pulpal cells, predentin matrix and dentinal tubules also showed a positive reaction for Dsp protein in addition to differentiated odontoblasts. In conclusion, nestin is valuable as a differentiation marker for odontoblasts, whereas Dsp mRNA is a functional marker for their secretory activity.