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-1ß-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.

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.

Peripheral nerve injury leads to sensory ganglion hyperexcitation, which increases neurotransmitter release and neuropathic pain. Botulinum toxin type A (BoNT/A) regulates pain transmission by reducing neurotransmitter release, thereby attenuating neuropathic pain. Despite multiple studies on the use of BoNT/A for managing neuropathic pain in the orofacial region, its exact mechanism of transport remains unclear. In this study, we investigated the effects of BoNT/A in managing neuropathic pain in two different animal models and its transport mechanism in the trigeminal nerve. Intraperitoneal administration of cisplatin induced bilateral neuropathic pain in the orofacial region, reducing the head withdrawal threshold to mechanical stimulation. Unilateral infraorbital nerve constriction (IONC) also reduced the ipsilateral head withdrawal threshold to mechanical stimulation. Unilateral peripheral administration of BoNT/A to the rat whisker pad attenuated cisplatin-induced pain behavior bilaterally. Furthermore, contralateral peripheral administration of BoNT/A attenuated neuropathy-induced behavior caused by IONC. We also noted the presence of BoNT/A in the blood using the mouse bioassay. In addition, the Alexa Fluor-488-labeled C-terminal half of the heavy chain of BoNT/A (BoNT/A-Hc) was localized in the neurons of the bilateral trigeminal ganglia following its unilateral administration. These findings suggest that axonal and hematogenous transport are involved in the therapeutic effects of peripherally administered BoNT/A in the orofacial region.

Conditioned medium from stem cells derived from human exfoliated deciduous teeth ameliorates NASH via the Gut-Liver 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-ß, 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.

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

OBJECTIVE: We aimed to identify the immunoregulatory role of the cyclin-dependent kinase inhibitor p21 in the homeostasis of mandibular condylar cartilage affected by mechanical stress. MATERIALS AND METHODS: Ten C57BL/6 wild-type (WT) and ten p21-/- 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. RESULTS: Treated p21-/- condyles with mechanical stress revealed more severe subchondral bone destruction, with thinner cartilage layers and smaller proteoglycan area relative to treated WT condyles; untreated WT and p21-/- condyles had smoother surfaces. Immunohistochemistry revealed significant increases in the numbers of caspase-3, interleukin-1ß, matrix metalloprotease (MMP)-9, and MMP-13 positive cells, and few aggrecan positive cells, in treated p21-/- than in treated WT samples. Moreover, the number of TUNEL positive cells markedly increased in p21-/- condyles affected by mechanical stress. CONCLUSIONS: 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.

Effectiveness of low-intensity pulsed ultrasound on osteoarthritis of the temporomandibular joint: A review.

Loading is indispensable for the growth, development, and maintenance of joint tissues, including mandibular condylar cartilage, but excessive loading or reduced host adaptive capacity can considerably damage the temporomandibular joint (TMJ), leading to temporomandibular joint osteoarthritis (TMJ-OA). TMJ-OA, associated with other pathological conditions and aging processes, is a highly degenerative disease affecting the articular cartilage. Many treatment modalities for TMJ-OA have been developed. Traditional clinical treatment includes mainly nonsurgical options, such as occlusal splints. However, non-invasive therapy does not achieve joint tissue repair and regeneration. Growing evidence suggests that low-intensity pulsed ultrasound (LIPUS) accelerates bone fracture healing and regeneration, as well as having extraordinary effects in terms of soft tissue repair and regeneration. The latter have received much attention, and various studies have been performed to evaluate the potential role of LIPUS in tissue regeneration including that applied to articular cartilage. The present article provides an overview of the status of LIPUS stimulation used to prevent the onset and progression of TMJ-OA and enhance the tissue regeneration of mandibular condylar cartilage. The etiology and management of TMJ-OA are explained briefly, animal models of TMJ-OA are described, and the effectiveness of LIPUS on cell metabolism and tissue regeneration in the TMJ is discussed.

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

The aim of this study was to investigate the association between RDW values and the prognosis of patients with Bell palsy in an effort to find a prognostic biomarker that predicts recovery from Bell palsy. We measured RDW and evaluated facial movement in 61 patients with Bell palsy aged 50 years and less. All patients were treated with a steroid plus an antiviral agent. Seven patients underwent surgery for facial nerve decompression. During the post-treatment period, patients with a Yanagihara grading score of 36 or more were regarded as having a satisfactory recovery. Patients were divided into two groups (recovered and unrecovered) according to their response to treatment, and several parameters, including the RDW, were measured for further analysis. RDW values were significantly higher in the unrecovered group than in the recovered group (13.5 ± 1.7 vs. 12.7 ± 0.7%, p = 0.046). In the multiple logistic regression model, RDW was the only factor associated with recovery from Bell palsy (odds ratio 1.93, 95% confidence interval 1.02-4.65, p = 0.042). Our preliminary study provides the first evidence that the red cell distribution width (RDW) can predict recovery from Bell palsy in patients aged 50 years and less. Further studies are necessary to elucidate the potential pathophysiological mechanisms for our findings.

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.

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.

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.

Multifaceted neuro-regenerative activities of human dental pulp stem cells for functional recovery after spinal cord injury.

Spinal cord injury (SCI) often leads to persistent functional deficits due to the loss of neurons and glia and to limited axonal regeneration after such injury. Recently, three independent groups have reported marked recovery of hindlimb locomotor function after the transplantation of human adult dental pulp stem cells (DPSCs) and stem cells from human exfoliated deciduous teeth (SHEDs) into rats or mice with acute, sub-acute or chronic SCI. This review summarizes the primary characteristics of human dental pulp stem cells and their therapeutic benefits for treating SCI. Experimental data from multiple preclinical studies suggest that pulp stem cells may promote functional recovery after SCI through multifaceted neuro-regenerative activities.