Comparing the Osteogenic Potentials and Bone Regeneration Capacities of Bone Marrow and Dental Pulp Mesenchymal Stem Cells in a Rabbit Calvarial Bone Defect Model.

The bone regeneration efficiency of bone marrow mesenchymal stem cells (BMSCs) and dental pulp mesenchymal stem cells (DPSCs) combined with xenografts in the craniofacial region remains unclear. Accordingly, this study commenced by comparing the cell morphology, cell proliferation, trilineage differentiation, mineral synthesis, and osteogenic gene expression of BMSCs and DPSCs in vitro. Four experimental groups (empty control, Bio-Oss only, Bio-Oss+BMSCs, and Bio-Oss+DPSCs) were then designed and implanted in rabbit calvarial defects. The BMSCs and DPSCs showed a similar morphology, proliferative ability, surface marker profile, and trilineage-differentiation potential in vitro. However, the BMSCs exhibited a higher mineral deposition and expression levels of osteogenic marker genes, including alkaline phosphatase (ALP), runt related transcription factor 2 (RUNX2), and osteocalcin (OCN). In the in vivo studies, the bone volume density in both MSC groups was significantly greater than that in the empty control or Bio-Oss only group. Moreover, the new bone formation and Collagen I / osteoprotegerin protein expressions of the scaffold+MSC groups were higher than those of the Bio-Oss only group. Finally, the Bio-Oss+BMSC and Bio-Oss+DPSC groups had a similar bone mineral density, new bone formation, and osteogenesis-related protein expression. Overall, the DPSCs seeded on Bio-Oss matched the bone regeneration efficacy of BMSCs in vivo and hence appear to be a promising strategy for craniofacial defect repair in future clinical applications.

Static magnetic field attenuates lipopolysaccharide-induced inflammation in pulp cells by affecting cell membrane stability.

One of the causes of dental pulpitis is lipopolysaccharide- (LPS-) induced inflammatory response. Following pulp tissue inflammation, odontoblasts, dental pulp cells (DPCs), and dental pulp stem cells (DPSCs) will activate and repair damaged tissue to maintain homeostasis. However, when LPS infection is too serious, dental repair is impossible and disease may progress to irreversible pulpitis. Therefore, the aim of this study was to examine whether static magnetic field (SMF) can attenuate inflammatory response of dental pulp cells challenged with LPS. In methodology, dental pulp cells were isolated from extracted teeth. The population of DPSCs in the cultured DPCs was identified by phenotypes and multilineage differentiation. The effects of 0.4 T SMF on DPCs were observed through MTT assay and fluorescent anisotropy assay. Our results showed that the SMF exposure had no effect on surface markers or multilineage differentiation capability. However, SMF exposure increases cell viability by 15%. In addition, SMF increased cell membrane rigidity which is directly related to higher fluorescent anisotropy. In the LPS-challenged condition, DPCs treated with SMF demonstrated a higher tolerance to LPS-induced inflammatory response when compared to untreated controls. According to these results, we suggest that 0.4 T SMF attenuates LPS-induced inflammatory response to DPCs by changing cell membrane stability.

Melatonin enhances osteogenic differentiation of dental pulp mesenchymal stem cells by regulating MAPK pathways and promotes the efficiency of bone regeneration in calvarial bone defects.

BACKGROUND: Mesenchymal stem cell (MSC)-based tissue engineering plays a major role in regenerative medicine. However, the efficiency of MSC transplantation and survival of engrafted stem cells remain challenging. Melatonin can regulate MSC biology. However, its function in the osteogenic differentiation of dental pulp-derived MSCs (DPSCs) remains unclear. We investigated the effects and mechanisms of melatonin on the osteogenic differentiation and bone regeneration capacities of DPSCs. METHODS: The biological effects and signaling mechanisms of melatonin with different concentrations on DPSCs were evaluated using a proliferation assay, the quantitative alkaline phosphatase (ALP) activity, Alizarin red staining, a real-time polymerase chain reaction, and a western blot in vitro cell culture model. The in vivo bone regeneration capacities were assessed among empty control, MBCP, MBCP + DPSCs, and MBCP + DPSCs + melatonin preconditioning in four-created calvarial bone defects by using micro-computed tomographic, histological, histomorphometric, and immunohistochemical analyses after 4 and 8 weeks of healing. RESULTS: In vitro experiments revealed that melatonin (1, 10, and 100 µM) significantly and concentration-dependently promoted proliferation, surface marker expression (CD 146), ALP activity and extracellular calcium deposition, and osteogenic gene expression of DPSCs (p < 0.05). Melatonin activated the protein expression of ALP, OCN, and RUNX-2 and inhibited COX-2/NF-κB expression. Furthermore, the phosphorylation of mitogen-activated protein kinase (MAPK) p38/ERK signaling was significantly increased in DPSCs treated with 100 µM melatonin, and their inhibitors significantly decreased osteogenic differentiation. In vivo experiments demonstrated that bone defects implanted with MBCP bone-grafting materials and melatonin-preconditioned DPSCs exhibited significantly greater bone volume fraction, trabecular bone structural modeling, new bone formation, and osteogenesis-related protein expression than the other three groups at 4 and 8 weeks postoperatively (p < 0.05). CONCLUSIONS: These results suggest that melatonin promotes the proliferation and osteogenic differentiation of DPSCs by regulating COX-2/NF-κB and p38/ERK MAPK signaling pathways. Preconditioning DPSCs with melatonin before transplantation can efficiently enhance MSCs function and regenerative capacities.

Three-dimensional Spheroid Culture Enhances Multipotent Differentiation and Stemness Capacities of Human Dental Pulp-derived Mesenchymal Stem Cells by Modulating MAPK and NF-kB Signaling Pathways.

BACKGROUND: Three-dimensional (3D) culture of mesenchymal stem cells has become an important research and development topic. However, comprehensive analysis of human dental pulp-derived mesenchymal stem cells (DPSCs) in 3D-spheroid culture remains unexplored. Thus, we evaluated the cellular characteristics, multipotent differentiation, gene expression, and related-signal transduction pathways of DPSCs in 3D-spheroid culture via magnetic levitation (3DM), compared with 2D-monolayer (2D) and 3D-aggregate (3D) cultures. METHODS: The gross morphology and cellular ultrastructure were observed in the 2D, 3D, and 3DM experimental groups using scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Surface markers and trilineage differentiation were evaluated using flow cytometry and staining analysis. Quantitative reverse transcription-polymerase chain reaction and immunofluorescence staining (IF) were performed to investigate the expression of differentiation and stemness markers. Signaling transduction pathways were evaluated using western blot analysis. RESULTS: The morphology of cell aggregates and spheroids was largely influenced by the types of cell culture plates and initial cell seeding density. SEM and TEM experiments confirmed that the solid and firm structure of spheroids was quickly formed in the 3DM-medium without damaging cells. In addition, these three groups all expressed multilineage differentiation capabilities and surface marker expression. The trilineage differentiation capacities of the 3DM-group were significantly superior to the 2D and 3D-groups. The osteogenesis, angiogenesis, adipogenesis, and stemness-related genes were significantly enhanced in the 3D and 3DM-groups. The IF analysis showed that the extracellular matrix expression, osteogenesis, and angiogenesis proteins of the 3DM-group were significantly higher than those in the 2D and 3D-groups. Finally, 3DM-culture significantly activated the MAPK and NF-kB signaling transduction pathways and ameliorated the apoptosis effects of 3D-culture. CONCLUSIONS: This study confirmed that 3DM-spheroids efficiently enhanced the therapeutic efficiency of DPSCs.

Dental Pulp Stem Cell Transplantation with 2,3,5,4'-Tetrahydroxystilbene-2-O-ß-D-glucoside Accelerates Alveolar Bone Regeneration in Rats.

INTRODUCTION: Although the therapeutic potential of human dental pulp stem cells (hDPSCs) has been studied for bone regeneration, the therapeutic efficiency needs further consideration and examinations for clinical applications. Thus, the aims of this study were to evaluate the effect of 2,3,5,4'-tetrahydroxystilbene-2-O-ß-D-glucoside (THSG) on the osteogenic differentiation of hDPSCs and to examine the therapeutic efficiency of the THSG-enhanced osseous potential of hDPSCs in alveolar bony defects of rats. METHODS: Expressions of osteogenic messenger RNAs (including ALP, RUNX2, BGLAP, and AMBN) were examined by quantitative real-time polymerase chain reaction. Alizarin red S staining was conducted to analyze THSG-induced mineralization of hDPSCs. To investigate the regenerative effects of THSG-treated hDPSCs on dental alveolar bone, bony defects were created in male Sprague-Dawley rats. Defects were treated with Matrigel (Corning Inc, Corning, NY), hDPSCs, or hDPSCs + THSG. After 2 weeks, defect healing was evaluated by micro-computed tomographic and histologic analyses. RESULTS: In the cell model, THSG induced osteogenesis-associated genes (ALP, RUNX2, and BGLAP) and an enamel-related gene (AMBN), resulting in mineralization as detected by alizarin red S staining after 2 weeks of treatment. In the animal model, THSG increased all parameters of bone formation (the relative bone volume, trabecular thickness, trabecular number, and trabecular separation) in alveolar bony defects of rats. THSG not only improved the quality of newly formed bone but also the quantity of new bone. CONCLUSIONS: These results showed important findings in revealing the THSG-enhanced osteogenic differentiation of hDPSCs and THSG-facilitated bone regeneration, which may provide an alternative option for cell-based regenerative therapy.

An evaluation of the biocompatibility and osseointegration of novel glass fiber reinforced composite implants: In vitro and in vivo studies.

OBJECTIVES: The aim of this study was to evaluate the in vitro biocompatibility and in vivo osseointegration of three novel bioactive glass fiber reinforced composite (GFRC) implants and to compare these with metal (Ti6Al4V) implants. METHODS: The surfaces of these experimental substrates were characterized by scanning electron microscopy (SEM), a 2D profilometer and by contact angle measurement. In vitro biological performance was assessed using MG-63 human osteoblast-like cell morphology, cell proliferation assays and the alkaline phosphatase (ALP) activity testing. Furthermore, in vivo osseointegration performance was examined by installing samples into rabbit femurs and evaluated the results using micro-CT, histology and histomorphometrical analysis; these assessments were carried out after 1, 2, 4 and 8 weeks of healing. RESULTS: The results showed that moderate surface roughness, moderate hydrophilic exposure and moderate homogenous exposure of bioactive glass fibers were present for all of the GFRC substrates. Furthermore, MG-63 cells, when cultured on all of the GFRC substrates, grew well and exhibited a more differentiated phenotype than cells grown on titanium alloy (Ti6Al4V) substrate. Histological evaluation revealed more newly-formed bone regeneration within the thread of the GFRC implants during the initial healing period. In addition, the novel GFRC implants with a bioactive Bio-fiber structure and glass particles within the epoxy resin matrix showed better bone volume/tissue volume (BV/TV) values at 4 weeks and this was accompanied by bone-implant contact (BIC) values at 8 weeks comparable to the Ti6Al4V group. SIGNIFICANCE: These findings demonstrated that novel GFRC implants seem to show improved osteogenesis and osseointegration functionality and have potential as a substitute for Ti6Al4V, or other metal-based materials, when used for clinically dental and orthopedic applications.

Monitoring the Changes of Material Properties at Bone-Implant Interface during the Healing Process In Vivo: A Viscoelastic Investigation.

The aim of this study was to monitor the changes of viscoelastic properties at bone-implant interface via resonance frequency analysis (RFA) and the Periotest device during the healing process in an experimental rabbit model. Twenty-four dental implants were inserted into the femoral condyles of rabbits. The animals were sacrificed immediately after implant installation or on day 14, 28, or 56 after surgery. Viscoelastic properties at bone-implant interface were evaluated by measuring the implant stability quotient (ISQ) using RFA and by measuring the Periotest values (PTVs) using the Periotest device. The bone/implant specimens were evaluated histopathologically and histomorphometrically to determine the degree of osseointegration (BIC%). The BIC% values at different time points were then compared with the corresponding ISQ values and PTVs. The mean ISQ value increased gradually and reached 81 ± 1.7 on day 56, whereas the mean PTV decreased over time, finally reaching -0.7 ± 0.5 on day 56. Significant correlations were found between ISQ and BIC% (r = 0.701, p < 0.001), PTV and BIC% (r = -0.637, p < 0.05), and ISQ and PTV (r = -0.68, p < 0.05). These results show that there is a positive correlation between implant stability parameters and peri-implant-bone healing, indicating that the RFA and Periotest are useful for measuring changes of viscoelastic properties at bone-implant interface and are reliable for indirectly predicting the degree of osseointegration.

Damping Factor as a Diagnostic Parameter for Assessment of Osseointegration during the Dental Implant Healing Process: An Experimental Study in Rabbits.

The purpose of this study was to evaluate the possibility of using damping factor (DF) analysis to provide additional information on osseointegration of dental implants during the healing period. A total of 30 dental implants were installed in the bilateral femoral condyles of 15 rabbits. A DF analyzer detected with an impulse-forced vibration method and a commercialized dental implant stability analyzer based on resonance frequency (RF) analysis were used to measure the implant stability immediately after implant placement and 1, 2, 4, and 8 weeks post-surgically. Results of DF and RF analyses at different time points were compared with the corresponding osseointegration performance of dental implants via micro-computed tomography (micro-CT), histological and histomorphometrical analysis. The DF values revealed a decrease with time and reached 0.062 ± 0.007 at 8 weeks after implantation, which is almost 50% lower than the initial value. Moreover, highly significant correlations between DF values and bone volume densities (R 2 = 0.9797) and percentages of bone-to-implant contact measured at trabecular bone area (R 2 = 0.9773) were also observed. These results suggested that DF analysis combined with RF analysis results in a more sensitive assessment of changes in the dental implant/bone complex during the healing period than RF analysis alone.