Caterina Fede

The cellular uptake of meta-tetra (hydroxyphenyl)chlorin entrapped in organically modified silica nanoparticles is mediated by serum proteins

Nanosized objects made of various materials are gaining increasing attention as promising vehicles for the delivery of therapeutic and diagnostic agents for cancer. Photodynamic therapy (PDT) appears to offer a very attractive opportunity to implement drug delivery systems since no release of the sensitizer is needed to obtain the therapeutic effect and the design of the nanovehicle should be much easier. The aim of our study was to investigate the use of organic-modified silica nanoparticles (NPs) for the delivery of the second-generation photosensitizer meta-tetra(hydroxyphenyl)chlorin (mTHPC) to cancer cells in vitro. mTHPC was entrapped in NPs (approximately 33 nm diameter) in a monomeric form which produced singlet oxygen with a high efficiency. In aqueous media with high salt concentrations, the NPs underwent aggregation and precipitation but their stability could be preserved in the presence of foetal bovine serum. The cellular uptake, localization and phototoxic activity of mTHPC was determined comparatively in human oesophageal cancer cells after its delivery by the NPs and the standard solvent ethanol/poly(ethylene glycol) 400/water (20:30:50, by vol). The NP formulation reduced the cellular uptake of mTHPC by about 50% in comparison to standard solvent while it did not affect the concentration-dependent photokilling activity of mTHPC and its intracellular localization. Fluorescence resonance energy transfer measurements, using NPs with mTHPC physically entrapped and a cyanine covalently linked, and ultracentrifugation experiments indicated that mTHPC is transferred from NPs to serum proteins when present in the medium. However, the coating of the NP surface with poly(ethylene glycol) largely prevented the transfer to proteins. In conclusion, mTHPC is rapidly transferred from the uncoated nanoparticles to the serum proteins and then internalized by the cells as a protein complex, irrespective of its modality of delivery.

Evaluation of gold nanoparticles toxicity towards human endothelial cells under static and flow conditions.

A new in vitro model system, adding advection and shear stress associated with a flowing medium, is proposed for the investigation of nanoparticles uptake and toxicity towards endothelial cells, since these processes are normally present when nanoparticles formulations are intravenously administered. In this model system, mechanical forces normally present in vivo, such as advection and shear stress were applied and carefully controlled by growing human umbilical vein endothelial cells inside a microfluidic device and continuously infusing gold nanoparticle (Au NPs) solution in the device. The tests performed in the microfluidic device were also run in multiwells, where no flow is present, so as to compare the two model systems and evaluate if gold nanoparticles toxicity differs under static and flow culture conditions. Full characterization of Au NPs in water and in culture medium was accomplished by standard methods. Two-photon fluorescence correlation spectroscopy was also employed to map the flow speed of Au NPs in the microfluidic device and characterize Au NPs before and after interactions with the cells. Au NPs uptake in both in vitro systems was investigated through electron and fluorescence microscopy and ICP-AES, and NPs toxicity measured through standard bio-analytical tests. Comparison between experiments run in multiwells and in microfluidic device plays a pivotal role for the investigation of nanoparticle-cell interaction and toxicity assessment: our work showed that administration of equal concentrations of Au NPs under flow conditions resulted in a reduced sedimentation of nanoparticle aggregates onto the cells and lower cytotoxicity with respect to experiments run in ordinary static conditions (multiwells).

An easy-to-handle microfluidic device suitable for immunohistochemical procedures in mammalian cells grown under flow conditions

Microfluidics, the technology that manipulates small amount of fluids in microscale complex devices, has undergone a remarkable development during the last decade, by targeting a significant range of applications, including biological tests and single-cell analysis, and by displaying many advantages such as reduced reagent consumption, decreased costs and faster analysis. Furthermore, the introduction of microfluidic tools has revolutionized the study of vascular functions, because the controlled three-dimensional environment and the continuous perfusion provided by the microdevice allow simulating the physiological characteristics of the circulatory system. Researchers interested in the study of vascular physiology, however, are often hampered by the difficulty in handling reduced number of cells after growth in these devices. This work shows how to apply different protocols commonly used in biology, such as the immunofluorescence technique, to cells grown in reversibly-bound microfluidic devices, obtaining results comparable to those retrieved under static conditions in multiwells. In this way, we are able to combine the advantages of microfluidic, i.e., application of continuous flow and shear stress, with classical protocols for the study of endothelial cells.

Expression of the endocannabinoid receptors in human fascial tissue.

Cannabinoid receptors have been localized in the central and peripheral nervous system as well as on cells of the immune system, but recent studies on animal tissue gave evidence for the presence of cannabinoid receptors in different types of tissues. Their presence was supposed also in myofascial tissue, suggesting that the endocannabinoid system may help resolve myofascial trigger points and relieve symptoms of fibromyalgia. However, until now the expression of CB1 (cannabinoid receptor 1) and CB2 (cannabinoid receptor 2) in fasciae has not yet been established. Small samples of fascia were collected from volunteers patients during orthopedic surgery. For each sample were done a cell isolation, immunohistochemical investigation (CB1 and CB2 antibodies) and real time RT-PCR to detect the expression of CB1 and CB2. Both cannabinoid receptors are expressed in human fascia and in human fascial fibroblasts culture cells, although to a lesser extent than the control gene. We can assume that the expression of mRNA and protein of CB1 and CB2 receptors in fascial tissue are concentrated into the fibroblasts. This is the first demonstration that the fibroblasts of the muscular fasciae express CB1 and CB2. The presence of these receptors could help to provide a description of cannabinoid receptors distribution and to better explain the role of fasciae as pain generator and the efficacy of some fascial treatments. Indeed the endocannabinoid receptors of fascial fibroblasts can contribute to modulate the fascial fibrosis and inflammation.

Altered gene transcription in human cells treated with Ludox® silica nanoparticles.

Silica (SiO2) nanoparticles (NPs) have found extensive applications in industrial manufacturing, biomedical and biotechnological fields. Therefore, the increasing exposure to such ultrafine particles requires studies to characterize their potential cytotoxic effects in order to provide exhaustive information to assess the impact of nanomaterials on human health. The understanding of the biological processes involved in the development and maintenance of a variety of pathologies is improved by genome-wide approaches, and in this context, gene set analysis has emerged as a fundamental tool for the interpretation of the results. In this work we show how the use of a combination of gene-by-gene and gene set analyses can enhance the interpretation of results of in vitro treatment of A549 cells with Ludox® colloidal amorphous silica nanoparticles. By gene-by-gene and gene set analyses, we evidenced a specific cell response in relation to NPs size and elapsed time after treatment, with the smaller NPs (SM30) having higher impact on inflammatory and apoptosis processes than the bigger ones. Apoptotic process appeared to be activated by the up-regulation of the initiator genes TNFa and IL1b and by ATM. Moreover, our analyses evidenced that cell treatment with Ludox® silica nanoparticles activated the matrix metalloproteinase genes MMP1, MMP10 and MMP9. The information derived from this study can be informative about the cytotoxicity of Ludox® and other similar colloidal amorphous silica NPs prepared by solution processes.

Hormone receptor expression in human fascial tissue

Many epidemiologic, clinical, and experimental findings point to sex differences in myofascial pain in view of the fact that adult women tend to have more myofascial problems with respect to men. It is possible that one of the stimuli to sensitization of fascial nociceptors could come from hormonal factors such as estrogen and relaxin, that are involved in extracellular matrix and collagen remodeling and thus contribute to functions of myofascial tissue. Immunohistochemical and molecular investigations (real-time PCR analysis) of relaxin receptor 1 (RXFP1) and estrogen receptor-alpha (ERα) localization were carried out on samples of human fascia collected from 8 volunteers patients during orthopedic surgery (all females, between 42 and 70 yrs, divided into pre- and post-menopausal groups), and in fibroblasts isolated from deep fascia, to examine both protein and RNA expression levels. We can assume that the two sex hormone receptors analyzed are expressed in all the human fascial districts examined and in fascial fibroblasts culture cells, to a lesser degree in the post-menopausal with respect to the pre-menopausal women. Hormone receptor expression was concentrated in the fibroblasts, and RXFP1 was also evident in blood vessels and nerves. Our results are the first demonstrating that the fibroblasts located within different districts of the muscular fasciae express sex hormone receptors and can help to explain the link between hormonal factors and myofascial pain. It is known, in fact, that estrogen and relaxin play a key role in extracellular matrix remodeling by inhibiting fibrosis and inflammatory activities, both important factors affecting fascial stiffness and sensitization of fascial nociceptors.

The fasciacytes: A new cell devoted to fascial gliding regulation: The Fasciacytes

Hyaluronan occurs between deep fascia and muscle, facilitating gliding between these two structures, and also within the loose connective tissue of the fascia, guaranteeing the smooth sliding of adjacent fibrous fascial layers. It also promotes the functions of the deep fascia. In this study a new class of cells in fasciae is identified, which we have termed fasciacytes, devoted to producing the hyaluronan‐rich extracellular matrix. Synthesis of the hyaluronan‐rich matrix by these new cells was demonstrated by Alcian Blue staining, anti‐HABP (hyaluronic acid binding protein) immunohistochemistry, and transmission electron microscopy. Expression of HAS2 (hyaluronan synthase 2) mRNA by these cells was detected and quantified using real time RT‐PCR. This new cell type has some features similar to fibroblasts: they are positive for the fibroblast marker vimentin and negative for CD68, a marker for the monocyte‐macrophage lineage. However, they have morphological features distinct from classical fibroblasts and they express the marker for chondroid metaplasia, S‐100A4. The authors suggest that these cells represent a new cell type devoted to the production of hyaluronan. Since hyaluronan is essential for fascial gliding, regulation of these cells could affect the functions of fasciae so they could be implicated in myofascial pain. Clin. Anat. 31:667–676, 2018.

In complete SCI patients, long-term functional electrical stimulation of permanent denervated muscles increases epidermis thickness

Abstract Our studies have shown that atrophic Quadriceps muscles from spinal cord injury patients suffering with permanent denervation-induced atrophy and degeneration of muscle fibers, were almost completely rescued to normal size after two years of home-based functional electrical stimulation (h-bFES). Because we used surface electrodes to stimulate the muscle, we wanted to know how the skin was affected by the treatments. Here, we report preliminary data from histological morphometry of Hematoxylin-Eosin-stained paraffin-embedded skin sections harvested from the legs of three SCI patients before and after two years of h-bFES. Despite the heterogeneity of gender and time from SCI, comparing pre vs post h-bFES in these three SCI patients, the data show that: (1) In one subject skin biopsies from both the right and left leg experienced a statistically significant increase in thickness of the epidermis after two years of H-bFES; (2) In the other two subjects, one leg showed a significant increase in epidermis thickness, while in the other leg there was either small positive or negative non-significant changes in epidermis thickness; and (3) more importantly, comparison of grouped data from the three subjects shows that there was a significant 28% increase in the thickness of the epidermis in response to two years of h-bFES rehabilitation. In conclusion, the three educational cases show a long-term positive modulation of epidermis thickness after two years of h-bFES, thus extending to skin the positive results previously demonstrated in skeletal muscle, specifically, a substantial recovery of muscle mass and contractile function after long-term h-bFES.

Morphometric and dynamic measurements of muscular fascia in healthy individuals using ultrasound imaging: a summary of the discrepancies and gaps in the current literature

PurposeThe objectives of this work was to conduct a comprehensive state-of-the art review of the current literature to identify any gaps or discrepancies and summarize the main challenges for obtaining a homogeneous evaluation of muscular fascia in healthy individuals.MethodsAn electronic document search using key words and MeSH terms was performed with various databases. Two independent investigators were tasked with the screening of articles and data extraction. A critical appraisal of what is known was then conducted.ResultsThe literature search identified 65 articles related to healthy facia in the various databases consulted and 20 articles were kept for the review. The thickest portion of the fascia lata (the iliotibial tract) and the plantar fascia are the most often studied muscular fasciae whereas there is paucity of studies on fascia related to other muscles in the body.ConclusionUS imaging is suitable to complement physical examination and for evaluating treatment outcomes. However, the small number of studies and the heterogeneity of the methods did not allow us to establish normal reference values for muscular fascia thickness and to provide strong recommendations about measurement protocols.

Quantification of hyaluronan in human fasciae: variations with function and anatomical site

Recently, alterations in fascial gliding‐like movement have been invoked as critical in the etiology of myofascial pain. Various methods have been attempted for the relief of this major and debilitating clinical problem. Paramount have been attempts to restore correct gliding between fascial layers and the movement over bone, joint, and muscular structures. One of the key elements that underlies such fascial movement is hyaluronan. However, until now, the precise content of hyaluronan within fasciae has been unknown. This study quantifies for the first time the hyaluronan content of human fascial samples obtained from a variety of anatomic sites. Here, we demonstrate that the average amount varies according to anatomic site, and according to the different kinds of sliding properties of the particular fascia. For example, the fascia lata has 35 μg of hyaluronan per gram of tissue, similar to that of the rectus sheath (29 μg g−1). However, the types of fascia adherent to muscle contain far less hyaluronan: 6 μg g−1 in the fascia overlying the trapezius and deltoid muscles. In the fascia that surrounds joints, the hyaluronan increases to 90 μg g−1, such as in the retinacula of the ankle, where greater degrees of movement occur. Surprisingly, no significant differences were detected at any site as a function of age or sex (P‐value > 0.05, t‐test) with the sole exception of the plantar fascia. This work can provide a better understanding of the role of hyaluronan in fascia. It will facilitate a better comprehension of the modulation of the hyaluronan‐rich layer that occurs in relation to the various conditions that affect fascia, and the diverse factors that underlie the attendant pathologies.