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This study assesses the impact of defoliation applied to three developmental stages across three cropping seasons from 2021 to 2023 on growth, yield and forage quality in maize. The experimental design included three treatments: defoliation of three expanded leaves at the 3rd–4th leaf stage (DF1), the 5th–6th expanded leaves by leaf punch (DF2) and expanding leaves with the DF2 treatment (DF3) at the 6th–7th leaf stages, compared with no defoliation (control). Over three years, the most significant decrease in dry matter (DM) yield occurred in DF1 during spring sowing, while in summer sowing, the largest reduction was in DF3, both of which were correlated with changes in the number of grains per ear. The DM yields at harvest were positively correlated with plant leaf areas at the silking stage. The digestibility of forage in in vitro DM decreased concomitantly with an increase in acid detergent fiber content, indicating a decrease in forage quality. Given the frequent severe damage observed in summer sown maize and the detrimental effects of early growth stage leaf feeding on quality and quantity of spring sown maize, the application of registered insecticides is advised to reduce pest damage to maize crops.

Background: Eosinophilic otitis media (EOM) is a refractory condition associated with eosinophilic chronic rhinosinusitis and bronchial asthma. EOM is characterized by type-2 inflammation and is refractory to various treatments. We investigated the efficacy of dupilumab, interleukin-4 receptor alpha antagonist, for patients with EOM complicated by eosinophilic chronic rhinosinusitis (ECRS). Methods: Between April 2017 and April 2022, we treated 124 patients with dupilumab for refractory CRS or bronchial asthma. Of these, 14 had EOM concurrently, and 10 of them who had been treated for >6 months were included in our study. We retrospectively evaluated the efficacy of dupilumab by the amount of systemic corticosteroid used, the frequency of exacerbations, severity score of EOM, computed tomography (CT) score of temporal bones, and pure tone audiometry. We also enrolled 8 EOM patients without dupilumab treatment as a control group. Results: Dupilumab significantly improved the amount of systemic corticosteroid used and the frequency of exacerbation and compared with before dupilumab was used (p = 0.01 and

Novel functional biomaterials are expected to bring about breakthroughs in developing immunotherapy and regenerative medicine through their application as drug delivery systems and scaffolds. Nanogels are defined as nanoparticles with a particle size of 100 nm or less and as having a gel structure. Nanogels have a three-dimensional network structure of cross-linked polymer chains, which have a high water content, a volume phase transition much faster than that of a macrogel, and a quick response to external stimuli. As it is possible to transmit substances according to the three-dimensional mesh size of the gel, a major feature is that relatively large substances, such as proteins and nucleic acids, can be taken into the gel. Furthermore, by organizing nanogels as a building block, they can be applied as a scaffold material for tissue regeneration. This review provides a brief overview of the current developments in nanogels in general, especially drug delivery, therapeutic applications, and tissue engineering. In particular, polysaccharide-based nanogels are interesting because they have excellent complexation properties and are highly biocompatible.

Polymethyl methacrylate (PMMA) bone cements are used as “glue” between orthopedic/orthodontic implants and bone, since they can be modeled and easily applied by the surgeon, while being chemically and biologically stable for many years after surgery. In this research, Y2O3 powder was added to commercial PMMA bone cement to produce a composite resin, which was then characterized and tested in vitro to evaluate the cell proliferation and the quality of osteoblastic bone formed in vitro on the composite. Biological assays showed an increase in cell proliferation on the Y2O3-PMMA composite as compared to the pristine sample. Alizarin Red staining (ARS) showed the amount of bone formed on a composite PMMA resin was about 30% higher than that on the pristine PMMA bone cement reference. The quality of bone tissue was evaluated using Raman spectroscopy, showing the bone tissue formed on the composite had a better degree of mineralization and a higher maturity as compared to the tissue grown on the control sample. These preliminary results suggest that Y2O3 plays a biologically active role in bone growth and Y2O3-composites are affordable, superior candidates as bone cement materials.

Introduction: Prevention of tooth loss contributes to an extended life expectancy, namely longevity. Aging-related oral hypofunction, including tooth loss, markedly increases the risks of functional disorder and mortality. Dysbiosis of the oral microbiome has recently been associated with various diseases, such as liver cirrhosis, pancreatic cancer, colorectal cancer, and inflammatory bowel disease. Therefore, the relationship between the oral microbiome and systemic health has been attracting increasing attention. In the present study, we examined oral function and the oral microbiome in the elderly in a world-leading longevity area. Materials and Methods: An oral examination, chewing ability/tongue-lip motor function/saliva tests, and a metagenomic analysis with a 16S rRNA gene-targeting next-generation sequencer were conducted on 78 subjects aged ≥80 years. Twenty-six healthy individuals aged between 20 and 39 years were also investigated as controls. The data obtained were statistically analyzed. The protocol of the present study was approved by the Ethics Review Board of our university (ERB-C-885). Results: Chewing ability, tongue–lip motor function, and saliva volume were normal in elderly subjects with a current tooth number ≥20, but were significantly lower in those with a current tooth number

Raman spectroscopy was applied to study the structural differences between herpes simplex virus Type I (HSV-1) and Epstein–Barr virus (EBV). Raman spectra were first collected with statistical validity on clusters of the respective virions and analyzed according to principal component analysis (PCA). Then, average spectra were computed and a machine-learning approach applied to deconvolute them into sub-band components in order to perform comparative analyses. The Raman results revealed marked structural differences between the two viral strains, which could mainly be traced back to the massive presence of carbohydrates in the glycoproteins of EBV virions. Clear differences could also be recorded for selected tyrosine and tryptophan Raman bands sensitive to pH at the virion/environment interface. According to the observed spectral differences, Raman signatures of known biomolecules were interpreted to link structural differences with the viral functions of the two strains. The present study confirms the unique ability of Raman spectroscopy for answering structural questions at the molecular level in virology and, despite the structural complexity of viral structures, its capacity to readily and reliably differentiate between different virus types and strains.

The aim of this study was to elucidate the chemistry of cellular degeneration in human neuroblastoma cells upon exposure to outer-membrane vesicles (OMVs) produced by Porphyromonas gingivalis (Pg) oral bacteria by monitoring their metabolomic evolution using in situ Raman spectroscopy. Pg-OMVs are a key factor in Alzheimer’s disease (AD) pathogenesis, as they act as efficient vectors for the delivery of toxins promoting neuronal damage. However, the chemical mechanisms underlying the direct impact of Pg-OMVs on cell metabolites at the molecular scale still remain conspicuously unclear. A widely used in vitro model employing neuroblastoma SH-SY5Y cells (a sub-line of the SK-N-SH cell line) was spectroscopically analyzed in situ before and 6 h after Pg-OMV contamination. Concurrently, Raman characterizations were also performed on isolated Pg-OMVs, which included phosphorylated dihydroceramide (PDHC) lipids and lipopolysaccharide (LPS), the latter in turn being contaminated with a highly pathogenic class of cysteine proteases, a key factor in neuronal cell degradation. Raman characterizations located lipopolysaccharide fingerprints in the vesicle structure and unveiled so far unproved aspects of the chemistry behind protein degradation induced by Pg-OMV contamination of SH-SY5Y cells. The observed alterations of cells’ Raman profiles were then discussed in view of key factors including the formation of amyloid β (Aβ) plaques and hyperphosphorylated Tau neurofibrillary tangles, and the formation of cholesterol agglomerates that exacerbate AD pathologies.

Hydrolytic reactions taking place at the surface of a silicon nitride (Si3N4) bioceramic were found to induce instantaneous inactivation of Human herpesvirus 1 (HHV-1, also known as Herpes simplex virus 1 or HSV-1). Si3N4 is a non-oxide ceramic compound with strong antibacterial and antiviral properties that has been proven safe for human cells. HSV-1 is a double-stranded DNA virus that infects a variety of host tissues through a lytic and latent cycle. Real-time reverse transcription (RT)-polymerase chain reaction (PCR) tests of HSV-1 DNA after instantaneous contact with Si3N4 showed that ammonia and its nitrogen radical byproducts, produced upon Si3N4 hydrolysis, directly reacted with viral proteins and fragmented the virus DNA, irreversibly damaging its structure. A comparison carried out upon testing HSV-1 against ZrO2 particles under identical experimental conditions showed a significantly weaker (but not null) antiviral effect, which was attributed to oxygen radical influence. The results of this study extend the effectiveness of Si3N4’s antiviral properties beyond their previously proven efficacy against a large variety of single-stranded enveloped and non-enveloped RNA viruses. Possible applications include the development of antiviral creams or gels and oral rinses to exploit an extremely efficient, localized, and instantaneous viral reduction by means of a safe and more effective alternative to conventional antiviral creams. Upon incorporating a minor fraction of micrometric Si3N4 particles into polymeric matrices, antiherpetic devices could be fabricated, which would effectively impede viral reactivation and enable high local effectiveness for extended periods of time.

High-precision poly(L-lactic acid) (PLLA) scaffold fabricated by melt electrowriting (MEW) as a bone substitute was investigated, however, the inherent fragility and the low bioactivity of the scaffold impede its clinical application. In this context, a biomimetic reinforced composite scaffold combining the MEW PLLA lattice with gelatin/genipin/bioglass hydrogel is firstly proposed. Incorporating with 1 wt% bioglass, the composite scaffold shows excellent interconnectivity, high storage modulus (121.3 kPa, 1.3 times of gelatin/genipin) and improved storage modulus (422.3 kPa). Moreover, this reinforced scaffold displays elevated in vitro osteoinduction/conduction capability, high amount of bone tissue formation and a prime Ca/P ratio of 1.69 (as compared with natural bone with Ca/P ratio of 1.67). The mechanisms of the enhanced mechanical properties and the variation of bone formation ability of various scaffolds are elucidated by a potential model. This study gives insights into some potential innovative strategies for using MEW PLLA scaffold to achieve unprecedented results in bone regeneration.

The multidrug-resistant Candida auris often defies treatments and presently represents a worldwide public health threat. Currently, the ergosterol-targeting Amphotericin B (AmB) and the DNA/RNA-synthesis inhibitor 5-flucytosine (5-FC) are the two main drugs available for first-line defense against life-threatening Candida auris infections. However, important aspects of their mechanisms of action require further clarification, especially regarding metabolic reactions of yeast cells. Here, we applied Raman spectroscopy empowered with specifically tailored machine-learning algorithms to monitor and to image in situ the susceptibility of two Candida auris clades to different antifungal drugs (LSEM 0643 or JCM15448T, belonging to the East Asian Clade II; and, LSEM 3673 belonging to the South African Clade III). Raman characterizations provided new details on the mechanisms of action against Candida auris Clades II and III, while also unfolding differences in their metabolic reactions to different drugs. AmB treatment induced biofilm formation in both clades, but the formed biofilms showed different structures: a dense and continuous biofilm structure in Clade II, and an extra-cellular matrix with a “fluffy” and discontinuous structure in Clade III. Treatment with 5-FC caused no biofilm formation but yeast-to-hyphal or pseudo-hyphal morphogenesis in both clades. Clade III showed a superior capacity in reducing membrane permeability to the drug through chemically tailoring chitin structure with a high degree of acetylation and fatty acids networks with significantly elongated chains. This study shows the suitability of the in situ Raman method in characterizing susceptibility and stress response of different C. auris clades to antifungal drugs, thus opening a path to identifying novel clinical solutions counteracting the spread of these alarming pathogens.

This study probed in vitro the mechanisms of competition/coexistence between Streptococcus sanguinis (known for being correlated with health in the oral cavity) and Streptococcus mutans (responsible for aciduric oral environment and formation of caries) by means of quantitative Raman spectroscopy and imaging. In situ Raman assessments of live bacterial culture/coculture focusing on biofilm exopolysaccharides supported the hypothesis that both species engaged in antagonistic interactions. Experiments of simultaneous colonization always resulted in coexistence, but they also revealed fundamental alterations of the biofilm with respect to their water-insoluble glucan structure. Raman spectra (collected at fixed time but different bacterial ratios) showed clear changes in chemical bonds in glucans, which pointed to an action by Streptococcus sanguinis to discontinue the impermeability of the biofilm constructed by Streptococcus mutans. The concurrent effects of glycosidic bond cleavage in water-insoluble α − 1,3–glucan and oxidation at various sites in glucans’ molecular chains supported the hypothesis that secretion of oxygen radicals was the main “chemical weapon” used by Streptococcus sanguinis in coculture.

Several species of egg parasitoids (Hymenoptera: Mymaridae and Trichogrammatidae) of the leafhopper pest of grapevines in Japan, Arboridia (Arboridia) apicalis (Nawa) (Hemiptera, Cicadellidae), were reared and identified for the first time. Using a combination of genetic and morphological evidence, Anagrus (Anagrus) arboridiae Triapitsyn & Adachi-Hagimori, sp. nov. (Mymaridae) is described and illustrated from Honshu Island (Shimane Prefecture) and Kyushu Island (Miyazaki Prefecture). It is shown to be different from Anagrus (Anagrus) japonicus Sahad and A. flaviapex Chiappini & Lin, to which it is most similar; the latter species was originally described from China and is newly recorded here from Okinawa Island, Japan. Mitochondrial and nuclear ribosomal DNA sequence data provide clear evidence for the separation of A. arboridiae from A. flaviapex, A. japonicus, and some other members of the Anagrus (Anagrus) atomus (L.) species group. Two other species of Anagrus Haliday, A. (Anagrus) avalae Soyka and A. atomus, are also identified in Japan from eggs of the leafhoppers Edwardsiana ishidae (Matsumura) and Eurhadina ? betularia Anufriev, respectively. An updated key to females of the Japanese species of Anagrus is given. Oligosita pallida Kryger (a new record for Japan), Oligosita sp., and an Aphelinoidea (Aphelinoidea) sp. (Trichogrammatidae) were the other, although much less abundant, apparent egg parasitoids of A. apicalis in Shimane Prefecture, mainly in non-organic vineyards.

Abstract The multiple mutations of the severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) virus have created variants with structural differences in both their spike and nucleocapsid proteins. While the functional relevance of these mutations is under continuous scrutiny, current findings have documented their detrimental impact in terms of affinity with host receptors, antibody resistance, and diagnostic sensitivity. Raman spectra collected on two British variant sub‐types found in Japan (QK002 and QHN001) are compared with that of the original Japanese isolate (JPN/TY/WK‐521), and found bold vibrational differences. These included: i) fractions of sulfur‐containing amino acid rotamers, ii) hydrophobic interactions of tyrosine phenol ring, iii) apparent fractions of RNA purines and pyrimidines, and iv) protein secondary structures. Building upon molecular scale results and their statistical validations, the authors propose to represent virus variants with a barcode specially tailored on Raman spectrum. Raman spectroscopy enables fast identification of virus variants, while the Raman barcode facilitates electronic recordkeeping and translates molecular characteristics into information rapidly accessible by users.

The thermally sensitive polymer, poly (l-lactic acid) PLLA, is first printed to a high-resolution object using an advanced 3D printing technology, called melt electrowriting (MEW). The MEW processing conditions are systematically investigated to manufacture PLLA scaffolds with adjustable filament diameter and pore size for the application of bone tissue engineering. To this end, the relationships among the rheological behavior of the molten polymer, the electro-hydrodynamics of the jet, and the processing parameters (applied voltage, collection speed, and flow rate) are illuminated. The scaffold with relatively high deposition accuracy and crystallinity of PLLA is achieved when the printing speed was set slightly above the critical translation speed. The MEW PLLA scaffold with filament diameter of 40 μm and pore size of 200 μm is evaluated with KUSA-A1 cells to prove its in vitro potential. This is a milestone work for MEW since it supplies one of the best strategies to design and manufacture an essential biobased polymer for tissue engineering.

Invasive fungal infections caused by yeasts of the genus Candida carry high morbidity and cause systemic infections with high mortality rate in both immunocompetent and immunosuppressed patients. Resistance rates against antifungal drugs vary among Candida species, the most concerning specie being Candida auris, which exhibits resistance to all major classes of available antifungal drugs. The presently available identification methods for Candida species face a severe trade-off between testing speed and accuracy. Here, we propose and validate a machine-learning approach adapted to Raman spectroscopy as a rapid, precise, and labor-efficient method of clinical microbiology for C. auris identification and drug efficacy assessments. This paper demonstrates that the combination of Raman spectroscopy and machine learning analyses can provide an insightful and flexible mycology diagnostic tool, easily applicable on-site in the clinical environment.

Human dermal fibroblasts (HDFs) were converted into osteoblasts using a ALK inhibitor II (inhibitor of transforming growth factor-β signal) on freeze-dried nanogel-cross-linked porous (FD-NanoClip) polysaccharide sheets or fibers. Then, the ability of these directly converted osteoblasts (dOBs) to produce calcified substrates and the expression of osteoblast genes were analyzed in comparison with osteoblasts converted by exactly the same procedure but seeded onto a conventional atelocollagen scaffold. dOBs exposed to FD-NanoClip in both sheet and fiber morphologies produced a significantly higher concentration of calcium deposits as compared to a control cell sample (i.e., unconverted fibroblasts), while there was no statistically significant difference in calcification level between dOBs exposed to atelocollagen sheets and the control group. The observed differences in osteogenic behaviors were interpreted according to Raman spectroscopic analyses comparing different polysaccharide scaffolds and Fourier transform infrared spectroscopy analyses of dOB cultures. This study substantiates a possible new path to repair large bone defects through a simplified transplantation procedure using FD-NanoClip sheets with better osteogenic outputs as compared to the existing atelocollagen scaffolding material.

The microstructural and molecular-scale variations induced by laser irradiation treatment on human teeth enamel in comparison with synthetic hydroxyapatite (HAp) were examined through Raman microprobe spectroscopy as a function of irradiation power. The results demonstrated that laser irradiation could modify stoichiometry, microstructure, and the population of crystallographic defects, as well as the hardness of the materials. These modifications showed strong dependences on both laser power and initial nonstoichiometric structure (defective content of HPO4), because of the occurrence of distinct reactions and structural reconstruction. The reported observations can redirect future trends in tooth whitening by laser treatment and the production of HAp coatings because of the important role of stoichiometric defects.

This study targets on-site/real-time taxonomic identification and metabolic profiling of seven different Candida auris clades/subclades by means of Raman spectroscopy and imaging. Representative Raman spectra from different Candida auris samples were systematically deconvoluted by means of a customized machine-learning algorithm linked to a Raman database in order to decode structural differences at the molecular scale. Raman analyses of metabolites revealed clear differences in cell walls and membrane structure among clades/subclades. Such differences are key in maintaining the integrity and physical strength of the cell walls in the dynamic response to external stress and drugs. It was found that Candida cells use the glucan structure of the extracellular matrix, the degree of α-chitin crystallinity, and the concentration of hydrogen bonds between its antiparallel chains to tailor cell walls’ flexibility. Besides being an effective ploy in survivorship by providing stiff shields in the α–1,3–glucan polymorph, the α–1,3–glycosidic linkages are also water-insoluble, thus forming a rigid and hydrophobic scaffold surrounded by a matrix of pliable and hydrated β–glucans. Raman analysis revealed a variety of strategies by different clades to balance stiffness, hydrophobicity, and impermeability in their cell walls. The selected strategies lead to differences in resistance toward specific environmental stresses of cationic/osmotic, oxidative, and nitrosative origins. A statistical validation based on principal component analysis was found only partially capable of distinguishing among Raman spectra of clades and subclades. Raman barcoding based on an algorithm converting spectrally deconvoluted Raman sub-bands into barcodes allowed for circumventing any speciation deficiency. Empowered by barcoding bioinformatics, Raman analyses, which are fast and require no sample preparation, allow on-site speciation and real-time selection of appropriate treatments.

Alumina (Al2O3) ceramic implants do not stimulate osteoblasts in vivo. Surface alterations targeted at changing the chemistry or topography have been proposed to enhance the bioactivity of alumina. This surface modification is intended to improve oxide bioceramic’s ability to integrate with the biological environment and, in particular, to rapidly osteointegrate. In this study, the surface of zirconia-toughened alumina (ZTA) was functionalized using two methods: (i) Surface laser-patterning and successive filling of patterned wells with powder mixtures of bioglass and Si3N4; and, (ii) Si3N4 coating by pulse-laser sintering. Functionalized ZTA surfaces were characterized with vibrational spectroscopy, biological testing, and laser microscopy. Both enhancements resulted in osteoblast activation, a property that is relevant to osteosynthesis.

Fairyfly (Hymenoptera, Mymaridae) egg parasitoids of the tea green leafhopper Empoasca (Matsumurasca) onukii Matsuda (Hemiptera, Cicadellidae), an economically important pest in Asia of the tea plant, Camellia sinensis, were identified from specimens reared in Japan. Using a combination of genetic and morphological evidence, Anagrus (Anagrus) rugmanjonesi Triapitsyn & Adachi-Hagimori, sp. n., is described and illustrated. It is shown to be different from the most similar A. turpanicus Triapitsyn & Hu, an egg parasitoid of a leafhopper pest of cultivated grapes which is known from Xinjiang Uyghur Autonomous Region in China. Mitochondrial and nuclear ribosomal DNA sequence data provide clear evidence for the separation of A. rugmanjonesi from A. turpanicus and other members of the Anagrus incarnatus Haliday species complex. A key to females of the Japanese species of Anagrus Haliday is given. Two other species of Mymaridae, Arescon enocki (Subba Rao & Kaur) and Stethynium empoascae Subba Rao, are also identified, albeit the latter one only tentatively. Both latter taxa are newly recorded from Japan, and E. onukii represents their new host association.

This study presents a set of vibrational characterizations on a nanogel-cross-linked porous freeze-dried gel (NanoCliP-FD gel) scaffold for tissue engineering and regenerative therapy. This scaffold is designed for the in vitro culture of high-quality cartilage tissue to be then transplanted in vivo to enable recovery from congenital malformations in the maxillofacial area or crippling jaw disease. The three-dimensional scaffold for in-plate culture is designed with interface chemistry capable of stimulating cartilage formation and maintaining its structure through counteracting the dedifferentiation of mesenchymal stem cells (MSCs) during the formation of cartilage tissue. The developed interface chemistry enabled high efficiency in both growth rate and tissue quality, thus satisfying the requirements of large volumes, high matrix quality, and superior mechanical properties needed in cartilage transplants. We characterized the cartilage tissue in vitro grown on a NanoCliP-FD gel scaffold by human periodontal ligament-derived stem cells (a type of MSC) with cartilage grown by the same cells and under the same conditions on a conventional (porous) atelocollagen scaffold. The cartilage tissues produced by the MSCs on different scaffolds were comparatively evaluated by immunohistochemical and spectroscopic analyses. Cartilage differentiation occurred at a higher rate when MSCs were cultured on the NanoCliP-FD gel scaffold compared to the atelocollagen scaffold, and produced a tissue richer in cartilage matrix. In situ spectroscopic analyses revealed the cell/scaffold interactive mechanisms by which the NanoCliP-FD gel scaffold stimulated such increased efficiency in cartilage matrix formation. In addition to demonstrating the high potential of human periodontal ligament-derived stem cell cultures on NanoCliP-FD gel scaffolds in regenerative cartilage therapy, the present study also highlights the novelty of Raman spectroscopy as a non-destructive method for the concurrent evaluation of matrix quality and cell metabolic response. In situ Raman analyses on living cells unveiled for the first time the underlying physiological mechanisms behind such improved chondrocyte performance.

Oral candidiasis, a common opportunistic infection of the oral cavity, is mainly caused by the following four Candida species (in decreasing incidence rate): Candida albicans, Candida glabrata, Candida tropicalis, and Candida krusei. This study offers in-depth Raman spectroscopy analyses of these species and proposes procedures for an accurate and rapid identification of oral yeast species. We first obtained average spectra for different Candida species and systematically analyzed them in order to decode structural differences among species at the molecular scale. Then, we searched for a statistical validation through a chemometric method based on principal component analysis (PCA). This method was found only partially capable to mechanistically distinguish among Candida species. We thus proposed a new Raman barcoding approach based on an algorithm that converts spectrally deconvoluted Raman sub-bands into barcodes. Barcode-assisted Raman analyses could enable on-site identification in nearly real-time, thus implementing preventive oral control, enabling prompt selection of the most effective drug, and increasing the probability to interrupt disease transmission.

In vitro and in vivo studies have recently shown that silicon nitride (Si3N4) is an osseoconductive and potential osseoinductive biomaterial. Even though the amount of bone mineral produced by Si3N4 was inferior when compared to commercial Bioglass®, it strongly stimulated the production of extracellular matrix and collagen tissues. Consequently, this study was designed to determine if appropriate mixtures of Bioglass® and Si3N4 might improve the ratio of mineral apatite to its biological matrix. Commercial 45S5 Bioglass® powder was mixed with 5 and 10 mol% Si3N4 powder, followed by melting, cooling, crushing, and comminution. The composite powders had modified morphologies and chemical structures including a reduced average particle size, a reduction of oxygen in the SiO backbone structure, and trapped CO2 gas which was converted into calcium carbonate. When exposed to human osteosarcoma cells within an osteogenic medium, the 5 mol% Si3N4 composite powder resulted in higher amounts of hydroxyapatite while the 10 mol% Si3N4 powder provided in a more balanced combination of collagen and mineral fractions closer to the natural composition of native human bone. Keywords: Bioglass, Si3N4, Osteosarcoma, Nitrogen, Spectroscopy, Osseointegration

The bone resorption inhibitor bisphosphonate (BP) is used to prevent fractures in patients with osteoporosis and bone metastases caused by cancer. However, BP induces apoptosis of osteoclasts and excessively suppresses bone turnover, so that side effects such as jawbone necrosis have become a problem. In the super-aging society that Japan is facing, it is expected that jawbone necrosis (Medication-related osteonecrosis of the jaw: MRONJ) will increase as the number of osteoporosis patients increases. There are many unclear points about the pathophysiology of jawbone necrosis, and there have been attempts to clarify it. Most of the research on osteoclasts so far has comprised destructive and invasive analyses, such as TRAP staining and PCR by culturing osteoclasts on a plastic plate, which is the original physiological function of osteoclasts. “Bone resorption” cannot be analyzed in real time. In this study, Raman spectroscopy is used to show the state of bone resorption of osteoclasts cultured on ivory sections or octacalcium phosphate plates noninvasively and without the need for colorimetric assays. This makes it possible to clarify the effect of BP on osteoclast metabolism in an environment closer to that of a living body. If this method is established, then we aim to elucidate the pathophysiology of bone pathologies and medical treatments that directly affect osteoclasts, such as medication-related osteonecrosis, and establish a diagnostic method.

Zirconia is the most common ceramic used in dental implants. Even if it possesses both mechanical strength and esthetics, its intrinsic bio-inertness often results in a lack of biological integration. On the other hand, Si3N4 has been proved to be bio-active and to be able to easily osteointegrate. In this work, a Si3N4 powder-based laser-cladding process has been developed in order to improve the biological response to biomedical zirconia. The process resulted in the formation of a composite coating with Si3N4 particles dispersed in nano-crystalline/amorphous silicon. Microscopic observation showed that the layer is adherent to the substrate. The application of the laser cladding treatment resulted in an increase of the roughness, further enhancing the probability of interaction with biological tissues and consequently the bioactivity. Testing with human osteosarcoma cell lines resulted in the formation of bone tissue with high collagen maturity, high carbonate to phosphate ratios and good tissue mineralization, but lower cell proliferation when compared to stoichiometric Si3N4. The bone tissue quality parameters, as measured by Raman spectroscopy, resulted to be comparable to healthy human bone tissue and suggest that laser-cladded Si3N4 treatments might be able to improve the stability of zirconia implants.

Silicon nitride (Si3N4) can facilitate bone formation; hence, it is used as a biomaterial in orthopedics. Nevertheless, its usability for dentistry is unexplored. The aim of the present study was to investigate the effect of Si3N4 granules for the proliferation and odontogenic differentiation of rat dental pulp cells (rDPCs). Four different types of Si3N4 granules were prepared, which underwent different treatments to form pristine as-synthesized Si3N4, chemically treated Si3N4, thermally treated Si3N4, and Si3N4 sintered with 3 wt.% yttrium oxide (Y2O3). rDPCs were cultured on or around the Si3N4 granular beds. Compared with the other three types of Si3N4 granules, the sintered Si3N4 granules significantly promoted cellular attachment, upregulated the expression of odontogenic marker genes (Dentin Matrix Acidic Phosphoprotein 1 and Dentin Sialophosphoprotein) in the early phase, and enhanced the formation of mineralization nodules. Furthermore, the water contact angle of sintered Si3N4 was also greatly increased to 40°. These results suggest that the sintering process for Si3N4 with Y2O3 positively altered the surface properties of pristine as-synthesized Si3N4 granules, thereby facilitating the odontogenic differentiation of rDPCs. Thus, the introduction of a sintering treatment for Si3N4 granules is likely to facilitate their use in the clinical application of dentistry.

Cellulose acetate (CA) is a synthetic compound that is derived from the acetylation of cellulose. CA is well known as it has been used for many commercial products such as textiles, plastic films, and cigarette filters. In this research, antibacterial CA composites were produced by addition of aluminum nitride (AlN) at different weight percentage, from 0 wt. % to 20 wt. %. The surface characterization was performed using laser microscope, Raman and FTIR spectroscopy. The mechanical and thermal properties of the composite were analyzed. Although the mechanical strength tended to decrease as the concentration of AlN increased and needed to be optimized, the melting temperature (Tm) and glass transition temperature (Tg) showed a shift toward higher values as the AlN concentration increased leading to an improvement in thermal properties. AlN additions in weight percentages >10 wt. % led to appreciable antibacterial properties against S. epidermidis and E. coli bacteria. Antibacterial CA/AlN composites with higher thermal stability have potential applications as alternative materials for plastic packaging in the food industry.

The present work investigated the effect of Polylactic acid (PLA) fibers produced by centrifugal spinning with incorporated BaTiO3 particles to improve their bacteriostatic behavior. The PLA matrix and three composites, presenting three different amounts of fillers, were subjected to UV/O3 treatment monitoring the possible modifications that occurred over time. The morphological and physical properties of the surfaces were characterized by different microscopic techniques, contact angle, and surface potential measurements. Subsequently, the samples were tested in vitro with human dermal fibroblasts (HDF) to verify the cytotoxicity of the substrates. No significant differences between the PLA matrix and composites emerged; the high hydrophobicity of the fibers, derived by the polymer structure, represented an obstacle limiting the fibroblast attachment. Samples underwent bacterial exposure (Staphylococcus epidermidis) for 12 and 24 h. Increasing the concentration of BT, the number of living bacteria and their distribution decreased in comparison with the PLA matrix suggesting an effect of the inorganic filler, which generates a neutralization effect leading to reactive oxygen species (ROS) generation and subsequently to bacterial damages. These results suggest that the barium titanate (BT) fillers clearly improve the antibacterial properties of PLA fibers after aging tests made before bacterial exposure, representing a potential candidate in the creation of composites for medical applications.

The surfaces of silicon nitride (β-Si3N4) and zirconia toughened alumina (ZTA) were patterned using a high-energy laser source, which operated at a wavelength of 1064 nm. The patterning procedure yielded a series regular, cylindrical cavities 500 and 300 μm in diameter and depth, respectively. These cavities were subsequently filled with bioglass mixed with different fractions of Si3N4 powder (0, 5, and 10 mol.%) to obtain bioactive functionalized bioceramic surfaces. The laser-patterned samples were first characterized using several spectroscopic techniques before and after functionalization, and then tested in vitro with respect to their osteoconductivity using a human osteosarcoma cell line (SaOS-2). After in vitro testing, fluorescence microscopy was used to address the biological response and to estimate osteopontin and osteocalcin protein contents and distributions. The presence of bioglass greatly enhanced the biological response of both ceramic surfaces, but mainly induced production of inorganic apatite. On the other hand, the addition of minor fraction of Si3N4 into the bioglass-filled holes greatly enhanced bio-mineralization and stimulated the SaOS-2 cells to produce higher amounts of bone extracellular matrix (collagen and proteins), thus enhancing the osteopontin to osteocalcin ratio. It was also observed that the presence of a fraction of Si3N4 in the powder mixture filling the holes bestowed more uniform cell colonization on the otherwise bioinert ZTA surface.

Tooth loss impairs mastication, deglutition and esthetics and affects systemic health through nutritional deficiency, weight loss, muscle weakness, delayed wound healing, and bone fragility. Approximately 90% of tooth loss is due to dental caries and periodontal disease. Accordingly, early treatment of dental caries is essential to maintaining quality of life. To date, the clinical diagnosis of dental caries has been based on each dentist’s subjective assessment, but this visual method lacks objectivity. To improve diagnostic ability, highly sensitive quantitative methods have been developed for the diagnosis and prevention of dental caries and are gradually becoming a mandatory item in modern dentistry. High-resolution Raman spectroscopy is a suitable tool for recognizing the subtle structural changes that occur in dental enamel in already developed or, more importantly, incipient dental caries. Raman analysis could soon emerge as a breakthrough in dentistry because of its high diagnostic sensitivity. In this study, we build upon our previous findings in a new analysis of dental caries using Raman spectroscopy imaging and discuss the possibility of using Raman photonic imaging in support of objective diagnostics in dentistry. Our findings support the Raman method of caries detection in comparison with other conventional or new approaches.

The transplantation of engineered three-dimensional (3D) bone graft substitutes is a viable approach to the regeneration of severe bone defects. For large bone defects, an appropriate 3D scaffold may be necessary to support and stimulate bone regeneration, even when a sufficient number of cells and cell cytokines are available. In this study, we evaluated the in vivo performance of a nanogel tectonic 3D scaffold specifically developed for bone tissue engineering, referred to as nanogel cross-linked porous-freeze-dry (NanoCliP-FD) gel. Samples were characterized by a combination of micro-computed tomography scanning, Raman spectroscopy, histological analyses, and synchrotron radiation–based Fourier transform infrared spectroscopy. NanoCliP-FD gel is a modified version of a previously developed nanogel cross-linked porous (NanoCliP) gel and was designed to achieve highly improved functionality in bone mineralization. Spectroscopic imaging of the bone tissue grown in vivo upon application of NanoCliP-FD gel enables an evaluation of bone quality and can be employed to judge the feasibility of NanoCliP-FD gel scaffolding as a therapeutic modality for bone diseases associated with large bone defects.

Functional coatings are commonly applied to biomaterials in order to improve their properties. In this work, polyethylene was coated with a silicon nitride (Si3N4) powder using a pulsed laser source in a nitrogen gas atmosphere. Several analytical techniques were used to characterize the functionalized surface of the polymer, including Raman spectroscopy, laser microscopy, scanning electron microscopy (SEM), and energy dispersive X-ray spectroscopy (EDS). Antibacterial properties were tested in vitro against Staphylococcus epidermidis. The Si3N4 coating sensibly reduced the amount of living bacteria when compared to the uncoated polymer. Osteoconductivity was also tested in vitro using SaOS-2 osteosarcoma cells. The presence of Si3N4 coating resulted in an increased amount of hydroxyapatite. Coating of polyethylene with silicon nitride may lead to improved performance of indwelling orthopaedic or less invasive medical devices.

This study investigated the efficiency of chitosan/polyethylene oxide (PEO)-based nanofibers with incorporated bioactive glass particles as a coating for titanium alloy, in order to improve the bacteriostatic behavior and, concurrently, promote the production of mineralized tissue. Nanofibers with and without bioglass powder were fabricated by electrospinning technique and characterized using several microscopic and spectroscopic techniques in order to study their morphological and physiochemical properties. Subsequently, the substrates were tested in vitro against Staphylococcus epidermidis and SaOS-2 human osteosarcoma cell line. After in vitro testing, viability and CFU counting assays combined with fluorescence microscopy showed a clear decrease in bacterial growth on all substrates with increasing time. However, this trend was stronger for substrates coated with nanofibers. Formation of mineralized matrix upon exposure to osteoblasts was confirmed by means of SEM/EDX and the content/distribution of osteocalcin and osteopontin estimated by fluorescence microscopy. Incorporation of bioglass promoted biomineralization and stimulated osteoblasts to produce a higher amount of bone extracellular matrix. The present results suggest that a chitosan/PEO/bioactive glass nanofiber composite applied as coating on titanium alloys could concurrently improve antibacterial and osteoconductive properties and could be a potential candidate for dental and orthopedic applications.

The availability of osteoinductive biomaterials has encouraged new therapies in bone regeneration and has potentially triggered paradigmatic shifts in the development of new implants in orthopedics and dentistry. Among several available synthetic biomaterials, bioceramics have gained attention for their ability to induce mesenchymal cell differentiation and successive bone formation when implanted in the human body. However, there is currently a lack of understanding regarding the fundamental biochemical mechanisms by which these materials can induce bone formation. Phenomenological studies of retrievals have clarified the final effect of bone formation, but have left the chemical interactions at the cell−material interface uncharted. Accordingly, the knowledge of the intrinsic material properties relevant for osteoblastogenesis and osteoinduction remains incomplete. Here, we systematically monitored in vitro the chemistry of mesenchymal cell metabolism and the ionic exchanges during osteoblastogenesis on selected substrates through conventional biological assays as well as via in situ and ex situ spectroscopic techniques. Accordingly, the chemical behavior of different bioceramic substrates during their interactions with mesenchymal cells could be unfolded and compared with that of biomedical titanium alloy. Our goal was to clarify the cascade of chemical equations behind the biological processes that govern osteoblastogenic effects on different biomaterial substrates.

Genome-wide gene-expression data from DNA-microarray technology and molecular-network data from computational text-mining have led to a paradigm shift in biological research. However, interpretation of the huge amount of data is a bottleneck. We have developed an informatics system, which we refer to as bioSpace Explorer, that can extract pathways and molecules of interest from genome-wide data and show the mutual relationships among these pathways and molecules. Differentiation of 3T3-L1 cells into adipocytes and the action of a peroxisome proliferator-activated receptor gamma (PPARγ) agonist or α-linolenic acid on this process was analyzed with bioSpace Explorer. The results suggested a biological basis for adipocyte differentiation and a strategy to enhance lipid oxidation in adipocytes. Clustered changes of molecules were apparent in the insulin, Wnt, and PPARγ signaling pathways and in the lipogenesis, lipid oxidation, and lipid transport pathways during cell differentiation. A PPARγ agonist enhanced lipid oxidation in adipocytes and α-linolenic acid gave similar results to the PPARγ agonist. An analysis of sex hormone and thyroid hormone, in addition to PPARγ signaling, suggested that these molecules are important for enhancement of lipid oxidation in adipocytes. The results indicate the utility of bioSpace Explorer for biological research on genome-wide molecular networks. Keywords:: DNA microarray, adipocyte, lipid oxidation, peroxisome proliferator-activated receptor gamma (PPARγ), text-mining, pharmacogenomics, network analysis

Background: β-agonists are frequently used as bronchodilators for asthma as not only a reliever but also a controller, and their utility has increased with the development of long-acting β2 selective drugs. Although antiinflammatory effects of β2 selective-agonists have been reported in vitro, side effects on augmentation of airway hyperresponsiveness by chronic use of β2 selective-agonists have been described in several reports. In this study, we investigated the effects of procaterol, a second-generation β2-agonist, on airway inflammation in vivo using an antigen-specific murine model of asthma. Methods: Mice immunized with ovalbumin (OVA) + alum and challenged with inhaled ovalbumin were orally administered procaterol during the challenge. After inhalation, the mice were tracheostomized and placed in a body box under controlled ventilation to measure airway resistance before and after acetylcholine inhalation. Results: Administration of procaterol at a clinical dose equivalent did not augment airway hyperresponsiveness, inflammation of the airway wall, or subsequent airway wall thickening induced by OVA inhalation. BALF cell analysis revealed that the eosinophil number in the BALF was significantly reduced in procaterol-treated mice compared to untreated mice. Conclusions: Oral administration of procaterol at a clinical dose did not augment airway responsiveness, but did reduce eosinophil inflammation.

Many intracellular microbial symbionts of arthropods are strictly vertically transmitted and manipulate their host's reproduction in ways that enhance their own transmission. Rare horizontal transmission events are nonetheless necessary for symbiont spread to novel host lineages. Horizontal transmission has been mostly inferred from phylogenetic studies but the mechanisms of spread are still largely a mystery. Here, we investigated transmission of two distantly related bacterial symbionts--Rickettsia and Hamiltonella--from their host, the sweet potato whitefly, Bemisia tabaci, to three species of whitefly parasitoids: Eretmocerus emiratus, Eretmocerus eremicus and Encarsia pergandiella. We also examined the potential for vertical transmission of these whitefly symbionts between parasitoid generations. Using florescence in situ hybridization (FISH) and transmission electron microscopy we found that Rickettsia invades Eretmocerus larvae during development in a Rickettsia-infected host, persists in adults and in females, reaches the ovaries. However, Rickettsia does not appear to penetrate the oocytes, but instead is localized in the follicular epithelial cells only. Consequently, Rickettsia is not vertically transmitted in Eretmocerus wasps, a result supported by diagnostic polymerase chain reaction (PCR). In contrast, Rickettsia proved to be merely transient in the digestive tract of Encarsia and was excreted with the meconia before wasp pupation. Adults of all three parasitoid species frequently acquired Rickettsia via contact with infected whiteflies, most likely by feeding on the host hemolymph (host feeding), but the rate of infection declined sharply within a few days of wasps being removed from infected whiteflies. In contrast with Rickettsia, Hamiltonella did not establish in any of the parasitoids tested, and none of the parasitoids acquired Hamiltonella by host feeding. This study demonstrates potential routes and barriers to horizontal transmission of symbionts across trophic levels. The possible mechanisms that lead to the differences in transmission of species of symbionts among species of hosts are discussed.