Chemical Properties of Human Dentin Blocks and Vertical Augmentation by Ultrasonically Demineralized Dentin Matrix Blocks on Scratched Skull without Periosteum of Adult-Aged Rats

Vertical augmentation is one of the most challenging techniques in bone engineering. Several parameters, such mechano-chemical characteristics, are important to optimize vertical bone regeneration using biomaterials. The aims of this study were to chemically characterize human dentin blocks (calcified demineralized dentin matrix: CDM, partially demineralized dentin matrix: PDDM and completely demineralized dentin matrix: CDDM) (2 × 2 × 1 mm3) chemically and evaluate the behavior of PDDM blocks on non-scratched or scratched skulls without periosteum of adult rats (10–12 months old, female) as a vertical augmentation model. The dissolved efficiency of CDM showed 32.3% after ultrasonic demineralization in 1.0 L of 2% HNO3 for 30 min. The 30 min-demineralized dentin was named PDDM. The SEM images of PDDM showed the opening of dentinal tubes, nano-microcracks and the smooth surface. In the collagenase digestion test, the weight-decreasing rates of CDM, PDDM and CDDM were 9.2%, 25.5% and 78.3% at 12 weeks, respectively. CDM inhibited the collagenase digestion, compared with PDDM and CDDM. In the PDDM onlay graft on an ultrasonically scratched skull, the bone marrow-space opening from original bone was found in the bony bridge formation between the human PDDM block and dense skull of adult senior rats at 4 and 8 weeks. On the other hand, in the cases of the marrow-space closing in both non-scratched skulls and scratched skulls, the bony bridge was not formed. The results indicated that the ultrasonic scratching into the compact parietal bone might contribute greatly to the marrow-space opening from skull and the supply of marrow cells, and then bony bridge formation could occur in the vertical augmentation model without a periosteum.

3D Bioprinting of Polycaprolactone-Based Scaffolds for Pulp-Dentin Regeneration: Investigation of Physicochemical and Biological Behavior

In this study, two structurally different scaffolds, a polycaprolactone (PCL)/45S5 Bioglass (BG) composite and PCL/hyaluronic acid (HyA) were fabricated by 3D printing technology and were evaluated for the regeneration of dentin and pulp tissues, respectively. Their physicochemical characterization was performed by field emission scanning electron microscopy (FESEM) equipped with energy dispersive spectroscopy (EDS), Fourier-transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), atomic force microscopy (AFM), contact angle, and compressive strength tests. The results indicated that the presence of BG in the PCL/BG scaffolds promoted the mechanical properties, surface roughness, and bioactivity. Besides, a surface treatment of the PCL scaffold with HyA considerably increased the hydrophilicity of the scaffolds which led to an enhancement in cell adhesion. Furthermore, the gene expression results showed a significant increase in expression of odontogenic markers, e.g., dentin sialophosphoprotein (DSPP), osteocalcin (OCN), and dentin matrix protein 1 (DMP-1) in the presence of both PCL/BG and PCL/HyA scaffolds. Moreover, to examine the feasibility of the idea for pulp-dentin complex regeneration, a bilayer PCL/BG-PCL/HyA scaffold was successfully fabricated and characterized by FESEM. Based on these results, it can be concluded that PCL/BG and PCL/HyA scaffolds have great potential for promoting hDPSC adhesion and odontogenic differentiation.

Tailoring Silicon Nitride Surface Chemistry for Facilitating Odontogenic Differentiation of Rat Dental Pulp Cells

Silicon nitride (Si3N4) can facilitate bone formation; hence, it is used as a biomaterial in orthopedics. Nevertheless, its usability for dentistry is unexplored. The aim of the present study was to investigate the effect of Si3N4 granules for the proliferation and odontogenic differentiation of rat dental pulp cells (rDPCs). Four different types of Si3N4 granules were prepared, which underwent different treatments to form pristine as-synthesized Si3N4, chemically treated Si3N4, thermally treated Si3N4, and Si3N4 sintered with 3 wt.% yttrium oxide (Y2O3). rDPCs were cultured on or around the Si3N4 granular beds. Compared with the other three types of Si3N4 granules, the sintered Si3N4 granules significantly promoted cellular attachment, upregulated the expression of odontogenic marker genes (Dentin Matrix Acidic Phosphoprotein 1 and Dentin Sialophosphoprotein) in the early phase, and enhanced the formation of mineralization nodules. Furthermore, the water contact angle of sintered Si3N4 was also greatly increased to 40°. These results suggest that the sintering process for Si3N4 with Y2O3 positively altered the surface properties of pristine as-synthesized Si3N4 granules, thereby facilitating the odontogenic differentiation of rDPCs. Thus, the introduction of a sintering treatment for Si3N4 granules is likely to facilitate their use in the clinical application of dentistry.

Effect of Biodentine Coated with Emdogain on Proliferation and Differentiation of Stem Cells from the Apical Papilla

Background: This study assessed the effect of Biodentine coated with Emdogain (Biodentine/Emdogain) on proliferation and differentiation of stem cells from the apical papilla (SCAP). Methods and Results: In this in vitro, experimental study, SCAP were isolated from two immature impacted third molars and cultured. After ensuring the stemness of the cells by assessing their cell surface markers, they were exposed to Biodentine, Emdogain, and Biodentine/Emdogain for 24 and 72 hours. The control cells did not receive any intervention. Cell viability was evaluated by the methyl thiazolyl tetrazolium (MTT) assay. Expression of odontogenic differentiation genes was analyzed by the quantitative reverse transcription polymerase chain reaction (qRT-PCR). Alkaline phosphatase (ALP) activity was quantified by the respective kit. Data were analyzed by one-way ANOVA, t-test, and Mann-Whitney test (α=0.05). Cell viability did not change after 24 hours of exposure to biomaterials. At 72 hours, the viability of the cells exposed to Biodentine and Biodentine/Emdogain decreased compared with the control group. The expression of dentin sialophosphoprotein (DSPP), dentin matrix protein 1 (DMP1), and bone sialoprotein (BSP) genes, and ALP activity significantly increased in all three experimental groups, compared with the control group at both 24 and 72 hours; this increase was significantly greater in Biodentine/Emdogain group. The number of mineralized nodules significantly increased in all groups after 72 hours with a greater rate in Biodentine/Emdogain group.Conclusions: All biomaterials increased the differentiation of SCAP, expression of odontogenic genes, and ALP activity, but Biodentine/Emdogain was significantly more effective for this purpose.

Dentin Matrix Protein 1 on Titanium Surface Facilitates Osteogenic Differentiation of Stem Cells

Dentin matrix protein 1 (DMP1) contains a large number of acidic domains, multiple phosphorylation sites, a functional arginine-glycine-aspartate (RGD) motif, and a DNA binding domain, and has been shown to play essential regulatory function in dentin and bone mineralization. DMP1 could also orchestrate bone matrix formation, but the ability of DMP1 on Ti to human mesenchymal stem cell (hMSC) conversion to osteoblasts has not been studied. There is importance to test if the DMP1 coated Ti surface would promote cell migration and attachment to the metal surface and promote the differentiation of the attached stem cells to an osteogenic lineage. This study aimed to study the human mesenchymal stem cells (hMSCs) attachment and proliferation on DMP1 coated titanium (Ti) disks compared to non-coated disks, and to assess possible osteoblastic differentiation of attached hMSCs. Sixty-eight Ti disks were divided into two groups. Group 1 disks were coated with dentin matrix protein 1 and group 2 disks served as control. Assessment with light microscopy was used to verify hMSC attachment and proliferation. Cell viability was confirmed through fluorescence microscopy and mitochondrial dehydrogenase activity. Real-time polymerase chain reaction analysis was done to study the gene expression. The proliferation assay showed significantly greater cell proliferation with DMP1 coated disks compared to the control group (p-value < 0.001). Cell vitality analysis showed a greater density of live cells on DMP1 coated disks compared to the control group. Alkaline phosphatase staining revealed higher enzyme activity on DMP1 coated disks and showed itself to be significantly higher than the control group (p-value < 0.001). von Kossa staining revealed higher positive areas for mineralized deposits on DMP1 coated disks than the control group (p-value < 0.05). Gene expression analysis confirmed upregulation of runt-related transcription factor 2, osteoprotegerin, osteocalcin, osteopontin, and alkaline phosphatase on DMP1 coated disks (p-value < 0.001). The dentin matrix protein promoted the adhesion, proliferation, facilitation differentiation of hMSC, and mineralized matrix formation.

Chondrogenic Potential of Dental-Derived Mesenchymal Stromal Cells

The field of tissue engineering has revolutionized the world in organ and tissue regeneration. With the robust research among regenerative medicine experts and researchers, the plausibility of regenerating cartilage has come into the limelight. For cartilage tissue engineering, orthopedic surgeons and orthobiologists use the mesenchymal stromal cells (MSCs) of various origins along with the cytokines, growth factors, and scaffolds. The least utilized MSCs are of dental origin, which are the richest sources of stromal and progenitor cells. There is a paradigm shift towards the utilization of dental source MSCs in chondrogenesis and cartilage regeneration. Dental-derived MSCs possess similar phenotypes and genotypes like other sources of MSCs along with specific markers such as dentin matrix acidic phosphoprotein (DMP) -1, dentin sialophosphoprotein (DSPP), alkaline phosphatase (ALP), osteopontin (OPN), bone sialoprotein (BSP), and STRO-1. Concerning chondrogenicity, there is literature with marginal use of dental-derived MSCs. Various studies provide evidence for in-vitro and in-vivo chondrogenesis by dental-derived MSCs. With such evidence, clinical trials must be taken up to support or refute the evidence for regenerating cartilage tissues by dental-derived MSCs. This article highlights the significance of dental-derived MSCs for cartilage tissue regeneration.

Bone regeneration of demineralized dentin matrix with platelet-rich fibrin and recombinant human bone morphogenetic protein-2 on the bone defects in rabbit calvaria

Background This study was to evaluate the bone formation ability of demineralized dentin matrix (DDM) combined with platelet-rich fibrinogen (PRF) and DDM combined with recombinant human bone morphogenetic protein-2 (rhBMP-2) to improve the osteoinductive ability of DDM. Methods After four bone defects with a diameter of 8mm were created in the calvarium of each rabbit, DDM was grafted into the first defect (experimental groups 1), a combination of DDM and PRF was grafted into the second defect (experimental groups 2), and DDM with absorbed rhBMP-2 was grafted into the third defect (experimental groups 3). The fourth defect was used as the control group. Twelve healthy male rabbits (New Zealand, white rabbits) weighing around 3.0–4.0 kg were used. Among 12 rabbits, 3 rabbits were sacrificed immediately after surgery and at 2, 4, and 8 weeks after surgery, respectively. Histopathologic analysis and histomorphometric analysis were conducted to evaluate bone formation in each group. Results The PRF/DDM group did not show a significantly higher degree of new bone formation in calvarial bone defects than the DDM group at 2, 4, and 8 weeks postoperatively in histopathological findings and histomorphometric results. On the other side, the rhBMP-2/DDM group showed higher degrees of new bone formation and calcification, and the lamellae of bone matrix, which are observed in mature bone tissue, were more distinctly visible in the rhBMP-2/DDM group. Moreover, the rhBMP-2/DDM group showed a significantly higher amount of new bone formation, compared to the DDM group at 4 and 8 weeks postoperatively (P<0.05) in histomorphometric results. Conclusion The DDM has great potential as a carrier for the maintenance and sustained release of rhBMP-2, which has been recently receiving wide attention as a type of signaling molecules to promote bone formation.

Plasma Membrane Ca2+–ATPase in Rat and Human Odontoblasts Mediates Dentin Mineralization

Intracellular Ca2+ signaling engendered by Ca2+ influx and mobilization in odontoblasts is critical for dentinogenesis induced by multiple stimuli at the dentin surface. Increased Ca2+ is exported by the Na+–Ca2+ exchanger (NCX) and plasma membrane Ca2+–ATPase (PMCA) to maintain Ca2+ homeostasis. We previously demonstrated a functional coupling between Ca2+ extrusion by NCX and its influx through transient receptor potential channels in odontoblasts. Although the presence of PMCA in odontoblasts has been previously described, steady-state levels of mRNA-encoding PMCA subtypes, pharmacological properties, and other cellular functions remain unclear. Thus, we investigated PMCA mRNA levels and their contribution to mineralization under physiological conditions. We also examined the role of PMCA in the Ca2+ extrusion pathway during hypotonic and alkaline stimulation-induced increases in intracellular free Ca2+ concentration ([Ca2+]i). We performed RT-PCR and mineralization assays in human odontoblasts. [Ca2+]i was measured using fura-2 fluorescence measurements in odontoblasts isolated from newborn Wistar rat incisor teeth and human odontoblasts. We detected mRNA encoding PMCA1–4 in human odontoblasts. The application of hypotonic or alkaline solutions transiently increased [Ca2+]i in odontoblasts in both rat and human odontoblasts. The Ca2+ extrusion efficiency during the hypotonic or alkaline solution-induced [Ca2+]i increase was decreased by PMCA inhibitors in both cell types. Alizarin red and von Kossa staining showed that PMCA inhibition suppressed mineralization. In addition, alkaline stimulation (not hypotonic stimulation) to human odontoblasts upregulated the mRNA levels of dentin matrix protein-1 (DMP-1) and dentin sialophosphoprotein (DSPP). The PMCA inhibitor did not affect DMP-1 or DSPP mRNA levels at pH 7.4–8.8 and under isotonic and hypotonic conditions, respectively. We also observed PMCA1 immunoreactivity using immunofluorescence analysis. These findings indicate that PMCA participates in maintaining [Ca2+]i homeostasis in odontoblasts by Ca2+ extrusion following [Ca2+]i elevation. In addition, PMCA participates in dentinogenesis by transporting Ca2+ to the mineralizing front (which is independent of non-collagenous dentin matrix protein secretion) under physiological and pathological conditions following mechanical stimulation by hydrodynamic force inside dentinal tubules, or direct alkaline stimulation by the application of high-pH dental materials.

Silencing VEGFR-2 Hampers Odontoblastic Differentiation of Dental Pulp Stem Cells

Dental pulp stem cells (DPSCs) are a source of postnatal stem cells essential for maintenance and regeneration of dentin and pulp tissues. Previous in vivo transplantation studies have shown that DPSCs are able to give rise to odontoblast-like cells, form dentin/pulp-like structures, and induce blood vessel formation. Importantly, dentin formation is closely associated to blood vessels. We have previously demonstrated that DPSC-induced angiogenesis is VEGFR-2-dependent. VEGFR-2 may play an important role in odontoblast differentiation of DPSCs, tooth formation and regeneration. Nevertheless, the role of VEGFR-2 signaling in odontoblast differentiation of DPSCs is still not well understood. Thus, in this study we aimed to determine the role of VEGFR-2 in odontoblast differentiation of DPSCs by knocking down the expression of VEGFR-2 in DPSCs and studying their odontoblast differentiation capacity in vitro and in vivo. Isolation and characterization of murine DPSCs was performed as previously described. DPSCs were induced by VEGFR-2 shRNA viral vectors transfection (MOI = 10:1) to silence the expression of VEGFR-2. The GFP+ expression in CopGFP DPSCs was used as a surrogate to measure the efficiency of transfection and verification that the viral vector does not affect the expression of VEGFR-2. The efficiency of viral transfection was shown by significant reduction in the levels of VEGFR-2 based on the Q-RT-PCR and immunofluorescence in VEGFR-2 knockdown DPSCs, compared to normal DPSCs. VEGFR-2 shRNA DPSCs expressed not only very low level of VEGFR-2, but also that of its ligand, VEGF-A, compared to CopGFP DPSCs in both transcriptional and translational levels. In vitro differentiation of DPSCs in osteo-odontogenic media supplemented with BMP-2 (100 ng/ml) for 21 days demonstrated that CopGFP DPSCs, but not VEGFR-2 shRNA DPSCs, were positive for alkaline phosphatase (ALP) staining and formed mineralized nodules demonstrated by positive Alizarin Red S staining. The expression levels of dentin matrix proteins, dentin matrix protein-1 (Dmp1), dentin sialoprotein (Dspp), and bone sialoprotein (Bsp), were also up-regulated in differentiated CopGFP DPSCs, compared to those in VEGFR-2 shRNA DPSCs, suggesting an impairment of odontoblast differentiation in VEGFR-2 shRNA DPSCs. In vivo subcutaneous transplantation of DPSCs with hydroxyapatite (HAp/TCP) for 5 weeks demonstrated that CopGFP DPSCs were able to differentiate into elongated and polarized odontoblast-like cells forming loose connective tissue resembling pulp-like structures with abundant blood vessels, as demonstrated by H&amp;E, Alizarin Red S, and dentin matrix staining. On the other hand, in VEGFR-2 shRNA DPSC transplants, odontoblast-like cells were not observed. Collagen fibers were seen in replacement of dentin/pulp-like structures. These results indicate that VEGFR-2 may play an important role in dentin regeneration and highlight the potential of VEGFR-2 modulation to enhance dentin regeneration and tissue engineering as a promising clinical application.