Bacteriostatic Behavior of PLA-BaTiO3 Composite Fibers Synthesized by Centrifugal Spinning and Subjected to Aging Test.

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.

Metabolic machinery encrypted in the Raman spectrum of influenza A virus-inoculated mammalian cells.

Raman spectroscopy was applied with a high spectral resolution to a structural study of Influenza (type A) virus before and after its inoculation into Madin-Darby canine kidney cells. This study exploits the fact that the major virus and cell constituents, namely DNA/RNA, lipid, and protein molecules, exhibit peculiar fingerprints in the Raman spectrum, which clearly differed between cells and viruses, as well as before and after virus inoculation into cells. These vibrational features, which allowed us to discuss viral assembly, membrane lipid evolution, and nucleoprotein interactions of the virus with the host cells, reflected the ability of the virus to alter host cells' pathways to enhance its replication efficiency. Upon comparing Raman signals from the host cells before and after virus inoculation, we were also able to discuss in detail cell metabolic reactions against the presence of the virus in terms of compositional variations of lipid species, the formation of fatty acids, dephosphorylation of high-energy adenosine triphosphate molecules, and enzymatic hydrolysis of the hemagglutinin glycoprotein.

Off-Stoichiometric Reactions at the Cell-Substrate Biomolecular Interface of Biomaterials: In Situ and Ex Situ Monitoring of Cell Proliferation, Differentiation, and Bone Tissue Formation.

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.

In vitro antibacterial activity of oxide and non-oxide bioceramics for arthroplastic devices: I. In situ time-lapse Raman spectroscopy.

Over the next two decades, a strong demographic demand for arthroplastic devices coupled with a decreased efficacy of antibiotics has been predicted to result in an exponential increase in the number of periprosthetic joint infections (PJIs). Advanced strategies are therefore required to improve the local peri-implant immune response and curb the pathogenic events of bacterial adhesion and biofilm formation. The use of biomaterials that autonomously counter infections is one approach to improve orthopedic outcomes. Using conventional molecular biology characterization methods and advanced Raman spectroscopy, this study examined the bacteriostatic response of two bioceramic materials commonly employed as prosthetic implants: zirconia-toughened alumina (ZTA) and silicon nitride (Si3N4). Unlike the ZTA, it was found that non-oxide Si3N4 possesses an inherently anti-infective surface chemistry, which acts in a responsive way against bacterial loading. The mechanistic details of its behavior are elucidated. Non-oxide bioceramics appear to be promising, but their full development requires a transitional approach that integrates the fundamental biochemical concepts with clinical outcomes.

In vitro antibacterial activity of oxide and non-oxide bioceramics for arthroplastic devices: II. Fourier transform infrared spectroscopy.

The metabolic response of Gram-positive Staphylococcus epidermidis (S. epidermidis) bacteria to bioceramic substrates was probed by means of Fourier transform infrared spectroscopy (FTIR). Oxide zirconia-toughened alumina (ZTA) and non-oxide silicon nitride (Si3N4) substrates were tested. Bacteria exposed to silica glass substrates were used as a control. S. epidermidis, a major cause of periprosthetic infections, was screened to obtain a precise time-lapse knowledge of its molecular composition and to mechanistically understand its interaction with different substrates. At the molecular level, the structure of proteins, lipids, nucleic acid, and aromatic amino acids evolved with time in response to different substrates. In combination with statistical validation and local pH measurements, a chemical lysis mechanism was spectroscopically observed in situ on the Si3N4 substrates. Utilization of FTIR in this study avoided fluorescence noise which occurred while probing the ZTA samples with Raman spectroscopy in a companion paper. The substrate-driven dynamics of polysaccharide and peptide variations in the bacterial cell wall, peculiar to Si3N4 bioceramics, are elucidated.

Green Tea Polyphenols Coupled with a Bioactive Titanium Alloy Surface: In Vitro Characterization of Osteoinductive Behavior through a KUSA A1 Cell Study.

A chemically-treated titanium alloy (Ti6Al4V) surface, able to induce hydroxyapatite precipitation from body fluids (inorganic mineralization activity), was functionalized with a polyphenolic extract from green tea (tea polyphenols, TPH). Considering that green tea polyphenols have stimulating effects on bone forming cells (biological mineralization), the aim was to test their osteoinductive behavior due to co-operation of inorganic and biological mineralization on mesenchymal stem cells KUSA A1. The functionalized surfaces were characterized by using the Folin⁻Ciocalteu method and X-ray photoelectron spectroscopy to confirm the successful outcome of the functionalization process. Two cell cultures of mesenchymal stem cells, KUSA A1 were performed, with or without osteoinductive factors. The cells and surfaces were characterized for monitoring cell viability and hydroxyapatite production: Fourier Transform Infrared Spectroscopy and Raman spectroscopy analyses showed deposition of hydroxyapatite and collagen due to the cell activity, highlighting differentiation of KUSA A1 into osteoblasts. A higher production of extracellular matrix was highlighted on the functionalized samples by laser microscope and the fluorescence images showed higher viability of cells and greater presence of osteocalcin in these samples. These results highlight the ability of polyphenols to improve cell differentiation and to stimulate biological mineralization, showing that surface functionalization of metal implants could be a promising way to improve osteointegrability.

Silicon Nitride Bioceramics Induce Chemically Driven Lysis in Porphyromonas gingivalis.

Organisms of Gram-negative phylum bacteroidetes, Porphyromonas gingivalis, underwent lysis on polished surfaces of silicon nitride (Si3N4) bioceramics. The antibacterial activity of Si3N4 was mainly the result of chemically driven principles. The lytic activity, although not osmotic in nature, was related to the peculiar pH-dependent surface chemistry of Si3N4. A buffering effect via the formation of ammonium ions (NH4(+)) (and their modifications) was experimentally observed by pH microscopy. Lysis was confirmed by conventional fluorescence spectroscopy, and the bacteria's metabolism was traced with the aid of in situ Raman microprobe spectroscopy. This latter technique revealed the formation of peroxynitrite within the bacterium itself. Degradation of the bacteria's nucleic acid, drastic reduction in phenilalanine, and reduction of lipid concentration were observed due to short-term exposure (6 days) to Si3N4. Altering the surface chemistry of Si3N4 by either chemical etching or thermal oxidation influenced peroxynitrite formation and affected bacteria metabolism in different ways. Exploiting the peculiar surface chemistry of Si3N4 bioceramics could be helpful in counteracting Porphyromonas gingivalis in an alkaline pH environment.

Protocol for extracting and isolating porcine bone-marrow-derived macrophages from ribs.

Due to anatomical and biological similarities with humans, pigs are increasingly used for inflammation- and immune-related studies in biomedical research, including the field of osteonecrosis and osteoimmunology. Here, we present a protocol for rib extraction, isolation of the bone marrow by centrifugation, and processing to obtain bone-marrow-derived macrophages (BMDMs). Then, we describe the procedures of in vitro experiments to evaluate the cell phenotype. For complete details on the use and execution of this protocol, please refer to Andre et al.1.

Simple Electrospinning Method for Biocompatible Polycaprolactone ß-Carotene Scaffolds: Advantages and Limitations.

In this study, electrospun scaffolds were fabricated using polycaprolactone (PCL) loaded with varying concentrations of ß-carotene (1.2%, 2.4%, and 3.6%) via the electrospinning technique. The electrospinning process involved the melting of PCL in acetic acid, followed by the incorporation of ß-carotene powder under constant stirring. Raman spectroscopy revealed a homogeneous distribution of ß-carotene within the PCL matrix. However, the ß-carotene appeared in particulate form, rather than being dissolved and blended with the PCL matrix, a result also confirmed by thermogravimetric analysis. Additionally, X-ray diffraction analysis indicated a decrease in crystallinity with increasing ß-carotene concentration. Mechanical testing of the scaffolds demonstrated an increase in ultimate strain, accompanied by a reduction in ultimate stress, indicating a potential plasticizing effect. Moreover, antimicrobial assays revealed a marginal antibacterial effect against Escherichia coli for scaffolds with higher ß-carotene concentrations. Conversely, preliminary biological assessment using KUSA-A1 mesenchymal cells indicated enhanced cellular proliferation in response to the scaffolds, suggesting the potential biocompatibility and cell-stimulating properties of ß-carotene-loaded PCL scaffolds. Overall, this study provides insights into the fabrication and characterization of electrospun PCL scaffolds containing ß-carotene, laying the groundwork for further exploration in tissue engineering and regenerative medicine applications.

Ex vivo study of detergent-assisted intraosseous bone wash treatment of osteonecrosis.

Avascular necrosis (AVN) involves ischemic cell death of the bone. AVN leaves an abundance of necrotic lipids and debris in the bone marrow, which instigates inflammatory bone repair. Consequently, the necrotic bone microenvironment stimulates excessive bone resorption, leading to joint deformities and osteoarthritis. Here, we performed a detergent-assisted bone wash using poloxamer 407 (P407) to clean the necrotic bone environment by removing lipids and necrotic debris. The new concept was tested using an established ex vivo AVN model of porcine cadaver humeral heads. The P407 wash was performed using P407 solution and followed with saline via two intraosseous needles. Visual inspection and image analyses of average pixel light intensity showed that the P407 wash produced a better-cleaned bone than the saline wash. Analyses of the collected bone wash solution showed a two-fold increase in triglycerides (101 vs. 53 mmol/head, p = 0.006) and a 10-fold increase in the dry weight of the removed debris (1.34 vs. 0.13 g/head, p = 0.02) with the P407 wash compared to saline. The histological evaluation showed significantly decreased Oil-Red-O (fats) staining in the P407-washed bone compared with the saline-washed bone. The in vitro assays of Alizarin red and qPCR showed the P407 wash neither altered the osteogenic behaviors of porcine bone marrow-derived mesenchymal cells (pBMMCs) nor raised inflammatory responses of porcine bone marrow-derived macrophages (pBMMs). In conclusion, detergent-assisted bone wash using P407 produced a better removal of nonsoluble debris from the bone marrow space than the saline wash without causing changes to osteogenesis or inflammatory reactions.

Raman spectroscopic insight into osteoarthritic cartilage regeneration by mRNA therapeutics encoding cartilage-anabolic transcription factor Runx1.

While joint arthroplasty remains nowadays the most popular option available to repair chronically degenerated osteoarthritic joints, possibilities are recently emerging for regeneration of damaged cartilage rather than its replacement with artificial biomaterials. This latter strategy could allow avoiding the quite intrusive surgical procedures associated with total joint replacement. Building upon this notion, we first apply Raman spectroscopy to characterize diseased cartilage in a mice model of instability-induced knee osteoarthritis (OA) upon medial collateral ligament (MCL) and medial meniscus (MM) transections. Then, we examine the same OA model after cartilage regeneration by means of messenger RNA (mRNA) delivery of a cartilage-anabolic runt-related transcription factor 1 (RUNX1). Raman spectroscopy is shown to substantiate at the molecular scale the therapeutic effect of the Runx1 mRNA cartilage regeneration approach. This study demonstrates how the Raman spectroscopic method could support and accelerate the development of new therapies for cartilage diseases.

Innovative electrospun PCL/fibroin/l-dopa scaffolds scaffolds supporting bone tissue regeneration.

Poly-caprolactone is one of the most promising biocompatible polymers on the market, in particular for temporary devices that are not subjected to high physiological loads. Even if completely resorbable in various biological environments, poly-caprolactione does not play any specific biological role in supporting tissue regeneration and for this reason has a limited range of possible applications. In this preliminary work, for the first time l-dopa and fibroin have been combined with electrospun poly-caprolactone fibers in order to induce bioactive effects and, in particular, stimulate the proliferation, adhesion and osteoconduction of the polymeric fibers. Results showed that addition of low-molecular weight fibroin reduces the mechanical strength of the fibers while promoting the formation of mineralized deposits, when testedin vitrowith KUSA-A1 mesenchymal cells. l-dopa, on the other hand, improved the mechanical properties and stimulated the formation of agglomerates of mineralized deposits containing calcium and phosphorous with high specific volume. The combination of the two substances resulted in good mechanical properties and higher amounts of mineralized deposits formedin vitro.

Cytotoxicity and osteogenic effects of aluminum ions.

In this study, we monitored the effect of Al3+ ions on mesenchymal cells (KUSA-A1) and human fibroblasts (NHDF) by means of in vitro experiments by culturing the cells with addition of small concentrations of aluminum ions (i.e., 0.1, 1, 10, and 100 ppm). Bone formation test was then conducted using KUSA-A1. Small concentrations of aluminum ions delayed but did not completely inhibit cell proliferation. The amount of bone tissue decreased as the concentration of Al3+ increased and crystallinity changes were also detected by Raman spectroscopic experiments. Moreover, Al3+ ions greatly affected both structure and chemistry of bone tissues with mineral nodules becoming larger and atomic substitution of Ca with Al in bone tissue being more preponderant with increasing Al3+ concentration. Such effects in turn impaired the balance between mineral and collagen in the formed bone tissue.

Enhancing the bioactivity of melt electrowritten PLLA scaffold by convenient, green, and effective hydrophilic surface modification.

As an emerging additive manufacturing (AM) technique, melt electrospinning writing (MEW) is used to fabricate three-dimensional (3D) submicron filament-based scaffolds with adjustable pore size and customized structure for bone regeneration. Poly(L-lactic acid) (PLLA) scaffold with excellent biodegradability and biocompatibility is first successfully manufactured using our self-assembled MEW device. However, the ultralow cell affinity and poor bioactivity severely hamper their practical applications in bone tissue engineering. These issues are caused by the severe inherent biologically inert, hydrophobicity as well as the smooth surface of the MEW PLLA filaments. In this study, a green and robust alkaline method is applied to modify the scaffold surface and to improve the bioactivity of the MEW PLLA scaffold. Without deterioration in mechanical property but robust surface hydrophilicity, the optimal MEW PLLA scaffold shows promoted surface roughness, enhanced filament tensile modulus (~ 2 folds of the as-prepared sample), and boosted crystallizability (ultrahigh WAXD intensity). Moreover, after being cultured with KUSA-A1 cells, the 0.5 M NaOH, 2 h treated MEW PLLA scaffold exhibits higher osteoinductive ability and increased immature bone tissue amounts (3 times of controlled scaffold). Thus, the flexible surface functionalization by the specific alkaline treatment was found to be an effective method for the preparation of bioactivated MEW PLLA scaffolds with promoted bone regeneration.

Raman Molecular Fingerprints of SARS-CoV-2 British Variant and the Concept of Raman Barcode.

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.

Silicon nitride: a potent solid-state bioceramic inactivator of ssRNA viruses.

Surface inactivation of human microbial pathogens has a long history. The Smith Papyrus (2600 ~ 2200 B.C.) described the use of copper surfaces to sterilize chest wounds and drinking water. Brass and bronze on doorknobs can discourage microbial spread in hospitals, and metal-base surface coatings are used in hygiene-sensitive environments, both as inactivators and modulators of cellular immunity. A limitation of these approaches is that the reactive oxygen radicals (ROS) generated at metal surfaces also damage human cells by oxidizing their proteins and lipids. Silicon nitride (Si3N4) is a non-oxide ceramic compound with known surface bacterial resistance. We show here that off-stoichiometric reactions at Si3N4 surfaces are also capable of inactivating different types of single-stranded RNA (ssRNA) viruses independent of whether their structure presents an envelop or not. The antiviral property of Si3N4 derives from a hydrolysis reaction at its surface and the subsequent formation of reactive nitrogen species (RNS) in doses that could be metabolized by mammalian cells but are lethal to pathogens. Real-time reverse transcription (RT)-polymerase chain reaction (PCR) tests of viral RNA and in situ Raman spectroscopy suggested that the products of Si3N4 hydrolysis directly react with viral proteins and RNA. Si3N4 may have a role in controlling human epidemics related to ssRNA mutant viruses.

Raman Molecular Fingerprints of Rice Nutritional Quality and the Concept of Raman Barcode.

The nutritional quality of rice is contingent on a wide spectrum of biochemical characteristics, which essentially depend on rice genome, but are also greatly affected by growing/environmental conditions and aging during storage. The genetic basis and related identification of genes have widely been studied and rationally linked to accumulation of micronutrients in grains. However, genetic classifications cannot catch quality fluctuations arising from interannual, environmental, and storage conditions. Here, we propose a quantitative spectroscopic approach to analyze rice nutritional quality based on Raman spectroscopy, and disclose analytical algorithms for the determination of: (i) amylopectin and amylose concentrations, (ii) aromatic amino acids, (iii) protein content and structure, and (iv) chemical residues. The proposed Raman algorithms directly link to the molecular composition of grains and allow fast/non-destructive determination of key nutritional parameters with minimal sample preparation. Building upon spectroscopic information at the molecular level, we newly propose to represent the nutritional quality of labeled rice products with a barcode specially tailored on the Raman spectrum. The Raman barcode, which can be stored in databases promptly consultable with barcode scanners, could be linked to diet applications (apps) to enable a rapid, factual, and unequivocal product identification based on direct molecular screening.

Antibacterial Property of Cellulose Acetate Composite Materials Reinforced with Aluminum Nitride.

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.

Osteogenic Response to Polysaccharide Nanogel Sheets of Human Fibroblasts After Conversion Into Functional Osteoblasts by Direct Phenotypic Cell Reprogramming.

Human dermal fibroblasts (HDFs) were converted into osteoblasts using a ALK inhibitor II (inhibitor of transforming growth factor-ß 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.

Antifungal activity of polymethyl methacrylate/Si3N4 composites against Candida albicans.

Previous studies using gram-positive and -negative bacteria demonstrated that hydrolysis of silicon nitride (Si3N4) in aqueous suspensions elutes nitrogen and produces gaseous ammonia while buffering pH. According to immunochemistry assays, fluorescence imaging, and in situ Raman spectroscopy, we demonstrate here that the antipathogenic surface chemistry of Si3N4 can be extended to polymethylmethacrylate (PMMA) by compounding it with a minor fraction (~8 vol.%) of Si3N4 particles without any tangible loss in bulk properties. The hydrolytic products, which were eluted from partly exposed Si3N4 particles at the composite surface, exhibited fungicidal action against Candida albicans. Using a specific nitrative stress sensing dye and highly resolved fluorescence micrographs, we observed in situ congestion of peroxynitrite (ONOO-) radicals in the mitochondria of the Candida cells exposed to the PMMA/Si3N4 composite, while these radicals were absent in the mitochondria of identical cells exposed to monolithic PMMA. These in situ observations suggest that the surface chemistry of Si3N4 mimics the antifungal activity of macrophages, which concurrently produce NO radicals and superoxide anions (O2•-) resulting in the formation of candidacidal ONOO-. The fungicidal properties of PMMA/Si3N4 composites could be used in dental appliances to inhibit the uncontrolled growth of Candida albicans and ensuing candidiasis while being synergic with chemoprophylaxis. STATEMENT OF SIGNIFICANCE: In a follow-up of previous studies of gram-positive and gram-negative bacteria, we demonstrate here that the antipathogenic surface chemistry of Si3N4 could be extended to polymethylmethacrylate (PMMA) containing a minor fraction (~8 vol.%) of Si3N4 particles without tangible loss in bulk properties. Hydrolytic products eluted from Si3N4 particles at the composite surface exhibited fungicidal action against Candida albicans. Highly resolved fluorescence microscopy revealed congestion of peroxynitrite (ONOO-) radicals in the mitochondria of the Candida cells exposed to the PMMA/Si3N4 composite, while radicals were absent in the mitochondria of identical cells exposed to monolithic PMMA. The fungicidal properties of PMMA/Si3N4 composites could be used in dental appliances to inhibit uncontrolled growth of Candida albicans and ensuing candidiasis in synergy with chemoprophylaxis.