Effect of 3D-printed polycaprolactone/osteolectin scaffolds on the odontogenic differentiation of human dental pulp cells.

Cell-based tissue engineering often requires the use of scaffolds to provide a 3-dimensional (3D) framework for cell proliferation and tissue formation. Polycaprolactone (PCL), a type of polymer, has good printability, favorable surface modifiability, adaptability, and biodegradability. However, its large-scale applicability is hindered by its hydrophobic nature, which affects biological properties. Composite materials can be created by adding bioactive materials to the polymer to improve the properties of PCL scaffolds (PSs). Osteolectin is an odontogenic factor that promotes the maintenance of the adult skeleton by promoting the differentiation of LepR+ cells into osteoblasts. Therefore, the aim of this study was to evaluate whether 3D-printed PCL/osteolectin scaffolds supply a suitable microenvironment for the odontogenic differentiation of human dental pulp cells (hDPCs). The hDPCs were cultured on 3D-printed PSs with or without pores. Cell attachment and cell proliferation were evaluated using EZ-Cytox. The odontogenic differentiation of hDPCs was evaluated by alizarin red S staining and alkaline phosphatase assays. Western blotting was used to evaluate the expression of the proteins DSPP and DMP-Results: The attachment of hDPCs to PSs with pores was significantly higher than to PSs without pores. The odontogenic differentiation of hDPCs was induced more in PCL/osteolectin scaffolds than in PSs, but there was no statistically significant difference. 3D-printed PSs with pores are suitable for the growth of hDPCs, and the PCL/osteolectin scaffolds can provide a more favorable microenvironment for the odontogenic differentiation of hDPCs. .

The potential bone regeneration effects of leptin- and osteolectin-coated 3D-printed PCL scaffolds: an in vivo study.

This study investigated the effectiveness of bone regeneration upon the application of leptin and osteolectin to a three-dimensional (3D) printed poly(ε-caprolactone) (PCL) scaffold. A fused deposition modeling 3D bioprinter was used to fabricate scaffolds with a diameter of 4.5 mm, a height of 0.5 mm, and a pore size of 420-520 nm using PCL (molecular weight: 43,000). After amination of the scaffold surface for leptin and osteolectin adhesion, the experimental groups were divided into the PCL scaffold (control), the aminated PCL (PCL/Amine) scaffold, the leptin-coated PCL (PCL/Leptin) scaffold, and the osteolectin-coated PCL (PCL/Osteo) scaffold. Next, the water-soluble tetrazolium salt-1 (WST-1) assay was used to assess cell viability. All groups exhibited cell viability rates of >100%. Female 7-week-old Sprague-Dawley rats were used for in vivo experiments. Calvarial defects were introduced on the rats' skulls using a 5.5 mm trephine bur. The rats were divided into the PCL (control), PCL/Leptin, and PCL/Osteo scaffold groups. The scaffolds were then inserted into the calvarial defect areas, and the rats were sacrificed after 8-weeks to analyze the defect area. Micro-CT analysis indicated that the leptin- and osteolectin-coated scaffolds exhibited significantly higher bone regeneration. Histological analysis revealed new bone and blood vessels in the calvarial defect area. These findings indicate that the 3D-printed PCL scaffold allows for patient-customized fabrication as well as the easy application of proteins like leptin and osteolectin. Moreover, leptin and osteolectin did not show cytotoxicity and exhibited higher bone regeneration potential than the existing scaffold.

Icariin negatively regulated lipopolysaccharide-induced inflammation and ameliorated the odontogenic activity of human dental pulp cells in vitro.

Alleviating inflammation and promoting dentine regeneration is critical for the healing of pulpitis. In this study, we investigated the anti-inflammatory, angiogenesis and odontogenesis function of icariin on Human dental pulp cells (HDPCs) under inflammatory state. Furthermore, the underlying mechanisms was also evaluated. Icariin attenuated the LPS-induced pro-inflammatory marker expression, such as interleukin-1ß (IL-1ß), IL-6 and IL-8. The immunoblotting and immunofluorescence staining results showed that icariin suppressed the inflammatory responses mediated by the protein kinase B (Akt) and nuclear factor kappa-B (NF-κB) signaling cascades. Additionally, icariin also upregulated the expression of odontogenic and angiogenic genes and proteins (namely dentin sialophosphoprotein (DSPP), dentin matrix protein 1 (DMP1), anti-collagen Ⅰ (COL-Ⅰ), and vascular endothelial growth factor (VEGF) and fibroblast growth factor-1 (FGF-1)), alkaline phosphatase activity, and calcium nodule deposition in LPS-exposed HDPCs. In a word, our findings indicated that icariin attenuated pulp inflammation and promoted odontogenic and angiogenic differentiation in the inflammatory state. Icariin may be a promising vital pulp therapy agent for the regenerative treatment of the inflamed dental pulp.

A leptin-loaded poly-ϵ-caprolactone 3D printing scaffold for odontoblastic differentiation in human dental pulp cells.

This study investigated the effects on odontoblast differentiation of a 3D-printed poly-ϵ-caprolactone (PCL) scaffold that incorporated leptin. Material extrusion-type 3D printing with a 43 000-molecular weight PCL material was used to fabricate a PCL scaffold with a 6 mm diameter, 1 mm height, and 270-340 µm pore size. The experimental groups were PCL scaffolds (control group), PCL scaffolds with aminated surfaces (group A), and PCL scaffolds with leptin on the aminated surface (group L). The aminated surface was treated with 1,6-hexanediamine and verified by ninhydrin analysis. Leptin loading was performed using Traut's reagent and 4-(N-Maleimidomethyl)cyclohexane-1-carboxylic acid 3-sulfo-N-hydroxysuccinimide ester sodium salt (Sulfo-SMCC). Groups A and L showed significantly higher surface wettability, pulp cell adhesion, and proliferation than the control group. Group L exhibited increased alkaline phosphatase, calcification deposits, and mRNA and protein expression of dentin sialophosphoprotein and dentin matrix acidic phosphoprotein 1 compared with the control group. In this study, a 3D-printed PCL scaffold containing leptin was enhanced odontoblast differentiation and dental pulp cells adhesion and proliferation.

A comparison of osteogenic effect of newly manufactured calcium silicate-based sealers in vitro.

This study aimed to assess the effect of different calcium silicate-based root canal sealers (CSRS) on osteogenic effect in human periodontal ligament cells (hPDLCs). hPDLCs were cultured in a medium containing extract of 5 types of CSRS. The specimens were assessed by the cell cytotoxicity test, alkaline phosphatase staining, alizarin red S staining, quantitative real-time PCR, Western blot analysis, and enzyme-linked immunosorbent assay. The diluted concentrations of extracted solutions had no significant effect on the viability of hPDLCs. There was a statistically significant difference in the mRNA expression level of bone sialoprotein (BSP), osteocalcin (OCN), and runt-related transcription factor 2 (RUNX2) among some groups. The protein expressions of BSP, OCN, and RUNX2 were significantly higher in some groups compared to the control group. The CSRS did not interfere with the osteogenic differentiation of hPDLCs, compared to the control group. CSRS are shown to have biocompatibility and osteogenic differentiation effect on hPDLCs.

Osteolectin Promotes Odontoblastic Differentiation in Human Dental Pulp Cells.

INTRODUCTION: Osteolectin is a secreted glycoprotein of the C-type lectin domain superfamily, expressed in bone tissues and is reported as a novel osteogenic factor that promotes bone regeneration. However, the effect of osteolectin on human dental pulp cells (hDPCs) has not been reported. Therefore, we aimed to investigate the odontoblastic differentiation of osteolectin in hDPCs and further attempt to reveal its underlying mechanism. METHODS: Cytotoxicity assays were used to detect the cytotoxicity of osteolectin. The odontoblastic differentiation of hDPCs and its underlying mechanisms were measured by the alkaline phosphatase (ALP) activity, mineralized spots formation, and the gene and protein expression of odontoblastic differentiation through ALP staining, Alizarin red S staining, quantitative real-time polymerase chain reaction, and Western blot analysis, respectively. RESULTS: WST-1 assay showed osteolectin at concentrations below 300 ng/ml was noncytotoxic and safe for hDPCs. The following experiment demonstrated that osteolectin could increase ALP activity, accelerate the mineralization process, and up-regulate the odontogenic differentiation markers in both gene and protein levels (P < .05). Osteolectin stimulated the phosphorylation of ERK, JNK, and Protein kinase B (AKT) in hDPCs. Extracellular signal-regulated kinase (ERK), Jun N-terminal kinase (JNK), and AKT inhibitors decreased ALP activity and mineralization capacity and suppressed the expression of dentin sialophosphoprotein and dentin matrix protein-1. CONCLUSION: Osteolectin can promote odontoblastic differentiation of hDPCs, and the whole process may stimulate ERK, JNK, and AKT signaling pathways by increasing p-ERK, p-JNK, and p-AKT signals.

Boeravinone B, a natural rotenoid, inhibits osteoclast differentiation through modulating NF-κB, MAPK and PI3K/Akt signaling pathways.

Osteoporosis is a major public health concern, which requires novel therapeutic strategies to prevent or mitigate bone loss. Natural compounds have attracted attention as potential therapeutic agents due to their safety and efficacy. In this study, we investigated the regulatory activities of boeravinone B (BOB), a natural rotenoid isolated from the medicinal plant Boerhavia diffusa, on the differentiation of osteoclasts and mesenchymal stem cells (MSCs), the two main cell components responsible for bone remodeling. We found that BOB inhibited osteoclast differentiation and function, as determined by TRAP staining and pit formation assay, with no significant cytotoxicity. Furthermore, our results showing that BOB ameliorates ovariectomyinduced bone loss demonstrated that BOB is also effective in vivo. BOB exerted its inhibitory effects on osteoclastogenesis by downregulating the RANKL/RANK signaling pathways, including NF-κB, MAPK, and PI3K/Akt, resulting in the suppression of osteoclast-specific gene expression. Further experiments revealed that, at least phenomenologically, BOB promotes osteoblast differentiation of bone marrow-derived MSCs but inhibits their differentiation into adipocytes. In conclusion, our study demonstrates that BOB inhibits osteoclastogenesis and promotes osteoblastogenesis in vitro by regulating various signaling pathways. These findings suggest that BOB has potential value as a novel therapeutic agent for the prevention and treatment of osteoporosis. [BMB Reports 2023; 56(10): 545-550].

Effects of Red LED Irradiation in Enhancing the Mineralization of Human Dental Pulp Cells In Vitro.

Dentin regeneration is the preferred method used to preserve dental pulp vitality after pulp exposure due to caries. Red light-emitting diode irradiation (LEDI), which is based on photobiomodulation (PBM), has been used to promote hard-tissue regeneration. However, the underlying mechanism still needs elucidation. This study aimed to explore the mechanism involved in red LEDI affecting dentin regeneration. Alizarin red S (ARS) staining revealed that red LEDI induced mineralization of human dental pulp cells (HDPCs) in vitro. We further distinguished the cell proliferation (0-6 d), differentiation (6-12 d), and mineralization (12-18 d) of HDPCs in vitro and treated cells either with or without red LEDI in each stage. The results showed that red LEDI treatment in the mineralization stage, but not the proliferation or differentiation stages, increased mineralized nodule formation around HDPCs. Western blot also indicated that red LEDI treatment in the mineralization stage, but not the proliferation or differentiation stages, upregulated the expression of dentin matrix marker proteins (dentin sialophosphoprotein, DSPP; dentin matrix protein 1, DMP1; osteopontin, OPN) and an intracellular secretory vesicle marker protein (lysosomal-associated membrane protein 1, LAMP1). Therefore, the red LEDI might enhance the matrix vesicle secretion of HDPCs. On the molecular level, red LEDI enhanced mineralization by activating the mitogen-activated protein kinase (MAPK) signaling pathways (ERK and P38). ERK and P38 inhibition reduced mineralized nodule formation and the expression of relevant marker proteins. In summary, red LEDI enhanced the mineralization of HDPCs by functioning to produce a positive effect in the mineralization stage in vitro.

Effects of CTHRC1 on odontogenic differentiation and angiogenesis in human dental pulp stem cells.

Objectives: This study aimed to determine whether collagen triple helix repeat containing-1 (CTHRC1), which is involved in vascular remodeling and bone formation, can stimulate odontogenic differentiation and angiogenesis when administered to human dental pulp stem cells (hDPSCs). Materials and Methods: The viability of hDPSCs upon exposure to CTHRC1 was assessed with the WST-1 assay. CTHRC1 doses of 5, 10, and 20 µg/mL were administered to hDPSCs. Reverse-transcription polymerase reaction was used to detect dentin sialophosphoprotein, dentin matrix protein 1, vascular endothelial growth factor, and fibroblast growth factor 2. The formation of mineralization nodules was evaluated using Alizarin red. A scratch wound assay was conducted to evaluate the effect of CTHRC1 on cell migration. Data were analyzed using 1-way analysis of variance followed by the Tukey post hoc test. The threshold for statistical significance was set at p < 0.05. Results: CTHRC1 doses of 5, 10, and 20 µg/mL had no significant effect on the viability of hDPSCs. Mineralized nodules were formed and odontogenic markers were upregulated, indicating that CTHRC1 promoted odontogenic differentiation. Scratch wound assays demonstrated that CTHRC1 significantly enhanced the migration of hDPSCs. Conclusions: CTHRC1 promoted odontogenic differentiation and mineralization in hDPSCs.

Periodontal ligament preloading and rapid prototyping of the donor tooth in the autotransplantation of premolars with complete root formation.

This case report describes the successful orthodontic treatment of an 11-year-old girl with skeletal Class II malocclusion and congenitally missing mandibular second premolars. To resolve her upper lip protrusion and restore the missing mandibular premolars, extraction of the maxillary first premolars and subsequent autotransplantation of the extracted premolars onto the site of the missing mandibular second premolars were performed. To ensure the success of the autotransplantation and subsequent orthodontic treatment, an orthodontic force was preapplied on the donor teeth, and the recipient sockets were prepared with the aid of replica teeth. Thereafter, comprehensive orthodontic treatment was performed to close the extraction space in the maxilla and align the mandibular dentition, including the transplants. The patient achieved a functional occlusion with an improved facial profile. Results of the orthodontic treatment and autotransplantation were stable during the 5-year follow-up. On the basis of this report, a management protocol for a biomechanically enhanced autotransplantation procedure was suggested. This approach would enable an effective treatment procedure, thereby increasing the usefulness of autotransplantation.

The Effects of 3-Dimensional Bioprinting Calcium Silicate Cement/Methacrylated Gelatin Scaffold on the Proliferation and Differentiation of Human Dental Pulp Stem Cells.

A calcium silicate cement/methacrylated gelatin (GelMa) scaffold has been applied in tissue engineering; however, the research on its applications in dental tissue regeneration remains lacking. We investigate the effect of this scaffold on human dental pulp stem cells (hDPSCs). hDPSCs were cultured in 3D-printed GelMa and MTA-GelMa scaffolds. Cell adhesion was evaluated using scanning electron microscopy images. Cells were cultured in an osteogenic differentiation medium, which contained a complete medium or α-MEM containing aqueous extracts of the 3D-printd GelMa or MTA-GelMa scaffold with 2% FBS, 10 mM ß-glycerophosphate, 50 µg/mL ascorbic acid, and 10 nM dexamethasone; cell viability and differentiation were shown by WST-1 assay, Alizarin Red S staining, and alkaline phosphatase staining. Quantitative real-time PCR was used to measure the mRNA expression of DSPP and DMP-1. One-way analysis of variance followed by Tukey's post hoc test was used to determine statistically significant differences, identified at p < 0.05. hDPSCs adhered to both the 3D-printed GelMa and MTA-GelMa scaffolds. There was no statistically significant difference between the GelMa and MTA-GelMa groups and the control group in the cell viability test. Compared with the control group, the 3D-printed MTA-GelMa scaffold promoted the odontogenic differentiation of hDPSCs. The 3D-printed MTA-GelMa scaffold is suitable for the growth of hDPSCs, and the scaffold extracts can better promote odontoblastic differentiation.

Odontogenic Effect of Icariin on the Human Dental Pulp Cells.

Background and Objectives: Human dental pulp cells (HDPCs) can be used for dentin regeneration due to its odontogenic differentiation property. Icariin can induce osteogenic differentiation of stem cells. However, its potential to induce odontogenic differentiation of HDPCs remains unclear. Thus, the aim of this study was to evaluate the capacity of icariin to induce odontogenic differentiation of HDPCs and investigate the underlying molecular mechanism. Materials and Methods: Cell viability assay was used to detect the cytotoxicity of icariin to HDPCs. Effect of icariin on HDPCs chemotaxis was measured by scratch migration assay. The mineralized and odontogenic differentiation of HDPCs was assessed by alkaline phosphatase (ALP) staining, alizarin red S (ARS) staining, real-time PCR, and Western blot of dentin matrix protein 1 (DMP 1) and dentin sialophosphoprotein (DSPP). In addition, Mitogen-activated protein kinase (MAPK) signaling pathway of icariin-induced biomineralization was investigated by Western blot. Results: Cells treated with icariin at all concentrations tested maintained viability, indicating that icariin was biocompatible. Icariin accelerated HDPCs chemotaxis (p < 0.05). Expression levels of related odontogenic markers were increased in the presence of icariin (p < 0.05). Icariin-induced odontogenic differentiation occurred via activation of the MAPK signaling pathway. Furthermore, MAPK inhibitors suppressed expression levels of DSPP and DMP 1 protein, ALP activity, and mineralization of HDPCs. Conclusions: Icariin can upregulate odontogenic differentiation of HDPCs by triggering the MAPK signaling pathway.

Inactivation of PI3K/Akt promotes the odontoblastic differentiation and suppresses the stemness with autophagic flux in dental pulp cells.

BACKGROUND/PURPOSE: Autophagy is involved in controlling differentiation of various cell types. The present study aimed to investigate the mechanism related to autophagy in regulating odontogenic differentiation of dental pulp cells. MATERIALS AND METHODS: Human dental pulp cells (HDPCs) were cultured in differentiation inductive medium (DM) and odontoblastic differentiation and mineralization were evaluated by alkaline phosphatase (ALP) staining and Alizarin red S staining, respectively. Tooth cavity preparation was made on the mesial surface of lower first molars in rat. The expression of autophagy-related signal molecules was detected using Western blot analysis and Immunohistochemistry. RESULTS: HDPCs cultured in DM showed increased autophagic flux and declined phosphorylation of phosphoinositide 3-kinases (PI3K), protein kinase B (Akt), and mTOR. Dentin matrix protein-1 (DMP-1) and dentin sialoprotein (DSP), markers of odontoblastic differentiation, were upregulated and autophagic activation showing increased LC3-II and decreased p62 levels was observed during odontogenic differentiation of HDPCs. However, PI3K blocker 3-methyladenine (3MA), lentiviral shLC3 and Akt activator SC79 attenuated the expression of LC3II as well as DMP-1, ALP activity and mineralization enhanced in HDPCs under DM condition. In addition, 3MA, shLC3 and SC79 recovered the expression of pluripotency factor CD146, Oct4 and Nanog downregulated in DM condition. In rat tooth cavity preparation model, the expression of LC3B and DMP-1 was elevated near odontoblast-dentin layer during reparative dentin formation, whereas 3MA significantly reduced the expression of LC3B and DMP-1. CONCLUSION: These findings indicated autophagy promotes the odontogenic differentiation of dental pulp cells modulating stemness via PI3K/Akt inactivation and the repair of pulp.

Anti-Inflammatory and Mineralization Effects of Bromelain on Lipopolysaccharide-Induced Inflammation of Human Dental Pulp Cells.

Background and Objectives: Bromelain is a mixture of protease obtained from pineapple fruits or stems. Even though the biological mechanism of action of bromelain has not been completely understood, it is well known that bromelain possesses anticancer, anti-inflammatory and immunomodulatory effects. This study investigated the anti-inflammatory effects of bromelain on lipopolysaccharide (LPS)-induced human dental pulp cells (hDPCs). Materials and Methods: Cell viability after bromelain treatment was measured using WST-1 assay. We exposed hDPCs to 5 µg/mL of LPS with 2.5 or 5 µg/mL of bromelain. We performed reverse-transcription polymerase chain reaction and enzyme-linked immunosorbent assay to detect interleukin-1ß, interleukin-6, and interleukin-8 levels. Western blots were used to detect intercellular adhesion molecules-1 (ICAM-1) and vascular cell adhesion molecules-1 (VCAM-1) levels. Immunofluorescence staining and Western blots were used to determine bromelain's anti-inflammatory mechanism. We also performed alkaline phosphatase and Alizarin red staining to verify mineralization nodule formation. Results: Bromelain at 2.5, 5, 10, or 20 µg/mL did not affect the viability of hDPCs significantly. LPS increased interleukin-1ß, interleukin-6, interleukin-8, ICAM-1 and VCAM-1 expression in hDPCs. Bromelain significantly decreased interleukin-1ß, interleukin-6, interleukin-8, ICAM-1, and VCAM-1 levels in hDPCs, which were stimulated by LPS. Bromelain treatment significantly reduced p65 phosphorylation in the cytoplasm and the nucleus. It also significantly decreased phosphorylation levels of extracellular signal-related kinases (ERK) and p38 mitogen-activated protein kinases (p38). Bromelain also promoted ALP activity and mineralized nodule formation. Conclusions: Bromelain inhibits the expression of inflammatory cytokines in LPS-stimulated hDPCs. The inhibitory effect of bromelain on inflammatory mediators is related to decreased NF-κB and the MAPK pathway. Therefore, bromelain might have the potential to be used for regenerative endodontics, including vital pulp therapy.

Hard tissue formation after direct pulp capping with osteostatin and MTA in vivo.

OBJECTIVES: In recent in vitro study, it was reported that osteostatin (OST) has an odontogenic effect and synergistic effect with mineral trioxide aggregate (MTA) in human dental pulp cells. Therefore, the aim of this study was to evaluate whether OST has a synergistic effect with MTA on hard tissue formation in vivo. MATERIALS AND METHODS: Thirty-two maxillary molars of Spraque-Dawley rats were used in this study. An occlusal cavity was prepared and the exposed pulps were randomly divided into 3 groups: group 1 (control; ProRoot MTA), group 2 (OST 100 µM + ProRoot MTA), group 3 (OST 10 mM + ProRoot MTA). Exposed pulps were capped with each material and cavities were restored with resin modified glass ionomer. The animals were sacrificed after 4 weeks. All harvested teeth were scanned with micro-computed tomography (CT). The samples were prepared and hard tissue formation was evaluated histologically. For immunohistochemical analysis, the specimens were sectioned and incubated with primary antibodies against dentin sialoprotein (DSP). RESULTS: In the micro-CT analysis, it is revealed that OST with ProRoot MTA groups showed more mineralized bridge than the control (p < 0.05). In the H&E staining, it is showed that more quantity of the mineralized dentin bridge was formed in the OST with ProRoot MTA group compared to the control (p < 0.05). In all groups, DSP was expressed in newly formed reparative dentin area. CONCLUSIONS: OST can be a supplementary pulp capping material when used with MTA to make synergistic effect in hard tissue formation.

Comparing volumetric and biological aspects of 3D-printed interim restorations under various post-curing modes.

PURPOSE: This study aims to compare the volumetric change, degree of conversion (DOC), and cytotoxicity of 3D-printed restorations post-cured under three different conditions. MATERIALS AND METHODS: 3D-printed interim restorations were post-cured under three different conditions and systems: 5 min, 30 min, and 24 h. Three-unit and six-unit fixed dental prostheses (n = 30 for each case) were printed; ten specimens from each group were post-cured and then scanned to compare their volumetric changes. Root-mean-squared (RMS) values of the data were acquired by superimposing the scanned files with original files. Thirty disk-shaped specimens were printed to evaluate the DOC ratio. Fourier transform infrared spectroscopy was used to compare the DOCs of 10 specimens from each group. Human gingival fibroblasts were used to measure the cell viability of every specimen (n = 7). The data from this experiment were employed for one-way analysis of variance and Tukey's post-hoc comparisons. RESULTS: Differences between the three-unit restorations were statistically insignificant, regardless of the post-curing conditions. However, for the six-unit restorations, a high RMS value was acquired when the post-curing duration was 30 min. The average DOC was approximately 56 - 62%; the difference between each group was statistically insignificant. All the groups exhibited cell viability greater than 70%, rendering them clinically acceptable. CONCLUSION: The post-curing conditions influenced the volume when the length of the restoration was increased. However, this deviation was found to be clinically acceptable. Additionally, post-curing did not significantly influence the DOC and cytotoxicity of the restorations.

Hybrid porous zirconia scaffolds fabricated using additive manufacturing for bone tissue engineering applications.

For the formation of new bone in critical-sized bone defects, bioactive scaffolds with an interconnected porous network are necessary. Herein, we fabricated three-dimensional (3D) porous hybrid zirconia scaffolds to promote hybrid functionality, i.e., excellent mechanical properties and bioactive performance. Specifically, the 3D printed scaffolds were subjected to Zn-HA/glass composite coating on glass-infiltrated zirconia (ZC). In addition, to pertain the extracellular matrix of bone, biopolymer (alginate/gelatine) was embedded in a developed 3D construct (ZB and ZCB). A zirconia-printed scaffold (Z) group served as a control. The structural and mechanical properties of the constructed scaffolds were studied using essential characterization techniques. Furthermore, the biological performance of the designed scaffolds was tested by a sequence of in vitro cell tests, including the attachment, proliferation, and osteogenic differentiation of dental pulp cells (DPCs). The ZC and ZCB scaffolds exhibited 20% higher compression strength than the zirconia (Z) scaffolds. More importantly, the ZC constructs exhibited superior cell-adhesion, distribution, and osteogenic differentiation ability due to the synergistic effects of the composite coating. In addition, the biopolymer-embedded scaffolds (ZB, ZCB) showed an excellent biological and mechanical performance. Thus, our results suggest that the Zn-HA/glass composite-coated glass-infiltrated zirconia (ZC, ZCB) scaffolds are a dynamic approach to designing bioactive 3D scaffolds for the load-bearing bone regeneration applications.

Effect of parathyroid hormone-related protein on odontogenic differentiation in human dental pulp cells.

BACKGROUND: Parathyroid hormone-related protein (PTHrP) plays an important role in many physiological processes, including bone regeneration. The function of PTHrP is similar to PTH. It promotes osteogenic differentiation in MC3T3-E1 cells. The aim of this study was to investigate whether PTHrP might have odontogenic differentiation ability in human dental pulp cells (hDPCs). METHODS: The viability of hDPCs after stimulation with PTHrP was measured. Real-time polymerase chain reaction and Western blot analysis were performed to evaluate the expression levels of odontogenic markers and activation of protein kinase B (PKB/AKT), extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 mitogen-activated protein kinase (MAPK). To evaluate mineralized nodule formation, alkaline phosphatase (ALP) staining and alizarin red S staining were performed. RESULTS: PTHrP promoted odontogenic differentiation as evidenced by the formation of mineralized nodules, the induction of ALP activity, and the upregulation of odontogenic markers (dentin sialophosphoprotein and dentin matrix protein-1). The phosphorylation of AKT, ERK, JNK, and p38 was increased by PTHrP. However, an AKT inhibitor (LY294002), an ERK inhibitor (U0126), a JNK inhibitor (SP600125), and a p38 inhibitor (SB203580) inhibited the increase of mineralization induced by PTHrP. CONCLUSION: The present study revealed that PTHrP could promote odontogenic differentiation and mineralization through activating the AKT, ERK, JNK, and p38 signaling pathways. These results provide novel insights into the odontogenic action of PTHrP.

In Vitro Characterization of Dental Pulp Stem Cells Cultured in Two Microsphere-Forming Culture Plates.

Various three-dimensional (3D) culture methods have been introduced to overcome the limitations of in vitro culture and mimic in vivo conditions. This study aimed to evaluate two microsphere-forming culture methods and a monolayer culture method. We evaluated cell morphology, viability, osteo-, adipo-, and chondrogenic differentiation potential of dental pulp stem cells (DPSCs) cultured in 3D culture plates: ultra-low attachment (ULA) and U-bottomed StemFit 3D (SF) plates, and a two-dimensional (2D) monolayer plate. RNA sequencing (RNA-seq) revealed differentially expressed gene (DEG) profiles of the DPSCs. In contrast to an increasing pattern in the 2D group, cell viability in 3D groups (ULA and SF) showed a decreasing pattern; however, high multilineage differentiation was observed in both the 3D groups. RNA-seq showed significantly overexpressed gene ontology categories including angiogenesis, cell migration, differentiation, and proliferation in the 3D groups. Hierarchical clustering analysis revealed a similar DEG regulation pattern between the 3D groups; however, a comparatively different DEG was observed between the 2D and 3D groups. Taken together, this study shows that DPSCs cultured in microsphere-forming plates present superior multilineage differentiation capacities and demonstrate higher DEG expression in regeneration-related gene categories compared to that in DPSCs cultured in a conventional monolayer plate.

Effect of autoclave sterilization on cyclic fatigue and torsional fracture resistance of NiTi rotary instruments.

The purpose of this study is to assess the effect of autoclave sterilization on the cyclic fatigue and torsional fracture resistance of ProTaper Universal (PTU), K3XF, HyFlex EDM (EDM), and TF adaptive (TFA). Sixty instruments from each file type were divided into two categories for cyclic fatigue group (CGr) and torsional fracture group (TGr). CGr and TGr were divided into three subgroups, respectively, consisting of ten instruments from each file type. Cyclic fatigue fracture test was performed using artificial canal made of stainless steel, and the mean number of cycles to failure (NCF) were determined. CGr1, the files were tested to establish baseline for NCF; CGr2, the files were tested cyclic fatigue after 10 cycles of autoclave; CGr3, instruments were autoclaved after being cycled to 25, 50, and 75% of corresponding NCF determined in CGr1, followed by cyclic fatigue test. The torsional fracture test was performed without autoclave (TGr1), after 3-cycle autoclave (TGr2), and 7-cycle autoclave (TGr3), respectively, which evaluated maximum torque and angular deflection. NCF, maximum torque and angular deflection were compared using one-way ANOVA with Bonferroni test. Two-way ANOVA was performed to determine the interaction between 'autoclave treatment' and 'type of NiTi file'. EDM showed highest NCF within the same autoclave treatment. TFA presented the lowest maximum torque and the highest angular deflection, and PTU presented the lowest angular deflection. Within the same NiTi file systems, most of NCF, maximum torque and angular deflection of tested files were not significantly influenced by autoclave condition.