Calcium-phosphate (CaP) scaffolds are widely regarded as the gold standard in orthopedics due to their biomimetic properties. However, next-generation bone substitutes should be multifunctional, promoting bone regeneration while simultaneously preventing post-surgical infections. This study investigates the interplay between physico-chemical properties, surface structure/-texture and hydrophilicity in modulating bacterial adhesion on two CaP-based scaffolds: a biomorphic non-sintered scaffold (GB) and a sintered MgSr-CaP ceramic scaffold. Surface morphology and topography were characterized by scanning electron microscopy (SEM) and atomic force microscopy (AFM), while ionic release profiles were quantified by inductively coupled plasma optical emission spectroscopy (ICP-OES) and related to the surface activity. A computer-vision-based approach using Haralick texture descriptors was applied to quantitatively analyze surface features. Antibacterial performance was evaluated against Staphylococcus aureus (including MRSA), Escherichia coli, and Pseudomonas aeruginosa by quantifying colony-forming units (CFUs) after 6 h and by SEM and time-lapse confocal imaging. The GB scaffold significantly reduced bacterial adhesion compared with MgSr-CaP scaffold. Pearson correlation analysis further demonstrated that Haralick-derived texture features are strong predictors of bacterial attachment behavior. These findings highlight the potential of biomorphic fabrication strategies combined with advanced surface texture analysis to guide the design of next-generation infection-resistant orthopedic biomaterials.

We present for the first time a hybrid platform combining a Thin-Film Transistor Microelectrode Array (TFT-MEA) and a Micro-Pillar Array (MPA) for simultaneous, label-free analysis of electrophysiological and contractile activity in cardiomyocytes. Validation with isoprenaline confirmed the system's sensitivity to pharmacological effects, supporting its use in drug screening and cardiotoxicity studies.

α-amylase enzyme molecules were conjugated to biocompatible and biodegradable ad-hoc synthetized polycaprolactone (PCL) nanoparticles, in order to fabricate an effective and innovative nanoplatform for the treatment of nosocomial Staphylococcus aureus contaminations. Indeed, PCL was chosen as the polymer matrix due to its features and the easy scalability of its synthesis, which enables to obtain a linear or star-shaped architecture with high functionality as in the polymer used.The developed nanoformulation underwent extensive structural and functional characterization to evaluate nanoparticles size and morphology before and after enzyme immobilization. Its antibiofilm effectiveness was then validated against bacterial strains isolated from hospital surfaces, demonstrating its potential for practical anti-biofilm applications. The obtained results demonstrated that the system prepared from the enzyme-conjugated nanoparticles exhibited a significant enzymatic activity and an efficient ability to degrade the protective bacterial biofilm. The proposed nanoformulation can therefore be considered an effective and completely environmentally friendly material for surface disinfection in healthcare facilities, which can be safely used in different environment (e.g. sinks and pipes) or medical equipment surfaces (e.g. touch screens).

In the orthopedics field, scientists are continuing to research scaffolds to be used as bone implants [1] capable on one side to guide and sustain the natural metabolism of cells towards tissue regrowth and on the other to confer antibacterial properties to face post-surgical infections. The aim of this study is to test the anti-adhesive efficacy of two different commercially available calcium phosphate bone substitutes. Hydroxyapatite-tricalcium phosphate bone substitutes were prepared from Rattan wood pieces following the procedure described in [2] and doped with Mg and Sr. A biphasic commercial scaffold was used as a comparison. The gamma sterilized scaffolds were characterized in terms of morphological and topographical properties [3] through scanning electron microscopy (SEM) and atomic force microscopy (AFM), respectively. A texture analysis of surfaces was conducted through the Haralick computer-based approach. The anti-adhesive properties of the surfaces were investigated using planktonic cultures of bacterial strains commonly found in bone infections, specifically a gram-positive ( Staphylococcus aureus ) and a gram-negative strain ( Escherichia coli ), in a time-dependent manner. Microscopy analyses (SEM) were performed on bacterial adhesion. The analyses of surfaces in terms of texture and topography demonstrated significant differences among the scaffolds. Preliminary results on bacterial adhesion exhibited a substantial reduction in the adhesion of both bacterial strains on the wood derived scaffolds. The obtained data demonstrated a difference in roughness and texture among all the scaffolds in terms of topography and morphology. Preliminary microbiological results indicated a reduced adhesion on surfaces of wood-derived scaffolds, especially at early time points. This finding suggests a correlation between surface properties, doping ions and bacterial adhesion behavior. The reduced adhesion at early time points could slow down biofilm formation and potentially enhance the effectiveness of antimicrobial treatments. Further microbiological and in vivo studies will be conducted to confirm the anti-adhesive properties of the scaffold.

Thermoplastic polyurethane (TPU) is a polymer used in a variety of fields, including medical applications. Here, we aimed to verify if the brush and bar coater deposition techniques did not alter TPU properties. The topography of the TPU-modified surfaces was studied via AFM demonstrating no significant differences between brush and bar coater-modified surfaces, compared to the un-modified TPU (TPU Film). The effect of the surfaces on planktonic bacteria, evaluated by MTT assay, demonstrated their anti-adhesive effect on E. coli, while the bar coater significantly reduced staphylococcal planktonic adhesion and both bacterial biofilms compared to other samples. Interestingly, Pearson's R coefficient analysis showed that Ra roughness and Haralick's correlation feature were trend predictors for planktonic bacterial cells adhesion. The surface adhesion property was evaluated against NIH-3T3 murine fibroblasts by MTT and against human fibrinogen and human platelet-rich plasma by ELISA and LDH assay, respectively. An indirect cytotoxicity experiment against NIH-3T3 confirmed the biocompatibility of the TPUs. Overall, the results indicated that the deposition techniques did not alter the antibacterial and anti-adhesive surface properties of modified TPU compared to un-modified TPU, nor its bio- and hemocompatibility, confirming the suitability of TPU brush and bar coater films in the biomedical and pharmaceutical fields.

Over the past 10 years, the number of dental implants has grown significantly. This increase has consequently led to an elevation of the statistics related to cases of peri-implantitis. Laser therapy has conquered a place among the therapies of excellence to treat peri-implantitis. However, the laser device used could influence the therapy’s success. The aim of this comparative experimental work was to highlight the differences in the work on grade 4 titanium surfaces of the most commonly used laser lights in this field, taking into consideration any structural damage that lasers could cause to implant surfaces. The lasers examined were a 980 nm diode laser; a 1064 nm Nd:YAG laser; and a new generation of 1064 nm Nd:YAG Q-switch nano pulsed laser. We evaluated the titanium temperature increase, the pre- and post-treatment two-dimensional surface appearance observed under the scanning electron microscope; finally, the three-dimensional pre- and post-treatment topographic analysis was assessed using atomic force microscopy. We showed that the 1064-nm Q-switch Nd:YAG nanosecond pulsed laser appears to be more suitable for the preservation of implant morphology because of the absence of the induction of metal damage.

In modern times, patients are not only seeking oral health but also aiming for a flawless smile. Despite the progress made in aesthetic dentistry, there are instances where insufficient teeth whitening results contrast with the considerable cost of professional treatments. Patient discomfort and the potential for tooth damage further compound these challenges. In this study, we conducted a comparative split-mouth ex vivo investigation to evaluate the innovative BlancOne ULTRA+ (IDS SpA) in comparison with established professional products: Opalescence Extraboost (ULTRADENT), Zoom WhiteSpeed (Philips Research Eindhoven High Tech), and Pola Office (SDI Limited). Our initial focus was on the whitening effectiveness of each product, which was measured using a spectrophotometer. Subsequently, we assessed any structural changes in enamel post-treatment using an atomic force microscope (AFM) and a nanoindentation procedure. All tested bleaching agents demonstrated teeth-brightening effects. BlancOne ULTRA+, Zoom WhiteSpeed, and Pola Office caused minor alterations in the texture of the enamel surface within nanometric limits. However, Opalescence Extraboost exhibited notably more pronounced changes, indicating significant modifications in surface roughness and potential reductions in material hardness due to consequential shifts in mechanical properties. BlancOne ULTRA+ appears to offer the most favourable cost–benefit outcome.

In recent years, demand for novel drug delivery systems has risen in order to minimize the adverse effects associated with systemic administration of drugs that affect both healthy and diseased tissues. Controlled and targeted drug release leads to an improvement in the effectiveness and safety of the treatment. In particular, there is an increasing interest in treatments controlled both in spatial and temporal terms. In this work we employed low intensity ultrasound to develop a protocol for the release of a model drug (doxorubicin) loaded into the volume of biopolymeric microcapsules. The proposed ultrasound-based release protocol was first characterized in terms of release efficiency and then tested in vitro using a cancer cell line (MCF7). We show that the appropriate mechanical stimulus enables the effective release of the drug from the capsules, yet maintaining safe conditions in terms of the resulting mechanical and thermal indexes.

We have developed a novel experimental set-up that simultaneously, (i) applies static and dynamic deformations to adherent cells in culture, (ii) allows the visualization of cells under fluorescence microscopy, and (iii) allows atomic force microscopy nanoindentation measurements of the mechanical properties of the cells. The cell stretcher device relies on a dielectric elastomer film that can be electro-actuated and acts as the cell culture substrate. The shape and position of the electrodes actuating the film can be controlled by design in order to obtain specific deformations across the cell culture chamber. By using optical markers we characterized the strain fields under different electrode configurations and applied potentials. The combined setup, which includes the cell stretcher device, an atomic force microscope, and an inverted optical microscope, can assess in situ and with sub-micron spatial resolution single cell topography and elasticity, as well as ion fluxes, during the application of static deformations. Proof of performance on fibroblasts shows a reproducible increase in the average cell elastic modulus as a response to applied uniaxial stretch of just 4%. Additionally, high resolution topography and elasticity maps on a single fibroblast can be acquired while the cell is deformed, providing evidence of long-term instrumental stability. This study provides a proof-of-concept of a novel platform that allows in situ and real time investigation of single cell mechano-transduction phenomena with sub-cellular spatial resolution.

The brain is the most complex organ of our body. Such a complexity spans from the single-cell morphology up to the intricate connections that hundreds of thousands of neurons establish to create dense neuronal networks. All these components are involved in the genesis of the rich patterns of electrophysiological activity that characterize the brain. Over the years, researchers coming from different disciplines developedin vitrosimplified experimental models to investigate in a more controllable and observable way how neuronal ensembles generate peculiar firing rhythms, code external stimulations, or respond to chemical drugs. Nowadays, suchin vitromodels are namedbrain-on-a-chippointing out the relevance of the technological counterpart as artificial tool to interact with the brain: multi-electrode arrays are well-used devices to record and stimulate large-scale developing neuronal networks originated from dissociated cultures, brain slices, up to brain organoids. In this review, we will discuss the state of the art of the brain-on-a-chip, highlighting which structural and biological features a realisticin vitrobrain should embed (and how to achieve them). In particular, we identified two topological features, namely modular and three-dimensional connectivity, and a biological one (heterogeneity) that takes into account the huge number of neuronal types existing in the brain. At the end of this travel, we will show how 'far' we are from the goal and how interconnected-brain-regions-on-a-chip is the most appropriate wording to indicate the current state of the art.

Evaluation and understanding the effect of drug delivery in in vitro systems is fundamental in drug discovery. We present an assay based on real-time electrical impedance spectroscopy (EIS) measurements that can be used to follow the internalisation and cytotoxic effect of a matrix metalloproteinase (MMP)–sensitive liposome formulation loaded with oxaliplatin (OxPt) on colorectal cancer cells. The EIS response identified two different cellular processes: (i) a negative peak in the cell index (CI) within the first 5 h, due to onset of liposome endocytosis, followed by (ii) a subsequent CI increase, due to the reattachment of cells until the onset of cytotoxicity with a decrease in CI. Free OxPt or OxPt-loaded Stealth liposomes did not show this two-stage EIS response; the latter can be due to the fact that Stealth cannot be cleaved by MMPs and thus is not taken up by the cells. Real-time bright-field imaging supported the EIS data, showing variations in cell adherence and cell morphology after exposure to the different liposome formulations. A drastic decrease in cell coverage as well as rounding up of cells during the first 5 h of exposure to OxPt-loaded (MMP)-sensitive liposome formulation is reflected by the first negative EIS response, which indicates the onset of liposome endocytosis.

Neuro-electronic interfaces play a fundamental role for studying brain functions and cellular mechanisms of information processing both in vitro and in vivo. Here we present a new concept in which electronic devices are integrated with models of neuronal networks of human origin together with nanotools to characterize the (patho)physiology of such in vitro complex systems. Large-scale CMOS micro-transducer arrays, coupled to 2D and 3D networks are presented together with new techniques for non-invasively modulate the cell activity. Specifically, piezo-electric nano particles and gold nano rods are used to excite-inhibit the neural activity that is recorded by the micro-transducer arrays. Concept, technology, and preliminary results are presented toward the implementation of a bio-hybrid brain-on-a-chip.

OBJECTIVE:The goal of this work is to develop and characterize an innovative experimental framework to design interconnected (i.e. modular) heterogeneous (cortical-hippocampal) neuronal cultures with a three-dimensional (3D) connectivity and to record their electrophysiological activity using micro-electrode arrays (MEAs).APPROACH:A two-compartment polymeric mask for the segregation of different neuronal populations (cortex and hippocampus) was coupled to the MEA surface. Glass microbeads were used as a scaffold to mimic the 3D brain micro-architecture.MAIN RESULTS:We built a fully functional heterogeneous 3D neuronal network. From an electrophysiological point of view, we found that the heterogeneity induces a global increase of the activity rate, while the 3D connectivity modulates the duration and the organization of the bursting activity.SIGNIFICANCE:In vivo, studies of network dynamics and interactions between neuronal populations are often time-consuming, low-throughput, complex, and suffer from reproducibility. On the other hand, most of the commonly used in vitro brain models are too simplified and thus far from the in vivo situation. The achieved results demonstrate the feasibility to build a more realistic and controllable experimental in vitro model of interconnected brain regions on-a-chip whose applications may have impacts on the study of neurological disorders that impair the connectivity between brain areas (e.g. Parkinson disease).

The increase of different types of cell cultures, which can be used for the in vitro studies of physiological and/or pathological processes, has introduced the need to improve culture techniques through the use of materials and culture media that promote growth, recreating a cellular micro-environment that can be asserted in in vivo condition. The standard methods for the functionalization of supports used for cell cultures are based on the use of synthetic or natural biopolymers, which generally have high costs, such as poly-lysine and polyornithine. The aim of this work is to demonstrate the alternative use of the polysaccharide chitosan as adhesion factor and structural component for 2D/3D neuronal cultures. Thanks to its versatility, it could be easily functionalized for the fabrication of personalized of in vitro models.

The high sensitivity of silicon microcantilever sensors has expanded their use in areas ranging from gas sensing to bio-medical applications. Photochromic molecules also represent promising candidates for a large variety of sensing applications. In this work, the operating principles of these two sensing methods are combined in order to detect the reversible conformational change of a molecular switch, spiropyran. Thus, arrays of silicon microcantilever sensors were functionalized with spiropyran on the gold covered side and used as test microcantilevers. The microcantilever deflection response was observed, in five sequential cycles, as the transition from the spiropyran (SP) (CLOSED) to the merocyanine (MC) (OPEN) state and vice-versa when induced by UV and white light LED sources, respectively, proving the reversibility capabilities of this type of sensor. The microcantilever deflection direction was observed to be in one direction when changing to the MC state and in the opposite direction when changing back to the SP state. A tensile stress was induced in the microcantilever when the SP to MC transition took place, while a compressive stress was observed for the reverse transition. These different type of stresses are believed to be related to the spatial conformational changes induced in the photochromic molecule upon photo-isomerisation.

Any Atomic Force Microscope (AFM) scanner based on piezoelectric ceramics is affected by nonlinear distortions, mainly due to rate-independent hysteresis and rate-dependent creep, two different phenomena often referred to, collectively, as rate-dependent hysteresis. To compensate for these distortions, especially in old or cheap instruments, empirical open-loop compensation techniques are frequently adopted. In this paper, a complete hardware/software toolchain is proposed, for data acquisition, identification of hysteresis and creep models, and real-time open-loop compensation of rate-dependent hysteresis in AFM scanners.

Malignancy diagnosis of uterine smooth muscle neoplasms can be challenging. Morphologic features are subjective, and the utility of immunohistochemistry is still debated.1Oliva E. Practical issues in uterine pathology from banal to bewildering: The remarkable spectrum of smooth muscle neoplasia.Mod Pathol. 2016; 29: S104-S120Crossref PubMed Scopus (34) Google Scholar, 2Ferenczy A. Richart R.M. Okagaki T. A comparative ultrastructural study of leiomyosarcoma, cellular leiomyoma, and leiomyoma of the uterus.Cancer. 1971; 28: 1004-1018Crossref PubMed Scopus (121) Google Scholar We propose to characterize and compare the ultrastructural and mechanical properties of leiomyoma and leiomyosarcoma with those of normal myometrium, using atomic force microscopy (AFM) technique. Three representative groups of samples were selected from the database of the Pathology Unit–San Martino Hospital, Genoa, Italy. Group 1 comprised 1 sample of normal myometrium and 1 sample of spindle cells leiomyoma from a 39-year-old patient and 1 sample of spindle cells leiomyosarcoma from a 62-year-old patient. Group 2 comprised 1 sample of normal myometrium and 1 sample of spindle cells leiomyoma from a 49-year-old patient and 1 sample of spindle cells leiomyosarcoma from a 67-year-old patient. Group 3 comprised 1 sample of normal myometrium and 1 sample of spindle cells leiomyoma from a 50-year-old patient and 1 sample of spindle cells leiomyosarcoma from a 55-year-old patient. Two shadowed sections (thickness of 5 μm for AFM-imaging and 15 μm for AFM-indentation testing) were collected separately from each paraffin-embedded tissue specimen. After being removed from the paraffin and dried, the specimens were left exposed for AFM-scanning. Sections were not stained. AFM-imaging was performed in contact mode at room temperature and in air on regions of interest, which were selected accurately through optical microscopy. The specimens for mechanical characterization were probed in liquid conservation medium (phosphate-buffered saline solution) at room temperature through AFM-nanoindentation on three 32×32-array/90×90-μm2 regions of interest. Image processing was made with the use of Fiji (https://doi.org/10.1038/nmeth.2019). Elastic modulus values that were calculated from the AFM-indentation test were analyzed statistically (P≤.05) with the use of MedCalc software (version 18.11; Ostend, Belgium). AFM-imaging evidences regularly oriented smooth muscle cells in normal myometrium. The nuclear pattern shows blunt-ended, thin ellipsoidal nuclei, centrally located in sarcoplasm. Larger myofibrils and smaller connective fibrils show similar positioning, bestowing high homogeneity to the interstitium (Figure 1, A). Similarities in regular orientation of smooth muscle cells, nuclei density, shape, and location in the sarcoplasm were observed between leiomyoma and normal myometrium. However, despite such similar directionality, myofibrils are narrower, and the interstitium is comparatively wider and devoid of small connective fibrils (Figure 1, B). Contrarily, leiomyosarcoma evidences irregularly oriented pleomorphic smooth muscle cells. Nuclei appear bigger (>50% of sarcoplasm), more rounded, and at a higher density than in leiomyoma and normal myometrium. Interstitium shows further loss of myofibrils and small connective fibrils that are replaced by amorphous, nonfibrillar material. This feature confers high irregularity and heterogeneity to the tissue ultrastructure (Figure 1, C). Concerning the AFM-indentation measurements, which were performed on each group of samples, The analysis of variance assay test indicates statistically significant differences between the elastic moduli of different samples (P<.0001), and a t-test showed a significantly lower average elastic modulus (ie, softer tissue) for leiomyosarcoma when compared with leiomyoma and an even lower average elastic modulus when compared with normal myometrium (Figure 1, D). Interestingly, nearly 80% of elastic modulus values that are measured on different points of leiomyoma and >95% of those measured in leiomyosarcoma are below the mean elastic value Ē of the normal myometrium (Figure 1, E). Leiomyoma, leiomyosarcoma, and normal myometrium show significant differences at the nanoscale that cannot be observed easily and measured at the microscale with clinically available techniques. Ultrastructural differences in myofibrils positioning and thickness, nuclear pattern, and interstitium are observed clearly. Similar to other tumor types, elastic modulus of tumorous myometrium decreases compared with normal myometrium.3Sokolov I. Atomic force microscopy in cancer cell research.in: Nalwa H.S. Webster T. Cancer nanotechnology-nanomaterials for cancer diagnosis and therapy. American Scientific Publishers, NY2006: 43-59Google Scholar, 4Zemła J. Danilkiewicz J. Orzechowska B. Pabijan J. Seweryn S. Lekka M. Atomic force microscopy as a tool for assessing the cellular elasticity and adhesiveness to identify cancer cells and tissues.Semin Cell Dev Biol. 2018; 73: 115-124Crossref PubMed Scopus (45) Google Scholar, 5Cui Y. Zhang X. You K. et al.Nanomechanical characteristics of cervical cancer and cervical intraepithelial neoplasia revealed by atomic force microscopy.Med Sci Monit. 2017; 23: 4205-4213Crossref PubMed Scopus (8) Google Scholar

The cardiac excitation-contraction coupling is the cellular process through which the heart absolves its blood pumping function, and it is directly affected when cardiac pathologies occur. Cardiomyocytes are the functional units in which this complex biomolecular process takes place: they can be represented as a two-stage electro-chemo and chemo-mechanical transducer, along which each stage can be probed and monitored via appropriate micro/nanotechnology-based tools. Atomic force microscopy (AFM), with its unique nanoresolved force sensitivity and versatile modes of extracting sample properties, can represent a key instrument to study time-dependent heart mechanics and topography at the single cell level. In this work, we show how the integrative possibilities of AFM allowed us to implement an in vitro system which can monitor cardiac electrophysiology, intracellular calcium dynamics, and single cell mechanics. We believe this single cell-sensitive and integrated system will unlock improved, fast, and reliable cardiac in vitro tests in the future.