Magneto-responsive Cell Culture Substrates that can be Modulated in situ.

Understanding the interaction between cells and their environment is fundamental for mechanobiology. To mimic the behavior of cells in physiological and pathological conditions, synthetic substrates must have topographical and/or mechanical properties that evolve in time. Dynamic substrates mainly rely on stimuli-responsive materials where an external stimulus induces controlled variations in topography or mechanics. Herein, we describe the development of a dynamic cell culture substrate where mechanical properties are reversibly tuned in situ using magnetically-responsive superparamagnetic iron oxide nanoparticles (SPIONs).

Artificial Lysosomal Platform to Study Nanoparticle Long-term Stability.

Nanoparticles (NPs) possess unique properties useful for designing specific functionalities for biomedi- cal applications. A prerequisite of a safe-by-design and effective use in any biomedical application is to study NP-cell interactions to gain a better understanding of cellular consequences upon exposure. Cellular uptake of NPs results mainly in the localization of NPs in the complex environment of lysosomes, a compartment which can be mimicked by artificial lysosomal fluid. In this work we showed the applicability of lysosomal fluid as a platform for a fast assessment of gold, iron oxide and silica NP stability over 24 h in a relevant biological fluid, by using multiple analytical methods.

Hypothesis Test of the Photon Count Distribution for Dust Discrimination in Dynamic Light Scattering.

Users of dynamic light scattering (DLS) are challenged when a sample of nanoparticles (NPs) contains dust. This is a frequently inevitable scenario and a major problem that critically affects the reproducibility and accuracy of DLS measurements. Current methods approach this problem via photon correlation spectroscopy, but remedy exists only for a few special cases. We introduce here a general criterion and a clearly defined measure to discriminate between NPs and dust particles. The experimental results show that, in contrast to photon correlation spectroscopy, hypothesis testing and the statistical moment analysis of the photon count distribution provides an accurate and precise way to characterize NPs and Brownian dynamics in the presence of dust. To demonstrate, analyses of silica, iron oxide, and gold NPs of low polydispersity are presented.

Probing nano-scale viscoelastic response in air and in liquid with dynamic atomic force microscopy.

We perform a comparative study of dynamic force measurements using an Atomic Force Microscope (AFM) on the same soft polymer blend samples in both air and liquid environments. Our quantitative analysis starts with calibration of the same cantilever in both environments. Intermodulation AFM (ImAFM) is used to measure dynamic force quadratures on the same sample. We validate the accuracy of the reconstructed dynamic force quadratures by numerical simulation of a realistic model of the cantilever in liquid. In spite of the very low quality factor of this resonance, we find excellent agreement between experiment and simulation. A recently developed moving surface model explains the measured force quadrature curves on the soft polymer, in both air and liquid.

Accuracy and prognostic value of sentinel lymph node biopsy in head and neck melanomas.

BACKGROUND: Debate remains around the accuracy and prognostic implications of sentinel lymph node biopsy (SLNB) for melanoma arising in the head and neck (HN) areas because several analyses have shown discordances between clinically predicted lymphatic drainage pathways and those identified by lymphoscintigraphy. This study assesses the accuracy and prognostic value of SLNB in this critical anatomic region. METHODS: Retrospective review of a prospectively collected melanoma database identified 331 patients with HN melanomas from January 2000 to December 2012. Primary end points included SLNB result, time to recurrence, site of recurrence, and survival. Multivariate models were constructed for analyses. RESULTS: A sentinel lymph node (SLN) was identified in all 331 patients. There were 59 patients with a positive SLN (17.8%) with a recurrence rate of 88.1% compared with 22.4% in SLN-negative patients (P < 0.0001). The 5-y overall survival was 91.2% for SLN-negative patients and 48.7% for SLN-positive patients (P < 0.0001). Patients with scalp melanoma had thicker lesions and an elevated risk of SLN positivity, recurrence, and death compared with those with other sites. Among the 272 SLN-negative patients, four patients developed regional nodal disease in the same basin and had undergone a previous SLNB procedure for a false-omission rate of 1.45%. Risks for false-negative SLN occurrences included thick and scalp melanomas. Multivariate analysis on prognostic factors affecting relapse-free survival showed positive SLNB status to be the most prognostic clinicopathologic predictor of recurrence (hazard ratio, 20.56; P < 0.0001). CONCLUSIONS: SLNB for patients with HN melanomas is an accurate procedure and has prognostic value.

Small nodular melanoma: the beginning of a life-threatening lesion. A clinical study on 11 cases.

AIMS AND BACKGROUND: Because of its high thickness, nodular melanoma often bears a poor prognosis. Thus, an earlier diagnosis of this type of lesion while it is still thin would be an important step in secondary prevention. The principal aim of the present study was to better define the initial clinical features of nodular melanoma to allow an early diagnosis. A secondary aim was to establish the prognosis of this type of lesion. METHODS: We retrospectively studied and illustrated the clinical features of 11 small (< or = 6 mm maximum diameter) cutaneous nodular melanomas seen and treated during a 10-year period. Prognostic characteristics of the various lesions were also described. RESULTS: The results of the study help to describe a small nodular melanoma as a dark and/or pink/red raised lesion, which may be evenly or unevenly colored, with well-defined borders, that often appears de novo. A correct clinical diagnosis was made in 7 of the cases. During a median follow-up of 6 years, none of the patients had local or distant relapses. CONCLUSIONS: Detection of small nodular melanoma is feasible by accurate visual inspection, provided that physicians are aware of this type of lesion and maintain the index of suspicion at a high level to bring about curative surgery.

Pure desmoplastic melanoma: a melanoma with distinctive clinical behavior.

OBJECTIVE: The purpose of the study was to evaluate the excision margin necessary for desmoplastic melanoma (DM). BACKGROUND: DM consists of 2 histologic subtypes, pure DM (PDM) and mixed DM (MDM), differing in extent of fibrotic component. We investigated clinical and therapeutic determinants of prognosis in these DM entities. METHODS: We reviewed 118 PDM and 124 MDM treated at our Institute over 25 years. Local relapse, distant metastasis, and survival were studied. RESULTS: Most (91.7%) distant metastases in PDM developed after 1 or more local recurrences; whereas distant metastasis usually (79.6%) occurred as first event in MDM. Overall mortality trends in relation to lesion-thickness-plus-excision-width differed for PDM (P = 0.014) but not MDM (P = 0.185). For PDM, 5-year crude cumulative incidence (CCI) of mortality was higher (40.0%) for thin tumors (≤ 2 mm thick) excised with 1 cm margin than those excised with 2 cm (14.8%); CCI of mortality for PDM > 2 mm thick excised with 2 cm margins (13.4%) was similar to that for thin PDM lesions excised with 2 cm (14.8%). CCI of local recurrence was also greater in PDM excised with 1 cm margins. In MDM, mortality increased with stage but was independent of excision width (CCI: 29.4% for ≤ 2 mm/2 cm, 31.3% for ≤ 2 mm/1 cm, and 48.3% for > 2 mm/2 cm); a similar trend was found for MDM distant metastases. CONCLUSIONS: In PDM, limited excision width is associated with significantly greater local recurrence and mortality; treatment should be excision with 2 cm margins even for thin lesions. MDM behaves similarly to other melanomas; treatment should follow guidelines on melanoma management.

Characterization of the Shape Anisotropy of Superparamagnetic Iron Oxide Nanoparticles during Thermal Decomposition.

Magnetosomes are near-perfect intracellular magnetite nanocrystals found in magnetotactic bacteria. Their synthetic imitation, known as superparamagnetic iron oxide nanoparticles (SPIONs), have found applications in a variety of (nano)medicinal fields such as magnetic resonance imaging contrast agents, multimodal imaging and drug carriers. In order to perform these functions in medicine, shape and size control of the SPIONs is vital. We sampled SPIONs at ten-minutes intervals during the high-temperature thermal decomposition reaction. Their shape (sphericity and anisotropy) and geometric description (volume and surface area) were retrieved using three-dimensional imaging techniques, which allowed to reconstruct each particle in three dimensions, followed by stereological quantification methods. The results, supported by small angle X-ray scattering characterization, reveal that SPIONs initially have a spherical shape, then grow increasingly asymmetric and irregular. A high heterogeneity in volume at the initial stages makes place for lower particle volume dispersity at later stages. The SPIONs settled into a preferred orientation on the support used for transmission electron microscopy imaging, which hides the extent of their anisotropic nature in the axial dimension, there by biasing the interpretation of standard 2D micrographs. This information could be feedback into the design of the chemical processes and the characterization strategies to improve the current applications of SPIONs in nanomedicine.

Biocompatible thermo- and magneto-responsive shape-memory polyurethane bionanocomposites.

Thermo- and magneto-responsive shape-memory bionanocomposites based on a bio-based polyurethane and magnetite nanoparticles were prepared. Due to the structure of the reactants, the behavior of the polyurethane matrix differs from common polyurethanes, since the soft segment was formed by a diisocyanate and a chain extender, whereas the macrodiol served as hard segment. The influence of the magnetite nanoparticles on the thermal and mechanical properties and the shape-memory behavior was studied. It was observed that magnetite nanoparticles interacted with macrodiol-rich domains and decreased the overall crystallinity of the material, although their presence did not affect the mechanical properties to a great extent. At the same time, the magnetite nanoparticles increased the shape fixity and contributed to shape recovery. The bionanocomposites exhibited magnetic behavior and could be easily heated in an alternating magnetic field, allowing fast and almost complete shape recovery. Preliminary cytotoxicity, hemocompatibility, and cell adhesion analysis suggest that the new materials are benign and potentially useful for biomedical applications.

Nanoparticle administration method in cell culture alters particle-cell interaction.

As a highly interdisciplinary field, working with nanoparticles in a biomedical context requires a robust understanding of soft matter physics, colloidal behaviors, nano-characterization methods, biology, and bio-nano interactions. When reporting results, it can be easy to overlook simple, seemingly trivial experimental details. In this context, we set out to understand how in vitro technique, specifically the way we administer particles in 2D culture, can influence experimental outcomes. Gold nanoparticles coated with poly(vinylpyrrolidone) were added to J774A.1 mouse monocyte/macrophage cultures as either a concentrated bolus, a bolus then mixed via aspiration, or pre-mixed in cell culture media. Particle-cell interaction was monitored via inductively coupled plasma-optical emission spectroscopy and we found that particles administered in a concentrated dose interacted more with cells compared to the pre-mixed administration method. Spectroscopy studies reveal that the initial formation of the protein corona upon introduction to cell culture media may be responsible for the differences in particle-cell interaction. Modeling of particle deposition using the in vitro sedimentation, diffusion and dosimetry model helped to clarify what particle phenomena may be occurring at the cellular interface. We found that particle administration method in vitro has an effect on particle-cell interactions (i.e. cellular adsorption and uptake). Initial introduction of particles in to complex biological media has a lasting effect on the formation of the protein corona, which in turn mediates particle-cell interaction. It is of note that a minor detail, the way in which we administer particles in cell culture, can have a significant effect on what we observe regarding particle interactions in vitro.

Nanoparticle-Cell Interaction: A Cell Mechanics Perspective.

Progress in the field of nanoparticles has enabled the rapid development of multiple products and technologies; however, some nanoparticles can pose both a threat to the environment and human health. To enable their safe implementation, a comprehensive knowledge of nanoparticles and their biological interactions is needed. In vitro and in vivo toxicity tests have been considered the gold standard to evaluate nanoparticle safety, but it is becoming necessary to understand the impact of nanosystems on cell mechanics. Here, the interaction between particles and cells, from the point of view of cell mechanics (i.e., bionanomechanics), is highlighted and put in perspective. Specifically, the ability of intracellular and extracellular nanoparticles to impair cell adhesion, cytoskeletal organization, stiffness, and migration are discussed. Furthermore, the development of cutting-edge, nanotechnology-driven tools based on the use of particles allowing the determination of cell mechanics is emphasized. These include traction force microscopy, colloidal probe atomic force microscopy, optical tweezers, magnetic manipulation, and particle tracking microrheology.

Combined approach for the biomechanical characterization of skin lesions.

Melanocytic nevi are common benign skin lesions, known as moles, due to proliferation of melanocytes, the pigmented skin cells. The uncontrolled growth of these cells leads instead to cutaneous malignant melanoma, an aggressive tumour whose rate of survival dramatically increases if early diagnosis is provided. Alteration on the mechanical properties of the skin in presence of lesions has been assessed. In this context, we aim at developing a combined approach consisting of an experimental and a computational study to biomechanically characterize the skin and both malign and benign skin lesions (i.e., nevi and malignant melanoma). In particular, the former study is performed to evaluate the biomechanical response of the different skin layers after the application of a displacement field and relies on a multi-scale strategy, ranging from the tissue level to the cellular level. Computational models will be tuned against experimental data (e.g., confocal laser scanning microscopy data) to estimate the mechanical properties of the different layers of the skin and the skin lesions. In particular, the confocal laser scanning microscopy is able to provide non-invasive histomorphological analysis of skin in vivo. The integration of the experimental and the computational results will allow the evaluation of possible alterations of the local mechanical properties occurring in case of pathological condition.

Transcriptional profiling of melanoma sentinel nodes identify patients with poor outcome and reveal an association of CD30(+) T lymphocytes with progression.

Sentinel lymph nodes set the stance of the immune system to a localized tumor and are often the first site to be colonized by neoplastic cells that metastasize. To investigate how the presence of neoplastic cells in sentinel lymph nodes may trigger pathways associated with metastatic progression, we analyzed the transcriptional profiles of archival sentinel node biopsy specimens obtained from melanoma patients. Biopsies from positive nodes were selected for comparable tumor infiltration, presence or absence of further regional node metastases, and relapse at 5-year follow-up. Unsupervised analysis of gene expression profiles revealed immune response to be a major gene ontogeny represented. Among genes upregulated in patients with progressing disease, the TNF receptor family member CD30/TNFRSF8 was confirmed in biopsy specimens from an independent group of patients. Immunohistochemical analysis revealed higher numbers of CD30(+) lymphocytes in nodes from progressing patients compared with nonprogressing patients. Phenotypic profiling demonstrated that CD30(+) lymphocytes comprised a broad population of suppressive or exhausted immune cells, such as CD4(+)Foxp3(+) or PD1(+) subpopulations and CD4(-)CD8(-) T cells. CD30(+) T lymphocytes were increased in peripheral blood lymphocytes of melanoma patients at advanced disease stages. Our findings reinforce the concept that sentinel nodes act as pivotal sites for determining progression patterns, revealing that the presence of CD30(+) lymphocytes at those sites associate positively with melanoma progression.

The use of polytetrafluoroethylene to facilitate the vascular access in recurrent melanoma to limbs.

INTRODUCTION: Melanoma with recurrent loco-regional metastases to limbs often makes difficult a second surgical approach because of the adhesions affecting the vascular access. Our aim was to evaluate whether the placement of a polytetrafluoroethylene (PTFE) membrane around vessels might facilitate a surgical re-approach. PRESENTATION OF CASE: We reported a case of a 64-year-old male with a melanoma on the left foot who developed in transit metastases after LND. While performing the inguinopelvic LND we coated the iliac vessels with PTFE patch to facilitate the vascular access in case of re-intervention for a ILP. In the second surgical approach we made a cutaneous incision in the left iliac region and we proceeded through the subcutaneous tissue until detection of iliac vessels, more clearly visible because of the PTFE patch fixed around vascular walls to minimize adhesions. We removed the PTFE coating and easily performed arteriotomy and venotomy for the completion of the ILP. DISCUSSION: This case report seems to demonstrate the efficacy of a PTFE membrane applied in a patient around iliac vessels during inguinopelvic dissection, to reduce adhesion density. In fact this membrane provided a barrier to adhesions of the iliac vessels, decreasing the risk of vascular injury thereby facilitating a subsequent vascular access. Re-coating of the iliac vessels with PTFE could be preparatory to a better identification of the vascular structures in cases of a surgical re-approach. CONCLUSION: The use of PTFE effectively simplifies the second approach to vessels in event of a melanoma metastasizing to limbs.

Radical dissection after positive groin sentinel biopsy in melanoma patients: rate of further positive nodes.

The aim of this retrospective study was to analyze the incidence of further nonsentinel node metastases at completion lymphadenectomy of the groin after a positive sentinel node biopsy to evaluate whether radical dissection remains the treatment of choice for these patients. Patients treated at the National Cancer Institute of Milan between January 1999 and December 2006 were reviewed retrospectively. The analysis included patients with a diagnosis of positive sentinel node biopsy of the groin (clinically negative) who underwent completion groin, iliac, and obturatory dissections. The primary melanoma was located on the lower extremities and trunk in 82.5 and 17.5%, respectively. The median follow-up was more than 30 months. The number of positive sentinel nodes was considered, as well as the size and location of the metastases (micro vs. macro). After radical dissection, the number and the location (groin, iliac, or groin+iliac nodes) of further nonsentinel node metastases were analyzed. The frequency of further nonsentinel node metastases at completion of groin dissection was correlated to Breslow's thickness and to the characteristics of the positive sentinel node biopsy. A total of 1581 patients with primary melanoma (>1 mm, or Clark's IV-V) underwent lymphatic mapping and sentinel node biopsy: 752 patients had sentinel node biopsy at the groin basin; among these, 150 (20%) patients presented positive sentinel node biopsy and underwent completion radical dissection (groin, obturatory, and external iliac+obturatory radical node dissections). We found further positive nonsentinel node metastases in 36 of 150 (24%) patients, 69% (25 of 36) of which were located in the iliac-obturator area and 31% in the groin area only: 16 patients (44.4%) had one additional metastatic node and seven patients (19.4%) had two, whereas 13 (36.1%) had three or more. In 22 cases (61.1%), the sentinel node showed a macrometastasis (>2 mm deposit in the node) and in 14 cases (38.9%) a micrometastasis (<2 mm deposit). In conclusion, there is clear evidence that patients with a positive sentinel node biopsy could have further positive nonsentinel node metastases (in our series, 24%). Although it is well known that the impact of sentinel node biopsy on survival of melanoma patients has yet to be defined, to obtain a clear nodal basin and regional control a groin+iliac-obturatory radical node dissection remains an appropriate procedure in the presence of a positive sentinel node biopsy at the groin level. This could be considered a standard treatment until new data, provided by ongoing studies, indicate new parameters for selecting patients for completion lymph node dissection.