Factors secreted from the stem cells of human exfoliated deciduous teeth inhibit osteoclastogenesis through the activation of the endogenous antioxidant system

Systemic administration of conditioned medium (CM) from stem cells derived from human exfoliated deciduous teeth (SHED-CM) in mouse models of rheumatoid arthritis, osteoporosis, and osteoarthritis suppresses excessive osteoclast activity and restores bone integrity. However, the mechanism through which SHED-CM regulates osteoclastogenesis remains largely unknown. In the present study, we examined the anti-osteoclastogenic mechanism of SHED-CM in vitro. Bone marrow macrophages and RAW264.7 cells were treated with receptor activator of nuclear factor kappa-Β ligand (RANKL) in the presence of SHED-CM or CM from bone marrow mesenchymal stem cells (BMSC-CM). Osteoclast differentiation was assessed using tartrate-resistant acid phosphatase staining, actin ring formation, and expression of osteoclast-specific markers. RANKL-induced reactive oxygen species (ROS) production was analyzed as a critical mediator of osteoclastogenesis. The activation of endogenous antioxidant gene expression was examined using reverse transcription quantitative PCR. Liquid chromatography with tandem mass spectrometry (LC-MS) was used to identify proteins enriched in SHED-CM, and neutralizing antibodies were used to evaluate their functional roles. Compared to BMSC-CM, SHED-CM effectively inhibited RANKL-induced early osteoclast differentiation and late maturation. Notably, SHED-CM but not BMSC-CM suppressed RANKL-induced ROS production. SHED-CM increased the expression of genes encoding antioxidant enzymes. The LC-MS analysis identified seven proteins uniquely enriched in SHED-CM that activated the endogenous antioxidant system. Neutralizing antibodies against some of these proteins restore RANKL-induced ROS production and osteoclast differentiation. SHED-CM inhibited osteoclastogenesis, partially through the activation of multiple antioxidant enzymes in osteoclast precursors, highlighting its potential for treating bone-destructive diseases.


Conditioned Medium From Stem Cells of Human Exfoliated Deciduous Teeth Alleviates Mouse Osteoarthritis by Inducing sFRP1-Expressing M2 Macrophages.

Intravenous administration of conditioned medium from stem cells of human exfoliated deciduous teeth (SHED-CM) regenerates mechanically injured osteochondral tissues in mouse temporomandibular joint osteoarthritis (TMJOA). However, the underlying therapeutic mechanisms remain unclear. Here, we showed that SHED-CM alleviated injured TMJ by inducing anti-inflammatory M2 macrophages in the synovium. Depletion of M2 by Mannosylated Clodrosome abolished the osteochondral repair activities of SHED-CM. Administration of CM from M2-induced by SHED-CM (M2-CM) effectively ameliorated mouse TMJOA by inhibiting chondrocyte inflammation and matrix degradation while enhancing chondrocyte proliferation and matrix formation. Notably, in vitro, M2-CM directly suppressed the catabolic activities while enhancing the anabolic activities of interleukin-1β-stimulated mouse primary chondrocytes. M2-CM also inhibited receptor activator of nuclear factor NF-κB ligand-induced osteoclastogenesis in RAW264.7 cells. Secretome analysis of M2-CM and M0-CM revealed that 5 proteins related to anti-inflammation and/or osteochondrogenesis were enriched in M2-CM. Of these proteins, the Wnt signal antagonist, secreted frizzled-related protein 1 (sFRP1), was the most abundant and played an essential role in the shift to anabolic chondrocytes, suggesting that M2 ameliorated TMJOA partly through sFRP1. This study suggests that secretome from SHED exerted remarkable osteochondral regeneration activities in TMJOA through the induction of sFRP1-expressing tissue-repair M2 macrophages.


Conditioned Medium from Stem Cells of Human Exfoliated Deciduous Teeth Partially Alters the Expression of Inflammation-associated Molecules of Mouse Condylar Chondrocytes via Secreted Frizzled-related Protein 1


Therapeutic benefits of factors derived from stem cells from human exfoliated deciduous teeth for radiation-induced mouse xerostomia

Radiation therapy for head and neck cancers is frequently associated with adverse effects on the surrounding normal tissue. Irreversible damage to radiation-sensitive acinar cells in the salivary gland (SG) causes severe radiation-induced xerostomia (RIX). Currently, there are no effective drugs for treating RIX. We investigated the efficacy of treatment with conditioned medium derived from stem cells from human exfoliated deciduous teeth (SHED-CM) in a mouse RIX model. Intravenous administration of SHED-CM, but not fibroblast-CM (Fibro-CM), prevented radiation-induced cutaneous ulcer formation (p < 0.0001) and maintained SG function (p < 0.0001). SHED-CM treatment enhanced the expression of multiple antioxidant genes in mouse RIX and human acinar cells and strongly suppressed radiation-induced oxidative stress. The therapeutic effects of SHED-CM were abolished by the superoxide dismutase inhibitor diethyldithiocarbamate (p < 0.0001). Notably, quantitative liquid chromatography-tandem mass spectrometry shotgun proteomics of SHED-CM and Fibro-CM identified eight proteins activating the endogenous antioxidant system, which were more abundant in SHED-CM than in Fibro-CM (p < 0.0001). Neutralizing antibodies against those activators reduced antioxidant activity of SHED-CM (anti-PDGF-D; p = 0.0001, anti-HGF; p = 0.003). Our results suggest that SHED-CM may provide substantial therapeutic benefits for RIX primarily through the activation of multiple antioxidant enzyme genes in the target tissue.


Conditioned Medium From the Stem Cells of Human Exfoliated Deciduous Teeth Ameliorates Neuropathic Pain in a Partial Sciatic Nerve Ligation Model.

In neuropathic pain (NP), injury or diseases of the somatosensory system often result in highly debilitating chronic pain. Currently, there is no effective drug for the complete and definitive treatment of NP. We investigated the therapeutic potential of conditioned medium (CM) derived from stem cells from human exfoliated deciduous teeth (SHED-CM) against NP using a mouse partial sciatic nerve ligation (PSL) model. Abnormal pain sensation, such as tactile allodynia and hyperalgesia, can be caused by PSL. In the behavioral test, intravenous administration of SHED-CM greatly improved the PSL-induced hypersensitivity. We found that treatment with SHED-CM resulted in the recruitment of M2 macrophages in the injured sciatic nerve and ipsilateral L4/L5 dorsal root ganglion and suppressed microglial activation in the spinal cord. Notably, specific depletion of the anti-inflammatory M2 macrophages by mannosylated-Clodrosome markedly reduced the antinociceptive effect of SHED-CM. Intravenous administration of CM from M2 induced by SHED-CM (M2-CM) ameliorated the PSL-induced hypersensitivity. We found that M2-CM directly suppressed the expression of nociceptive receptors as well as proinflammatory mediators in Schwann cells. Taken together, our data suggest that SHED-CM ameliorates NP through the induction of the analgesic anti-inflammatory M2 macrophages. Thus, SHED-CM may be a novel therapeutic candidate for NP.


Peripherally Administered Botulinum Toxin Type A Localizes Bilaterally in Trigeminal Ganglia of Animal Model


Conditioned medium from stem cells derived from human exfoliated deciduous teeth ameliorates NASH via the GutLiver axis

Non-alcoholic steatohepatitis (NASH) occurrence has been increasing and is becoming a major cause of liver cirrhosis and liver cancer. However, effective treatments for NASH are still lacking. We examined the benefits of serum-free conditioned medium from stem cells derived from human exfoliated deciduous teeth (SHED-CM) on a murine non-alcoholic steatohepatitis (NASH) model induced by a combination of Western diet (WD) and repeated administration of low doses of carbon tetrachloride intraperitoneally, focusing on the gut-liver axis. We showed that repeated intravenous administration of SHED-CM significantly ameliorated histological liver fibrosis and inflammation in a murine NASH model. SHED-CM inhibited parenchymal cell apoptosis and reduced the activation of inflammatory macrophages. Gene expression of pro-inflammatory and pro-fibrotic mediators (such as Tnf-α, Tgf-β, and Ccl-2) in the liver was reduced in mice treated with SHED-CM. Furthermore, SHED-CM protected intestinal tight junctions and maintained intestinal barrier function, while suppressing gene expression of the receptor for endotoxin, Toll-like receptor 4, in the liver. SHED-CM promoted the recovery of Caco-2 monolayer dysfunction induced by IFN-γ and TNF-α in vitro. Our findings suggest that SHED-CM may inhibit NASH fibrosis via the gut-liver axis, in addition to its protective effect on hepatocytes and the induction of macrophages with unique anti-inflammatory phenotypes.


Low-intensity pulsed ultrasound ameliorates neuropathic pain induced by partial sciatic nerve ligation via regulating macrophage polarization


The immunoregulatory role of p21 in the development of the temporomandibular joint-osteoarthritis.

Our findings indicate that p21 in chondrocytes contributes to regulate matrix synthesis via the control ofm aggrecan and MMP-13 expression under mechanical stress. Thus, p21 might regulate the pathogenesis of osteoarthritis in the TMJ.

mice aged 8 weeks were divided into the untreated and treated groups. In the treated groups, mechanical stress was applied to the temporomandibular joint (TMJ) through forced mouth opening for 3 hr/day for 7 days. The dissected TMJs were assessed using micro-CT, histology, and immunohistochemistry.

Clinical and Experimental Dental Research


Factors Secreted from Dental Pulp Stem Cells Show Multifaceted Benefits for Treating Experimental Temporomandibular Joint Osteoarthritis

Temporomandibular joint osteoarthritis (TMJOA) is a degenerative disease characterized by progressive cartilage degeneration, abnormal bone remodeling, and chronic pain. In this study, we aimed to investigate effective therapies to reverse or suppress TMJOA progression. To this end, we performed intravenous administration of serum free conditioned media from human exfoliated deciduous teeth stem cells (SHED-CM) into a mechanical-stress induced murine TMJOA model. SHED-CM administration markedly suppressed temporal muscle inflammation, and improved bone integrity and surface smoothness of the destroyed condylar cartilage. Moreover, SHED-CM treatment decreased the number of IL-1β, iNOS, and MMP-13 expressing chondrocytes, whereas it specifically increased PCNA-positive cells in the multipotent polymorphic cell layer. Notably, the numbers of TUNEL-positive apoptotic chondrocytes in the SHED-CM treated condyles were significantly lower than in those treated with DMEM, whereas the proteoglycan positive area was restored to a level similar to that of the sham treated group, demonstrating that SHED-CM treatment regenerated the mechanical-stress injured condylar cartilage and subchondral bone. Secretome analysis revealed that SHED-CM contained multiple therapeutic factors that act in osteochondral regeneration. Our data demonstrated that SHED-CM treatment promoted the regeneration and repair of mechanical-stress induced mouse TMJOA. Our observations suggest that SHED-CM has potential to be a potent tissue-regenerating therapeutic agent for patients with severe TMJOA.


Oral self-injury associated with septicaemia in a case of Cornelia de Lange syndrome

Journal of Oral and Maxillofacial Surgery, Medicine, and Pathology


Conditioned Media from Human Dental Pulp Stem Cells Prevent Radiation-induced Skin Injury.


Monocyte chemoattractant protein-1 and secreted ectodomain of sialic acid-binding Ig-like lectin-9 enhance bone regeneration by inducing M2 macrophages


Third molar pericoronitis in neutropenia

We report the case of a 40 year-old woman who presented third molar pericoronitis in neutropenia. she had undergone extraction of the right third mandibular molar. This may have been due to an infection in the extraction socket or part of the crown. There was no inflammation seen after transplant of umbilical cord blood.


Diagnostic role of mean platelet volume in tonsillitis with and without peritonsillar abscess

To assess the diagnostic role of mean platelet volume in tonsillitis with and without peritonsillar abscess. Mean platelet volume and other laboratory data were retrospectively investigated. Mean platelet volume was significantly lower in the tonsillitis group (7.8 per cent ± 0.7 per cent) than in the control group (8.7 per cent ± 0.6 per cent; p < 0.0001), and it was significantly lower in the abscess group (7.5 per cent ± 0.6 per cent) than in the no abscess group (8.0 per cent ± 0.7 per cent; p = 0.0277). White blood cell counts and C-reactive protein levels were not significantly different between patients with an abscess and those without. The mean platelet volume cut-off values for the diagnosis of tonsillitis and peritonsillar abscess were 7.95 fl and 7.75 fl, respectively. Our results suggest that a decreased mean platelet volume is associated with the development and severity of tonsillitis. This finding provides useful diagnostic information for physicians treating patients with tonsillitis.


顎下腺に発生したびまん性大細胞型B細胞リンパ腫を含むMALTリンパ腫の1例.

顎下腺に発生したびまん性大細胞型B細胞リンパ腫を含むMALTリンパ腫の1例.

Japanese Journal of Oral & Maxillofacial Surgery


Nasal hemangiopericytoma successfully treated with a combination of rIL-2 and extranasal approaches

Hemangiopericytomas (HPCs) are uncommon vascular tumors originating from extracapillary cells called pericytes, and rarely occur in the nose or paranasal sinuses. We treated a 57-year-old man with nasal HPC who presented with nasal obstruction and hemorrhage. Nasal endoscopy showed a readily bleeding mass between the right nasal septum and inferior turbinate. Enhanced CT revealed a heterogeneous mass lesion with an enhancement effect that filled the right nasal cavity. A biopsy specimen was proved to exhibit a HPC histopathology. Recombinant interleukin-2 (rIL-2) was administered with a measurement of natural killer cell (NK cell) activity. Afterwards, wide excision with an extranasal approach was performed. The use of rIL-2 caused not only increased NK cell activity but also a reduction in the tumor size. With a combination of rIL-2 and wide excision with extranasal approaches, no local recurrence or metastasis has occurred over the last 4 years.


Efficacy of the red blood cell distribution width for predicting the prognosis of Bell palsy: a pilot study


Efficacy of the red blood cell distribution width for predicting the prognosis of Bell palsy: a pilot study.

European Archives of Oto-Rhino-Laryngology


Secreted Ectodomain of Sialic Acid-Binding Ig-Like Lectin-9 and Monocyte Chemoattractant Protein-1 Synergistically Regenerate Transected Rat Peripheral Nerves by Altering Macrophage Polarity

Peripheral nerves (PNs) exhibit remarkable self-repairing reparative activity after a simple crush or cut injury. However, the neuronal transection involving a nerve gap overwhelms their repairing activity and causes persistent paralysis. Here, we show that an implantation of the serum-free conditioned medium from stem cells from human exfoliated deciduous teeth (SHED-CM) immersed in a collagen sponge into the nerve gap formed by rat facial nerves transection restored the neurological function. In contrast, SHED-CM specifically depleted of a set of anti-inflammatory M2 macrophage inducers, monocyte chemoattractant protein-1 (MCP-1) and the secreted ectodomain of sialic acid-binding Ig-like lectin-9 (sSiglec-9) lost the ability to restore neurological function in this model. Notably, the combination of MCP-1 and sSiglec-9 induced the polarization of M2 macrophages in vitro, resulting in the expression of multiple trophic factors that enhanced proliferation, migration, and differentiation of Schwann cells, blood vessel formation, and nerve fiber extension. Furthermore, the implantation of a collagen graft containing MCP-1/sSiglec-9 into the nerve gap induced anti-inflammatory M2 macrophage polarization, generated a Schwann-cell bridge instead of fibrotic scar, induced axonal regrowth, and restored nerve function. The specific elimination of M2 macrophages by Mannosylated-Clodrosome suppressed the MCP-1/sSiglec-9-mediated neurological recovery. Taken together, our data suggest that MCP-1/sSiglec-9 regenerates PNs by inducing tissue-repairing M2 macrophages and may provide therapeutic benefits for severe peripheral nerve injuries. Stem Cells 2017;35:641-653.


Secreted ectodomain of sialic acid-binding Ig-like lectin-9 and monocyte chemoattractant protein-1 promote recovery after rat spinal cord injury by altering macrophage polarity.

Engrafted mesenchymal stem cells from human deciduous dental pulp (SHEDs) support recovery from neural insults via paracrine mechanisms that are poorly understood. Here we show that the conditioned serum-free medium (CM) from SHEDs, administered intrathecally into rat injured spinal cord during the acute postinjury period, caused remarkable functional recovery. The ability of SHED-CM to induce recovery was associated with an immunoregulatory activity that induced anti-inflammatory M2-like macrophages. Secretome analysis of the SHED-CM revealed a previously unrecognized set of inducers for anti-inflammatory M2-like macrophages: monocyte chemoattractant protein-1 (MCP-1) and the secreted ectodomain of sialic acid-binding Ig-like lectin-9 (ED-Siglec-9). Depleting MCP-1 and ED-Siglec-9 from the SHED-CM prominently reduced its ability to induce M2-like macrophages and to promote functional recovery after spinal cord injury (SCI). The combination of MCP-1 and ED-Siglec-9 synergistically promoted the M2-like differentiation of bone marrow-derived macrophages in vitro, and this effect was abolished by a selective antagonist for CC chemokine receptor 2 (CCR2) or by the genetic knock-out of CCR2. Furthermore, MCP-1 and ED-Siglec-9 administration into the injured spinal cord induced M2-like macrophages and led to a marked recovery of hindlimb locomotor function after SCI. The inhibition of this M2 induction through the inactivation of CCR2 function abolished the therapeutic effects of both SHED-CM and MCP-1/ED-Siglec-9. Macrophages activated by MCP-1 and ED-Siglec-9 extended neurite and suppressed apoptosis of primary cerebellar granule neurons against the neurotoxic effects of chondroitin sulfate proteoglycans. Our data suggest that the unique combination of MCP-1 and ED-Siglec-9 repairs the SCI through anti-inflammatory M2-like macrophage induction.


The development of new therapy facial nerve injury using the m2 macrophage induction factors


Analysis of the neuroregenerative activities of mesenchymal stem cells in functional recovery after rat spinal cord injury.

Spinal cord injury (SCI) involves concurrent, interacting pathological processes, and requires a multifaceted therapeutic strategy. Stem cell-based transplantation holds great promise as such an approach. We have reported that stem cells derived from human dental pulp have remarkable neuroregenerative activity, and that when transplanted into animal models of SCI, these cells promote functional recovery by inhibiting massive SCI-induced apoptosis, preserving neural fibers and myelin, regenerating transected axons, and replacing damaged cells by differentiating into oligodendrocytes. Here, we introduce some details of our experimental procedures, which may serve as a guide for designing experiments to evaluate the therapeutic benefits of various types of stem cells.