Melatonin's effect on hepatic differentiation of stem cells remains unclear. The aim of this study was to investigate the action of melatonin on hepatic differentiation as well as its related signaling pathways of human dental pulp stem cells (hDPSCs) and to examine the therapeutic effects of a combination of melatonin and hDPSC transplantation on carbon tetrachloride (CCl4 )-induced liver fibrosis in mice. In vitro hepatic differentiation was assessed by periodic acid-Schiff (PAS) staining and mRNA expression for hepatocyte markers. Liver fibrosis model was established by injecting 0.5 mL/kg CCl4 followed by treatment with melatonin (5 mg/kg, twice a week) and hDPSCs. In vivo therapeutic effects were evaluated by histopathology and by means of liver function tests including measurement of alanine transaminase (ALT), aspartate transaminase (AST), and ammonia levels. Melatonin promoted hepatic differentiation based on mRNA expression of differentiation markers and PAS-stained glycogen-laden cells. In addition, melatonin increased bone morphogenic protein (BMP)-2 expression and Smad1/5/8 phosphorylation, which was blocked by the BMP antagonist noggin. Furthermore, melatonin activated p38, extracellular signal-regulated kinase (ERK), and nuclear factor-κB (NF-κB) in hDPSCs. Melatonin-induced hepatic differentiation was attenuated by inhibitors of BMP, p38, ERK, and NF-κB. Compared to treatment of CCl4 -injured mice with either melatonin or hDPSC transplantation alone, the combination of melatonin and hDPSC significantly suppressed liver fibrosis and restored ALT, AST, and ammonia levels. For the first time, this study demonstrates that melatonin promotes hepatic differentiation of hDPSCs by modulating the BMP, p38, ERK, and NF-κB pathway. Combined treatment of grafted hDPSCs and melatonin could be a viable approach for the treatment of liver cirrhosis.

Recently, the involvement of PIN1, a peptidyl-prolyl cis/trans isomerase, has been reported in age-related bone homeostasis and adipogenesis. However, the role of PIN1 during odontogenic and adipogenic differentiation remains to be fully understood, particularly regarding human dental pulp stem cells (HDPSCs). Thus, in the present study, we have investigated the role of PIN1 in odontogenic and adipogenic differentiation of HDPSCs and signaling pathways possibly involved. PIN1 mRNA and protein level were upregulated in a time-dependent manner during adipogenic differentiation, increasing until 1 day of odontogenic induction and then steadily declined during odontogenic differentiation. Treatment of a known PIN1 inhibitor, juglone, significantly increased odontogenic differentiation as confirmed by alkaline phosphatase (ALP) activity, calcium deposition, and mRNAs induction of odontogenic markers [ALP, osteopontin (OPN), osteocalcin (OCN), dentin sialophosphoprotein (DSPP), and dentin matrix protein 1 (DMP-1)]. On the contrary, adipogenic differentiation was dramatically reduced upon juglone treatment, with concomitant downregulation of lipid droplet accumulation and adipogenic marker genes [peroxisome proliferation-activated receptor gamma (PPARγ), lipoprotein lipase (LPL), and adipocyte fatty acid-binding protein (AP2)]. In contrast to PIN1 inhibition, the overexpression of PIN1 via adenoviral infection (Ad-PIN1) in HDPSCs inhibited odontogenic differentiation but increased adipogenic differentiation, in which stem cell property markers such as stage-specific embryonic antigen-4 (SSEA-4) and STRO-1 were upregulated during odontogenic differentiation but downregulated in adiopogenic differentiation. Consistently, juglone-mediated inhibition of PIN1 augmented the osteogenic medium (OM)-induced activation of bone morphogenetic protein (BMP), Wnt/β-catenin, extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and nuclear factor-kappa B (NF-κB) pathway, which response was reversed by Ad-PIN1. Moreover, juglone blocked the adipogenic induction medium-induced activation of PPARγ, C/EBPα, C/EBPβ, ERK, and NF-κB pathways, which was rescued by Ad-PIN1 infection. In summary, the present study shows for the first time that PIN1 acts as an important modulator of odontogenic and adipogenic differentiation of HDPSCs and may have clinical implications for regenerative dentistry.

This study assesses the cytoprotective effects of human dental pulp stem cells (hDPSCs) and conditioned medium from hDPSCs (CM-hDPSCs) on ischemic human astrocytes (hAs) in vitro compared with human bone marrow-derived mesenchymal stem cells (hMSCs). Ischemia of hAs was induced by oxygen-glucose deprivation (OGD). CM-hDPSCs and hMSCs were collected after 48 hr of culture. Cell death was determined by 3-[4,5-dimethylthialzol-2-yl]-2,5-diphenyltetrazolium bromide and cellular ATP assays. The expression of glial fibrillary acidic protein (GFAP) and musashi-1 as markers of reactive astrogliosis was examined with immunochemical staining. mRNA expression and reactive oxygen species (ROS) were analyzed by RT-PCR and flow cytometry, respectively. OGD increased cytotoxicity in a time-dependent manner and decreased cellular ATP content concomitantly in hAs. Pretreatment and posttreatment with hDPSCs were associated with greater recovery from OGD-induced cytotoxicity in hAs compared with hMSCs. Similarly, CM-hDPSCs had a greater effect on OGD-induced cytotoxicity in a dose-dependent manner. Pre- and posttreatment with CM-hDPSCs or CM-hMSCs attenuated OGD-induced GFAP, nestin, and musashi-1 expression in hAs. Furthermore, treatment of cells with CM-hDPSCs and hMSCs blocked OGD-induced ROS production and interleukin-1ß upregulation. This study demonstrates for the first time that hDPSCs and CM-hDPSCs confer superior cytoprotection against cell death in an in vitro OGD model compared with hMSCs as shown by cell viability assay. Reactive gliosis, ROS production, and inflammatory mediators might contribute to this protective effect. Therefore, hDPSCs could represent an alternative source of cell therapy for ischemic stroke.

Inflammatory responses and osteoclast differentiation play pivotal roles in the pathogenesis of osteolytic bone diseases such as periodontitis. Although overexpression or inhibition of peptidyl-prolyl cis/trans isomerase NIMA-interacting 1 (PIN1) offers a possible therapeutic strategy for chronic inflammatory diseases, the role of PIN1 in periodontal disease is unclear. The aim of the present study was to evaluate PIN1 expression in periodontitis patients as well as the effects of PIN1 inhibition by juglone or PIN1 small-interfering RNA (siRNA) and of PIN1 overexpression using a recombinant adenovirus encoding PIN1 (Ad-PIN1) on the inflammatory response and osteoclastic differentiation in lipopolysaccharide (LPS)-and nicotine-stimulated human periodontal ligament cells (PDLCs). PIN1 was up-regulated in chronically inflamed PDLCs from periodontitis patients and in LPS-and nicotine-exposed PDLCs. Inhibition of PIN1 by juglone or knockdown of PIN1 gene expression by siRNA markedly attenuated LPS-and nicotine-stimulated prostaglandin E 2 (PGE 2 ) and nitric oxide (NO) production, as well as cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) expression, whereas PIN1 overexpression by Ad-PIN1 increased it. LPS-and nicotine-induced nuclear factor (NF)-κB activation was blocked by juglone and PIN1 siRNA but increased by Ad-PIN1. Conditioned medium prepared from LPS-and nicotinetreated PDLCs increased the number of tartrate-resistant acid phosphatase-stained osteoclasts and osteoclast-specific gene expression. These responses were blocked by PIN1 inhibition and silencing but stimulated by Ad-PIN1. Furthermore, juglone and PIN1 siRNA inhibited LPS-and nicotine-induced osteoclastogenic cytokine expression in PDLCs. This study is the first to demonstrate that PIN1 inhibition exhibits anti-inflammatory effects and blocks osteoclastic differentiation in LPS-and nicotine-treated PDLCs. PIN1 inhibition may be a therapeutic strategy for inflammatory osteolysis in periodontal disease.

Within the limits of this study, EMD enhanced the proliferation of hMSCs. After evaluation with ARS staining, EMD seemed to enhance mineralization, and the RT-PCR test revealed that EMD promoted early-stage osteoblast differentiation by enhancing Col I A2 expression, but exerted an inhibitory effect on the mineralization by lowering the gene expression of BSP and BGLAP. Mineralized nodules formed with EMD may be composed of substances other than normal bone. Because most of the organic matrix of bone is type I collagen, which acts as the mineralization site, bone or bone-like mineralized mass might have been formed in spite of the different components of the non-collagenous proteins.

A supplemental appendix to this article is published electronically only at http://jdr.sagepub.com/supplemental. AbstrAct A large proportion of pulpal nociceptors are known to contain neuropeptides such as CGRP. However, the projection of non-peptidergic nociceptors to tooth pulp is controversial. Recently, the nonpeptidergic subset of nociceptors has been implicated in mechanical pain in the skin. Since mechanical irritation of pulpal nociceptors is critical for evoking tooth pain under pathophysiological conditions, we investigated whether the non-peptidergic afferents project to tooth pulp as potential mechanotransducing afferents. For clear visualization of the non-peptidergic afferents, we took advantage of a recently generated knock-in mouse model in which an axonal tracer, farnesylated green fluorescence protein (GFP), is expressed from the locus of a sensory neuron-specific gene, Mrgprd. In the trigeminal ganglia (TG), we demonstrated that GFP is exclusively expressed in afferents binding to isolectin B4 (IB4), a neurochemical marker of non-peptidergic nociceptors, but is rarely co-localized with CGRP. Retrograde labeling of pulpal afferents demonstrated that a low proportion of pulpal afferents was co-localized with GFP. Immunohistochemical detection of the axonal tracer revealed that GFP-positive afferent terminals were densely projected into the tooth pulp. These results provide convincing evidence that non-peptidergic nociceptors are projected into the tooth pulp and suggest a potential role for these afferents in tooth pain.

Objective: To evaluate the localization of osteopontin (OPN) and osterix in periodontal tissue during experimental tooth movement with heavy force in rats. Materials and Methods: Nickel-titanium closed-coil springs were used to create a 100 g mesial force to the maxillary first molars. On days 3, 7, 10, and 14 after force application, histological changes in periodontium were examined by immunohistochemistry using proliferating cell nuclear antigen (PCNA), OPN, and osterix. Results: PCNA-positive cells were found close to the alveolar bone and cementum on both sides. OPN-positive cells were observed along the cementing line of the cementum and bone on both sides and also were visible along with newly formed fibers in the periodontal ligament on the tension side. Osterix-positive cells were strongly detected on the surface of the alveolar bone and cementum on both sides. Conclusions: During tooth movement, periodontal remodeling occurs on both sides. These results indicate that OPN and osterix may play an important role of differentiation and osteoblasts and cementoblasts matrix formation during periodontal tissue remodeling.

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