Luteolin Is a Potential Immunomodulating Natural Compound against Pulpal Inflammation.

Aim: The present study evaluated the therapeutic effects of luteolin in alleviating pulpitis of dental pulp- (DP-) derived microvesicles (MVs) via the inhibition of protein kinase R- (PKR-) mediated inflammation. Methodology. Proteomic analysis of immortalized human dental pulp (DP-1) cell-derived MVs was performed to identify PKR-associated molecules. The effect of luteolin on PKR phosphorylation in DP-1 cells and the expression of tumor necrosis factor-α (TNF-α) in THP-1 macrophage-like cells were validated. The effect of luteolin on cell proliferation was compared with that of chemical PKR inhibitors (C16 and 2-AP) and the unique commercially available sedative guaiacol-parachlorophenol. In the dog experimental pulpitis model, the pulps were treated with (1) saline, (2) guaiacol-parachlorophenol, and (3) luteolin. Sixteen teeth from four dogs were extracted, and the pulp tissues were analyzed using hematoxylin and eosin staining. Immunohistochemical staining was performed to analyze the expression of phosphorylated PKR (pPKR), myeloperoxidase (MPO), and CD68. Experimental endodontic-periodontal complex lesions were established in mouse molar through a silk ligature and simultaneous MV injection. MVs were prepared from DP-1 cells with or without pretreatment with 2-AP or luteolin. A three-dimensional microcomputed tomography analysis was performed on day 7 (n = 6). Periodontal bone resorption volumes were calculated for each group (nonligated-ligated), and the ratio of bone volume to tissue volume was measured. Results: Proteomic analysis identified an endogenous PKR activator, and a protein activator of interferon-induced PKR, also known as PACT, was included in MVs. Luteolin inhibited the expressions of pPKR in DP-1 cells and TNF-α in THP-1 cells with the lowest suppression of cell proliferation. In the dog model of experimental pulpitis, luteolin treatment suppressed the expression of pPKR-, MPO-, and CD68-positive cells in pulp tissues, whereas guaiacol-parachlorophenol treatment caused coagulative necrosis and disruption. In a mouse model of endodontic-periodontal complex lesions, luteolin treatment significantly decreased MV-induced alveolar bone resorption. Conclusion: Luteolin is an effective and safe compound that inhibits PKR activation in DP-derived MVs, enabling pulp preservation.

Scaffold-free bone-like 3D structure established through osteogenic differentiation from human gingiva-derived stem cells.

Introduction & objectives: Stem cell therapy for regenerative medicine has been sincerely investigated, but not still popular although some clinical trials show hopeful results. This therapy is suggested to be a representative candidate such as bone defect due to the accident, iatrogenic resection oncological tumor, congenital disease, and severe periodontitis in oral region. Recently, the Bio-3D printer "Regenova®" has been introduced as an innovative three-dimensional culture system, equipped scaffold-free bio-assembling techniques without any biomaterials. Therefore, we expected a mount of bone defect could be repaired by the structure established from this Bio-3D printer using osteogenic potential stem cells. Material & methods: The gingival tissue (1x1 mm) was removed from the distal part of the lower wisdom tooth of the patients who agreed our study. Human Gingival Mesenchymal Stem Cells (hGMSCs) were isolated from this tissue and cultured, since we confirmed the characteristics such as facile isolation and accelerated proliferation, further, strong potential of osteogenic-differentiation. Spheroids were formed using hGMSC in 96-well plates designed for low cell adhesion. The size of the spheroids was measured, and fluorescent immunostaining was employed to verify the expression of stem cell and apoptosis marker, and extracellular matrix. Following four weeks of bone differentiation, µCT imaging was performed. Calcification was confirmed by alizarin red and von Kossa staining. Fluorescent immunostaining was utilized to assess the expression of markers indicative of advanced bone differentiation. Results: We have established and confirmed the spheroids (∼600 µm in diameter) constructed from human GMSCs (hGMSCs) still maintain stem cell potentials and osteogenic differentiation abilities from the results that CD73 and not CD34 were expressed as stem cell positive and negative marker, respectively. These spheroids were pilled up like cylindal shape to the "Kenzan" platform of Bio-3D printer and cultured for 7days. The cylindal structure originated from compound spheroids were tried to differentiate into bone four weeks with osteogenic induction medium. The calcification of bio-3D printed bone-like structures was confirmed by alizarin red and Von Kossa staining. In addition, µCT analysis revealed that the HU (Hounsfield Unit) of the calcified structures was almost identical to that of trabecular bone. Immunofluorescent staining detected osteocalcin expression, a late-stage bone differentiation marker. Conclusion: For the first time, we have achieved the construction of a scaffold-free, bone-like luminal structure through the assembly of spheroids comprised of this hGMSCs. This success is sure to be close to the induction of clinical application against regenerative medicine especially for bone defect disease.

Inhibition of non-canonical NF-κB signaling suppresses periodontal inflammation and bone loss.

Periodontal disease is an infectious disease that affects many people worldwide. Disease progression destroys the alveolar bone and causes tooth loss. We have previously shown that alymphoplasia (aly/aly) mice harboring a loss-of-function mutation in the map3k14 gene, which is involved in p100 to p52 processing of the alternative NF-κB pathway, exhibited mild osteopetrosis due to decreased number of osteoclasts, suggesting the alternative NF-κB pathway as a potential drug target for the amelioration of bone disease. In the present study, wild-type (WT) and aly/aly mice were subjected to silk ligation to establish a periodontitis model. Alveolar bone resorption was suppressed in aly/aly mice by decreased numbers of osteoclasts in the alveolar bone in comparison to WT mice. Furthermore, the expression of receptor activator of NF-κB ligand (RANKL) and TNFα (cytokines involved in osteoclast induction in periligative gingival tissue) was decreased. When primary osteoblasts (POBs) and bone marrow cells (BMCs) derived from WT and aly/aly mice were prepared and co-cultured, osteoclasts were induced from WT-derived BMCs, regardless of the origin of the POBs, but hardly formed from aly/aly mouse-derived BMCs. Furthermore, the local administration of an NIK inhibitor, Cpd33, inhibited osteoclast formation and thereby inhibited alveolar bone resorption in the periodontitis model. Therefore, the NIK-mediated NF-κB alternative pathway can be a therapeutic target for periodontal disease.

Inhibition of the ATG4-LC3 pathway suppressed osteoclast maturation.

Autophagy is a non-selective action in which cells degrade parts of themselves, reusing degraded cellular components. Among autophagy-related gene (ATG) family members, ATG4 proteins play crucial roles in the microtubule-associated protein 1 light chain 3 (LC3) phosphatidylethanolamine (PE) system which is essential for autophagosome maturation. Although autophagy has been shown to be involved in osteoclastic bone resorption, the role of ATG4/LC3 in bone resorption remains unclear. When mouse bone marrow cells were treated with various concentrations of NSC185058 (NSC), a specific inhibitor of ATG4B, 1 h prior to treatment with receptor activator of NF-κB ligand (RANKL) in the presence of macrophage colony stimulating factor (M-CSF), NSC inhibited osteoclastogenesis in a dose-dependent manner. Addition of NSC in the late stages of osteoclast differentiation suppressed multinucleation and reduced the expression of markers for mature osteoclasts such as Dc-stamp, Mmp9, and Ctsk. NSC also suppressed actin ring formation and pit formation in mature osteoclasts. When a periodontitis model involving eight-week-old male mice in which the right maxillary second molar had been ligated with silk thread was injected with or without NSC, alveolar bone resorption was suppressed by a decrease in the number of osteoclasts in the NSC-treated group. These results suggest that LC3 is important for the maturation of osteoclasts and that LC3 inhibition is a new therapeutic strategy for periodontal disease.

Application of a new scan body for face-driven fixed prosthetics.

OBJECTIVE: The current method of digitally designing dental prostheses mainly focuses on intra-oral soft and hard tissues, although the harmony of the facial soft tissue and the prosthesis is crucial, especially for esthetics. Here, we introduce a new method of digitally designing dental prostheses using a new device that generates a virtual patient and incorporates facial features into the prosthetic design. MATERIALS AND METHODS: A new extra-oral scan body for facial scanning was designed and developed. A definitive edentulous maxilla implant cast with four extra-oral scan bodies (regions: maxillary left and right lateral incisors, maxillary left and right premolars) was placed in the mouth of a dental mannequin. The dental mannequin was scanned with and without the extra-oral scan bodies. For reference data, an impression of the maxilla was taken and scanned with a laboratory scanner. By superimposing each acquired data, a virtual patient was generated, and the spatial location of the abutments relative to the face was clarified. Identifying the accurate location of the abutments enabled to design face-driven dental prosthesis. RESULTS: Based on the color-coded deviation map created by the data acquired from conventional and extra-oral scan bodies, the divergence of the two data was mostly within 0.1 mm, which proves that the extra-oral scan bodies were as accurate as conventional scan bodies. Therefore, the facial scan data and the scan data of the oral cavity were successfully superimposed, which allowed to generate a virtual patient to design face-driven prosthesis. CONCLUSION: The new method is effective for designing high-quality face-driven prostheses, especially when treating a patient with a full-arch implant-fixed prosthesis.

[Functional neurosurgery for spasticity].

The basic concept of neurosurgical procedures to treat spasticity is to decrease the hyperactivity of the stretch reflex. Selective peripheral neurotomy is a method to partially resect the peripheral motor nerve. The alpha motor and Ia afferent nerves are resected, but the latter is essential owing to its lasting effect in reducing spasticity. Focal spasticity in adult patients can be effectively treated using peripheral neurotomy. Functional posterior rhizotomy, mostly used to treat paraplegic spasticity in children with cerebral palsy, involves the sectioning of posterior rootlets associated with abnormal motor responses to electrical stimulation. Intrathecal baclofen therapy is useful in treating diffuse spasticity. Baclofen inhibits the activity of alpha motor neurons both pre and post synaptically at the level of the spinal cord. A decrease in Hmax/Mmax in the H-reflex electrophysiologically represents the effectiveness of these procedures. Good clinical results can be achieved by appropriate indication depending on the clinical features of spasticity in each patient.

Cerebral venous thrombosis after ventriculoperitoneal shunting: a case report.

Ventriculoperitoneal shunting (VPS) is a simple procedure, but there are several potential complications. We describe the first reported case of cerebral venous thrombosis (CVT) after VPS. A 69-year-old man suffering from normal pressure hydrocephalus underwent left VPS. Two months later he developed CVT and cerebral venous hemorrhage in the left frontal lobe. Computed tomography (CT) and magnetic resonance imaging (MRI) revealed the thrombus formation just adjacent to the shunt tube. One possible cause is compression of the cortical vein after brain shift and/or tension of the cortical vein due to intracranial hypotension. A protein C deficiency was also detected. Surgeons should be aware that cerebral venous thrombosis can occur after VPS.

The patient had a normal magnetic resonance imaging and temporal lobe epilepsy secondary to a porencephalic cyst but showed structural lesions (hippocampal sclerosis).

Patients with a porencephalic cyst frequently develop intractable temporal lobe epilepsy (TLE). We report a surgically-treated male patient with intractable mesial TLE (mTLE) secondary to a porencephalic cyst. Although magnetic resonance imaging showed no hippocampal abnormalities, long-term video-electrocorticography revealed seizure onset discharges in the hippocampus. Temporal lobectomy brought an end to the patient's seizures. Hippocampal sclerosis was histopathologically confirmed (dual pathology). Careful evaluation of hippocampal epileptogenicity is required, and temporal lobectomy, which is less invasive than hemispherectomy, can be a treatment of choice for patients with mTLE secondary to a porencephalic cyst.

[Laparoscopy-assisted ventriculoperitoneal and lumboperitoneal shunt surgery].

Recently, laparoscopy (also referred to as minimally invasive surgery) has been used during peritoneal catheter implantation in shunt placement for hydrocephalus; however, the procedure and devices for this technique have not yet been well established. We adopted umbilical and paraumbilical laparoscopy for peritoneal catheter insertion. In this paper, we describe the technique we used and its clinical results and benefits. Ten consecutive patients with hydrocephalus who underwent laparoscopic shunt surgery (6 cases of ventriculoperitoneal shunt and 4 of lumboperitoneal shunt) were enrolled for this study. The follow-up period ranged from 21 to 434 days (mean, 263 days). After a standard cranial/spinal procedure, an approximately 5-mm incision was made in the lateral side of the umbilicus, where the abdominal catheter was introduced subcutaneously. Thereafter, we inserted a laparoscope into the peritoneal cavity via a small incision beneath or just on the umbilicus. A shunt catheter was laparoscopically inserted through a peel-off cannula and placed after taking note of the outflow of cerebrospinal fluid (CSF) from the catheter tip. In all patients, the shunt was inserted with no complications, and good patency was achieved. Laparoscopy allows implantation of the catheter into the peritoneal cavity, and the outflow of CSF can be confirmed intraoperatively. Furthermore, the abdominal surgical wounds are minimal, even for obese patients, and fascia/muscle incisions are not needed. Laparoscopy-assisted shunt surgery for hydrocephalus is effective and safe and also has cosmetic advantages.

Steroidal glycosides from the rhizomes of Ruscus hypophyllum.

Seven steroidal glycosides, along with one known glycoside, were isolated from the rhizomes of Ruscus hypophyllum (Liliaceae). Comprehensive spectroscopic analysis, including 2D NMR spectroscopy, and the results of acid hydrolysis allowed the chemical structures of the compounds to be assigned as (23S,25R)-23-hydroxyspirost-5-en-3beta-yl O-alpha-l-rhamnopyranosyl-(1-->4)-beta-d-glucopyranoside (1), 1beta-hydroxyspirosta-5,25(27)-dien-3beta-yl O-alpha-l-rhamnopyranosyl-(1-->4)-beta-d-glucopyranoside (2), (22S)-16beta,22-dihydroxycholest-5-en-3beta-yl O-alpha-l-rhamnopyranosyl-(1-->4)-beta-d-glucopyranoside (3), (22S)-16beta-[(beta-d-glucopyranosyl)oxy]-22-hydroxycholest-5-en-3beta-yl O-alpha-l-rhamnopyranosyl-(1-->4)-beta-d-glucopyranoside (4), (22S)-16beta-[(beta-d-glucopyranosyl)oxy]-22-hydroxycholest-5-en-3beta-yl beta-d-glucopyranoside (5), (22S)-16beta-[(beta-d-glucopyranosyl)oxy]-3beta,22-dihydroxycholest-5-en-1beta-yl O-alpha-l-rhamnopyranosyl-(1-->2)-(3,4-di-O-acetyl-beta-d-xylopyranoside) (6), and (22S)-16beta-[(beta-d-glucopyranosyl)oxy]-3beta,22-dihydroxycholest-5-en-1beta-yl O-alpha-l-rhamnopyranosyl-(1-->2)-O-[beta-d-xylopyranosyl-(1-->3)]-beta-d-xylopyranoside (7), respectively. This is the first isolation of a series of cholestane glycosides from a Ruscus species.

Primary cerebral angiitis containing marked xanthoma cells with massive intraparenchymal involvement--case report--.

A 27-year-old woman was referred to our hospital with mild disorientation, bilateral abducens nerve palsy, and mild left hemiparesis. Magnetic resonance (MR) imaging revealed diffuse mass lesions resembling malignant glioma in the right frontal intraparenchymal region, with enhancement of multiple meningeal and intraparenchymal nodules. Partial resection of the frontal lesion was performed. Histological examination revealed that the specimens consisted of brain tissue, with marked perivascular infiltration of histiocytes and sheets of xanthomatous cells. The diagnosis was primary cerebral angiitis containing marked xanthoma cells. Steroid therapy was administered over 1 week. MR imaging showed that the remaining lesions resolved gradually, and had disappeared 2 years after surgery. No neurological symptoms or recurrence of the tumor has been observed during the 6-year period since the operation.