A novel microplate-based spectrophotometric method for the quantitative assessment of freshwater bacterial coaggregation kinetics.

Coaggregation, the specific recognition and adhesion of genetically distinct bacteria, is proposed to contribute to the development of freshwater biofilms. This work aimed to develop a microplate-based system to measure and model the kinetics of freshwater bacterial coaggregation. Blastomonas natatoria 2.1 and Micrococcus luteus 2.13 were evaluated for coaggregation ability using 24-well microplates containing novel dome shaped wells (DSWs) and standard flat-bottom wells. Results were compared to a tube-based visual aggregation assay. The DSWs facilitated the reproducible detection of coaggregation via spectrophotometry and the estimation of coaggregation kinetics using a linked mathematical model. Quantitative analysis using DSWs was more sensitive than the visual tube aggregation assay and subject to substantially less variation than flat-bottom wells. Collectively these results demonstrate the utility of the DSW-based method and improve upon the current toolkit for studying freshwater bacterial coaggregation.

Development of Rat Caries-Induced Pulpitis Model for Vital Pulp Therapy.

Rodent animal models for vital pulp therapy are commonly used in dental research because their tooth anatomy and cellular processes are similar to the anatomy and processes in humans. However, most studies have been conducted using uninfected sound teeth, which makes it difficult to adequately assess the inflammatory shift after vital pulp therapy. In the present study, we aimed to establish a caries-induced pulpitis model based on the conventional rat caries model and then evaluate inflammatory changes during the wound-healing process after pulp capping in a model of reversible pulpitis induced by carious infection. To establish the caries-induced pulpitis model, the pulpal inflammatory status was investigated at different stages of caries progression by immunostaining targeted to specific inflammatory biomarkers. Immunohistochemical staining revealed that both Toll-like receptor 2 and proliferating cell nuclear antigen were expressed in moderate and severe caries-stimulated pulp, indicating that an immune reaction occurred at both stages of caries progression. M2 macrophages were predominant in moderate caries-stimulated pulp, whereas M1 macrophages were predominant in the severe caries-stimulated pulp. Pulp capping in teeth with moderate caries (i.e., teeth with reversible pulpitis) led to complete tertiary dentin formation within 28 d after treatment. Impaired wound healing was observed in teeth with severe caries (i.e., teeth with irreversible pulpitis). During the wound-healing process in reversible pulpitis after pulp capping, M2 macrophages were predominant at all time points; their proliferative capacity was upregulated in the early stage of wound healing compared with healthy pulp. In conclusion, we successfully established a caries-induced pulpitis model for studies of vital pulp therapy. M2 macrophages have an important role in the early stages of the wound-healing process in reversible pulpitis.

Novel Functional Peptide for Next-Generation Vital Pulp Therapy.

Although vital pulp therapy should be performed by promoting the wound-healing capacity of dental pulp, existing pulp-capping materials were not developed with a focus on the pulpal repair process. In previous investigations of wound healing in dental pulp, we found that organic dentin matrix components (DMCs) were degraded by matrix metalloproteinase-20, and DMC degradation products containing protein S100A7 (S100A7) and protein S100A8 (S100A8) promoted the pulpal wound-healing process. However, the direct use of recombinant proteins as pulp-capping materials may cause clinical problems or lead to high medical costs. Thus, we hypothesized that functional peptides derived from recombinant proteins could solve the problems associated with direct use of such proteins. In this study, we identified functional peptides derived from the protein S100 family and investigated their effects on dental pulp tissue. We first performed amino acid sequence alignments of protein S100 family members from several mammalian sources, then identified candidate peptides. Next, we used a peptide array method that involved human dental pulp stem cells (hDPSCs) to evaluate the mineralization-inducing ability of each peptide. Our results supported the selection of 4 candidate functional peptides derived from proteins S100A8 and S100A9. Direct pulp-capping experiments in a rat model demonstrated that 1 S100A8-derived peptide induced greater tertiary dentin formation compared with the other peptides. To investigate the mechanism underlying this induction effect, we performed liquid chromatography-tandem mass spectrometry analysis using hDPSCs and the S100A8-derived peptide; the results suggested that this peptide promotes tertiary dentin formation by inhibiting inflammatory responses. In addition, this peptide was located in a hairpin region on the surface of S100A8 and could function by direct interaction with other molecules. In summary, this study demonstrated that a S100A8-derived functional peptide promoted wound healing in dental pulp; our findings provide insights for the development of next-generation biological vital pulp therapies.

M. avium complex pulmonary disease: does the diagnostic method affect severity and progression?

SETTING: Diagnosis of Mycobacterium avium complex pulmonary disease (MAC-PD) requires positive culture of expectorated sputum or specimens acquired by bronchoscopy. Whether patients diagnosed using bronchoscopy have milder disease and milder progression than those diagnosed using sputum remains uncertain.OBJECTIVE: To clarify whether disease severity and progression differ according to the diagnostic method.METHODS: We retrospectively analysed 92 patients with MAC-PD. We compared characteristics of patients and disease progression according to the diagnostic methods used: sputum or bronchoscopy. Additionally, we investigated the impact of these methods on disease progression using multivariate analysis.RESULTS: Patients diagnosed using sputum were younger than those diagnosed using bronchoscopy; however, there were small differences from the viewpoint of clinical practice in disease severity, and estimated progression-free survival rate did not differ significantly. The predictors of disease progression were disease forms other than non-cavitary nodular/bronchiectatic disease, hypoalbuminemia and severe radiographic scores.CONCLUSION: The diagnostic methods had no significant impact on disease severity and disease progression of MAC-PD. If the diagnosis cannot be established by sputum culture or if sputum cannot be obtained in the patients with risk factors for disease progression, bronchoscopy would be useful to provide opportunity of treatment for MAC-PD.

South American rattlesnake cationic polypeptide crotamine trafficking dynamic in Plasmodium falciparum-infected erythrocytes: Pharmacological inhibitors, parasite cycle and incubation time influences in uptake.

Malaria is a parasitic infectious disease caused by Plasmodium sp, which was responsible for about 409 thousand deaths only in 2019. The clinical manifestations in patients with malaria, which may include fever and anemia and that can occasionally lead to the death of the host, are mainly associated to the asexual blood stage of parasite. The discovery of novel compounds active against stages of the intraerythrocytic cell cycle has been the focus of many researches seeking for alternatives to the control of malaria. The antimalarial effect of a native cationic polypeptide from the venom of a South American rattlesnake named crotamine, with ability of targeting and disrupting the acidic compartments of Plasmodium falciparum parasite, was previously described by us. Herein, we extended our previous studies by investigating the internalization and trafficking of crotamine in P. falciparum-infected erythrocytes at different blood-stages of parasites and periods of incubation. In addition, the effects of several pharmacological inhibitors in the uptake of this snake polypeptide with cell-penetrating properties were also assessed, showing that crotamine internalization was dependent on ATP generated via glycolytic pathway. We show here that crotamine uptake is blocked by the glycolysis inhibitor 2-deoxy-D-glucose, and the most efficient internalization is observed at trophozoite stage of parasite after at least 30 min of incubation. The present data provide important insights into biochemical pathway and cellular features determined by the parasite cycle, which may be underlying the internalization and effects of cationic antimalarials as crotamine.

Fabrication of Vascularized DPSC Constructs for Efficient Pulp Regeneration.

Dental pulp regeneration is a promising approach to restore the vitality of necrotic teeth. We have previously reported the fabrication of scaffold-free cell constructs containing only dental pulp stem cells (DPSCs) and their ability to form pulp-like tissue in the pulpless tooth. However, the DPSC construct could not build pulp-like tissue with a full root length because it is difficult to induce blood vessels from a small root canal foramen. Therefore, we hypothesized that vascular structure could be preformed in the DPSC construct by employing endothelial differentiation capability of DPSCs, and vascularized constructs might facilitate dental pulp regeneration in the pulpless tooth. In this study, vascularized DPSC constructs were fabricated by inducing endothelial differentiation, and then we investigated the behavior of differentiated DPSCs, the internal structure of cell constructs, and their pulp regenerative ability in vivo. We observed that DPSCs positive for CD31 and von Willebrand factor were localized at the outer layer of constructs and formed a reticulated lumen structure. The cells constituting the outer layer of the construct expressed endothelial differentiation markers at higher levels than cells in the inner part. These results indicated that DPSCs in the outer layer differentiated into endothelial cells and formed vascular-like structures in the cell construct. Next, a vascularized DPSC construct was transplanted into the human pulpless tooth that was implanted into immunodeficient mice in the subcutaneous space. After 6 wk of implantation, the vascularized construct formed pulp-like tissues with higher density of human CD31-positive blood vessels when compared with specimens implanted with a DPSC construct without prevascularization. These results suggest that the vascular structure formed in the DPSC construct facilitated the blood supply and enhanced pulp regeneration. This study demonstrates that a vascularized DPSC construct is a prospective biomaterial as an implant for novel dental pulp regeneration.

Phase I/II study of adding intraperitoneal paclitaxel in patients with pancreatic cancer and peritoneal metastasis.

BACKGROUND: Intraperitoneal chemotherapy using paclitaxel is considered an experimental approach for treating peritoneal carcinomatosis. This study aimed to determine the recommended dose, and to evaluate the clinical efficacy and safety, of the combination of intravenous gemcitabine, intravenous nab-paclitaxel and intraperitoneal paclitaxel in patients with pancreatic cancer and peritoneal metastasis. METHODS: The frequencies of dose-limiting toxicities were evaluated, and the recommended dose was determined in phase I. The primary endpoint of the phase II analysis was overall survival rate at 1 year. Secondary endpoints were antitumour effects, symptom-relieving effects, safety and overall survival. RESULTS: The recommended doses of intravenous gemcitabine, intravenous nab-paclitaxel and intraperitoneal paclitaxel were 800, 75 and 20 mg/m2 respectively. Among 46 patients enrolled in phase II, the median time to treatment failure was 6·0 (range 0-22·6) months. The response and disease control rates were 21 of 43 and 41 of 43 respectively. Ascites disappeared in 12 of 30 patients, and cytology became negative in 18 of 46. The median survival time was 14·5 months, and the 1-year overall survival rate was 61 per cent. Conversion surgery was performed in eight of 46 patients, and those who underwent resection survived significantly longer than those who were not treated surgically (median survival not reached versus 12·4 months). Grade 3-4 haematological toxicities developed in 35 of 46 patients, whereas non-haematological adverse events occurred in seven patients. CONCLUSION: Adding intraperitoneal paclitaxel had clinical efficacy with acceptable tolerability.

ANTECEDENTES: La quimioterapia intraperitoneal con paclitaxel se considera una terapia experimental para el tratamiento de la carcinomatosis peritoneal. Este estudio tuvo como objetivo determinar la dosis recomendada y evaluar la eficacia clínica y la seguridad de la combinación de gemcitabina intravenosa, nab-paclitaxel intravenoso y paclitaxel intraperitoneal en pacientes con cáncer de páncreas y metástasis peritoneales. MÉTODOS: Se evaluaron las frecuencias de las toxicidades limitantes de la dosis, y la dosis recomendada se determinó en la fase I. El objetivo principal de la fase II fue la tasa de supervivencia global a 1 año. Los objetivos secundarios fueron los efectos antitumorales, los efectos de alivio de los síntomas, la seguridad y la supervivencia global. RESULTADOS: Las dosis recomendadas de gemcitabina intravenosa, nab-paclitaxel intravenoso y paclitaxel intraperitoneal fueron de 800, 75 y 20 mg/m2 , respectivamente. De los 46 pacientes incluidos en la fase II del estudio, la mediana de tiempo hasta el fracaso del tratamiento fue de 6,0 meses (rango, 0-22,6). Las tasas de respuesta y de control de la enfermedad fueron del 45% y 95%, respectivamente. La ascitis desapareció en el 40% de los pacientes, y la citología se negativizó en el 39% de los pacientes. La mediana del tiempo de supervivencia fue de 14,5 meses y la tasa de supervivencia global a 1 año del 60,9%. La cirugía de rescate se realizó en ocho (17%) pacientes, y los que se sometieron a cirugía sobrevivieron significativamente más tiempo que los que no fueron tratados quirúrgicamente (mediana de supervivencia no alcanzada versus 12,4 meses). Las toxicidades hematológicas de grado 3/4 ocurrieron en el 76% de los pacientes, mientras que los eventos adversos no hematológicos se presentaron en el 15% de los pacientes. CONCLUSIÓN: Agregar paclitaxel intraperitoneal tuvo eficacia clínica con una tolerabilidad aceptable. (UMIN000018878).

Association between dexamethasone treatment and alterations in serum concentrations of trace metals.

Previously, a significant elevation in the serum levels of iron (Fe) was observed within a few days after the initiation of cisplatin (CDDP)-based chemotherapy. To clarify the underlying mechanisms, the serum concentration of hepcidin, a negative regulator of Fe release, was determined in the clinical samples obtained from six patients with cancer. The result showed that the serum concentration of hepcidin in patients receiving CDDP-based chemotherapy was significantly increased after 4-6 days of treatment, in comparison to the baseline level, suggesting that aforementioned excessive systemic Fe was not explained by the change of serum hepcidin level. All these patients received antiemetic premedication. We next evaluated of the effects of Pt-containing drugs and prophylactic antiemetic dexamethasone medication on the serum concentration of trace metals in mice, and on the hepatic and renal concentration of trace metals. The serum concentrations of Fe, Cu, and Zn in the CDDP-treated and oxaliplatin-treated mice were not significantly altered in comparison to those of the vehicle-treated control group. The serum concentrations of Fe, Cu, and Zn were increased after 24 h of dexamethasone treatment, compared to those of the control group (P < 0.05). The hepatic concentration of Mn was significantly reduced, whereas those of Fe and Cu inclined to diminish. The present findings suggest that dexamethasone can partly contribute to the changes in the serum concentrations of trace metals during anticancer chemotherapy.

The effect of two composite placement techniques on fracture resistance of MOD restorations with various resin composites.

OBJECTIVE: The aim of this in vitro study was to compare the effect of two restorative placement techniques, centripetal incremental technique (CIT) and bulk-fill technique (BT) on the fracture resistance of Class II MOD restorations with various resin composites in molar teeth. MATERIALS AND METHODS: Fifty-six extracted, caries free third molars were prepared with MOD preparations and restored with resin composites. The specimens were divided into two groups by placement technique, centripetal incremental technique (CIT) and bulk-fill technique (BT). Each group was subdivided into four groups according to resin composite: hybrid (Aelite LS), nano-hybrid (Virtuoso Universal), bulk fill (Filtek One Bulk Fill) and the micro-hybrid (Herculite XRV) as the control. RESULTS: Two-way analysis of variance test (ANOVA) followed by the multiple comparison procedure, Student-Newman-Keuls Method showed no a statistically significant difference between placement techniques and fracture resistance of Class II resin composite restorations (P > 0.05). Herculite XRV resisted a significantly higher load before fracture than the other three materials at a 0.05 level of significance, while Virtuoso Universal scored the lowest load. CONCLUSIONS: There was no significant effect of the two placement techniques on the fracture resistance of Class II resin composite restorations CLINICAL SIGNIFICANCE: Resin composite restorations in Class II MODs using a simplified bulk fill placement technique showed no significant difference in fracture resistance with the centripetal technique in molar teeth.

In silico experiments of bone remodeling explore metabolic diseases and their drug treatment.

Bone structure and function are maintained by well-regulated bone metabolism and remodeling. Although the underlying molecular and cellular mechanisms are now being understood, physiological and pathological states of bone are still difficult to predict due to the complexity of intercellular signaling. We have now developed a novel in silico experimental platform, V-Bone, to integratively explore bone remodeling by linking complex microscopic molecular/cellular interactions to macroscopic tissue/organ adaptations. Mechano-biochemical couplings modeled in V-Bone relate bone adaptation to mechanical loading and reproduce metabolic bone diseases such as osteoporosis and osteopetrosis. V-Bone also enables in silico perturbation on a specific signaling molecule to observe bone metabolic dynamics over time. We also demonstrate that this platform provides a powerful way to predict in silico therapeutic effects of drugs against metabolic bone diseases. We anticipate that these in silico experiments will substantially accelerate research into bone metabolism and remodeling.

Minimally invasive oesophagectomy with extended lymph node dissection and thoracic duct resection for early-stage oesophageal squamous cell carcinoma.

BACKGROUND: Oesophageal squamous cell carcinoma is an aggressive disease owing to early and widespread lymph node metastases. Multimodal therapy and radical surgery may improve prognosis. Few studies have investigated the efficacy of radical lymph node and thoracic duct resection. METHODS: Patients with oesophageal squamous cell carcinoma who underwent transthoracic minimally invasive oesophagectomy (TMIE) for cancer at Keio University Hospital between January 2004 and December 2016 were selected. Between 2004 and 2008, TMIE was performed in the lateral decubitus position without thoracic duct resection (standard TMIE). From 2009 onwards, TMIE with extended lymph node and thoracic duct resection was introduced (extended TMIE). Demographics, co-morbidity, number of retrieved lymph nodes, pathology, postoperative complications and recurrence-free survival (RFS) were compared between groups. RESULTS: Forty-four patients underwent standard TMIE and 191 extended TMIE. There were no significant differences in clinical and pathological tumour stage or postoperative complications. The extended-TMIE group had more lymph nodes removed at nodal stations 106recL and 112. Among patients with cT1 N0 disease, RFS was better in the extended-TMIE group (P < 0·001), whereas there was no difference in RFS between groups in patients with advanced disease. CONCLUSION: Extended TMIE including thoracic duct resection increased the number of lymph nodes retrieved and was associated with improved survival in patients with cT1 N0 oesophageal squamous cell carcinoma.

UVA-activated riboflavin promotes collagen crosslinking to prevent root caries.

Root caries is an increasingly problem in aging societies with severe implications for the general health and wellbeing of large numbers of people. Strengthening type-I collagen, a major organic component of human dentin, has proved effective in preventing root caries. This study sought to determine whether exposure to riboflavin followed by UVA irradiation (RF/UVA) could promote additional collagen crosslinking, and thus improve the acid and enzymatic resistance of human dentin under simulated oral environments. If so, it could offer potential for treatment of the intractable problem of root caries. The greatest flexural strengths were found in dentin exposed to a 0.1% riboflavin solution for 1 minute followed by 1,600 mW/cm2 UVA irradiation for 10 minutes. Mineral loss and lesion depth were significantly lower in the RF/UVA group than in the control group. The microstructures of dentinal tubules and collagen networks after RF/UVA treatment retained their original forms after acidic and enzymatic degradation. In conclusion, RF/UVA treatment may be a new method for preventing root caries with promising prospects for clinical application.

Effect of tissue inhibitor of metalloprotease 1 on human pulp cells in vitro and rat pulp tissue in vivo.

AIM: To evaluate the dentinogenetic effects of tissue inhibitor of metalloprotease (TIMP1) on human pulp cells in vitro and rat pulp tissue in vivo. METHODOLOGY: The effect of TIMP1 on pulp cell functions related to hard tissue formation as part of the wound healing process (i.e. biocompatibility, proliferation, differentiation and mineralized nodule formation) was evaluated in vitro and using a direct pulp capping experimental animal model in vivo. The effects of different-sized cavity preparations on hard tissue formation induced by ProRoot MTA at 2 weeks were evaluated using micro-computed tomography (micro-CT). Tertiary dentine formation quality and quantity after pulp capping using TIMP1, ProRoot MTA and phosphate-buffered saline (PBS) was also evaluated after 4 weeks using micro-CT in term of dentine volume (DV), dentine mineral density (DVD) and histological analysis. The data were evaluated by Student's t-test, one-way ANOVA with Tukey's post hoc test, the Kruskal-Wallis test or the Steel-Dwass test. P values < 0.05 were considered statistically significant. RESULTS: TIMP1 significantly stimulated dental pulp stem cell proliferation, differentiation, and mineralization and was more biocompatible compared with the PBS control (P < 0.05). In the pulp capping model, the amount of tertiary dentine that formed was directly proportional to the size of the pulp exposure; greater amounts of tertiary dentine were observed in pulps with larger exposures after 2 weeks. 4-week samples of TIMP1 and ProRoot MTA had similar characteristics, but both sample significantly induced tertiary dentine formation beneath the cavity compared with PBS (P <  0.05) under standardized cavity preparations. CONCLUSIONS: TIMP1 has an important role in pulpal wound healing, which makes it a potential biological pulp capping material and candidate molecule for regenerative endodontic therapy.

Interleukin 4 induces rapid mucin transport, increases mucus thickness and quality and decreases colitis and Citrobacter rodentium in contact with epithelial cells.

Citrobacter rodentium infection is a murine model for pathogenic intestinal Escherichia coli infection. C. rodentium infection causes an initial decrease in mucus layer thickness, followed by an increase during clearance. We aimed to identify the cause of these changes and to utilize this naturally occurring mucus stimulus to decrease pathogen impact and inflammation. We identified that mucin production and speed of transport from Golgi to secretory vesicles at the apical surface increased concomitantly with increased mucus thickness. Of the cytokines differentially expressed during increased mucus thickness, IFN-γ and TNF-α decreased the mucin production and transport speed, whereas IL-4, IL-13, C. rodentium and E. coli enhanced these aspects. IFN-γ and TNF-α treatment in combination with C. rodentium and pathogenic E. coli infection negatively affected mucus parameters in vitro, which was relieved by IL-4 treatment. The effect of IL-4 was more pronounced than that of IL-13, and in wild type mice, only IL-4 was present. Increased expression of Il-4, Il-4-receptor α, Stat6 and Spdef during clearance indicate that this pathway contributes to the increase in mucin production. In vivo IL-4 administration initiated 10 days after infection increased mucus thickness and quality and decreased colitis and pathogen contact with the epithelium. Thus, during clearance of infection, the concomitant increase in IL-4 protects and maintains goblet cell function against the increasing levels of TNF-α and IFN-γ. Furthermore, IL-4 affects intestinal mucus production, pathogen contact with the epithelium and colitis. IL-4 treatment may thus have therapeutic benefits for mucosal healing.

Synaptotagmin XIII expression and peritoneal metastasis in gastric cancer.

BACKGROUND: Peritoneal metastasis is a frequent cause of death in patients with gastric cancer. The aim of this study was to identify molecules responsible for mediating peritoneal metastasis of gastric cancer. METHODS: Transcriptome and bioinformatics analyses were conducted to identify molecules associated with peritoneal metastasis. The therapeutic effects of intraperitoneally administered small interfering (si) RNA were evaluated using mouse xenograft models. Expression of mRNA and protein was determined in gastric tissues from patients with gastric cancer. RESULTS: Synaptotagmin XIII (SYT13) was expressed at significantly higher levels in patients with peritoneal recurrence, but not in those with hepatic or distant lymph node recurrence. Inhibition of SYT13 expression in a gastric cancer cell line transfected with SYT13-specific siRNA (siSYT13) was associated with decreased invasion and migration ability of the cells, but not with proliferation and apoptosis. Intraperitoneal administration of siSYT13 significantly inhibited the growth of peritoneal nodules and prolonged survival in mice. In an analysis of 200 patients with gastric cancer, SYT13 expression in primary gastric cancer tissues was significantly greater in patients with peritoneal recurrence or metastasis. A high level of SYT13 expression in primary gastric cancer tissues was an independent risk factor for peritoneal recurrence. CONCLUSION: SYT13 expression in gastric cancer is associated with perioneal metatases and is a potential target for treatment.

Pulp Regeneration by 3-dimensional Dental Pulp Stem Cell Constructs.

Dental pulp regeneration therapy for the pulpless tooth has attracted recent attention, and clinical trial studies are underway with the tissue engineering approach. However, there remain many concerns, including the extended period for regenerating the dental pulp. In addition, the use of scaffolds increases the risk of inflammation and infection. To establish a basic technology for novel dental pulp regenerative therapy that allows transplant of pulp-like tissue, we attempted to fabricate scaffold-free 3-dimensional (3D) cell constructs composed of dental pulp stem cells (DPSCs). Furthermore, we assessed viability of these 3D DPSC constructs for dental pulp regeneration through in vitro and in vivo studies. For the in vitro study, we obtained 3D DPSC constructs by shaping sheet-like aggregates of DPSCs with a thermoresponsive hydrogel. DPSCs within constructs remained viable even after prolonged culture; furthermore, 3D DPSC constructs possessed a self-organization ability necessary to serve as a transplant tissue. For the in vivo study, we filled the human tooth root canal with DPSC constructs and implanted it subcutaneously into immunodeficient mice. We found that pulp-like tissues with rich blood vessels were formed within the human root canal 6 wk after implantation. Histologic analyses revealed that transplanted DPSCs differentiated into odontoblast-like mineralizing cells at sites in contact with dentin; furthermore, human CD31-positive endothelial cells were found at the center of regenerated tissue. Thus, the self-organizing ability of 3D DPSC constructs was active within the pulpless root canal in vivo. In addition, blood vessel-rich pulp-like tissues can be formed with DPSCs without requiring scaffolds or growth factors. The technology established in this study allows us to prepare DPSC constructs with variable sizes and shapes; therefore, transplantation of DPSC constructs shows promise for regeneration of pulpal tissue in the pulpless tooth.