Angiogenesis during coronal pulp regeneration using rat dental pulp cells: Neovascularization in rat molars in vivo and proangiogenic dental pulp cell-endothelial cell interactions in vitro.

Background/purpose: Angiogenesis is considered a crucial event for dental pulp regeneration. The purpose of this study was to demonstrate neovascularization during coronal pulp regeneration in rat molars using rat dental pulp cells (rDPCs) and to examine whether rDPC-endothelial cell interactions promote proangiogenic capacity in vitro. Materials and methods: Maxillary first molars of Wistar rats (n = 42) were pulpotomized and rDPCs isolated from incisors were implanted with a porous poly (l-lactic acid) (PLLA) scaffold and hydrogel (Matrigel). After 3, 7, and 14 days, coronal pulp tissues were examined histologically and by nestin and CD146 immunohistochemistry. rDPCs and rat dermal microvascular endothelial cells (rDMECs) were cocultured for 4 days and vascular endothelial growth factor (VEGF) synthesis and angiogenic factor gene expression were determined by enzyme-linked immunosorbent assays and real-time polymerase chain reaction, respectively. Effects of cocultured medium on tube formation by rDMECs were also evaluated. Results: Implantation of rDPC/PLLA/Matrigel induced coronal pulp regeneration with dentin bridge formation and arrangement of nestin-positive odontoblast-like cells at 14 days. PLLA/Matrigel without rDPCs did not induce pulp regeneration. CD146-positive blood vessels increased in density in the remaining pulp tissues at 3 and 7 days, and in the regenerated pulp tissue at 14 days. rDPC/DMEC coculture significantly promoted VEGF secretion and mRNA expression of nuclear factor-kappa B, angiogenic chemokine CXCL1, and chemokine receptor CXCR1. Cocultured medium significantly promoted tube formation. Conclusion: Coronal pulp regeneration with rDPC/PLLA/Matrigel was accompanied by neovascularization. rDPC-rDMEC interactions may promote angiogenic activity represented by proangiogenic factor upregulation and tube formation in vitro.

Pulp inflammation induces Kv1.1 K+ channel down-regulation in rat thalamus.

OBJECTIVES: Signals from inflamed tooth pulp activate thalamic neurons to evoke central sensitization. We aimed to gain insights into the mechanisms mediating the early phase of pulpal inflammation-induced thalamic neural and glial activation. MATERIALS AND METHODS: Pulpal inflammation was induced via the application of mustard oil (MO) to the upper first molar of Wistar rats with local anesthesia (LA) or saline injection. After 0.5, 1, 2, and 24 hr, contralateral thalami were subjected to microarrays, a real-time polymerase chain reaction and immunohistochemistry to identify differentially expressed genes and assess potassium voltage-gated channel subfamily A member 1 (Kv1.1)-expressing axons and glial fibrillary acidic protein (GFAP)-expressing astrocytes. RESULTS: The Kv1.1 gene (Kcna1) was down-regulated and the density of Kv1.1-expressing axons decreased in non-anesthetized rats, but not in anesthetized rats 1 hr after the MO treatment. The density of GFAP-expressing astrocytes increased in both groups until 24 hr after the MO treatment, with a greater increase being observed in the saline-injection group than in the LA group. CONCLUSIONS: MO induced the transient down-regulation of Kcna1, transiently reduced the density of Kv1.1-expressing axons, and increased astrocytes in thalami within 1 hr of pulpal application. These results suggest central sensitization represented by neuronal hyperexcitability and astrocyte activation.

Neural Regeneration/Remodeling in Engineered Coronal Pulp Tissue in the Rat Molar.

INTRODUCTION: This study aimed to examine the process of reinnervation during coronal pulp tissue regeneration in a rat model in which rat bone marrow mesenchymal stem cells were implanted in pulpotomized molars. METHODS: The maxillary first molars of Wistar rats were pulpotomized, and preformed biodegradable porous poly L-lactic acid scaffolds and hydrogel carrying rat bone marrow mesenchymal stem cells were implanted in the pulp chamber. After 3, 7, and 14 days, the implanted teeth were processed for histologic analysis; immunoperoxidase staining for protein gene product 9.5 (a general neuronal marker), calcitonin gene-related peptide (CGRP), or substance P (SP); and real-time polymerase chain reaction for nerve growth factor (NGF) and growth-associated protein 43 (GAP-43) messenger RNA (mRNA) expression. RESULTS: Histologic analysis of the implanted region revealed sparse cellular distribution at 3 days, pulplike tissue with a thin dentin bridge-like structure at 7 days, and dentin bridge-like mineralized tissue formation and resorption of most scaffolds at 14 days. Protein gene product 9.5 and CGRP-immunoreactive nerve fibers showed the lowest density at 3 days and significantly increased until 14 days when the CGRP-immunoreactive fibers reached normal levels. SP-immunoreactive nerve fibers showed the highest density at 7 days and decreased to normal levels at 14 days. NGF mRNA increased with time, whereas GAP-43 mRNA levels peaked at 3 days and subsequently dropped until 14 days. CONCLUSIONS: Regeneration/remodeling of SP-immunoreactive and CGRP-immunoreactive nerve fibers with increased mRNA expression of NGF and GAP-43 occurred in a rat model of coronal pulp tissue engineering with bone marrow mesenchymal stem cells.

Crosstalk between dental pulp stem cells and endothelial cells augments angiogenic factor expression.

OBJECTIVES: We aimed to investigate whether the mesenchymal stem cell-endothelial cell crosstalk enhances angiogenic factor expression via nuclear factor-kappa B (NF-κB)-dependent mechanisms. MATERIALS AND METHODS: Human dermal microvascular endothelial cells (HDMECs) and stem cells from human exfoliated deciduous teeth (SHEDs) were cocultured for 96 hr, in the presence of NF-κB decoy oligodeoxynucleotides (ODNs) or scramble (control). Vascular endothelial cell growth factor (VEGF) and phospho-NF-κB p65 were measured with enzyme-linked immunosorbent assay. Angiogenesis-related gene expression was analyzed with microarray analysis followed by real-time polymerase chain reaction. Tube formation assay was conducted in the presence of NF-κB decoy. RESULTS: The VEGF and phospho-NF-κB p65 levels were significantly higher in the coculture with NF-κB decoy scramble than in single culture and coculture with NF-κB decoy ODN. Microarray analysis of SHEDs and HDMECs with NF-κB decoy scramble showed higher expression of proangiogenic genes, Bcl-2, NF-κB1, VEGFA, CXCL8, and CXCR1, and lower expression of proapoptotic genes, Bax and Caspase 9, compared to cells with NF-κB decoy ODN. Real-time PCR results for Bcl-2 and CXCL8 showed a similar trend. Tube formation assay showed more tube development in the presence of NF-κB decoy scramble. CONCLUSION: The SHED-HDMEC crosstalk enhanced proangiogenic factor expression via NF-κB-dependent pathways.

In vivo fate of bone marrow mesenchymal stem cells implanted into rat pulpotomized molars.

In our previous work, we established an in vivo coronal pulp regeneration model in which biodegradable hydrogel-made scaffolds carrying rat bone marrow mesenchymal stem cells (BM-MSCs) were implanted in the coronal pulp chamber of pulpotomized rat maxillary first molars. In this study, we investigated the in vivo fate of LacZ-labeled BM-MSCs in our coronal pulp regeneration model. BM-MSCs were nucleofected with pVectOZ-LacZ plasmid encoding ß-galactosidase 1 day before implantation, and the LacZ-transfected BM-MSCs were implanted into the pulpotomized pulp chamber with biodegradable preformed scaffold-hydrogel constructs. Empty vector was used as a control. After 3 and 14 days, the molars were retrieved and subjected to ß-galactosidase staining. At 3 days, ß-galactosidase-expressing cells with a round profile were located mainly around the scaffold. At 14 days, when the pulp-like tissue had been generated, the majority of ß-galactosidase-expressing cells were detected under the newly formed dentin bridge-like structure, where nestin-expressing odontoblast-like cells were arranged. Immunoreactivity for dentin sialoprotein, a marker of mature odontoblasts, was strongly detected under the original dentin. No ß-galactosidase staining was observed in the control group. Thus, we demonstrated that BM-MSCs survived for 2 weeks after implantation and colonized within the site of potential cytodifferentiation. Our findings indicated that BM-MSCs could differentiate into cells involved in mineralized tissue formation in the functionally relevant region.

Dental Pulp Tissue Engineering Using Mesenchymal Stem Cells: a Review with a Protocol.

Mesenchymal stem cells (MSCs) are adult stem cells that can be isolated from human and animal sources such as rats. Recently, an in vivo protocol for pulp tissue engineering using implantation of bone marrow MSCs into rat pulpotomized molars was established by our research group. This coronal pulp regeneration model showed almost complete regeneration/healing with dentin bridge formation when the cavity was sealed with mineral trioxide aggregate (MTA) to create a biocompatible seal of the pulp. This method is a powerful tool for elucidating the processes of dental pulp tissue regeneration following implantation of MSCs. In the present review, we discuss the literature in the field of dental pulp tissue engineering using MSCs including dental pulp stem cells and stem cells from exfoliated deciduous teeth. In addition, we present a brief step-by-step protocol of the coronal pulp regeneration model focusing on the implantation of rat bone marrow MSCs, biodegradable scaffolds, and hydrogels in pulpotomized rat molars. The protocol may lay the foundation for studies aiming at defining further histological and molecular mechanism of the rat pulp tissue engineering.