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Hair follicles, mammalian mini-organs that grow hair, miniaturize during aging, leading to hair thinning and loss. Here we report that hair follicle stem cells (HFSCs) lose their regenerative capabilities during aging owing to the adoption of an atypical cell division program. Cell fate tracing and cell division axis analyses revealed that while HFSCs in young mice undergo typical symmetric and asymmetric cell divisions to regenerate hair follicles, upon aging or stress, they adopt an atypical ‘stress-responsive’ type of asymmetric cell division. This type of division is accompanied by the destabilization of hemidesmosomal protein COL17A1 and cell polarity protein aPKCλ and generates terminally differentiating epidermal cells instead of regenerating the hair follicle niche. With the repetition of these atypical divisions, HFSCs detach from the basal membrane causing their exhaustion, elimination and organ aging. The experimentally induced stabilization of COL17A1 rescued organ homeostasis through aPKCλ stabilization. These results demonstrate that distinct stem cell division programs may govern tissue and organ aging. The authors identify an atypical type of stem cell division that regulates hair follicle organ homeostasis and aging in mice. These ‘stress-responsive-type’ asymmetrical cell divisions cause hair follicle stem cells to detach from the basement membrane leading to their exhaustion, elimination and organ aging.

Stem cells underlie tissue homeostasis, but their dynamics during ageing—and the relevance of these dynamics to organ ageing—remain unknown. Here we report that the expression of the hemidesmosome component collagen XVII (COL17A1) by epidermal stem cells fluctuates physiologically through genomic/oxidative stress-induced proteolysis, and that the resulting differential expression of COL17A1 in individual stem cells generates a driving force for cell competition. In vivo clonal analysis in mice and in vitro 3D modelling show that clones that express high levels of COL17A1, which divide symmetrically, outcompete and eliminate adjacent stressed clones that express low levels of COL17A1, which divide asymmetrically. Stem cells with higher potential or quality are thus selected for homeostasis, but their eventual loss of COL17A1 limits their competition, thereby causing ageing. The resultant hemidesmosome fragility and stem cell delamination deplete adjacent melanocytes and fibroblasts to promote skin ageing. Conversely, the forced maintenance of COL17A1 rescues skin organ ageing, thereby indicating potential angles for anti-ageing therapeutic intervention. COL17A1-driven stem cell competition and symmetric cell divisions initially govern skin homeostasis, but the same mechanisms result in skin ageing later in life.

Some plus-stranded RNA viruses generate double-membrane vesicles (DMVs), one type of the membrane replication factories, as replication sites. Little is known about the lipid components involved in the biogenesis of these vesicles. Sphingomyelin (SM) is required for hepatitis C virus (HCV) replication, but the mechanism of SM involvement remains poorly understood. SM biosynthesis starts in the endoplasmic reticulum (ER) and gives rise to ceramide, which is transported from the ER to the Golgi by the action of ceramide transfer protein (CERT), where it can be converted to SM. In this study, inhibition of SM biosynthesis, either by using small-molecule inhibitors or by knockout (KO) of CERT, suppressed HCV replication in a genotype-independent manner. This reduction in HCV replication was rescued by exogenous SM or ectopic expression of the CERT protein, but not by ectopic expression of nonfunctional CERT mutants. Observing low numbers of DMVs in stable replicon cells treated with a SM biosynthesis inhibitor or in CERT-KO cells transfected with either HCV replicon or with constructs that drive HCV protein production in a replication-independent system indicated the significant importance of SM to DMVs. The degradation of SM of the in vitro-isolated DMVs affected their morphology and increased the vulnerability of HCV RNA and proteins to RNase and protease treatment, respectively. Poliovirus, known to induce DMVs, showed decreased replication in CERT-KO cells, while dengue virus, known to induce invaginated vesicles, did not. In conclusion, these findings indicated that SM is an essential constituent of DMVs generated by some plus-stranded RNA viruses. IMPORTANCE Previous reports assumed that sphingomyelin (SM) is essential for HCV replication, but the mechanism was unclear. In this study, we showed for the first time that SM and ceramide transfer protein (CERT), which is in the SM biosynthesis pathway, are essential for the biosynthesis of double-membrane vesicles (DMVs), the sites of viral replication. Low numbers of DMVs were observed in CERT-KO cells transfected with replicon RNA or with constructs that drive HCV protein production in a replication-independent system. HCV replication was rescued by ectopic expression of the CERT protein, but not by CERT mutants, that abolishes the binding of CERT to vesicle-associated membrane protein-associated protein (VAP) or phosphatidylinositol 4-phosphate (PI4P), indicating new roles for VAP and PI4P in HCV replication. The biosynthesis of DMVs has great importance to replication by a variety of plus-stranded RNA viruses. Understanding of this process is expected to facilitate the development of diagnosis and antivirus.

Supplemental Digital Content is available in the text., Background: The authors developed a noncontact low-frequency ultrasound device that delivers high-intensity mechanical force based on phased-array technology. It may aid wound healing because it is likely to be associated with lower risks of infection and heat-induced pain compared with conventional ultrasound methods. The authors hypothesized that the microdeformation it induces accelerates wound epithelialization. Its effects on key wound-healing processes (angiogenesis, collagen accumulation, and angiogenesis-related gene transcription) were also examined. Methods: Immediately after wounding, bilateral acute wounds in C57BL/6J mice were noncontact low-frequency ultrasound– and sham-stimulated for 1 hour/day for 3 consecutive days (10 Hz/90.6 Pa). Wound closure (epithelialization) was recorded every 2 days as the percentage change in wound area relative to baseline. Wound tissue was procured on days 2, 5, 7, and 14 (five to six per time point) and subjected to histopathology with hematoxylin and eosin and Masson trichrome staining, CD31 immunohistochemistry, and quantitative polymerase-chain reaction analysis. Results: Compared to sham-treated wounds, ultrasound/phased-array–treated wounds exhibited significantly accelerated epithelialization (65 ± 27 percent versus 30 ± 33 percent closure), angiogenesis (4.6 ± 1.7 percent versus 2.2 ± 1.0 percent CD31+ area), and collagen deposition (44 ± 14 percent versus 28 ± 13 percent collagen density) on days 5, 2, and 5, respectively (all p < 0.05). The expression of Notch ligand delta-like 1 protein (Dll1) and Notch1, which participate in angiogenesis, was transiently enhanced by treatment on days 2 and 5, respectively. Conclusions: The authors’ noncontact low-frequency ultrasound phased-array device improved the wound-healing rate. It was associated with increased early neovascularization that was followed by high levels of collagen-matrix production and epithelialization. The device may expand the mechanotherapeutic proangiogenesis field, thereby helping stimulate a revolution in infected wound care.

Osseointegrated implants have been recognized as being very reliable and having long-term predictability. However, host defense mechanisms against infection have been known to be impaired around a dental implant because of the lack of a periodontal ligament (PDL). The purpose of our experimental design was to produce cementum and PDL on the implant surface adopting cell sheet technology. To this aim we used PDL-derived cells, which contain multipotential stem cells, as the cell source and we cultured them on an implant material constituted of commercially pure titanium treated with acid etching, blasting, and a calcium phosphate (CaP) coating to improve cell attachment. Implants with adhered human PDL cell sheets were transplanted into bone defects in athymic rat femurs as a xenogeneic model. Implants with adhered canine PDL-derived cell sheets were transplanted into canine mandibular bone as an autologous model. We confirmed that PDL-derived cells cultured with osteoinductive medium had the ability to induce cementum formation. The attachment of PDL cells onto the titanium surface with three surface treatments was accelerated, compared with that onto the smooth titanium surface, at 40 min after starting incubation. Results in the rat model showed that cementum-like and PDL-like tissue was partly observed on the titanium surface with three surface treatments in combination with adherent PDL-derived cell sheets. On the other hand, osseointegration was observed on almost all areas of the smooth titanium surface that had PDL-derived cell sheets, but did not have the three surface treatments. In the canine model, histological observation indicated that formation of cementum-like and PDL-like tissue was induced on the titanium surface with surface treatments and that the PDL-like tissue was perpendicularly oriented between the titanium surface with cementum-like tissue and the bone. Results demonstrate that a periodontal-like structure was formed around a titanium implant, which is similar to the environment existing around a natural tooth. The clinical application of dental implants combined with a cell sheet technique may be feasible as an alternative implant therapy. Furthermore, application of this methodology may play an innovative role in the periodontal, prosthetic, and orthodontic fields in dentistry.

In vitro chondrogenesis of mesenchymal stem cells (MSCs) mimics in vivo chondrogenesis of MSCs. However, the size of the cartilage pellets that can be attained in vitro is limited by current methods; therefore, some modifications are required to obtain larger pellets. Petaloid pieces of recombinant peptide (petaloid RCP) have the advantage of creating spaces between cells in culture. The RCP used here is based on the alpha‐1 sequence of human collagen type I and contains 12 Arg‐Gly‐Asp motifs. We examined the effect and mechanisms of adding petaloid RCP on the in vitro chondrogenesis of human synovial MSCs by culturing 125k cells with or without 0.125 mg petaloid RCP in chondrogenic medium for 21 days. The cartilage pellets were sequentially analyzed by weight, sulfated glycosaminoglycan content, DNA retention, and histology. Petaloid RCP significantly increased the weight of the cartilage pellets: The petaloid RCP group weighed 7.7 ± 1.2 mg (n = 108), whereas the control group weighed 5.3 ± 1.6 mg. Sulfated glycosaminoglycan and DNA contents were significantly higher in the petaloid RCP group than in the control group. Light and transmission electron microscopy images showed that the petaloid RCP formed the framework of the pellet at day 1, the framework was broken by production of cartilage matrix by the synovial MSCs at day 7, and the cartilage pellet grew larger, with diffuse petaloid RCP remaining, at day 21. Therefore, petaloid RCP formed a framework for the pellet, maintained a higher cell number, and promoted in vitro cartilage formation of synovial MSCs. © 2018 The Authors. Journal of Orthopaedic Research® Published by Wiley Periodicals, Inc. J Orthop Res 37:1350–1357, 2019.

Summary: Autophagy is a lysosomal degradation pathway with important roles in physiological homeostasis and disease. However, the role of autophagy in intestinal stem cells (ISCs) is unclear. Here, we show that intrinsic autophagy in ISCs is important for ISC homeostasis. Mice lacking autophagy protein 5 (ATG5) in intestinal epithelial cells (iECs) (Villin-Cre: Atg5fl/fl, hereafter Atg5ΔIEC mice) or in all iECs except Paneth cells (Ah-Cre: Atg5fl/fl mice) had significantly fewer ISCs than did control mice and showed impaired ISC-dependent intestinal recovery after irradiation. Crypt ISCs from Atg5ΔIEC mice had significantly higher reactive oxygen species (ROS) levels than did those from control mice. A ROS-inducing reagent decreased the ISC number and impaired ISC regenerative capacity ex vivo, and treating Atg5ΔIEC mice with an antioxidant rescued their defects. Our results show that intrinsic autophagy supports ISC maintenance by reducing excessive ROS. Optimizing autophagy may lead to autophagy-based therapies for intestinal injuries. : Autophagy is a lysosomal degradation pathway with important roles in physiological homeostasis and disease. Asano et al. find that intrinsic autophagy is important for the maintenance of intestinal stem cells by reducing excessive reactive oxygen species. This stem cell maintenance is necessary to provide damage-induced intestinal regeneration. Keywords: intestinal epithelial cell (iEC), intestinal stem cell (ISC), autophagy, reactive oxygen species (ROS), regeneration, irradiation, 5-fluorouracil (5-FU)

Myopia is one of the most common visual disorders, and is characterized by a progressive axial elongation of the eye. Several methods have been tried to reduce the progression of axial elongation and myopia, but there are still no well-accepted procedures. We hypothesized that transplantation of fibroblasts on the sclera would lead to the synthesis of collagen fibrils on the sclera and reinforce it, and reduce the degree of axial elongation of eyes with form deprivation myopia. To examine this, we developed a form deprivation myopia model in albino Wistar rats and examined the effects of human fibroblasts (hFbs) transplantation on the sclera in the progression of myopia and axial elongation. We found that the form deprivation by eyelid suture induced a myopic shift and axial elongation associated with a thinner sclera and smaller-diameter collagen fibrils in Wistar rats. We also found that the transplanted hFbs synthesized type 1 collagen fibrils on the rat sclera, and these eyes with form deprivation had significantly reduced ocular elongation and myopic shift than the eyes without hFbs transplantation. Some of the synthesized collagen fibrils migrated into the sclera and had a bundle-like appearance and a stripe-like pattern, indicating they had mature characteristics. These findings suggest that the rat sclera was reinforced by the newly synthesized collagen fibrils and the axial elongation was reduced. These results can provide important information for the development of a therapy targeting myopia in humans. Copyright © 2017 John Wiley & Sons, Ltd.

The trigger for bone remodeling is bone resorption by osteoclasts. Osteoclast differentiation only occurs on the old bone, which needs to be repaired under physiological conditions. However, uncontrolled bone resorption is often observed in pro-inflammatory bone diseases, such as rheumatoid arthritis. Mature osteoclasts are multinuclear cells that differentiate from monocyte/macrophage lineage cells by cell fusion. Although Osteoclast precursors should migrate across osteoblast layer to reach bone matrix before maturation, the underlying mechanisms have not yet been elucidated in detail. We herein found that osteoclast precursors utilize two routes to migrate across osteoblast layer by confocal- and electro-microscopic observations. The osteoclast supporting activity of osteoblasts inversely correlated with osteoblast density and was positively related to the number of osteoclast precursors under the osteoblast layer. Osteoclast differentiation was induced by IL-1s, but not by PGE2 in high-density osteoblasts. Osteoblasts and osteoclast precursors expressed CX3CL1 and CX3CR1, respectively, and the expression of CX3CL1 increased in response to interleukin-1s. An anti-CX3CL1-neutralizing antibody inhibited the migration of osteoclast precursors and osteoclast differentiation. These results strongly suggest the involvement of CX3CL1 in the migration of osteoclast precursors and osteoclastogenesis, and will contribute to the development of new therapies for bone diseases. J. Cell. Physiol. 232: 1739-1745, 2017. © 2016 Wiley Periodicals, Inc.

The arrangement of immature germ cells changes regularly and periodically along the axis of the seminiferous tubule, and is used to describe the progression of spermatogenesis. This description is based primarily on the changes in the acrosome and the nuclear morphology of haploid spermatids. However, such criteria cannot be applied under pathological conditions with arrested spermatid differentiation. In such settings, the changes associated with the differentiation of premeiotic germ cells must be analyzed. Here, we found that the unique bipolar motor protein, KIF11 (kinesin-5/Eg5), which functions in spindle formation during mitosis and meiosis in oocytes and early embryos, is expressed in premeiotic germ cells (spermatogonia and spermatocytes). Thus, we aimed to investigate whether KIF11 could be used to describe the progression of incomplete spermatogenesis. Interestingly, KIF11 expression was barely observed in haploid spermatids and Sertoli cells. The KIF11 staining allowed us to evaluate the progression of meiotic processes, by providing the time axis of spindle formation in both normal and spermatogenesis-arrested mutant mice. Accordingly, KIF11 has the potential to serve as an excellent marker to describe spermatogenesis, even in the absence of spermatid development.

Energy stress, such as ischemia, induces mitochondrial damage and death in the heart. Degradation of damaged mitochondria by mitophagy is essential for the maintenance of healthy mitochondria and survival. Here, we show that mitophagy during myocardial ischemia was mediated predominantly through autophagy characterized by Rab9-associated autophagosomes, rather than the well-characterized form of autophagy that is dependent on the autophagy-related 7 (Atg) conjugation system and LC3. This form of mitophagy played an essential role in protecting the heart against ischemia and was mediated by a protein complex consisting of unc-51 like kinase 1 (Ulk1), Rab9, receptor-interacting serine/thronine protein kinase 1 (Rip1), and dynamin-related protein 1 (Drp1). This complex allowed the recruitment of trans-Golgi membranes associated with Rab9 to damaged mitochondria through S179 phosphorylation of Rab9 by Ulk1 and S616 phosphorylation of Drp1 by Rip1. Knockin of Rab9 (S179A) abolished mitophagy and exacerbated the injury in response to myocardial ischemia, without affecting conventional autophagy. Mitophagy mediated through the Ulk1/Rab9/Rip1/Drp1 pathway protected the heart against ischemia by maintaining healthy mitochondria.

There is a strong association between periodontal disease (PD) and atherosclerosis. However, it remains unknown whether PD is also involved in myocardial damage. We hypothesized that infection with periodontal pathogens could cause an adverse outcome after myocardial infarction (MI). C57BL/6J mice were inoculated with Porphyromonas gingivalis (P.g.), a major periodontal pathogen, or injected with phosphate-buffered saline (PBS) into a subcutaneously-implanted steelcoil chamber before and after coronary artery ligation. A significant increase in mortality, due to cardiac rupture, was observed in the P.g.-inoculated MI mice. Ultrastructural examinations revealed that P.g. invaded the ischemic myocardium of the P.g.-inoculated MI mice. The expression of p18 Bax, an active form of pro-apoptotic Bax protein, markedly increased in the P.g.-inoculated MI hearts. In vitro experiments demonstrated that gingipain, a protease uniquely secreted from P.g., cleaved wild type Bax at Arg34, as evidenced by the observation that the cleavage of Bax by gingipain was completely abolished by the Arg34Ala mutation in Bax. Treatment with immunoglobulin Y against gingipain significantly decreased the mortality of the P.g.-inoculated MI mice caused by cardiac rupture. Furthermore, inoculation of P.g. also resulted in an increase of MMP-9 activity in the post-MI myocardium by enhancing oxidative stress, possibly through impairing the selective autophagy-mediated clearance of damaged mitochondria. In conclusion, infection with P.g. during MI plays a detrimental role in the healing process of the infarcted myocardium by invasion of P.g. into the myocardium, thereby promoting apoptosis and the MMP-9 activity of the myocardium, which, in turn, causes cardiac rupture.

BACKGROUND: Recently decellularized nerves with various methods are reported as highly functional nerve grafts for the treatment of nerve defects. OBJECTIVE: To evaluate the efficacy of decellularized allogeneic nerve, compared with oriented chitosan mesh tube, and an autologous nerve. METHODS: Sciatic nerves harvested from Sprague-Dawley (SD) rats were decellularized in combination with Sodium dodecyl sulfate and Triton X-100. A graft into the sciatic nerve in Wistar rats was performed with the decellularized SD rat sciatic nerves or oriented chitosan nonwoven nanofiber mesh tubes (15 mm in length, N = 5 in each group). A portion of sciatic nerve of Wistar rat was cut, reversed and re-sutured in-situ as a control. Nerve functional and histological evaluations were performed 25 weeks postoperatively. RESULTS: It was revealed that functional, electrophysiological and histological recoveries in the decellularized nerve group match those in the autograft group. Recovery of sensory function and nerve maturation in the decellularized nerve group were superior to those in the chitosan mesh tube group. CONCLUSIONS: Nerve regeneration in the decellularized nerves could match that in the autografts and is somehow superior to artificial chitosan mesh tube. Detergents wash of SDS and Triton X-100 could obtain highly functional nerve grafts from allografts.

Mechanoforces experienced by an organ are translated into biological information for cellular sensing and response. In mammals, the tendon connective tissue experiences and resists physical forces, with tendon-specific mesenchymal cells called tenocytes orchestrating extracellular matrix (ECM) turnover. We show that Mohawk (Mkx), a tendon-specific transcription factor, is essential in mechanoresponsive tenogenesis through regulation of its downstream ECM genes such as type I collagens and proteoglycans such as fibromodulin both in vivo and in vitro Wild-type (WT) mice demonstrated an increase in collagen fiber diameter and density in response to physical treadmill exercise, whereas in Mkx(-/-) mice, tendons failed to respond to the same mechanical stimulation. Furthermore, functional screening of the Mkx promoter region identified several upstream transcription factors that regulate Mkx In particular, general transcription factor II-I repeat domain-containing protein 1 (Gtf2ird1) that is expressed in the cytoplasm of unstressed tenocytes translocated into the nucleus upon mechanical stretching to activate the Mkx promoter through chromatin regulation. Here, we demonstrate that Gtf2ird1 is essential for Mkx transcription, while also linking mechanical forces to Mkx-mediated tendon homeostasis and regeneration.

Background and Objective Mid-infrared erbium: yttrium-aluminum-garnet (Er:YAG) and erbium, chromium: yttrium-scandium-gallium-garnet (Er,Cr:YSGG) lasers (2.94- and 2.78-μm, respectively) are utilized for effective dental hard tissue treatment because of their high absorption in water, hydroxide ion, or both. Recently, a mid-infrared tunable, nanosecond pulsed, all-solid-state chromium-doped: cadmium-selenide (Cr:CdSe) laser system was developed, which enables laser oscillation in the broad spectral range around 2.9 μm. The purpose of this study was to evaluate the ablation of dental hard tissue by the nanosecond pulsed Cr:CdSe laser at a wavelength range of 2.76–3.00 μm. Study Design/Materials and Methods Enamel, dentin, and cementum tissue were irradiated at a spot or line at a fluence of 0–11.20 J/cm2/pulse (energy output: 0–2.00 mJ/pulse) with a repetition rate of 10 Hz and beam diameter of ∼150 μm on the target (pulse width ∼250 ns). After irradiation, morphological changes, ablation threshold, depth, and efficiency, and thickness of the structurally and thermally affected layer of irradiated surfaces were analyzed using stereomicroscopy, scanning electron microscopy (SEM), and light microscopy of non-decalcified histological sections. Results The nanosecond pulsed irradiation without water spray effectively ablated dental hard tissue with no visible thermal damage such as carbonization. The SEM analysis revealed characteristic micro-irregularities without major melting and cracks in the lased tissue. The ablation threshold of dentin was the lowest at 2.76 μm and the highest at 3.00 μm. The histological analysis revealed minimal thermal and structural changes ∼20 μm wide on the irradiated dentin surfaces with no significant differences between wavelengths. The efficiency of dentin ablation gradually increased from 3.00 to 2.76 μm, at which point the highest ablation efficiency was observed. Conclusion The nanosecond pulsed Cr:CdSe laser demonstrated an effective ablation ability of hard dental tissues, which was remarkably wavelength-dependent on dentin at the spectral range of 2.76–3.00 μm. These results demonstrate the potential feasibility of the use of pulsed Cr:CdSe laser as a novel laser system for dental treatment. Lasers Surg. Med. © 2016 Wiley Periodicals, Inc.

Purpose The aim of this study was to compare the effect of ligature-induced periimplantitis on dental implants with and without hydroxyapatite (HA) coat. Methods Thirty-two dental implants (3.3 mm wide, 13 mm long) with 4 surface treatments (8 implant/group) (M: machined, SA: sandblasted acid etched, S: sputter HA coat and P: plasma-sprayed HA coat) were inserted into canine mandibles. After 12 weeks, oral hygiene procedures were stopped and silk ligatures were placed around the implant abutments to allow plaque accumulation for the following 16 weeks. Implants with the surrounding tissues were retrieved and prepared for histological examination. Bone-to-implant contact (BIC) and implant surfaces were examined using scanning electron microscopy and energy dispersive x-ray spectroscopy. Results Histological observation revealed marginal bone loss and large inflammatory cell infiltrates in the periimplant soft tissue. Sputter HA implants showed the largest BIC (98.1%) and machined implant showed the smallest values (70.4%). After 28 weeks, thin sputter HA coat was almost completely dissolved, whereas plasma-sprayed HA coat showed complete thickness preservation. Conclusion Thin sputter HA-coated implants showed more bone implant contact and less marginal bone loss than thick HA-coated implants under periimplantitis condition.

Though the Er:YAG laser (ErL) has been used in periodontal therapy, the irradiated tip damage has not been studied in detail. In this study, the change in the energy output, surface morphology, and temperature of quartz tips was evaluated following contact irradiation. Soft tissue, calculus on extracted human teeth, and porcine bone were irradiated by ErL for 60 min at 14.2 or 28.3 J/cm(2)/pulse and 20 Hz with or without water spray. The energy output ratio declined the most in the calculus group, followed by the bone and soft tissue groups with and/or without water spray. Carbon contamination was detected in all groups, and contamination by P, Ca, and/or other inorganic elements was observed in the calculus and bone groups. The rate of energy output reduction and the degree of surface alteration/contamination is variously influenced by the targeting tissue, temperature elevation of the tip and water spray.

Background Synovial mesenchymal stem cells (MSCs) are an attractive cell source for cartilage and meniscus regeneration. Synovial tissue can be histologically classified into three regions; surface, stromal and perivascular region, but the localization of synovial MSCs has not been fully investigated. We identified markers specific for each region, and compared properties of MSCs derived from each region in the synovium. Methods The intensity of immunostaining with 19 antibodies was examined for surface, stromal, and perivascular regions of human synovium from six osteoarthritis patients. Specific markers were identified and synovial cells derived from each region were sorted. Proliferation, surface marker expression, chondrogenesis, calcification and adipogenesis potentials were compared in synovial MSCs derived from the three regions. Results We selected CD55+ CD271− for synovial cells in the surface region, CD55− CD271− in the stromal region, and CD55− CD271+ in the perivascular region. The ratio of the sorted cells to non-hematopoietic lineage cells was 5% in the surface region, 70% in the stromal region and 15% in the perivascular region. Synovial cells in the perivascular fraction had the greatest proliferation potential. After expansion, surface marker expression profiles and adipogenesis potentials were similar but chondrogenic and calcification potentials were higher in synovial MSCs derived from the perivascular region than in those derived from the surface and stromal regions. Conclusions We identified specific markers to isolate synovial cells from the surface, stromal, and perivascular regions of the synovium. Synovial MSCs in the perivascular region had the highest proliferative and chondrogenic potentials among the three regions.

A taxonomic study was performed on 15 bacterial isolates from the caeca of gnotobiotic mice that had been fed with thermophile-fermented compost. The 15 isolates were thermophilic, Gram-stain-positive, facultatively anaerobic, endospore-forming bacteria, and were most closely related to Bacillus thermoamylovorans CNCM I-1378T. The 16S rRNA gene sequence of strain N-11T, selected as representative of this new group, showed a similarity of 99.4 % with Bacillus thermoamylovorans CNCM I-1378T, 94.7 % with Bacillus thermolactis R-6488T, and 94.4 % with Bacillus kokeshiiformis MO-04T. The isolates were then classified into two distinct groups based on a (GTG)5-fingerprint analysis. Two isolates, N-11T and N-21, were the representatives of these two groups, respectively.` The N-11T and N-21 isolates showed 66–71 % DNA–DNA relatedness with one other, but had less than 37 % DNA–DNA relatedness with B. thermoamylovorans LMG 18084T. The other 13 isolates showed DNA–DNA relatedness values above 74 % with the N-11T isolate. All 15 isolates grew at 25–60 °C (optimum 50 °C), pH 6–8 (optimum pH 7) and were capable of growing on a medium containing 6 % (w/v) NaCl (optimum 0.5 %). The 15 isolates could be distinguished from B. thermoamylovorans LMG 18084T because they showed Tween 80 hydrolysis activity and did not produce acid from melibiose. The major fatty acids were anteiso-C15 : 0, C16 : 0, iso-C15 : 0, iso-C14 : 0 and iso-C16 : 0. The major polar lipids were diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol and several unidentified phospholipids. The diagnostic diamino acid in the cell-wall peptidoglycan was meso-diaminopimelic acid. The menaquinone was MK-7. The DNA G+C content was 37.9 mol%. Based on the phenotypic properties, the 15 strains represent a novel species of the genus Bacillus, for which the name Bacillus hisashii sp. nov. is proposed. The type strain is N-11T ( = NRBC 110226T = LMG 28201T).

The aim of interfacial nanoleakage evaluation is to gain a better understanding of degradation of the adhesive–dentin interface. The acid–base resistant zone (ABRZ) is recognized at the bonded interface under the hybrid layer (HL) in self-etch adhesive systems after an acid–base challenge. The purpose of this study was to evaluate nanoleakage in HL and ABRZ using three self-etch adhesives; Clearfil SE Bond (SEB), Clearfil SE One (SEO), and G-Bond Plus (GBP). One of the three adhesives was applied on the ground dentin surface and light cured. The specimens were longitudinally divided into two halves. One half remained as the control group. The others were immersed in ammoniacal silver nitrate solution, followed by photo developing solution under fluorescent light. Following this, the specimens were subjected to acid–base challenges with an artificial demineralization solution (pH4.5) and sodium hypochlorite, and prepared in accordance with common procedures for transmission electron microscopy (TEM) examination. The TEM images revealed silver depositions in HL and ABRZ due to nanoleakage in all the adhesives; however, the extent of nanoleakage was material dependent. Funnel-shaped erosion beneath the ABRZ was observed only in the all-in-one adhesive systems; SEO and GBP, but not in the two-step self-etch adhesive system; SEB.

Salivary phosphoproteins are essential in tooth mineral regulation but are often overlooked in vitro. This study aimed to evaluate the effect of casein, as a salivary phosphoprotein homologue, on the deposition and growth of hydroxyapatite (HA) on tooth surfaces. Hydroxyapatite growth was quantified using seeded crystal systems. Artificial saliva (AS) containing HA powder and 0, 10, 20, 50, or 100 μg ml(-1) of casein, or 100 μg ml(-1) of dephosphorylated casein (Dcasein), was incubated for 0-8 h at 37°C, pH 7.2. Calcium concentrations were measured using atomic absorption spectroscopy (AAS). Surface precipitation of HA on bovine enamel and dentine blocks, incubated in similar conditions for 7 d, was examined using field emission scanning electron microscopy (FE-SEM) and transmission electron microscopy (TEM) with selected area electron diffraction (SAED). Casein adsorption was assessed using modified Lowry assays and zeta-potential measurements. The AAS results revealed a concentration-dependent inhibition of calcium consumption. Hydroxyapatite precipitation occurred when no casein was present, whereas precipitation of HA was apparently completely inhibited in casein-containing groups. Adsorption data demonstrated increasingly negative zeta-potential with increased casein concentration and an affinity constant similar to proline-rich proteins with Langmuir modelling. Casein inhibited the deposition and growth of HA primarily through the binding of esterized phosphate to HA active sites, indicating its potential as a mineral-regulating salivary phosphoprotein homologue in vitro.

Background: The brain vascular system arises from the perineural vascular plexus (PNVP) which sprouts radially into the neuroepithelium and subsequently branches off laterally to form a secondary plexus in the subventricular zone (SVZ), the subventricular vascular plexus (SVP). The process of SVP formation remains to be fully elucidated. We investigated the role of Foxc1 in early stage vascular formation in the ventral telencephalon. Results: The Foxc1 loss of function mutant mouse, Foxc1ch/ch, showed enlarged telencephalon and hemorrhaging in the ventral telencephalon by embryonic day 11.0. The mutant demonstrated blood vessel dilation and aggregation of endothelial cells in the SVZ after the invasion of endothelial cells through the radial path, which lead to failure of SVP formation. During this early stage of vascular development, Foxc1 was expressed in endothelial cells and pericytes, as well as in cranial mesenchyme surrounding the neural tube. Correspondingly, abnormal deposition pattern of basement membrane proteins around the vessels and increased strong Vegfr2 staining dots were found in the aggregation sites. Conclusions: These observations reveal an essential role for Foxc1 in the early stage of vascular formation in the telencephalon. Developmental Dynamics 244:703–711, 2015. © 2015 Wiley Periodicals, Inc.

The aim of this study is to elucidate the structure of the resin-dentin interface formed by photochemical dentin treatment using an argon fluoride (ArF) excimer laser.The ArF excimer laser processes material by photochemical reaction without generating heat, while also providing surface conditioning that enhances material adhesion. In the case of bonding between resin and dentin, we demonstrated in a previous study that laser etching using an ArF excimer laser produced bonding strength comparable to that of the traditional bonding process; however, conditions of the bonding interface have not been fully investigated.A dentin surface was irradiated in air with an ArF excimer laser followed by bonding treatment. Cross sections were observed under light microscope, transmission electron microscope (TEM), and scanning electron microscope, then analyzed using an energy dispersive X-ray spectroscope (EDS): EDS line profiles of the elements C, O, Si, Cl, P, and Ca at the resin-dentin interface were obtained.The density of C in resin decreased as it approached the interface, reaching its lowest level within the dentin at a depth of 2 μm from the resin-dentin interface on EDS. There was no hybrid layer observed at the interface on TEM. Therefore, it was suggested that the resin monomer infiltrated into the microspaces produced on the dentin surface by laser abrasion.The monomer infiltration without hybrid layer is thought to be the adhesion mechanism after laser etching. Therefore, the photochemical processes at the bonding interface achieved using the ArF excimer laser has great potential to be developed into a new bonding system in dentistry.

The purpose of this study was to evaluate the role of 10-methacryloyloxydecyl dihydrogen phosphate (MDP) contained in the bonding resin of a two-step self-etch adhesive system. An experimental adhesive (M0) containing MDP only in the primer, but not in the bonding resin was prepared. Clearfil SE Bond (MM) and M0 were compared in terms of microtensile bond strength to dentin, ultimate tensile strength of the bonding resin, and dentin-resin bonding interface morphology under SEM and TEM. The immediate µTBS values of MM significantly decreased after thermal cycles while M0 were stable even after 10,000 cycles. In the SEM observations, formation of erosion was observed beneath the acid-base resistant zone only in M0. The results suggested that MDP in the bonding resin of the two-step self-etching system; 1) improved the immediate bond strength, but caused reduction in long-term bond durability; 2) offered the advantages of acid-base resistance at the ABRZ forefront area.

The disturbance of cellular attachment to dentin by sodium hypochlorite (NaOCl) may hamper pulp tissue regeneration. The aims of this study were to examine the recovering effect of EDTA on the attachment/differentiation of stemlike cells and to address the mechanisms of EDTA-induced recovery under the hypothesis that attachment to the exposed dentin matrix and the subsequent activation of integrin/phosphatidylinositol 3-kinase (PI3K) signaling play a crucial role.Mouse dental papilla (MDP) cells were cultured on bovine dentin disks treated with NaOCl (0%, 1.5%, or 6%) followed by EDTA (0%, 3%, or 17%). Cell attachment was evaluated by cell density, viability, and scanning and transmission electron microscopy. Odonto-/osteoblastic gene expression in attached MDP cells was analyzed with or without a pan-PI3K inhibitor (LY294002) using real-time polymerase chain reaction.NaOCl treatment (1.5%, 10 minutes) significantly diminished attached MDP cells (P .00001), but EDTA treatment (3% and 17%, ≥10 minutes) of NaOCl-pretreated dentin induced a significant increase in attached cells (P .05). Ultrastructurally, MDP cells on EDTA-treated dentin showed attachment to exposed collagen fibers. MDP cells cultured on EDTA-treated disks (with or without 1.5% NaOCl pretreatment) showed significant up-regulation of alkaline phosphatase, dentin matrix protein 1, and dentin sialophosphoprotein messenger RNAs (P .05). Alkaline phosphatase expression was down-regulated by LY294002 (P .05).Under the present experimental conditions, 10 minutes of EDTA treatment was sufficient to recover attachment/differentiation of MDP cells on 1.5% NaOCl-pretreated dentin. EDTA-induced exposure of collagen fibers and subsequent activation of integrin/PI3K signaling may contribute, at least partly, to the recovery.

Background Recently, the occurrence of peri-implantitis has been increasing. However, a suitable method to debride the contaminated surface of titanium implants has not been established. The aim of this study was to investigate the morphologic changes of the microstructured fixture surface after erbium laser irradiation, and to clarify the effects of the erbium lasers when used to remove calcified deposits from implant fixture surfaces. Methods In experiment 1, sandblasted, large grit, acid etched surface implants were treated with Er:YAG laser or Er,Cr:YSGG laser at 30 to 60 mJ/pulse and 20 Hz with water spray. In experiments 2 and 3, the effects of erbium lasers used to remove calcified deposits (artificially prepared deposits on virgin implants and natural calculus on failed implants) were investigated and compared with mechanical debridement using either a titanium curette or cotton pellets. After the various debridement methods, all specimens were analyzed by stereomicroscopy (SM), scanning electron microscopy (SEM), and energy dispersive X-ray spectroscopy (EDS). Results Stereomicroscopy and SEM showed that erbium lasers with optimal irradiation parameters did not influence titanium microstructures. Compared with mechanical debridement, erbium lasers were more capable of removing calcified deposits on the microstructured surface without surface alteration using a noncontact sweeping irradiation at 40 mJ/pulse (ED 14.2 J/cm2 /pulse) and 20 Hz with water spray. Conclusion These results indicate that Er:YAG and Er,Cr:YSGG lasers are more advantageous in removing calcified deposits on the microstructured surface of titanium implants without inducing damage, compared to mechanical therapy by cotton pellet or titanium curette.

We investigated the biological effects of Er:YAG laser (2940-nm; DELight, HOYA ConBio, Fremont, California) irradiation at fluences of 3.6, 4.2, 4.9, 6.3, 8.1 or 9.7 J cm-2 at 20 or 30 Hz for 20 or 30 seconds on primary human gingival fibroblasts (HGFs). Irradiation at 6.3 J cm-2 promoted maximal cell proliferation, determined by WST-8 assay and crystal violet staining, but was accompanied by lactate dehydrogenase release, on day 3 post-irradiation. Elevation of ATP level, Ki67 staining, and cyclin-A2 mRNA expression confirmed that Er:YAG affected the cell cycle and increased the number of proliferating cells. Transmission electron microscopy showed alterations of mitochondria and ribosomal endoplasmic reticulum (ER) at 3 hours post-irradiation at 6.3 J cm-2 , and the changes subsided after 24 hours, suggesting transient cellular injury. Microarray analysis revealed up-regulation of 21 genes involved in heat-related biological responses and ER-associated degradation. The mRNA expression of heat shock protein 70 family was increased, as validated by Real-time PCR. Surface temperature measurement confirmed that 6.3 J cm-2 generated heat (40.9°C post-irradiation). Treatment with 40°C-warmed medium increased proliferation. Laser-induced proliferation was suppressed by inhibition of thermosensory transient receptor potential channels. Thus, despite causing transient cellular damage, Er:YAG laser irradiation at 6.3 J cm-2 strongly potentiated HGF proliferation via photo-thermal stress, suggesting potential wound-healing benefit.

Objectives Smear layer deproteinizing was proved to reduce the organic phase of smear layer covered on dentin surface. It was shown to eliminate hybridized smear layer and nanoleakage expression in resin–dentin bonding interface of two-step self-etch adhesive. This study aimed to investigate those effects on various one-step self-etch adhesives. Methods Four different one-step self-etch adhesives were used in this study; SE One (SE), Scotchbond™ Universal (SU), BeautiBond Multi (BB), and Bond Force (BF). Flat human dentin surfaces with standardized smear layer were prepared. Smear layer deproteinizing was carried out by the application of 50 ppm hypochlorous acid (HOCl) on dentin surface for 15 s followed by Accel® (p-toluenesulfinic acid salt) for 5 s prior to adhesive application. No surface pretreatment was used as control. Microtensile bond strength (μTBS) and nanoleakage under TEM observation were investigated. The data were analyzed by two-way ANOVA and Tukey’s post-hoc test and t-test at the significant level of 0.05. Results Smear layer deproteinizing significantly improved μTBS of SE, SU, and BB (p Significance Smear layer deproteinizing by HOCl and Accel® application could enhance the quality of dentin for bonding to one-step self-etch adhesives, resulting in the improving μTBS, eliminating hybridized smear layer and preventing reticular nanoleakage formation in resin–dentin bonding interface.

Synovial mesenchymal stem cells (MSCs) are an attractive cell source for cartilage and meniscus regeneration. Use of primary MSCs is the preferable because these cells are safer than cells passaged several times in terms of probability of chromosome abnormalities. The effect of hypoxia on the proliferation of MSCs is controversial and remains unknown in primary synovial MSCs. Primary synovial MSCs were cultured at normoxia or hypoxia, and colony number, cell number, surface epitopes, mitochondria activity, TEM finding, and chondrogenic potential were analyzed. To investigate the effect of hypoxia on attachment of synovial MSCs, cells were cultured at hypoxia for the first 3 days, then cultured at normoxia. To investigate the effect of hypoxia on proliferation, cells were also cultured at hypoxia for the last 11 days. Hypoxia increased colony number and cell number per dish in primary synovial MSCs. Hypoxia did not affect cell number per colony, surface epitopes, mitochondria activity, TEM finding or chondrogenic potential. Hypoxia for the first 3 days did not alter colony number per dish or cell number per dish, while hypoxia for the last 11 days increased. Hypoxia enhanced proliferation through increase of colony formation rate with chondrogenic potential in primary synovial MSCs.

This study aimed to investigate deproteinizing effect of sodium-hypochlorite (NaOCl) and mild acidic hypochlorous-acid (HOCl) pretreatment on smear layer-covered dentine and to evaluate their effects on morphological characteristics of resin-dentine interface with self-etch adhesive.Human coronal-dentine discs with standardized smear layer were pretreated with 6% NaOCl or 50ppm HOCl for 15s or 30s. Their deproteinizing effects at the treated smear layer-covered dentine surfaces were determined by the measurement of amide:phosphate ratio using ATR-FTIR analysis. In addition, using TEM, micromorphological alterations of hybridized complex and nanoleakage expression were evaluated at the interface of a self-etch adhesive (Clearfil SE Bond) to the pretreated dentine surface with or without subsequent application of a reducing agent (p-Toluenesulfinic acid salt; Accel(®)).Both pretreatments of NaOCl and HOCl significantly reduced the amide:phosphate ratio as compared with the no-pretreated group (p0.05), coincident with the elimination of the hybridized smear layer on their bonded interfaces. Nanoleakage within the hybrid layer was found in the no-pretreated and NaOCl-pretreated groups, whereas the subsequent reducing agent application changed the reticular nanoleakage to spotted type. HOCl-pretreated groups showed less nanoleakage expression in a spotted pattern, regardless of reducing agent application.NaOCl and HOCl solutions could remove the organic component on the smear layer-covered dentine, which could eliminate the hybridized smear layer created by self-etch adhesive, leading to the reduction of nanoleakage expression within hybrid layer.Smear layer deproteinizing could modify dentine surface, giving an appropriate substrate for bonding to self-etch adhesive system.

Accordion nerve conduit of poly(lactide-co-glycolide-co-e-caprolactone) with perforations was developed by excimer laser processing. We evaluated its in vivo function for nerve repairing and discussed the influence of pore size and density. It was found that perforations help inner nerve regeneration remarkably, which effect is unrelated to pore size or density, and is not parallel with revascularization increment. Inducing of permeability only to allow substance exchange but not vessel ingrowth could facilitate nerve regeneration too. Perforating micropores with the size of 100 μm and the density of 25/cm provides permeability and vessel ingrowth both, therefore promotes the axon extension the best, larger, and more pores do not advance axon regeneration more. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 102B: 674–680, 2014.