Determination of the compressive modulus of elasticity of periodontal ligament derived from human first premolars.

Purpose: There are two commonly cited modulus of elasticity of the human periodontal ligament (EPDL), i.e., 6.89 ✕ 10-5 GPa (E1) and 6.89 ✕ 10-2 GPa (E2), which are exactly 1000-fold different from each other. This study aims to clarify the ambiguity of the two EPDL used for simulations and determine a more accurate EPDL value of human first premolars using experimental and simulation approaches. Methods: Numerical simulations using finite element analysis were performed to analyze PDL deformation under an average Asian occlusal force. To confirm the results, simple and multi-component, true-scale 3D models of a human first premolar were used in the simulations. Finally, a compression test using a universal testing machine on PDL specimens was conducted to identify the compressive EPDL of human first premolars. Results: The simulation results from both models revealed that E1 was inaccurate, because it resulted in excessive PDL deformation under the average occlusal force, which should not occur during mastication. Although the E2 did not lead to excessive PDL deformation, it was obtained by an error in unit conversion with no scientific backing. In contrast, the compression test results indicated that the compressive EPDL was 9.64 ✕ 10-4 GPa (E3). In the simulation, E3 did not cause excessive PDL deformation. Conclusion: The simulation results demonstrated that both commonly cited EPDL values (E1 and E2) were incorrect. Based on the experimental and simulation results, the average compressive EPDL of 9.64 ✕ 10-4 GPa is proposed as a more accurate value for human first premolars. Clinical significance: The proposed more accurate EPDL would contribute to more precise and reliable FEA simulation results and provide a better understanding of the stress distribution and deformation of dental materials, which will be beneficial to precision dentistry, orthodontics and restoration designs.

Anti-inflammatory effects of the prostacyclin analogue iloprost in an in vitro model of inflamed human dental pulp cells.

Iloprost's anti-inflammatory effects on human dental pulp stem cells (HDPCs) are currently unknown. We hypothesized that iloprost could downregulate the expression of inflammatory-related genes and protein in an inflamed HDPC in vitro model. To induce inflammation, the HDPCs were treated with a cocktail of interleukin-1 beta, interferon-gamma, and tumour necrosis alpha, at a ratio of 1:10:100. Iloprost (10-6  M) was then added or not to the cultures. Interleukin-6 (IL-6) and interleukin-12 (IL-12) mRNA expression were assessed by real-time polymerase chain reaction. IL-6 protein expression was assessed by enzyme-linked immunosorbent assay. The results were analysed using one-way ANOVA or the Kruskal-Wallis test. The cytokine cocktail induced more robust IL-6 expression than LPS treatment. Iloprost slightly, yet significantly, downregulated IL-6 and IL-12 mRNA expression. These findings suggest that iloprost might be used as a beneficial component in vital pulp therapy.

Wnt3a promotes odonto/osteogenic differentiation in vitro and tertiary dentin formation in a rat model.

AIM: To investigate the effect of Wnt3a on odonto/osteogenic differentiation of stem cells isolated from human exfoliated deciduous teeth (SHEDs) and reparative dentine formation in a rat model. METHODOLOGY: Stem cells isolated from human exfoliated deciduous teeth were cultured in media with Wnt3a (50-200 ng/ml). Wnt activation was confirmed by ß-catenin immunocytochemistry. Colony-forming unit assay (normalized percentage area), osteogenic gene expression analysis by real-time polymerase chain reaction and mineralization assays measured by the absorption at 540 nm were performed. Tertiary dentine formation in vivo was evaluated using 8-week-old, male Wistar rats. Cavities with pinpoint pulp exposure by a sharp instrument were prepared at the mesial surface of the first molars. Teeth were divided into (n = 6): (1) distilled water (negative control), (2) phosphate-buffered saline (PBS), (3) lithium chloride in DI (20 µM), and (4) Wnt3a in PBS (200 ng/ml). Collagen sponge was used as a scaffold. The cavity was sealed with glass ionomer restoration. Four weeks later, animals were euthanized by sodium pentobarbital (120 mg/kg body weight). Hard tissue formation was evaluated using micro-computerized tomography. Sixty consecutive slides from the initial plane were analysed and calculated as bone/dentine volume per total tissue volume. Paraffin sections (2 µm) were stained with haematoxylin and eosin and Masson's trichrome for morphological evaluation. Data are presented as the mean ± standard error. Mann-Whitney U test was used for two-group comparison. Kruskal Wallis followed by pairwise comparison was employed for three or more group comparisons. Statistical analysis was performed using GraphPad Prism 7. Differences were considered significant at p < .05. RESULTS: Wnt3a decreased SHEDs colony formation and increased OSX, BMP2, and DMP1 expression, corresponding to an increase in mineralization. Additionally, a significant increase in dentine/bone volume per total tissue volume was observed in Wnt3a treated defects. Dentine bridge formation at the exposure sites treated with Wnt3a demonstrated, while fibrous tissues were observed in the control. CONCLUSIONS: Wnt3a suppressed proliferation, increased osteogenic differentiation of SHEDs and promotes tertiary dentine formation. Wnt3a could be utilized as biological molecule for vital pulp therapy.

Application of shear stress for enhanced osteogenic differentiation of mouse induced pluripotent stem cells.

The self-organizing potential of induced pluripotent stem cells (iPSCs) represents a promising tool for bone tissue engineering. Shear stress promotes the osteogenic differentiation of mesenchymal stem cells, leading us to hypothesize that specific shear stress could enhance the osteogenic differentiation of iPSCs. For osteogenesis, embryoid bodies were formed for two days and then maintained in medium supplemented with retinoic acid for three days, followed by adherent culture in osteogenic induction medium for one day. The cells were then subjected to shear loading (0.15, 0.5, or 1.5 Pa) for two days. Among different magnitudes tested, 0.5 Pa induced the highest levels of osteogenic gene expression and greatest mineral deposition, corresponding to upregulated connexin 43 (Cx43) and phosphorylated Erk1/2 expression. Erk1/2 inhibition during shear loading resulted in decreased osteogenic gene expression and the suppression of mineral deposition. These results suggest that shear stress (0.5 Pa) enhances the osteogenic differentiation of iPSCs, partly through Cx43 and Erk1/2 signaling. Our findings shed light on the application of shear-stress technology to improve iPSC-based tissue-engineered bone for regenerative bone therapy.

The Prostacyclin Analog Iloprost Promotes Cementum Formation and Collagen Reattachment of Replanted Molars and Up-regulates Mineralization by Human Periodontal Ligament Cells.

INTRODUCTION: This study evaluated the use of the prostacyclin analog iloprost as a root surface treatment agent in promoting acellular cementum (AC) formation and collagen reattachment after tooth replantation in vivo. In addition, its effect on human periodontal ligament cell (hPDLC) mineralization was assessed in vitro. METHODS: First molars of 8-week-old Wistar rats were extracted. In 1 group, the root surfaces were treated with Hank's balanced salt solution (HBSS), and the other group's root surfaces were treated with 10-6 mol/L iloprost before replantation. At day 30, maxillae were prepared for micro-computed tomographic imaging and histomorphometric analysis. The effect of iloprost on mineralization by hPDLCs was analyzed by mineralized nodule formation and quantitative polymerase chain reaction at 7 and 14 days. RESULTS: Micro-computed tomographic imaging demonstrated a significant higher bone volume in the iloprost groups, whereas the HBSS groups had extensive bone and root resorption. Histologic analysis revealed deposition of a thick AC layer along the root in the iloprost group with well-organized periodontal ligament fibers inserted into the cementum. The HBSS group demonstrated more osteoclasts than the iloprost group. In vitro, iloprost-treated hPDLCs had a significantly increased RUNX2, OSX, BSP, and ALP gene expression that coincided with an increased deposition of mineralized nodules. These effects were abrogated by a PGI2 receptor inhibitor. CONCLUSIONS: Our results revealed that iloprost promoted PDL regeneration in replanted molars. Furthermore, resorption of the roots was decreased, whereas AC deposition was stimulated. Iloprost-treatment increased hPDLC mineralization and was mediated by PGI2 receptor activation. These observations indicate that iloprost may be a promising root surface treatment agent.

Mechanical loading and the control of stem cell behavior.

OBJECTIVE: Mechanical stimulation regulates many cell responses. The present study describes the effects of different in vitro mechanical stimulation approaches on stem cell behavior. DESIGN: The narrative review approach was performed. The articles published in English language that addressed the effects of mechanical force on stem cells were searched on Pubmed and Scopus database. The effects of extrinsic mechanical force on stem cell response was reviewed and discussed. RESULTS: Cells sense mechanical stimuli by the function of mechanoreceptors and further transduce force stimulation into intracellular signaling. Cell responses to mechanical stimuli depend on several factors including type, magnitude, and duration. Further, similar mechanical stimuli exhibit distinct cell responses based on numerous factors including cell type and differentiation stage. Various mechanical applications modulate stemness maintenance and cell differentiation toward specific lineages. CONCLUSIONS: Mechanical force application modulates stemness maintenance and differentiation. Modification of force regimens could be utilized to precisely control appropriate stem cell behavior toward specific applications.

Experimental data on mechanical behavior and numerical data on tensile stress distribution of a hyperelastic Polydimethysiloxane (PDMS) based membrane for cell culture.

The data contained within this article relate to experimental data on mechanical behavior of an in-house cast hyperelastic Polydimethysiloxane (PDMS) based membrane (SilasticⓇ T-4, Dow Corning) for cell culture, which was used as a tool for applying tensile stress on cells and tissues. With the experimentally obtained material constants, tensile stress distribution over the membrane surface was numerically assessed using a finite element analysis (FEA). The membrane area having a uniform tensile stress distribution under different strain loading conditions of 1-20% was suggested for cell culture.

Comparison of ultimate force revealed by compression tests on extracted first premolars and FEA with a true scale 3D multi-component tooth model based on a CBCT dataset.

OBJECTIVE: The aim of this study was to develop a new method for creating a multi-component and true scale 3-dimensional (3D) model of a human tooth based on cone-beam computed tomography (CBCT) images. MATERIALS AND METHODS: First maxillary premolar tooth model was reconstructed from a patient's CBCT images. The 2D serial sections were used to create the 3D model. This model was used for finite element analysis (FEA). Model validation was performed by comparing the ultimate compressive force (UF) obtained experimentally using a universal testing machine and from simulation. The simulations of three component-omitting models (silicone, cementum, and omitting both) were performed to analyze the maximum (max.) principal stress and stress distribution. RESULTS: The simulation-based UF indicating tooth fracture was 637 N, while the average UF in the in vitro loading was 651 N. The discrepancy between the simulation-based UF and the experimental UF was 2.2%. From the simulation, the silicone-omitting models showed a significant change in max. principal stress, resulting in a UF error of 26%, whereas there was no notable change in the cementum-omitting model. CONCLUSION: This study, for the first time, developed a true scale multi-component 3D model from CBCT for predicting stress distribution in a human tooth. CLINICAL RELEVANCE: This study proposed a method to create 3D modeling from CBCT in a true scale and multi-component manner. The PDL-like component-omitting simulation led to a higher error value of UF, indicating the importance of multi-component tooth modeling in FEA. Tooth 3D modeling could help determine mechanical failure in dental treatments in a more precise manner.

Prostacyclin Analog Promotes Human Dental Pulp Cell Migration via a Matrix Metalloproteinase 9-related Pathway.

INTRODUCTION: During dental pulp healing, progenitor cells migrate to the injured site. This study investigated the effect of iloprost (an exogenous prostacyclin [PGI2]) on enhancing human dental pulp cell (HDPC) migration and its underlying mechanism. METHODS: HDPC migration was analyzed using a wound scratch assay. HDPCs were obtained from extracted teeth and cultured in the presence of iloprost for 24 and 72 hours. Immunofluorescent staining for matrix metalloproteinase 9 (MMP-9), quantitative polymerase chain reaction gene expression analysis, gelatin zymography, and enzyme-linked immunosorbent assay of MMP-9 expression were performed. A PGI2 (IP) antagonist, protein kinase A (PKA) inhibitor, and MMP-9 inhibitor were used to inhibit the IP receptor, PKA signaling pathway, and MMP-9 activity, respectively. RESULTS: A mechanically applied scratch in HDPC cultures closed more rapidly in the presence of iloprost. This result coincided with increased MMP-9 messenger RNA and protein expression and higher gelatinase activity. These iloprost-enhanced effects were inhibited by an IP receptor antagonist or a PKA inhibitor. Forskolin, a PKA activator, increased MMP-9 expression concomitant with increased migration. The application of a selective MMP-9 inhibitor resulted in decreased iloprost-induced migration. CONCLUSIONS: MMPs play an important role in cell migration by degrading components of the extracellular matrix. In this study, iloprost accelerated HDPC migration in a wound scratch assay. MMP-9 expression was increased concomitantly by iloprost and appeared to be mediated by the IP-PKA pathway. These observations suggest that iloprost may enhance dental pulp tissue healing by up-regulating MMP-9. The PGI2 analog might be a promising biomolecule in dental pulp regenerative treatment.

Numerical data on the shear stress distribution generated by a rotating rod within a stationary ring over a 35-mm cell culture dish.

The data contained within this article relate to a rotating rod within a stationary ring that was used to generate shear stress on cells and tissues via a medium. The geometry of the rotating rod within a stationary ring was designed to work with a 35-mm diameter culture dish. The data of the shear stress distribution are presented in terms of area-weighted average shear stress and the uniformity index, which were calculated for medium volumes of 4 and 5 ml at various rotational speeds ranging from 0 to 1000 rpm.

Iloprost induces tertiary dentin formation.

INTRODUCTION: Prostacyclin (PGI2), a member of the prostaglandin family, can promote angiogenesis and cell proliferation. METHODS: In this study, the effect of the application of a PGI2 analog (iloprost) on dentin repair was examined in vitro and in vivo. RESULTS: Iloprost significantly stimulated the expression of vascular endothelial growth factor and osteo-/odontogenic marker messenger RNA in human dental pulp cells (HDPCs) under osteoinductive conditions in vitro. In addition, iloprost enhanced HDPC alkaline phosphatase enzymatic activity and mineral deposition. An in vivo study was performed using a rat molar mechanical pulp exposure model. After 30 days, histologic analysis revealed that there was a dramatic tertiary dentin formation in the iloprost-treated group compared with the calcium hydroxide and the untreated control groups. Furthermore, vascular endothelial growth factor protein expression in dental pulp tissue was increased in the iloprost-treated group as determined by immunohistochemical staining. CONCLUSIONS: Taken together, the present study, for the first time, shows that iloprost induces the expression of osteo-/odontogenic markers in vitro and promotes angiogenic factor expression and enhances tertiary dentin formation in vivo. This implies the potential clinical usefulness of iloprost in vital pulp therapy.

Iloprost up-regulates vascular endothelial growth factor expression in human dental pulp cells in vitro and enhances pulpal blood flow in vivo.

INTRODUCTION: Prostacyclin (PGI2) is a biomolecule capable of enhancing angiogenesis and cellular proliferation. METHODS: We investigated the influence of a PGI2 analogue (iloprost) on dental pulp revascularization in vitro and in vivo by using human dental pulp cells (HDPCs) and a rat tooth injury model, respectively. Iloprost stimulated the human dental pulp cell mRNA expression of vascular endothelial growth factor (VEGF), fibroblast growth factor-2 (FGF-2), and platelet-derived growth factor (PDGF) in a significant dose-dependent manner. This mRNA up-regulation was significantly inhibited by pretreatment with a PGI2 receptor antagonist and forskolin (a protein kinase A activator). In contrast, a protein kinase A inhibitor significantly enhanced the iloprost-induced mRNA expression of VEGF, FGF-2, and PDGF. Pretreatment with a fibroblast growth factor receptor inhibitor attenuated the VEGF, FGF-2, and PDGF mRNA expression, indicating opposing regulatory mechanisms. RESULTS: The effect of iloprost on the dental pulp was investigated in vivo by using a rat molar pulp injury model. The iloprost-treated group exhibited a significant increase in pulpal blood flow at 72 hours compared with control. CONCLUSIONS: The present study indicates that iloprost may be a candidate agent to promote neovascularization in dental pulp tissue, suggesting the potential clinical use of iloprost in vital pulp therapy.