Gerardo Marino

Smart materials as scaffolds for tissue engineering

In this review, we focused our attention on the more important natural extracellular matrix (ECM) molecules (collagen and fibrin), employed as cellular scaffolds for tissue engineering and on a class of semi‐synthetic materials made from the fusion of specific oligopeptide sequences, showing biological activities, with synthetic materials. In particular, these new “intelligent” scaffolds may contain oligopeptide cleaving sequences specific for matrix metalloproteinases (MMPs), integrin binding domains, growth factors, anti‐thrombin sequences, plasmin degradation sites, and morphogenetic proteins. The aim was to confer to these new “intelligent” semi‐synthetic biomaterials, the advantages offered by both the synthetic materials (processability, mechanical strength) and by the natural materials (specific cell recognition, cellular invasion, and the ability to supply differentiation/proliferation signals). Due to their characteristics, these semi‐synthetic biomaterials represent a new and versatile class of biomimetic hybrid materials that hold clinical promise in serving as implants to promote wound healing and tissue regeneration.

A critical overview of ESEM applications in the biological field

Scanning Electron Microscope (SEM) is a powerful research tool, but since it requires high vacuum conditions, the wet materials and biological samples must undergo a complex preparation that limits the application of SEM on this kind of specimen and often causes the introduction of artifacts. The introduction of Environmental Scanning Electron Microscope (ESEM), working in gaseous atmosphere, represented a new perspective in biological research. Despite the fact that many biological applications have demonstrated the convenience of ESEM, the full potentialities of this technology are still under investigation. In this review, the exploration of the recent literature data confronted with the first results obtained in our experimental work suggest that ESEM represents an important extension of conventional scanning microscopy.

Therapy with autologous adipose-derived regenerative cells for the care of chronic ulcer of lower limbs in patients with peripheral arterial disease.

BACKGROUND An ulcer is a trophic lesion with loss of tissue that often has a multifactorial genesis. It typically diverges from the physiologic processes of regeneration because it rarely tends to heal spontaneously. In this study, we used purified adipose-derived stem and regenerative cells (ADRCs) extracted from autologous fat, for the care of chronic ulcers of the lower limbs of arteriopathic patients. The primary objective of this study was complete re-epithelization of chronic ulcers; the secondary objective was a decrease in diameter and depth. METHODS From January 2010 to January 2012, 20 patients with peripheral arterial disease, with an ankle-brachial index between 0.30-0.40, in the age range 60-70 y (14 men and six women), with chronic ulcers of the lower limb, were involved in the study. Only 10 arteriopathic patients (seven men and three women) with chronic ulcers of the lower limb were surgically treated. Using the Celution system, we isolated a solution of ADRCs in about 150 min. The isolated cells were injected through a 10-mL syringe into the edges of the ulcer, taking care to spread it in all directions. Using a small amount of Celution extract, we performed cell characterization by flow cytometry analysis and cell viability assay. RESULTS We monitored patients treated with ADRC or untreated at 4, 10, 20, 60, and 90 d. In all cases treated with ADRC, we found a reduction in both diameter and depth of the ulcer, which led to a decrease in pain associated with the ulcer process. In six of 10 cases there was complete healing of the ulcer. Characterization of the cells by FACS clearly showed that the ADRC cells contained adipose-derived stem cells. Viability assays demonstrated that partial or total closure of the ulcer was attributable exclusively to ADRC cells present in the Celution extract, and not to growth factors extracted during the process of purification of the Celution and injected together with the cells. CONCLUSIONS For the first time, the Celution method has been applied for the care of chronic ulcers in the lower extremity of patients with peripheral arterial disease. Our results demonstrate that the technique is feasible for autologous cell application and is not associated with adverse events. Moreover, the transplantation of autologous stem cells extracted with Celution may represent a valuable method for the treatment of chronic ulcers in lower limbs of arteriopathic patients.

Growth and endothelial differentiation of adipose stem cells on polycaprolactone

Adipose tissue is a readily available source of multipotent adult stem cells for use in tissue engineering/regenerative medicine. Various growth factors have been used to stimulate acquisition of endothelial characteristics by adipose-derived stem cells (ADSC). Herein, we study the growth and endothelial differentiation potential of ADSC seeded onto a porous polycaprolactone (PCL) scaffold. The objective of this study is to demonstrate that PCL is a good material to be used as a scaffold to support reconstruction of new endothelial tissue using adipose stem cells. We found that undifferentiated ADSC adhere and grow on PCL. We show that, after culture in endothelial differentiation medium, ADSC were positive to LDL uptake and expressed molecular markers characteristic of endothelial cells (CD31; eNOS and vWF). In addition, our study defines the time required for the differentiation of ADSC directly onto PCL. This study suggests that PCL can be used as a scaffold to generate endothelial tissue in vitro. PLC has excellent mechanical properties and a slow degradation rate. Moreover, based on our results, we propose that PCL could be used to graft scaffolds coated with endothelial cells derived from ADSC stem cells. Endothelial cells-coated PCL could find several applications to replace damaged area of the body; for example, a possible use could be the generation of vascular grafts.

Cell surface protein detection with immunogold labelling in ESEM: Optimisation of the method and semi-quantitative analysis

In this work we used a combination of immunogold labelling (IGL) and environmental scanning electron microscopy (ESEM) to detect the presence of a protein on the cell surface. To achieve this purpose we chose as experimental system 3T3 Swiss Albino Mouse Fibroblasts and galectin‐3. This protein, whose sub‐cellular distribution is still under discussion, is involved in a large number of cell physiological and pathological processes. IGL technique has been utilised by many authors in combination with SEM and TEM to obtain the identification/localisation of receptors and antigens, both in cells and tissues. ESEM represents an important tool in biomedical research, since it does not require any severe processing of the sample, lowering the risk of generating artefacts and interfere with IGL procedure. The absence of metal coating could yield further advantages for our purpose as the labelling detection is based on the atomic number difference between Nanogold spheres and the biological material. Using the gaseous secondary electron detector (GSED) compositional contrast is easily revealed by the backscattered electrons component of the signal. In spite of this fact, only few published papers present a combination of ESEM and IGL. Hereby we present our method, optimised to improve the intensity and the specificity of the labelling signal, in order to obtain a semi‐quantitative evaluation of the labelling signal. J. Cell. Physiol. 214: 769–776, 2008.

Adhesion and proliferation of fibroblasts on RF plasma‐deposited nanostructured fluorocarbon coatings: Evidence of FAK activation

We used combined plasma‐deposition process to deposit smooth and nanostructured fluorocarbon coatings on polyethylenethereftalate (PET) substrates, to obtain surfaces with identical chemical composition and different roughness, and investigate the effect of surface nanostructures on adhesion and proliferation of 3T3 Swiss Albino Mouse fibroblasts. Untreated PET and polystyrene (PS) were used as controls for cell culture. We have found that the statistically significant increase of cell proliferation rate and FAK (a nonreceptor tyrosine kinase) activation detected on ROUGH fluorocarbon surfaces is due to the presence of nanostructures. Changes in cytoskeletal organization and phospho FAK (tyr 397) localization were evident after 60 min on cells adhering to ROUGH surfaces. This change was characterized by the formation of actin stress fibers along lamellar membrane protrusion instead of usual focal contacts. Also the morphology of the adhering fibroblasts (60 min) adhering on ROUGH surfaces was found quite different compared to cells adhering on smooth ones. J. Cell. Physiol.

Use of polycaprolactone (PCL) as scaffolds for the regeneration of nerve tissue

Adipose tissue is an easily accessible source of stem cells for use in tissue regenerative medicine. In the literature, different methods have been used to stimulate acquisition of neuronal characteristics by adipose-derived stem cells (ADSC). Herein we study the growth and neuronal differentiation potential of ADSC seeded onto a porous polycaprolactone (PCL) scaffold. The objective of this study is to demonstrate that PCL can be used as a scaffold to support reconstruction of new nervous tissue using adipose stem cells. We have previously shown that undifferentiated ADSC adhere and grow on PCL. Herein we show that, after culture on PCL in neuronal differentiation medium, ADSC expressed molecular markers characteristic of neuronal cells (β-tubulin-III, Neuron-Specific Enolase (NSE), Nestin) and secrete brain derived neurotrophic factor (BDNF) and nerve growth factor (NGF). This study suggests that PCL can be used as a scaffold to generate nervous tissue in vitro. PLC has excellent mechanical properties and a slow degradation rate. Moreover, on the basis of our results, we propose that PCL could be used for to make in vitro, scaffold coated with neuronal cells derived from Adipose stem cells (ADSC). Neuronal cells-coated PCL could find several applications to replace damaged area of ​​the body; for example, a possible use could be the generation of nerves.

Galectin-3 expression in thyroid fine needle cytology (t-FNAC) uncertain cases: validation of molecular markers and technology innovation.

Thyroid cancer is not very common, accounting for 1–2% of all cancers, with a population incidence of about 0.004%. Currently, the ability to discriminate between follicular adenoma and carcinoma represents the major challenge in preclinical diagnosis of thyroid proliferative lesions. Better discrimination between the two would help avoid unnecessary thyroidectomy and save valuable resources. Over the years, galectin‐3 (Gal‐3) has been proposed as a diagnostic marker with varied success. In this paper, we used Environmental Scanning Electron Microscopy Immunogold Labelling (ESEM‐IGL) to investigate the expression of Gal‐3 on Thin‐Prep fine needle aspiration cytology (FNAC). We optimized the ESEM‐IGL method on thyroid cell lines (RO‐82 and FTC‐133) comparing our membrane Gal‐3 labeling data with Western blot. We evaluated 183 thyroid FNAC from Italian patients with a uncertain pre‐surgical diagnosis. ESEM‐IGL method marker sensitivity is 71.2%, while specificity is 53.3% and diagnostic efficacy is 61.2%. Our results confirmed that Gal‐3 expression is associated with situations of hypertrophy and/or cellular hyperproliferation, pathophysiological situations common both to adenomas and to thyroid carcinomas. The innovation of thyroid FNAC Thin‐Prep ESEM‐IGL shows the levels of Gal‐3 immunolabeling clearly, even through the individual cells of a thyroid nodule. However, Gal‐3 alone, as a molecular marker of thyroid cancer, can still have a limited application in pre‐surgery diagnosis. J. Cell. Physiol.

Immunogold labelling in environmental scanning electron microscopy: applicative features for complementary cytological interpretation

We have combined environmental scanning electron microscopy (ESEM) and immunogold labelling (IGL) for the analysis of cell morphology and surface protein detection on human fine needle aspiration, which is processed in thin uniform monolayer (a single layer of cells) on a glass slide by Thin Prep technology.

Galactosyl derivative of Nω-nitro-L-arginine: Study of antiproliferative activity on human thyroid follicular carcinoma cells

The methyl ester prodrug of Nω‐nitro‐L‐arginine (L‐NAME) has been reported to exert anticancer effects against several human tumors, including thyroid carcinoma, by inhibiting nitric oxide synthase (NOS). However, chronic administration of L‐NAME has often led to adverse events causing cardiovascular alterations due to its potential toxic effect. Here we report for the first time the synthesis of the galactosyl ester prodrug of Nω‐nitro‐L‐arginine, NAGAL, a prodrug capable of inhibiting NOS more efficiently and with fewer adverse events than its parent drug. For this purpose RO82‐W‐1, a thyroid cell line derived from human follicular carcinoma, was used. MTT test results showed that NAGAL affected cell viability to a significantly greater extent than did L‐NAME. Moreover, fluorescence activated cell sorter (FACS) analyses revealed that NAGAL, compared to L‐NAME, was able to reduce nitric oxide (NO) production as well as increase the percentage of apoptotic thyreocytes. Western blot further confirmed the reduction in NOS‐II expression by NAGAL. Finally, by using the LC–MS technique, we found that NAGAL elicited a higher increase in Nω‐nitro‐L‐arginine (NA) concentration than did L‐NAME. Thus, this study suggests that NAGAL could be considered a potential therapeutic tool for those pathologies involving an overproduction of NO, including thyroid carcinoma. J. Cell. Physiol. 221: 440–447, 2009.