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.

Inhibition of Nuclear Factor Kappa B Prevents the Development of Experimental Periapical Lesions.

INTRODUCTION: Nuclear factor kappa B (NF-κB) is an important transcriptional regulator of angiogenesis involving B-cell lymphoma 2 (Bcl-2) and Bcl-2-associated X protein (Bax) signaling pathways. Thus, inhibition of NF-κB may suppress the development of periapical lesions via blockage of angiogenesis. Accordingly, we examined the effects of NF-κB decoy oligodeoxynucleotide (ODN) treatment on experimentally induced periapical lesions. METHODS: Periapical lesions were induced in the mandibular first molars of 5-week-old male Wistar rats by the application of lipopolysaccharide to the pulp. NF-κB decoy ODN or NF-κB decoy scramble (control) was injected intraperitoneally every 7 days, starting 1 day before pulp exposure. After 28 days, the samples were retrieved, and digital radiographs were taken for radiomorphometry. Samples were processed for (1) immunohistochemistry of CD31, Bcl-2, and Bax; (2) laser capture microdissection to analyze Bcl-2, Bax, chemokine (C-X-C motif) ligand 1 (CXCL1), CXC receptor 2 (CXCR2), and vascular endothelial cell growth factor receptor 2 (VEGFR2) messenger RNA (mRNA) expression in CD31+ endothelial cells; (3) enzyme-linked immunosorbent assay to determine NF-κB/p65 activity; and (4) Western blotting for vascular endothelial growth factor expression. RESULTS: NF-κB decoy ODN treatment significantly reduced lesion size, NF-κB/p65 activity, and the density of CD31+ endothelial cells in the lesion. NF-κB decoy ODNs also down-regulated CXCL1, CXCR2, and VEGFR2 mRNAs and up-regulated Bax mRNA in endothelial cells but did not affect Bcl2 mRNA in endothelial cells. Vascular endothelial growth factor protein expression in the lesions was significantly decreased. CONCLUSIONS: The inhibition of NF-κB activity by decoy ODN treatment suppressed the development of experimentally induced periapical lesions with a concomitant reduction in angiogenic responses in endothelial cells.

Implantation of Endothelial Cells with Mesenchymal Stem Cells Accelerates Dental Pulp Tissue Regeneration/Healing in Pulpotomized Rat Molars.

INTRODUCTION: This study aimed to examine whether the implantation of mesenchymal stem cells (MSCs) with endothelial cells (ECs) accelerates pulp tissue regeneration/healing and induces dentin bridge formation in a rat model of molar coronal pulp regeneration. METHODS: The maxillary first molars of Wistar rats were subjected to pulpotomy. Then, pulp chambers were implanted with biodegradable hydrogel-made scaffolds carrying MSCs together or without dermal microvascular ECs, and the cavities were sealed with mineral trioxide aggregate. After 14 days, pulp samples were analyzed by immunohistochemistry; messenger RNA expression of B-cell lymphoma 2 (Bcl-2), chemokine (C-X-C motif) ligand 1 (Cxcl1), CXC receptor 2 (Cxcr2), and dentin sialophosphoprotein (Dspp) by quantitative polymerase chain reaction, and protein expression of nestin and vascular endothelial growth factor by Western blotting. RESULTS: Teeth coimplanted with MSCs and ECs showed pulp healing with complete dentin bridge formation, whereas those implanted with MSCs alone had incomplete dentin bridges. Bcl-2, Cxcl1, Cxcr2, and Dspp messenger RNA levels were significantly up-regulated in the pulp of MSC/EC-implanted teeth compared with those in MSC-implanted teeth. Immunohistochemical analysis revealed the expression of nestin in odontoblastlike cells under dentin bridges in the MSC/EC coimplanted group. The density of CD31-expressing ECs and the expression of nestin and vascular endothelial growth factor proteins were significantly up-regulated in the MSC/EC-implanted pulp compared with the MSC-implanted pulp. CONCLUSIONS: The implantation of ECs with MSCs accelerated pulp tissue regeneration/healing and dentin bridge formation, up-regulated the expression of proangiogenic factors, and increased the density of ECs in pulpotomized rat molars.

Dental pulp tissue engineering of pulpotomized rat molars with bone marrow mesenchymal stem cells.

The major goal of dental pulp tissue engineering is to enable the healing of inflamed tissue or to replace necrotic pulp tissue with newly formed dental pulp tissue. Here, we report a protocol for pulp tissue engineering in vivo in pulpotomized rat teeth using constructs of rat bone marrow mesenchymal stem cells, preformed biodegradable scaffolds, and hydrogel. The constructs were implanted into pulpotomized pulp chambers for 3, 7, or 14 days. At 3 days, cells were located mainly along the preformed scaffolds. At 7 days, pulp tissue regeneration was observed in almost the entire implanted region. At 14 days, pulp tissue regeneration further progressed throughout the implanted region. In immunohistochemistry, at 3 days, a number of small and round macrophages immunoreactive to CD68 were predominantly distributed around the scaffolds. The density of CD68+ macrophages decreased until 14 days. On the other hand, nestin-expressing odontoblast-like cells beneath the dentin at the border of implanted region increased until 14 days. Quantitative gene expression analysis revealed that odontoblast differentiation marker dentin sialophosphoprotein mRNA in the implanted region gradually increased until 14 days. Together, the results suggested that regeneration of dental pulp tissue had occurred. Thus, our study provides a novel experimental rat model of dental pulp regeneration.