281 results on '"Ghersi, G."'
Search Results
152. Activation of EDTA-resistant Gelatinases in Malignant Human Tumors
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Masako Mitsumata, Yoshiyuki Mori, Donghai Chen, Jahn M. Nesland, Wei Zeng, Alanna Kennedy, Zhenhe Suo, Qiang Zhao, Wen-Tien Chen, Giulio Ghersi, Michael L. Pearl, Naoko Hirashima, Ah-Kau Ng, Tetsu Yamane, Jaw-Yuan Wang, Yuhuan Qiao, Meng-zhen Zhang, DONGHAI CHEN, ALANNA KENNEDY, JAW-YUAN WANG, WEI ZENG, QIANG ZHAO, MICHAEL PEARL, MENGZHEN ZHANG, ZHENHE SUO, JAHN M NESLAND, YUHUAN QIAO, AH-KAU NG, NAOKO HIRASHIMA, TETSU YAMANE, YOSHIYUKI MORI, MASAKO MITSUMATA, GHERSI G, and WEN-TIEN CHEN
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Models, Molecular ,Cancer Research ,Proteases ,Protein Conformation ,Dipeptidyl-peptidase activity ,In situ hybridization ,Biology ,Dipeptidyl peptidase ,Article ,Cell Line ,Fibroblast activation protein, alpha ,Neoplasms ,Endopeptidases ,medicine ,Gelatinase ,Animals ,Humans ,Dipeptidyl-Peptidases and Tripeptidyl-Peptidases ,Edetic Acid ,Serine Endopeptidases ,Membrane Proteins ,Haplorhini ,medicine.disease ,Recombinant Proteins ,seprase, fibroblast activation protein alpha (FAP-α), gelatinase, activation, malignant tumor ,Enzyme Activation ,Oncology ,Biochemistry ,Gelatinases ,Cancer research ,Immunohistochemistry ,Adenocarcinoma - Abstract
Among the many proteases associated with human cancer, seprase or fibroblast activation protein α, a type II transmembrane glycoprotein, has two types of EDTA-resistant protease activities: dipeptidyl peptidase and a 170-kDa gelatinase activity. To test if activation of gelatinases associated with seprase could be involved in malignant tumors, we used a mammalian expression system to generate a soluble recombinant seprase (r-seprase). In the presence of putative EDTA-sensitive activators, r-seprase was converted into 70- to 50-kDa shortened forms of seprase (s-seprase), which exhibited a 7-fold increase in gelatinase activity, whereas levels of dipeptidyl peptidase activity remained unchanged. In malignant human tumors, seprase is expressed predominantly in tumor cells as shown by in situ hybridization and immunohistochemistry. Proteins purified from experimental xenografts and malignant tumors using antibody- or lectin-affinity columns in the presence of 5 mmol/L EDTA were assayed for seprase activation in vivo. Seprase expression and activation occur most prevalently in ovarian carcinoma but were also detected in four other malignant tumor types, including adenocarcinoma of the colon and stomach, invasive ductal carcinoma of the breast, and malignant melanoma. Together, these data show that, in malignant tumors, seprase is proteolytically activated to confer its substrate specificity in collagen proteolysis and tumor invasion. (Cancer Res 2006; 66(20): 9977-85)
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- 2006
153. Espressione ed associazione di enzimi proteolitici durante la transizione epitelio/mesenchima in cellule endoteliali
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SALAMONE, Monica, VESCO, Viviana, NASCA, Mario, GHERSI, Giulio, SALAMONE M, VESCO V, NASCA M, and GHERSI G
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- 2006
154. Differential expression of proteolytic enzymes during epithelial-mesenchymal transaction of endothelial cells
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VESCO, Viviana, GIANCARLO, Raffaele, GHERSI, Giulio, SIRAGUSA A, AND, VESCO V, SIRAGUSA A, GIANCARLO R, AND, and GHERSI G
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- 2006
155. The protease complex consisting of dipeptidyl peptidase IV and seprase plays a role in the migration and invasion of human endothelial cells in collagenous matrices
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Yunyun Yeh, Monica Salamone, Stanley Zucker, Qiang Zhao, Giulio Ghersi, Wen-Tien Chen, GHERSI, G, ZHAO, Q, SALAMONE, M, YEH, Y, ZUCKER, S, and AND CHEN, WT
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Cancer Research ,Proteases ,Dipeptidyl Peptidase 4 ,medicine.medical_treatment ,Biology ,Article ,Dipeptidyl peptidase ,Extracellular matrix ,Fibroblast activation protein, alpha ,Cell Movement ,medicine ,Humans ,Serine protease ,Protease ,Serine Endopeptidases ,Antibodies, Monoclonal ,Endothelial Cells ,Cell migration ,dipeptidyl peptidase IV, CD26, seprase, fibroblast activation protein α, endothelial cell, migration, angiogenesis ,Extracellular Matrix ,Up-Regulation ,Endothelial stem cell ,Oncology ,Biochemistry ,biology.protein ,Gelatin ,Cell Surface Extensions ,Collagen ,Peptide Hydrolases - Abstract
Dipeptidyl peptidase IV (DPP4/CD26) and seprase/fibroblast activation protein α are homologous type II transmembrane, homodimeric glycoproteins that exhibit unique prolyl peptidase activities. Human DPP4 is ubiquitously expressed in epithelial and endothelial cells and serves multiple functions in cleaving the penultimate positioned prolyl bonds at the NH2 terminus of a variety of physiologically important peptides in the circulation. Recent studies showed a linkage between DPP4 and down-regulation of certain chemokines and mitogenic growth factors, and degradation of denatured collagens (gelatin), suggesting a role of DPP4 in the cell invasive phenotype. Here, we found the existence of a novel protease complex consisting of DPP4 and seprase in human endothelial cells that were activated to migrate and invade in the extracellular matrix in vitro. DPP4 and seprase were coexpressed with the three major protease systems (matrix metalloproteinase, plasminogen activator, and type II transmembrane serine protease) at the cell surface and organize as a complex at invadopodia-like protrusions. Both proteases were colocalized at the endothelial cells of capillaries, but not large blood vessels, in invasive breast ductal carcinoma in vivo. Importantly, monoclonal antibodies against the gelatin-binding domain of DPP4 blocked the local gelatin degradation by endothelial cells in the presence of the major metallo- and serine protease systems that modified pericellular collagenous matrices and subsequent cell migration and invasion. Thus, we have identified a novel mechanism involving the DPP4 gelatin-binding domain of the DPP4-seprase complex that facilitates the local degradation of the extracellular matrix and the invasion of the endothelial cells into collagenous matrices. (Cancer Res 2006; 66(9); 4652-61)
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- 2006
156. Proteolytic enzymes expression during epithelial/mesenchymal transaction of endothelial cells
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GHERSI, Giulio and GHERSI G
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- 2006
157. Comparative analyses of endothelial and tumoral cells cultured in 2D and 3D type-1 collagen fibril gels
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SALAMONE M, VESCO, Viviana, TAVERNA, Simona, VITTORELLI, Maria Letizia, GHERSI, Giulio, SALAMONE M, VESCO V, TAVERNA S, VITTORELLI ML, and GHERSI G
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- 2006
158. Clustering of type-II transmembrane serine proteases (TTSPs) and matrix metalloproteases (MMPs) in the cell surface and shed membrane vesicles of active endothelial cells
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SALAMONE, Monica, NASCA, Mario, VITTORELLI, Maria Letizia, GHERSI, Giulio, L. PITARRESI, W. T. CHEN, SALAMONE M, M NASCA, L PITARRESI, ML VITTORELLI, W-T CHEN, and GHERSI G
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- 2005
159. Nourrir 9 milliards d'hommes
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Maire, Bernard, Delpeuch, Francis, and Ghersi, G. (dir.)
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OBESITE ,SECURITE ALIMENTAIRE ,TRANSITION ALIMENTAIRE ,COUTUME ALIMENTAIRE ,CHANGEMENT ALIMENTAIRE ,MALNUTRITION ,DESEQUILIBRE ALIMENTAIRE ,CANCER ,NIVEAU DE VIE ,CONSOMMATION ALIMENTAIRE ,MALADIE CHRONIQUE ,DIABETE ,INDUSTRIALISATION ,MONDIALISATION - Published
- 2005
160. Proteolytic enzymes in membrane domains of endothelial cells cultured in type-I collagen 3D gels
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SALAMONE, Monica, NASCA, Mario, VESCO, Viviana, GHERSI, Giulio, VITTORELLI, Maria Letizia, SALAMONE M, NASCA M, VESCO V, GHERSI G, and VITTORELLI M L
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- 2005
161. Nourrir 9 milliards d'hommes
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Le Bihan, Geneviève, Delpeuch, Francis, and Ghersi, G. (dir.)
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BESOIN NUTRITIONNEL ,DROIT ,SYSTEME ALIMENTAIRE ,ALIMENTATION HUMAINE ,SECURITE ALIMENTAIRE ,DISPONIBILITE DES ALIMENTS ,ALLAITEMENT MATERNEL ,MALNUTRITION ,PRIX ,POLITIQUE ALIMENTAIRE - Published
- 2005
162. Meccanismi non convenzionali di secrezione dell’FGF-2
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TAVERNA, Simona, RIGOGLIUSO, Salvatrice, GHERSI, Giulio, VITTORELLI, Maria Letizia, and Taverna, S, Rigogliuso, S, Ghersi, G, Vittorelli, ML
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FGF-2 - Published
- 2004
163. CLUSTERING OF PROTEOLYTIC ENZYMES ON SPECIALIZED PLASMA MEMBRANE DOMAINS OF ENDOTHELIAL CELLS. ROLE IN ANGIOGENESIS
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GHERSI, Giulio, NASCA, Mario, VITTORELLI, Maria Letizia, SALAMONE M, PITARRESI L, GHERSI G, SALAMONE M, NASCA M, PITARRESI L, and VITTORELLI ML
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- 2004
164. Identification and characterization of a constitutive HSP75 in sea urchin embryos
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Gabriella Sconzo, Giuseppina Capra, D. Cascino, Giulio Ghersi, Giovanni Giudice, Gabriele Amore, Sconzo, G, Amore, G, Capra, G, Giudice, G, Cascino, D, and Ghersi, G
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Male ,Cytoplasm ,Embryo, Nonmammalian ,Recombinant Fusion Proteins ,Blotting, Western ,Molecular Sequence Data ,Biophysics ,Embryonic Development ,Receptors, Cell Surface ,HSP, sea urchin, embryos ,Biology ,Biochemistry ,Oogenesis ,biology.animal ,Cell cortex ,Animals ,HSP70 Heat-Shock Proteins ,Amino Acid Sequence ,Isoelectric Point ,education ,Molecular Biology ,Sea urchin ,Peptide sequence ,education.field_of_study ,Sequence Homology, Amino Acid ,Ovary ,Embryo ,Cell Biology ,Sperm receptor ,Immunohistochemistry ,Spermatozoa ,Molecular biology ,Sperm ,Fusion protein ,Molecular Weight ,Gastrulation ,Sea Urchins ,Oocytes ,Electrophoresis, Polyacrylamide Gel ,Female ,Plasmids - Abstract
An antiserum against a hsp of the 70-kDa family was prepared, by means of a fusion protein, which was able to detect a constitutive 75-kDa hsc in the sea urchinP. lividus.This hsc was present both during oogenesis and at all developmental stages. A two-dimensional electrophoresis has revealed four isolectric forms of this 75-kDa hsc. The amino acid sequence of the fragment used to prepare the anti-hsp70 antibodies revealed a 43% identity with the corresponding part of sea urchin sperm receptor, and in mature eggs a brighter immunofluorescence was seen all around the cell cortex where the receptor for sea urchin sperm is localized. In oocytes the hsp75 was localized in the cytoplasms but not in the nuclei. In the embryos a higher hsp75 concentration was found in the portion facing the lumen of the cells which invaginate at gastrulation.
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- 1997
165. The role of poly(ethylenglycol) molecular weight on viability and cell adhesion in polylactide based scaffolds
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Lo Re, G., Roberto Scaffaro, Salvatrice Rigogliuso, Giulio Ghersi, Lo Re, G, Scaffaro, R, Rigogliuso, S, and Ghersi, G
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Scaffolds, PLA, PEG, cell viability
166. Bi-layer PCL/PLA scaffold prepared by melt for interface tissue engineering
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Luigi Botta, Roberto Scaffaro, Francesco Lopresti, Salvatrice Rigogliuso, Giulio Ghersi, Botta, L., Scaffaro, R., Lopresti, F., Rigogliuso, S., and Ghersi, G.
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Melt mixing ,Particulate leaching ,Interface tissue engineering ,Pore size gradient ,Functionally graded scaffold - Abstract
The development of porous multilayer devices allow controlling chemical, physical and mechanical properties by tuning the properties of each single layer. For instance, this feature is of main concern for the production of porous devices designed to regenerate diseased zones at the interface of tissue presenting intrinsic anisotropic structures that gradually change from one tissue to another. In this context, synthetic biodegradable polymers commonly used biomedical applications include polylactic acid (PLA) and polycaprolactone (PCL). In this work, a novel bi-layered multiphasic scaffold (BLS) is presented. It is composed by a PLA-layer presenting pore size in the range of 90-110 μm while the other layer was made of PCL with pores ranging from 5 to 40 μm. The sponges were prepared by combining melt mixing and particulate leaching. In brief, PLA or PCL with PEG and NaCl were fed to a batch mixer and processed until a constant value of torque was achieved. The blends were then compression molded in a laboratory press in appropriate cylindrical molds. Finally, the porogen parts of the blends (NaCl and PEG) were removed by selective leaching in demineralized water. BLS have been characterized from a morphological and a mechanical point of view. Fibroblasts and osteoblasts were cultured and co-cultured in order to investigate the cells permeation trough the different layers.
167. Chemical hydrogels based on a hyaluronic acid-graft-α-elastin derivative as potential scaffolds for tissue engineering
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Giulio Ghersi, Gaetano Giammona, Salvatrice Rigogliuso, Calogero Fiorica, Giovanna Pitarresi, Fabio Salvatore Palumbo, Palumbo, FS, Pitarresi, G, Fiorica, C, Rigogliuso, S, Ghersi, G, and Giammona, G
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Materials science ,Magnetic Resonance Spectroscopy ,Bioengineering ,Biomaterials ,chemistry.chemical_compound ,Hydrolysis ,Tissue engineering ,Enzymatic hydrolysis ,Hyaluronic acid ,Polymer chemistry ,medicine ,Animals ,Humans ,Hyaluronic Acid ,Skin ,biology ,Tissue Engineering ,Tissue Scaffolds ,Hydrogels ,Fibroblasts ,Grafting ,Elastin ,Rats ,chemistry ,hydrogel, hyaluronic acid, elastin, scaffold, tissue engineering ,Mechanics of Materials ,Settore CHIM/09 - Farmaceutico Tecnologico Applicativo ,Self-healing hydrogels ,biology.protein ,Microscopy, Electron, Scanning ,Swelling ,medicine.symptom - Abstract
In this work hyaluronic acid (HA) functionalized with ethylenediamine (EDA) has been employed to graft α-elastin. In particular a HA-EDA derivative bearing 50 mol% of pendant amino groups has been successfully employed to produce the copolymer HA-EDA-g-α-elastin containing 32% w/w of protein. After grafting with α-elastin, remaining free amino groups reacted with ethylene glycol diglycidyl ether (EGDGE) for producing chemical hydrogels, proposed as scaffolds for tissue engineering. Swelling degree, resistance to chemical and enzymatic hydrolysis, as well as preliminary biological properties of HA-EDA-g-α-elastin/EGDGE scaffold have been evaluated and compared with a HA-EDA/EGDGE scaffold. The presence of α-elastin grafted to HA-EDA improves attachment, viability and proliferation of primary rat dermal fibroblasts and human umbilical artery smooth muscle cells. Biological performance of HA-EDA-g-α-elastin/EGDGE scaffold resulted comparable to that of a commercial collagen type I sponge (Antema®), chosen as a positive control.
168. EVALUATION OF STABILITY AND ENZYMATIC ACTIVITIES OF PROTEOLYTIC ENZYMES USED IN PANCREATIC ISLET TRANSPLANTATION
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Salamone, M., Gregorio Seidita, Cuttitta, A., Salvatrice Rigogliuso, Bertuzzi, F., Giulio Ghersi, Salamone, M, Seidita, G, Cuttitta, A, Rigogliuso, S, Bertuzzi, F, and Ghersi, G
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Diabet type 1 ,Settore BIO/10 - Biochimica ,Collagenase ,Clostridium hystoliticum ,Cell transplantation - Abstract
In pancreatic islets purification, for cell therapy applications, the major enzymes used are obtained from Clostridium hystoliticum; class I and class II collagenases (Coll-G and Coll-H). In a well defined composition Coll-G/Coll-H together enzymes working on hydrophobic amminoacid, the neutral protease (Dispase) or the thermolysin (Thermostable Neutral Protease), are used in Langerhans islets purification. By electrophoresis and gelatin zymography approaches, in combination to densitometry quantitative valuation we have compared in composition, stability and autodigestion processes C. hystoliticum collagenases, Neutral protease and Thermolysin from two different producers, Roche and Serva. On analyzed enzymes we have, also, explored about their stability when in solution in relation to storage processes at different temperatures (-20°C; 0°C and r.t.) and on their type-I collagen degradation activity when used at different temperatures (working temperatures: 25°C; 30°C; 37°C and 42 °C). Our results revealed an heterogeneous composition of the different blend of collagenases respect the HPLC profiles publish by vendors and an heterogeneity by lot to lot; moreover, we observed contamination from other enzymes in analysed samples. In fact, in gelatin zymographyes several digestive bands were catalytic active showing very high complex degradative patterns. Additionally, neutral protease from Serva contains gelatinolytic activities; not detected in thermolysin Roche. These data strongly suggest that a not controlled digestive processes can be associate to contaminants present in the blends and also due to auto-catalytic processes. In particular, the presence of low molecular weight gelatine/degradative activities denote the islets digestion process can not be control. Generation of recombinant collagenases probably could be the solution to overcome the variability in the extractive processes.
169. PLLA-fibrin scaffolds for Vascular Tissue Engineering
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Francesco Carfì Pavia, Salvatrice Rigogliuso, Vincenzo La Carrubba, Giulio Ghersi, Valerio Brucato, Carfì Pavia, F, Rigogliuso, S, La Carrubba, V, Ghersi, G, and Brucato, V
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Fibrin ,Settore ING-IND/24 - Principi Di Ingegneria Chimica ,Settore ING-IND/22 - Scienza E Tecnologia Dei Materiali ,Settore BIO/10 - Biochimica ,Poly Lactic Acid ,Vascular Tissue Engineering
170. PROCEDIMENTO DI PREPARAZIONE DI SCAFFOLD POLIMERICI PREANGIOGENIZZATI
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Valerio Brucato, Vincenzo La Carrubba, Giulio Ghersi, Francesco Carfì Pavia, Brucato, VMB, La Carrubba, V, Ghersi, G, and Carfì Pavia, F
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Tissue Engineering, TIPS, DIPS, Phase Separation, Biomedical Devices
171. Pore size graded scaffold for selective cellular permeation
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Roberto Scaffaro, Francesco Lopresti, Andrea Maio, Luigi Botta, Giulio Ghersi, Salvatrice Rigogliuso, Scaffaro, R, Lopresti, F, Maio, A, Botta, L, Ghersi, G, and Rigogliuso, S.
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tissue engineering, pore size graded scaffold, PLA, PEG
172. Patent number RM2009A000073: Procedimento di preparazione di scaffold polimerici preangiogenizzati
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Valerio Brucato, Vincenzo La Carrubba, Giulio Ghersi, Francesco Carfì Pavia, BRUCATO, V, LA CARRUBBA, V, GHERSI, G, and CARFÌ PAVIA, F
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Settore ING-IND/24 - Principi Di Ingegneria Chimica ,Settore ING-IND/22 - Scienza E Tecnologia Dei Materiali ,Settore BIO/10 - Biochimica ,Scaffold, Ingegneria tissutale, polimeri biodegradabili, angiogenesi - Abstract
Classe proposta A61F - Filtri da inserire nei vasi sanguigni; protesi; apparecchi ortopedici e curativi per contraccezione; fomentazione; trattamento o protezione degli occhi e delle orecchie; bendaggi, garze per medicazioni e assorbenti; necessario per il pronto soccorso
173. Comparative study on enzymatic activity and molecules stability of some commercial proteolytic enzymes used in pancreatic islet isolation
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Salamone, M., Salvatrice Rigogliuso, Gregorio Seidita, Cuttitta, A., Adamo, G., Mazzola, S., Bertuzzi, F., Giulio Ghersi, Salamone, M, Rigogliuso, S, Seidita, G, Cuttitta, A, Adamo, G, Mazzola, S, Bertuzzi, F, and Ghersi, G
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Neutral protease ,Settore BIO/10 - Biochimica ,Collagenases, Proteolytic enzymes, Pancreatic islet isolation ,Collagenase ,Dencitometry assay ,Proteolytic enzyme - Abstract
In pancreatic islets purification, for cell therapy applications, the major enzymes used are obtained from Clostridium hystoliticum; class I and class II collagenases (COLL G and COLL H). They are used in a defined tissue dissociation enzyme (TDE) mixtures together neutral protease (Dispase) or thermolysin (Thermostable Neutral Protease). The TDE mixtures were in part responsible for the success of the Edmonton protocol; however, just to now, people working in islets purification found discrepancy in an application to another one. This variability in application see in the enzymatic blend composition the higher accused, such as the contamination from endotoxines due to extractive production methods to obtain them. Using electrophoresis and gelatin zymography approaches; together densitometry evaluation assays we compared in composition, stability and auto-digestion processes C. hystoliticum collagenases, Neutral protease and Thermolysin from Roches and Serva. Moreover, we have investigated about the stability of analyzed enzymes when they are in solution in relation to their storage processes at different temperatures (-20°C; 0°C and r.t.); such as, concerning their digestive activity when applied at different temperatures (working temperatures: 25°C; 30°C; 37°C and 42 °C). Our results shown an heterogeneous composition of the different blend of collagenases from Roches and Serva, furthermore, heterogeneity is observed by lot to lot; on the other hand, we have found several more proteins and/or fragments respect the HPLC profiles publish by vendors. In gelatin zymographyes several digestive bands were catalytic active showing very high complex degradative patterns. Additionally, in neutral protease from Serva contaminants with gelatinolytic activities were detected; not in thermolysin. In stability experiments we investigated an inactivation/auto-digestive processes of analyzed enzymes; nevertheless, at different working temperatures, a more stable activity of analyzed molecules at 25°C were observed; activity that can be compared to that detectable at 37°C at the beginning of digestive process, at this temperature in the times inactivation enzymes were observed. These data take together strongly imply a not controlled digestive processes due to several contaminants present in the blends and to autocatalytic processes. Moreover, the presence of low molecular weight gelatine/degradative activities mean the impossibility to control the islets digestion process due to aspecific catalytic activities. Generation of recombinant collagenases probably could be of help to overcome the variability in the extractive processes. In pancreatic islets isolation for cell therapy the major enzymes used are obtained from Clostridium hystoliticum: class I and class II collagenases. They are used in a defined tissue dissociation enzyme mixture together with neutral protease (Dispase) or thermolysin (Thermostable Neutral Protease). However, just to now, people working in islets production found variable outcomes in isolation procedures mainly due to large variability in enzymatic blend composition and efficacy. Using electrophoresis and gelatin zymography approaches together with densitometry evaluation assays we compared the composition, stability and auto-digestion processes of C. hystoliticum collagenases, Neutral protease and Thermolysin from Roches and Serva. Moreover, we have assessed: –the stability of enzymes when they are in solution at different temperatures (-20 C; 0 C and room temperature); –their digestive activity when applied at different working temperatures (25 C; 30 C; 37 C and 42 C). Our results shown a heterogeneous composition of the different enzymatic blend enzymes analyzed. Furthermore, heterogeneity is observed among different batch enzymes; we found several more proteins and/or fragments compare to HPLC profiles published by vendors. In gelatin zymographyes several digestive bands were catalytic, showing very high complex degradative patterns, in part active even in condition of calcium deprivation. Additionally, in neutral protease from Serva (and not in Thermolysin) contaminants with gelatinolytic activities were detected. An auto-digestive/inactivation processes of enzymes occurred at different working temperatures, reduced by lowering temperature up to 25 C. These data taken together strongly imply a not controlled digestive processes due to several contaminants in enzyme blends and to autocatalytic processes. Moreover, the presence of low molecular weight gelatine/degradative activities obstacles the possibility to control islet digestion due to aspecific catalytic activities. Generation of recombinant collagenases probably could be of help to overcome the variability in the extractive processes.
174. A new method to valued efficiency of enzyme blend for pancreas tissue digestion
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Salamone, M., Gregorio Seidita, Cuttitta, A., Salvatrice Rigogliuso, La Venuta, G., Mazzola, S., Bertuzzi, F., Giulio Ghersi, Salamone, M, Seidita, G, Cuttitta, A, Rigogliuso, S, La Venuta, G, Mazzola, S, Bertuzzi, F, and Ghersi, G
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Islets transplantation ,Settore BIO/10 - Biochimica ,Collagenase ,Tissue digestion ,Langerhan - Abstract
One of the best successful example of cell therapy is represented by islet transplantation since the '90. However islet isolation methods are not completely standardized yet. More than half of isolation procedures failed to isolate adequate islets for transplantation, due to variable pancreas condition, and to unpredictable enzymatic blend efficiency. Enzymes used for pancreas digestion are purified from Clostridium histolyticum; these enzymes has a broad substrate specificity and potent collagenolytic activity compared to vertebrate collagenases. However, a major obstacle in human islet isolation successful is due to the variability in composition and concentration of the collagenases used during digestion phase. Another protease involved in islet isolation is the neutral protease, it plays an important role, however, should be considered as a double-edged sword: it accelerates tissue dissociation but, on the other hand, it could result in a decrease in islet yield, through islet fragmentation and breakdown. Therefore, protease activity should be carefully adjusted in a narrow range, in fact, there are not sufficient studies showing optimal dosage and composition of proteases that must be use in human pancreas islets extraction procedure. The current parameters available to characterize enzymatic blend are not predictive their efficiency in pancreas digestion and therefore are not useful to select which enzyme bathes can be successfully used for islet isolation purpose. Our goal is in direction to maximize the yield of functional islets; therefore we set up a new in vitro method to better in vitro characterize enzymatic blend before its use in human pancreas. In our experimental approach we have growth human immortalized cells (ECV-304) or human islets, obtained using a canonical extractive approach, within a 3-D type-I collagen gel in 96 wells plate. After one culture day, cells and/or islets were treated with different commercial enzymes (Liberase, Serva NB1 premium grade, Collagenase type P, Thermolysin, Neutral proteases) from different batches at different concentrations and for different times. Digestion of 3-D type-I collagen fibril gels were monitored by optical dense absorption to a fix l; while, morphology of released cells and/or islets were valued by confocal microscopy analyses. Cells were immunostained about expression of some adhesion molecules, like: integrins, cadherins and associated molecules, catenins, to appreciate cells morphology and islets aggregation modifications. Moreover, using SYTO 13/Et/Br in viability assayes we have quantify the toxiticy or not of tested enzymes in the different experimental conditions. We found that Neutral proteases is less pure and more toxic than Thermolysin: it has collagenase activity and it significantly decrease cell viability. Isolated cells by Neutral proteases appeared to be 77,5 % died. Even viable cells showed an alterated morphology with an impairment of cell to cell communications. We, also, observed an higher efficiency in extraction by Serva NB1 compared to Liberase and Collagenasi type P, used in the same experimental conditions. By this in vitro method we were also able to compare the minimal active enzyme concentration of different enzyme blends: we found that Liberase has its best value as nuber of extracted cell/alive cells in 130 mg/ml; while, in actual protocols is used to 1,3 mg/ml. Preliminary results showed a correlation between data achieved in cell lines with those of human islets, thus confirming the predictive role of this method for the selection of enzymes for human pancreas digestion purpose.
175. Water-borne polymeric nanoparticles for glutathione-mediated intracellular delivery of anticancer drugs
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Grimaldi, Natascia, Sabatino, Maria Antonietta, Bulone, D., Adamo, G., Salvatrice Rigogliuso, Giulio Ghersi, Clelia Dispenza, Grimaldi, N, Sabatino, MA, Bulone, D, Adamo, G, Rigogliuso, S, Ghersi, G, and Dispenza, C
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nanogels, e-beam radiation, disulphide linkage, doxorubicin - Abstract
A new family of water-borne, biocompatible and carboxyl- functionalized nanogels was developed for glutathione- mediated delivery of anticancer drugs. Poly(N-vinyl- pyrrolidone)-co-acrylic acid nanogels were generated by e- beam irradiation of aqueous solutions of a crosslinkable polymer, using industrial-type linear accelerators and set- ups. Nanogels physico-chemical properties and colloidal stability, in a wide pH range, were investigated. In vitro cell studies proved that the nanogels are fully biocompatible and able to quantitatively bypass cellular membrane. An anticancer drug, doxorubicin (DOX), was linked to the carboxyl groups of NGs through a spacer containing a disulphide cleavable linkage. In vitro release studies showed that glutathione is able to trigger the release of DOX through the reduction of the S-S linkage at a concentration comparable to its levels in the cytosol.
176. Large-scale manufacturing of radiation sculptured therapeutic nanogels
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Clelia Dispenza, Grimaldi, Natascia, Sabatino, Maria Antonietta, Adamo, G., Salvatrice Rigogliuso, Giulio Ghersi, Dispenza, C, Sabatino, MA, Grimaldi, N, Ditta, L, Alessi, S, Spadaro, G, DISPENZA, C, GRIMALDI, N, SABATINO, MA, ADAMO, G, RIGOGLIUSO, S, and GHERSI, G
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e-beam irradiation, PVP, NAnogels, antibody ,Nanogels ,Settore CHIM/07 - Fondamenti Chimici Delle Tecnologie - Abstract
Nanogels (NGs), or small particles formed by physically or chemically crosslinked polymer networks, represent a niche in the development of “smart” nanoparticles for drug delivery and diagnostics. Yet, they offerunique advantages over other systems, including a large and flexible surface for multivalent bio-conjugation; an internal 3D aqueous environment for incorporation and protection of (bio)molecular drugs; the possibility to entrap light-activemolecules, metal or mineral nanoparticles for imaging or phototherapeutic purposes; stimuli-responsiveness to achieve temporal and/or site control of the release function and biocompatibility. The availability of inexpensive, robust and versatile synthetic methodologies is at the basis of the development of effective nanogel-based theragnostic devices. In particular, we have established that nanogels can be produced with high yields and through-puts by pulsede-beam irradiation of dilute aqueous solutions of water-soluble biocompatible polymers e.g. poly(N-vinyl pyrrolidone), and functional acrylic monomers, such as acrylic acid or (3-aminopropyl) methacrylamide hydrochloride, using industrial electron accelerators and set-ups (see Figure 1). [1-4] Nanogels are the result of chemical follow-up reactions initiated by a continuous series of electron pulse-generated hydroxyl radicals in de-aerated water. A number of radical sites are generated on the polymer chains,which may have a different fate depending onthe system composition and irradiation conditions.Intra-molecular and inter-molecular radical recombination as well as disproportionation, chain scission and monomer or short polymer segments grafting may occur up to different extent. As a result, crosslinked-core nanoparticles with multi-armed surfaces can be generated, with controlled size, crosslinking density, surface electric charge density, number and nature of functional groups.No recourse to organic solvents, toxic initiators or catalysts and surfactants is made, therefore expensive or time-consuming purification procedures are not required. Simultaneous sterilization can be achieved depending on the irradiation doses. Long-term colloidal stability in the formof aqueous dispersions and redispersability from the freeze-dried form are advantageous properties especially in the view of a pharmaceutical use. Nanogels have been decorated with fluorescent probes, peptides, antibodies or oligonucleotides and/or conjugated to both molecular and macromolecular model drugs to demonstrate their amenability to be transformed into bio-hybrid, smart drug nanocarriers. All the base nanogels have been proved to benot cytotoxic or genotoxic at the cellular level. Indeed, they showed a good affinity for cells, as they rapidly and quantitatively bypass the cellularcompartments, to accumulate in specific cell portions for the first hours, to bethen completely released from the cells after 24 h.[5] In particular, active targeting features toward specific cell types and smart delivery functions of model chemotherapeutics of purposely designed bio-hybrid nanogels will be presented.
177. Vibrio Proteases for Biomedical Applications: Modulating the Proteolytic Secretome of V. alginolyticus and V. parahaemolyticus for Improved Enzymes Production
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Aldo Nicosia, Marcello Tagliavia, Giulio Ghersi, Monica Salamone, Salamone M., Nicosia A., Ghersi G., and Tagliavia M.
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Microbiology (medical) ,Proteases ,V. alginolyticus ,proteases production ,Microbiology ,Article ,V. parahaemolyticus ,03 medical and health sciences ,V. Alginolyticu ,Settore BIO/10 - Biochimica ,Virology ,medicine ,lcsh:QH301-705.5 ,030304 developmental biology ,2. Zero hunger ,chemistry.chemical_classification ,Vibrio alginolyticus ,0303 health sciences ,biology ,030306 microbiology ,Chemistry ,Vibrio parahaemolyticus ,Proteolytic enzymes ,Substrate (chemistry) ,biology.organism_classification ,Vibrio ,collagenase ,Enzyme ,Biochemistry ,lcsh:Biology (General) ,proteolytic secretome ,Collagenase ,medicine.drug - Abstract
Proteolytic enzymes are of great interest for biotechnological purposes, and their large-scale production, as well as the discovery of strains producing new molecules, is a relevant issue. Collagenases are employed for biomedical and pharmaceutical purposes. The high specificity of collagenase-based preparations toward the substrate strongly relies on the enzyme purity. However, the overall activity may depend on the cooperation with other proteases, the presence of which may be essential for the overall enzymatic activity, but potentially harmful for cells and tissues. Vibrios produce some of the most promising bacterial proteases (including collagenases), and their exo-proteome includes several enzymes with different substrate specificities, the production and relative abundances of which strongly depend on growth conditions. We evaluated the effects of different media compositions on the proteolytic exo-proteome of Vibrio alginolyticus and its closely relative Vibrio parahaemolyticus, in order to improve the overall proteases production, as well as the yield of the desired enzymes subset. Substantial biological responses were achieved with all media, which allowed defining culture conditions for targeted improvement of selected enzyme classes, besides giving insights in possible regulatory mechanisms. In particular, we focused our efforts on collagenases production, because of the growing biotechnological interest due to their pharmaceutical/biomedical applications.
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178. Nucleic Acids-Based Biomarkers for Alzheimer's Disease Diagnosis and Novel Molecules to Treat the Disease.
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Bivona G, Sammataro S, and Ghersi G
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- Humans, Nucleic Acids cerebrospinal fluid, Alzheimer Disease diagnosis, Alzheimer Disease metabolism, Alzheimer Disease cerebrospinal fluid, Biomarkers cerebrospinal fluid, tau Proteins cerebrospinal fluid, tau Proteins metabolism, Amyloid beta-Peptides cerebrospinal fluid, Amyloid beta-Peptides metabolism
- Abstract
Alzheimer's disease (AD) represents the most common form of dementia and affects million people worldwide, with a high social burden and considerable economic costs. AD diagnosis benefits from a well-established panel of laboratory tests that allow ruling-in patients, along with FDG and amyloid PET imaging tools. The main laboratory tests used to identify AD patients are Aβ40, Aβ42, the Aβ42/Aβ40 ratio, phosphorylated Tau 181 (pTau181) and total Tau (tTau). Although they are measured preferentially in the cerebrospinal fluid (CSF), some evidence about the possibility for blood-based determination to enter clinical practice is growing up. Unfortunately, CSF biomarkers for AD and, even more, the blood-based ones, present a few flaws, and twenty years of research in this field did not overcome these pitfalls. The tale even worsens when the issue of treating AD is addressed due to the lack of effective strategies despite the many decades of attempts by pharmaceutic industries and scientists. Amyloid-based drugs failed to stop the disease, and no neuroinflammation-based drugs have been demonstrated to work so far. Hence, only symptomatic therapy is available, with no disease-modifying treatment on hand. Such a desolate situation fully justifies the active search for novel biomarkers to be used as reliable tests for AD diagnosis and molecular targets for treating patients. Recently, a novel group of molecules has been identified to be used for AD diagnosis and follow-up, the nuclei acid-based biomarkers. Nucleic acid-based biomarkers are a composite group of extracellular molecules consisting of DNA and RNA alone or in combination with other molecules, including proteins. This review article reports the main findings from the studies carried out on these biomarkers during AD, and highlights their advantages and limitations.
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- 2024
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179. Effects of Cerebrospinal Fluids from Alzheimer and Non-Alzheimer Patients on Neurons-Astrocytes-Microglia Co-Culture.
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Iemmolo M, Bivona G, Piccoli T, Nicosia A, Schiera G, Di Liegro CM, Di Pietra F, and Ghersi G
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- Humans, Microglia metabolism, Coculture Techniques, Astrocytes metabolism, tau Proteins metabolism, Amyloid beta-Peptides metabolism, Neurons metabolism, Alzheimer Disease metabolism
- Abstract
Alzheimer's disease (AD) is the most common form of dementia, characterized by the accumulation of β-amyloid plaques, tau tangles, neuroinflammation, and synaptic/neuronal loss, the latter being the strongest correlating factor with memory and cognitive impairment. Through an in vitro study on a neurons-astrocytes-microglia (NAM) co-culture system, we analyzed the effects of cerebrospinal fluid (CSF) samples from AD and non-AD patients (other neurodegenerative pathologies). Treatment with CSF from AD patients showed a loss of neurofilaments and spheroids, suggesting the presence of elements including CX3CL1 (soluble form), destabilizing the neurofilaments, cellular adhesion processes, and intercellular contacts. The NAM co-cultures were analyzed in immunofluorescence assays for several markers related to AD, such as through zymography, where the expression of proteolytic enzymes was quantified both in cell extracts and the co-cultures' conditioned medium (CM). Through qRT-PCR assays, several genes involved in the formation of β-amyloid plaque, in phosphorylation of tau, and in inflammation pathways and MMP expression were investigated.
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- 2024
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180. Improvement of Osteogenic Differentiation of Mouse Pre-Osteoblastic MC3T3-E1 Cells on Core-Shell Polylactic Acid/Chitosan Electrospun Scaffolds for Bone Defect Repair.
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Lopresti F, Campora S, Rigogliuso S, Nicosia A, Lo Cicero A, Di Marco C, Tornabene S, Ghersi G, and La Carrubba V
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- Mice, Animals, Tissue Scaffolds chemistry, Osteogenesis, Polyesters chemistry, Chitosan chemistry
- Abstract
Electrospun hybrid scaffolds composed of synthetic and natural polymers have gained increasing interest in tissue engineering applications over the last decade. In this work, scaffolds composed of polylactic acid electrospun fibers, either treated (P-PLA) or non-treated (PLA) with air-plasma, were coated with high molecular weight chitosan to create a core-shell microfibrous structure. The effective thickness control of the chitosan layer was confirmed by gravimetric, spectroscopic (FTIR-ATR) and morphological (SEM) investigations. The chitosan coating increased the fiber diameter of the microfibrous scaffolds while the tensile mechanical tests, conducted in dry and wet environments, showed a reinforcing action of the coating layer on the scaffolds, in particular when deposited on P-PLA samples. The stability of the Chi coating on both PLA and P-PLA substrates was confirmed by gravimetric analysis, while their mineralization capacity was evaluated though scanning electron microscopy (SEM) and energy-dispersive spectroscopy (EDS) after immersing the scaffolds in simulated body fluids (SBF) at 37 °C for 1 week. Sample biocompatibility was investigated through cell viability assay and SEM analysis on mouse pre-osteoblastic MC3T3-E1 cells grown on scaffolds at different times (1, 7, 14 and 21 days). Finally, Alizarin Red assay and qPCR analysis suggested that the combination of plasma treatment and chitosan coating on PLA electrospun scaffolds influences the osteoblastic differentiation of MC3T3-E1 cells, thus demonstrating the great potential of P-PLA/chitosan hybrid scaffolds for bone tissue engineering applications.
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- 2024
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181. Collagen-Based Scaffolds for Chronic Skin Wound Treatment.
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La Monica F, Campora S, and Ghersi G
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Chronic wounds, commonly known as ulcers, represent a significant challenge to public health, impacting millions of individuals every year and imposing a significant financial burden on the global health system. Chronic wounds result from the interruption of the natural wound-healing process due to internal and/or external factors, resulting in slow or nonexistent recovery. Conventional medical approaches are often inadequate to deal with chronic wounds, necessitating the exploration of new methods to facilitate rapid and effective healing. In recent years, regenerative medicine and tissue engineering have emerged as promising avenues to encourage tissue regeneration. These approaches aim to achieve anatomical and functional restoration of the affected area through polymeric components, such as scaffolds or hydrogels. This review explores collagen-based biomaterials as potential therapeutic interventions for skin chronic wounds, specifically focusing on infective and diabetic ulcers. Hence, the different approaches described are classified on an action-mechanism basis. Understanding the issues preventing chronic wound healing and identifying effective therapeutic alternatives could indicate the best way to optimize therapeutic units and to promote more direct and efficient healing.
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- 2024
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182. Cerebrospinal and Blood Biomarkers in Alzheimer's Disease: Did Mild Cognitive Impairment Definition Affect Their Clinical Usefulness?
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Bivona G, Iemmolo M, and Ghersi G
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- Humans, Amyloid beta-Peptides cerebrospinal fluid, tau Proteins cerebrospinal fluid, Biomarkers cerebrospinal fluid, Peptide Fragments cerebrospinal fluid, Alzheimer Disease diagnosis, Alzheimer Disease cerebrospinal fluid, Cognitive Dysfunction diagnosis, Cognitive Dysfunction cerebrospinal fluid
- Abstract
Despite Alzheimer's Disease (AD) being known from the times of Alois Alzheimer, who lived more than one century ago, many aspects of the disease are still obscure, including the pathogenesis, the clinical spectrum definition, and the therapeutic approach. Well-established biomarkers for AD come from the histopathological hallmarks of the disease, which are Aβ and phosphorylated Tau protein aggregates. Consistently, cerebrospinal fluid (CSF) Amyloid β (Aβ) and phosphorylated Tau level measurements are currently used to detect AD presence. However, two central biases affect these biomarkers. Firstly, incomplete knowledge of the pathogenesis of diseases legitimates the search for novel molecules that, reasonably, could be expressed by neurons and microglia and could be detected in blood simpler and earlier than the classical markers and in a higher amount. Further, studies have been performed to evaluate whether CSF biomarkers can predict AD onset in Mild Cognitive Impairment (MCI) patients. However, the MCI definition has changed over time. Hence, the studies on MCI patients seem to be biased at the beginning due to the imprecise enrollment and heterogeneous composition of the miscellaneous MCI subgroup. Plasma biomarkers and novel candidate molecules, such as microglia biomarkers, have been tentatively investigated and could represent valuable targets for diagnosing and monitoring AD. Also, novel AD markers are urgently needed to identify molecular targets for treatment strategies. This review article summarizes the main CSF and blood AD biomarkers, underpins their advantages and flaws, and mentions novel molecules that can be used as potential biomarkers for AD.
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- 2023
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183. Nanoformulations based on collagenases loaded into halloysite/Veegum® clay minerals for potential pharmaceutical applications.
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Massaro M, Ghersi G, de Melo Barbosa R, Campora S, Rigogliuso S, Sànchez-Espejo R, Viseras-Iborra C, and Riela S
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- Clay, Excipients, Hydrogels, Minerals, Collagenases
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The design and development of nanomaterials capable of penetrate cancer cells is fundamental when anticancer therapy is involved. The use of collagenase (Col) is useful since this enzyme can degrade collagen, mainly present in the tumor extracellular matrix. However, its use is often limited since collagenase suffers from inactivation and short half-life. Use of recombinant ultrapure collagenase or carrier systems for their delivery are among the strategies adopted to increase the enzyme stability. Herein, based on the more stability showed by recombinant enzymes and the possibility to use them in anticancer therapy, we propose a novel strategy to further increase their stability by using halloysite nanotubes (HNTs) as carrier. ColG and ColH were supramolecularly loaded onto HNTs and used as fillers for Veegum gels. The systems could be used for potential local administration of collagenases for solid tumor treatment. All techniques adopted for characterization showed that halloysite interacts with collagenases in different ways depending with the Col considered. Furthermore, the hydrogels showed a very slow release of the collagenases within 24 h. Finally, biological assays were performed by studying the digestion of a type-I collagen matrix highlighting that once released the Col still possessed some activity. Thus we developed carrier systems that could further increase the high recombinant collagenases stability, preventing their inactivation in future in vivo applications for potential local tumor treatment., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)
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- 2023
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184. Mimicking Molecular Pathways in the Design of Smart Hydrogels for the Design of Vascularized Engineered Tissues.
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Nicosia A, Salamone M, Costa S, Ragusa MA, and Ghersi G
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- Tissue Engineering, Biocompatible Materials chemistry, Cell Adhesion, Hydrogels chemistry, Extracellular Matrix metabolism
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Biomaterials are pivotal in supporting and guiding vascularization for therapeutic applications. To design effective, bioactive biomaterials, understanding the cellular and molecular processes involved in angiogenesis and vasculogenesis is crucial. Biomaterial platforms can replicate the interactions between cells, the ECM, and the signaling molecules that trigger blood vessel formation. Hydrogels, with their soft and hydrated properties resembling natural tissues, are widely utilized; particularly synthetic hydrogels, known for their bio-inertness and precise control over cell-material interactions, are utilized. Naturally derived and synthetic hydrogel bases are tailored with specific mechanical properties, controlled for biodegradation, and enhanced for cell adhesion, appropriate biochemical signaling, and architectural features that facilitate the assembly and tubulogenesis of vascular cells. This comprehensive review showcases the latest advancements in hydrogel materials and innovative design modifications aimed at effectively guiding and supporting vascularization processes. Furthermore, by leveraging this knowledge, researchers can advance biomaterial design, which will enable precise support and guidance of vascularization processes and ultimately enhance tissue functionality and therapeutic outcomes.
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- 2023
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185. Monitoring the macrophage response towards biomaterial implants using label-free imaging.
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Lu CE, Levey RE, Ghersi G, Schueller N, Liebscher S, Layland SL, Schenke-Layland K, Duffy GP, and Marzi J
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Understanding the immune system's foreign body response (FBR) is essential when developing and validating a biomaterial. Macrophage activation and proliferation are critical events in FBR that can determine the material's biocompatibility and fate in vivo. In this study, two different macro-encapsulation pouches intended for pancreatic islet transplantation were implanted into streptozotocin-induced diabetes rat models for 15 days. Post-explantation, the fibrotic capsules were analyzed by standard immunohistochemistry as well as non-invasive Raman microspectroscopy to determine the degree of FBR induced by both materials. The potential of Raman microspectroscopy to discern different processes of FBR was investigated and it was shown that Raman microspectroscopy is capable of targeting ECM components of the fibrotic capsule as well as pro and anti-inflammatory macrophage activation states, in a molecular-sensitive and marker-independent manner. In combination with multivariate analysis, spectral shifts reflecting conformational differences in Col I were identified and allowed to discriminate fibrotic and native interstitial connective tissue fibers. Moreover, spectral signatures retrieved from nuclei demonstrated changes in methylation states of nucleic acids in M1 and M2 phenotypes, relevant as indicator for fibrosis progression. This study could successfully implement Raman microspectroscopy as complementary tool to study in vivo immune-compatibility providing insightful information of FBR of biomaterials and medical devices, post-implantation., Competing Interests: The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Katja Schenke-Layland reports financial support was provided by German Research Foundation. Katja Schenke-Layland & Garry Duffy reports financial support was provided by 10.13039/501100000780European Union. Chuan-En Lu reports equipment, drugs, or supplies was provided by Boston Scientific Ireland Ltd. Katja Schenke-Layland reports financial support was provided by State of Baden-Wurttemberg Ministry for Science Research and Art. Giulio Ghersi reports a relationship with Abiel Sr that includes: employment., (© 2023 The Authors.)
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- 2023
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186. CX3CL1 Pathway as a Molecular Target for Treatment Strategies in Alzheimer's Disease.
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Bivona G, Iemmolo M, and Ghersi G
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- Microglia physiology, CX3C Chemokine Receptor 1 metabolism, Alzheimer Disease physiopathology, Alzheimer Disease therapy, Molecular Targeted Therapy trends, Chemokine CXCL1 metabolism, Signal Transduction
- Abstract
Alzheimer's disease (AD) is a scourge for patients, caregivers and healthcare professionals due to the progressive character of the disease and the lack of effective treatments. AD is considered a proteinopathy, which means that aetiological and clinical features of AD have been linked to the deposition of amyloid β (Aβ) and hyperphosphorylated tau protein aggregates throughout the brain, with Aβ and hyperphosphorylated tau representing classical AD hallmarks. However, some other putative mechanisms underlying the pathogenesis of the disease have been proposed, including inflammation in the brain, microglia activation, impaired hippocampus neurogenesis and alterations in the production and release of neurotrophic factors. Among all, microglia activation and chronic inflammation in the brain gained some attention, with researchers worldwide wondering whether it is possible to prevent and stop, respectively, the onset and progression of the disease by modulating microglia phenotypes. The following key points have been established so far: (i) Aβ deposition in brain parenchyma represents repeated stimulus determining chronic activation of microglia; (ii) chronic activation and priming of microglia make these cells lose neuroprotective functions and favour damage and loss of neurons; (iii) quiescent status of microglia at baseline prevents chronic activation and priming, meaning that the more microglia are quiescent, the less they become neurotoxic. Many molecules are known to modulate the quiescent baseline state of microglia, attracting huge interest among scientists as to whether these molecules could be used as valuable targets in AD treatment. The downside of the coin came early with the observation that quiescent microglia do not display phagocytic ability, being unable to clear Aβ deposits since phagocytosis is crucial for Aβ clearance efficacy. A possible solution for this issue could be found in the modulation of microglia status at baseline, which could help maintain both neuroprotective features and phagocytic ability at the same time. Among the molecules known to influence the baseline status of microglia, C-X3-chemokine Ligand 1 (CX3CL1), also known as Fractalkine (FKN), is one of the most investigated. FKN and its microglial receptor CX3CR1 are crucial players in the interplay between neurons and microglia, modulating the operation of some neural circuits and the efficacy and persistence of immune response against injury. In addition, CX3CL1 regulates synaptic pruning and plasticity in the developmental age and in adulthood, when it strongly impacts the hippocampus neurogenesis of the adult. CX3CL1 has an effect on Aβ clearance and tau phosphorylation, as well as in microglia activation and priming. For all the above, CX3CL1/CX3CR1 signalling has been widely studied in relation to AD pathogenesis, and its biochemical pathway could hide molecular targets for novel treatment strategies in AD. This review summarizes the possible role of CX3CL1 in AD pathogenesis and its use as a potential target for AD treatment.
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- 2023
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187. The Cytokine CX3CL1 and ADAMs/MMPs in Concerted Cross-Talk Influencing Neurodegenerative Diseases.
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Iemmolo M, Ghersi G, and Bivona G
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- Humans, Cytokines metabolism, Neuroinflammatory Diseases, Chemokine CX3CL1 metabolism, Central Nervous System metabolism, Microglia metabolism, Neurodegenerative Diseases metabolism, Alzheimer Disease metabolism
- Abstract
Neuroinflammation plays a fundamental role in the development and progression of neurodegenerative diseases. It could therefore be said that neuroinflammation in neurodegenerative pathologies is not a consequence but a cause of them and could represent a therapeutic target of neuronal degeneration. CX3CL1 and several proteases (ADAMs/MMPs) are strongly involved in the inflammatory pathways of these neurodegenerative pathologies with multiple effects. On the one hand, ADAMs have neuroprotective and anti-apoptotic effects; on the other hand, they target cytokines and chemokines, thus causing inflammatory processes and, consequently, neurodegeneration. CX3CL1 itself is a cytokine substrate for the ADAM, ADAM17, which cleaves and releases it in a soluble isoform (sCX3CL1). CX3CL1, as an adhesion molecule, on the one hand, plays an inhibiting role in the pro-inflammatory response in the central nervous system (CNS) and shows neuroprotective effects by binding its membrane receptor (CX3CR1) present into microglia cells and maintaining them in a quiescent state; on the other hand, the sCX3CL1 isoform seems to promote neurodegeneration. In this review, the dual roles of CX3CL1 and ADAMs/MMPs in different neurodegenerative diseases, such as Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (MH), and multiple sclerosis (MS), are investigated.
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- 2023
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188. Recovery of Bioactive Compounds from Marine Organisms: Focus on the Future Perspectives for Pharmacological, Biomedical and Regenerative Medicine Applications of Marine Collagen.
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Rigogliuso S, Campora S, Notarbartolo M, and Ghersi G
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- Animals, Ecosystem, Regenerative Medicine, Collagen, Aquatic Organisms, Biological Products pharmacology, Biological Products therapeutic use
- Abstract
Marine environments cover more than 70% of the Earth's surface and are among the richest and most complex ecosystems. In terms of biodiversity, the ocean represents an important source, still not widely exploited, of bioactive products derived from species of bacteria, plants, and animals. However, global warming, in combination with multiple anthropogenic practices, represents a serious environmental problem that has led to an increase in gelatinous zooplankton, a phenomenon referred to as jellyfish bloom. In recent years, the idea of "sustainable development" has emerged as one of the essential elements of green-economy initiatives; therefore, the marine environment has been re-evaluated and considered an important biological resource. Several bioactive compounds of marine origin are being studied, and among these, marine collagen represents one of the most attractive bio-resources, given its use in various disciplines, such as clinical applications, cosmetics, the food sector, and many other industrial applications. This review aims to provide a current overview of marine collagen applications in the pharmacological and biomedical fields, regenerative medicine, and cell therapy.
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- 2023
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189. Microglial Activation and Priming in Alzheimer's Disease: State of the Art and Future Perspectives.
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Bivona G, Iemmolo M, Agnello L, Lo Sasso B, Gambino CM, Giglio RV, Scazzone C, Ghersi G, and Ciaccio M
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- Humans, Amyloid beta-Peptides metabolism, Anti-Inflammatory Agents pharmacology, Cytokines metabolism, Alzheimer Disease immunology, Microglia immunology
- Abstract
Alzheimer's Disease (AD) is the most common cause of dementia, having a remarkable social and healthcare burden worldwide. Amyloid β (Aβ) and protein Tau aggregates are disease hallmarks and key players in AD pathogenesis. However, it has been hypothesized that microglia can contribute to AD pathophysiology, as well. Microglia are CNS-resident immune cells belonging to the myeloid lineage of the innate arm of immunity. Under physiological conditions, microglia are in constant motion in order to carry on their housekeeping function, and they maintain an anti-inflammatory, quiescent state, with low expression of cytokines and no phagocytic activity. Upon various stimuli (debris, ATP, misfolded proteins, aggregates and pathogens), microglia acquire a phagocytic function and overexpress cytokine gene modules. This process is generally regarded as microglia activation and implies that the production of pro-inflammatory cytokines is counterbalanced by the synthesis and the release of anti-inflammatory molecules. This mechanism avoids excessive inflammatory response and inappropriate microglial activation, which causes tissue damage and brain homeostasis impairment. Once the pathogenic stimulus has been cleared, activated microglia return to the naïve, anti-inflammatory state. Upon repeated stimuli (as in the case of Aβ deposition in the early stage of AD), activated microglia shift toward a less protective, neurotoxic phenotype, known as "primed" microglia. The main characteristic of primed microglia is their lower capability to turn back toward the naïve, anti-inflammatory state, which makes these cells prone to chronic activation and favours chronic inflammation in the brain. Primed microglia have impaired defence capacity against injury and detrimental effects on the brain microenvironment. Additionally, priming has been associated with AD onset and progression and can represent a promising target for AD treatment strategies. Many factors (genetics, environmental factors, baseline inflammatory status of microglia, ageing) generate an aberrantly activated phenotype that undergoes priming easier and earlier than normally activated microglia do. Novel, promising targets for therapeutic strategies for AD have been sought in the field of microglia activation and, importantly, among those factors influencing the baseline status of these cells. The CX3CL1 pathway could be a valuable target treatment approach in AD, although preliminary findings from the studies in this field are controversial. The current review aims to summarize state of the art on the role of microglia dysfunction in AD pathogenesis and proposes biochemical pathways with possible targets for AD treatment.
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- 2023
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190. High Cerebrospinal Fluid CX3CL1 Levels in Alzheimer's Disease Patients but Not in Non-Alzheimer's Disease Dementia.
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Bivona G, Iemmolo M, Piccoli T, Agnello L, Lo Sasso B, Ciaccio M, and Ghersi G
- Abstract
Alzheimer's disease (AD) is the most common form of cognitive decline worldwide, occurring in about 10% of people older than 65 years. The well-known hallmarks of AD are extracellular aggregates of amyloid β (Aβ) and intracellular neurofibrillary tangles (NFTs) of tau protein. The evidence that Aβ overproduction leads to AD has paved the way for the AD pathogenesis amyloid cascade hypothesis, which proposes that the neuronal damage is sustained by Aβ overproduction. Consistently, AD cerebrospinal fluid (CSF) biomarkers used in clinical practice, including Aβ 1-42, Aβ 1-40, Aβ 42/40 ratio, and pTau, are related to the amyloid hypothesis. Recently, it was suggested that the Aβ deposition cascade cannot fully disclose AD pathogenesis, with other putative players being involved in the pathophysiology of the disease. Among all, one of the most studied factors is inflammation in the brain. Hence, biomarkers of inflammation and microglia activation have also been proposed to identify AD. Among them, CX3 chemokine ligand 1 (CX3CL1) has taken center stage. This transmembrane protein, also known as fractalkine (FKN), is normally expressed in neurons, featuring an N-terminal chemokine domain and an extended mucin-like stalk, following a short intra-cytoplasmatic domain. The molecule exists in both membrane-bound and soluble forms. It is accepted that the soluble and membrane-bound forms of FKN evoke differential signaling within the CNS. Given the link between CX3XL1 and microglial activation, it has been suggested that CX3CL1 signaling disruption could play a part in the pathogenesis of AD. Furthermore, a role for chemokine as a biomarker has been proposed. However, the findings collected are controversial. The current study aimed to describe the cerebrospinal fluid (CSF) levels of CX3XL1 and classical biomarkers in AD patients.
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- 2022
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191. Continuous Microfiber Wire Mandrel-Less Biofabrication for Soft Tissue Engineering Applications.
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Adamo A, Bartolacci JG, Pedersen DD, Traina MG, Kim S, Pantano A, Ghersi G, Watkins SC, Wagner WR, Badylak SF, and D'Amore A
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- Animals, Rats, Tendons, Tensile Strength, Wound Healing, Sutures, Tissue Engineering methods
- Abstract
Suture materials are the most common bioimplants in surgical and clinical practice, playing a crucial role in wound healing and tendon and ligament repair. Despite the assortment available on the market, sutures are still affected by significant disadvantages, including failure in mimicking the mechanical properties of the tissue, excessive fibrosis, and inflammation. This study introduces a mandrel-less electrodeposition apparatus to fabricate continuous microfiber wires of indefinite length. The mandrel-less biofabrication produces wires, potentially used as medical fibers, with different microfiber bundles, that imitate the hierarchical organization of native tissues, and tailored mechanical properties. Microfiber wire morphology and mechanical properties are characterized by scanning electron microscopy, digital image processing, and uniaxial tensile test. Wires are tested in vitro on monocyte/macrophage stimulation and in vivo on a rat surgical wound model. The wires produced by mandrel-less deposition show an increased M2 macrophage phenotype in vitro. The in vivo assessment demonstrates that microfiber wires, compared to the medical fibers currently used, reduce pro-inflammatory macrophage response and preserve their mechanical properties after 30 days of use. These results make this microfiber wire an ideal candidate as a suture material for soft tissue surgery, suggesting a crucial role of microarchitecture in more favorable host response., (© 2022 The Authors. Advanced Healthcare Materials published by Wiley-VCH GmbH.)
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- 2022
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192. Functionalized Poly( N -isopropylacrylamide)-Based Microgels in Tumor Targeting and Drug Delivery.
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Campora S, Mohsen R, Passaro D, Samir H, Ashraf H, Al-Mofty SE, Diab AA, El-Sherbiny IM, Snowden MJ, and Ghersi G
- Abstract
Over the past several decades, the development of engineered small particles as targeted and drug delivery systems (TDDS) has received great attention thanks to the possibility to overcome the limitations of classical cancer chemotherapy, including targeting incapability, nonspecific action and, consequently, systemic toxicity. Thus, this research aims at using a novel design of Poly( N -isopropylacrylamide) p(NIPAM)-based microgels to specifically target cancer cells and avoid the healthy ones, which is expected to decrease or eliminate the side effects of chemotherapeutic drugs. Smart NIPAM-based microgels were functionalized with acrylic acid and coupled to folic acid (FA), targeting the folate receptors overexpressed by cancer cells and to the chemotherapeutic drug doxorubicin (Dox). The successful conjugation of FA and Dox was demonstrated by dynamic light scattering (DLS), Fourier-transform infrared (FTIR) spectroscopy, thermogravimetric analysis (TGA), UV-VIS analysis, and differential scanning calorimetry (DSC). Furthermore, viability assay performed on cancer and healthy breast cells, suggested the microgels' biocompatibility and the cytotoxic effect of the conjugated drug. On the other hand, the specific tumor targeting of synthetized microgels was demonstrated by a co-cultured (healthy and cancer cells) assay monitored using confocal microscopy and flow cytometry. Results suggest successful targeting of cancer cells and drug release. These data support the use of pNIPAM-based microgels as good candidates as TDDS.
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- 2021
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193. The Foreign Body Response to an Implantable Therapeutic Reservoir in a Diabetic Rodent Model.
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Beatty R, Lu CE, Marzi J, Levey RE, Carvajal Berrio D, Lattanzi G, Wylie R, O'Connor R, Wallace E, Ghersi G, Salamone M, Dolan EB, Layland SL, Schenke-Layland K, and Duffy GP
- Subjects
- Animals, Prostheses and Implants, Rodentia, X-Ray Microtomography, Diabetes Mellitus, Foreign Bodies diagnostic imaging
- Abstract
Advancements in type 1 diabetes mellitus treatments have vastly improved in recent years. The move toward a bioartificial pancreas and other fully implantable systems could help restore patient's glycemic control. However, the long-term success of implantable medical devices is often hindered by the foreign body response. Fibrous encapsulation "walls off" the implant to the surrounding tissue, impairing its functionality. In this study we aim to examine how streptozotocin-induced diabetes affects fibrous capsule formation and composition surrounding implantable drug delivery devices following subcutaneous implantation in a rodent model. After 2 weeks of implantation, the fibrous capsule surrounding the devices were examined by means of Raman spectroscopy, micro-computed tomography (μCT), and histological analysis. Results revealed no change in mean fibrotic capsule thickness between diabetic and healthy animals as measured by μCT. Macrophage numbers (CCR7 and CD163 positive) remained similar across all groups. True component analysis also showed no quantitative difference in the alpha-smooth muscle actin and extracellular matrix proteins. Although principal component analysis revealed significant secondary structural difference in collagen I in the diabetic group, no evidence indicates an influence on fibrous capsule composition surrounding the device. This study confirms that diabetes did not have an effect on the fibrous capsule thickness or composition surrounding our implantable drug delivery device. Impact Statement Understanding the impact diabetes has on the foreign body response (FBR) to our implanted material is essential for developing an effective drug delivery device. We used several approaches (Raman spectroscopy and micro-computed tomography imaging) to demonstrate a well-rounded understanding of the diabetic impact on the FBR to our devices, which is imperative for its clinical translation.
- Published
- 2021
- Full Text
- View/download PDF
194. Core-shell PLA/Kef hybrid scaffolds for skin tissue engineering applications prepared by direct kefiran coating on PLA electrospun fibers optimized via air-plasma treatment.
- Author
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Lopresti F, Campora S, Tirri G, Capuana E, Carfì Pavia F, Brucato V, Ghersi G, and La Carrubba V
- Subjects
- Polyesters, Polysaccharides, Tissue Engineering, Tissue Scaffolds
- Abstract
Over the recent years, there is a growing interest in electrospun hybrid scaffolds composed of synthetic and natural polymers that can support cell attachment and proliferation. In this work, the physical and biological properties of polylactic acid (PLA) electrospun mats coated with kefiran (Kef) were evaluated. Gravimetric, spectroscopic (FTIR-ATR) and morphological investigations via scanning electron microscopy confirmed the effective formation of a thin kefiran layer wrapped on the PLA fibers with an easy-tunable thickness. Air plasma pre-treatment carried out on PLA (P-PLA) affected both the morphology and the crystallinity of Kef coating as confirmed by differential scanning calorimetry and X-ray diffraction analyses. Scaffolds were mechanically characterized with tensile tests to evaluate the reinforcing action of the Kef coating. The water resistance of Kefiran coating in distilled water at 37 °C evaluated on both PLA/Kef and P-PLA/Kef was carried out by gravimetric and morphological analyses. Finally, cell culture assays with embryonic fibroblast cells were conducted on selected hybrid scaffolds to compare the cell proliferation, morphology, and collagen production with PLA and P-PLA electrospun scaffolds. Based on the results, we can demonstrate that direct coating of PLA from Kef/water solutions is an effective approach to prepare hybrid scaffolds with tunable properties and that the plasma pre-treatment enhances the affinity between PLA and Kefiran. In vitro tests demonstrated the great potential of PLA/Kef hybrid scaffolds for skin tissue engineering., (Copyright © 2021 Elsevier B.V. All rights reserved.)
- Published
- 2021
- Full Text
- View/download PDF
195. Additive Manufacturing of Multi-Scale Porous Soft Tissue Implants That Encourage Vascularization and Tissue Ingrowth.
- Author
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Coulter FB, Levey RE, Robinson ST, Dolan EB, Deotti S, Monaghan M, Dockery P, Coulter BS, Burke LP, Lowery AJ, Beatty R, Paetzold R, Prendergast JJ, Bellavia G, Straino S, Cianfarani F, Salamone M, Bruno CM, Moerman KM, Ghersi G, Duffy GP, and O'Cearbhaill ED
- Subjects
- Animals, Humans, Materials Testing, Porosity, Swine, Prostheses and Implants, Silicones
- Abstract
Medical devices, such as silicone-based prostheses designed for soft tissue implantation, often induce a suboptimal foreign-body response which results in a hardened avascular fibrotic capsule around the device, often leading to patient discomfort or implant failure. Here, it is proposed that additive manufacturing techniques can be used to deposit durable coatings with multiscale porosity on soft tissue implant surfaces to promote optimal tissue integration. Specifically, the "liquid rope coil effect", is exploited via direct ink writing, to create a controlled macro open-pore architecture, including over highly curved surfaces, while adapting atomizing spray deposition of a silicone ink to create a microporous texture. The potential to tailor the degree of tissue integration and vascularization using these fabrication techniques is demonstrated through subdermal and submuscular implantation studies in rodent and porcine models respectively, illustrating the implant coating's potential applications in both traditional soft tissue prosthetics and active drug-eluting devices., (© 2021 The Authors. Advanced Healthcare Materials published by Wiley-VCH GmbH.)
- Published
- 2021
- Full Text
- View/download PDF
196. 3D Collagen Hydrogel Promotes In Vitro Langerhans Islets Vascularization through ad-MVFs Angiogenic Activity.
- Author
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Salamone M, Rigogliuso S, Nicosia A, Campora S, Bruno CM, and Ghersi G
- Abstract
Adipose derived microvascular fragments (ad-MVFs) consist of effective vascularization units able to reassemble into efficient microvascular networks. Because of their content in stem cells and related angiogenic activity, ad-MVFs represent an interesting tool for applications in regenerative medicine. Here we show that gentle dissociation of rat adipose tissue provides a mixture of ad-MVFs with a length distribution ranging from 33-955 μm that are able to maintain their original morphology. The isolated units of ad-MVFs that resulted were able to activate transcriptional switching toward angiogenesis, forming tubes, branches, and entire capillary networks when cultured in 3D collagen type-I hydrogel. The proper involvement of metalloproteases (MMP2/MMP9) and serine proteases in basal lamina and extracellular matrix ECM degradation during the angiogenesis were concurrently assessed by the evaluation of alpha-smooth muscle actin (αSMA) expression. These results suggest that collagen type-I hydrogel provides an adequate 3D environment supporting the activation of the vascularization process. As a proof of concept, we exploited 3D collagen hydrogel for the setting of ad-MVF-islet of Langerhans coculture to improve the islets vascularization. Our results suggest potential employment of the proposed in vitro system for regenerative medicine applications, such as the improving of the islet of Langerhans engraftment before transplantation.
- Published
- 2021
- Full Text
- View/download PDF
197. Tuning of the Mg Alloy AZ31 Anodizing Process for Biodegradable Implants.
- Author
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Zaffora A, Di Franco F, Virtù D, Carfì Pavia F, Ghersi G, Virtanen S, and Santamaria M
- Subjects
- Animals, Cell Line, Corrosion, Electrodes, Magnesium Compounds chemistry, Materials Testing, Mice, Phosphates chemistry, Surface Properties, Absorbable Implants, Alloys chemistry, Coated Materials, Biocompatible chemistry
- Abstract
Coatings were grown on the AZ31 Mg alloy by a hard anodizing process in the hot glycerol phosphate-containing electrolyte. Anodizing conditions were optimized, maximizing corrosion resistance estimated by impedance measurements carried out in Hank's solution at 37 °C. A post anodizing annealing treatment (350 °C for 24 h) allowed us to further enhance the corrosion resistance of the coatings mainly containing magnesium phosphate according to energy-dispersive X-ray spectroscopy and Raman analyses. Gravimetric measurements revealed a hydrogen evolution rate within the limits acceptable for application of AZ31 in biomedical devices. In vitro tests demonstrated that the coatings are biocompatible with a preosteoblast cell line.
- Published
- 2021
- Full Text
- View/download PDF
198. Production of Injectable Marine Collagen-Based Hydrogel for the Maintenance of Differentiated Chondrocytes in Tissue Engineering Applications.
- Author
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Rigogliuso S, Salamone M, Barbarino E, Barbarino M, Nicosia A, and Ghersi G
- Subjects
- Animals, Cattle, Cell Line, Cell Survival drug effects, Chondrocytes drug effects, Cytoskeleton drug effects, Cytoskeleton metabolism, Gene Expression Regulation drug effects, Glycosaminoglycans metabolism, Mice, Rats, Scyphozoa chemistry, Aquatic Organisms chemistry, Cell Differentiation drug effects, Chondrocytes cytology, Collagen pharmacology, Hydrogels pharmacology, Injections, Tissue Engineering methods
- Abstract
Cartilage is an avascular tissue with limited ability of self-repair. The use of autologous chondrocyte transplants represent an effective strategy for cell regeneration; however, preserving the differentiated state, which ensures the ability to regenerate damaged cartilage, represents the main challenge during in vitro culturing. For this purpose, we produced an injectable marine collagen-based hydrogel, by mixing native collagen from the jellyfish Rhizostoma pulmo with hydroxy-phenyl-propionic acid (HPA)-functionalized marine gelatin. This biocompatible hydrogel formulation, due to the ability of enzymatically reticulate using horseradish peroxidase (HPR) and H
2 O2 , gives the possibility of trap cells inside, in the absence of cytotoxic effects, during the cross-linking process. Moreover, it enables the modulation of the hydrogel stiffness merely varying the concentration of H2 O2 without changes in the concentration of polymer precursors. The maintenance of differentiated chondrocytes in culture was then evaluated via morphological analysis of cell phenotype, GAG production and cytoskeleton organization. Additionally, gene expression profiling of differentiation/dedifferentiation markers provided evidence for the promotion of the chondrogenic gene expression program. This, combined with the biochemical properties of marine collagen, represents a promising strategy for maintaining in vitro the cellular phenotype in the aim of the use of autologous chondrocytes in regenerative medicine practices.- Published
- 2020
- Full Text
- View/download PDF
199. Development of injectable and durable kefiran hydro-alcoholic gels.
- Author
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Sabatino MA, Carfì Pavia F, Rigogliuso S, Giacomazza D, Ghersi G, La Carrubba V, and Dispenza C
- Subjects
- Alcohols chemistry, Cell Proliferation drug effects, Cell Survival drug effects, Humans, Hydrogels pharmacology, Propylene Glycol chemistry, Rheology, Drug Delivery Systems, Hydrogels chemistry, Polysaccharides chemistry
- Abstract
Injectable, in-situ forming kefiran gels have been developed for potential applications as implantable drug delivery devices or scaffolds for tissue regeneration. Concentrated solutions (4, 5 and 6%w) of kefiran, extracted from kefir grains, have been assessed in term of viscosity and injectability through G26 syringe needles, and for their ability to undergo gelation upon mixing with different alcohols. Propylene glycol (PG) has been selected as gelling agent because it ensures homogenous gelation in relatively short times (from few minutes up to 6 h). The investigation of the rheological behavior of kefiran/PG gels varying polymer concentration and temperature (25 °C and 37 °C) has provided interesting hints to support a possible gelation mechanism that accounts also for the observed influence of the alcohol type. Finally, the study of kefiran/PG gels has been complemented with the investigation on selected formulations of the swelling/degradation behavior upon immersion in isotonic buffer solution for up to 40 days at 37 °C; of the ability of the gels to retain and/or release two model molecules; and within vitro cell viability and cytotoxicity tests, to support the absence of toxic effects on cells induced by direct contact with the gels or by leached components from these gels., (Copyright © 2020 Elsevier B.V. All rights reserved.)
- Published
- 2020
- Full Text
- View/download PDF
200. Neural Crest-Derived Chondrocytes Isolation for Tissue Engineering in Regenerative Medicine.
- Author
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Salamone M, Rigogliuso S, Nicosia A, Tagliavia M, Campora S, Cinà P, Bruno C, and Ghersi G
- Subjects
- Animals, Cattle, Cell Differentiation, Chondrocytes cytology, Humans, Chondrocytes metabolism, Neural Crest metabolism, Regenerative Medicine methods, Tissue Engineering methods
- Abstract
Chondrocyte transplantation has been successfully tested and proposed as a clinical procedure aiming to repair articular cartilage defects. However, the isolation of chondrocytes and the optimization of the enzymatic digestion process, as well as their successful in vitro expansion, remain the main challenges in cartilage tissue engineering. In order to address these issues, we investigated the performance of recombinant collagenases in tissue dissociation assays with the aim of isolating chondrocytes from bovine nasal cartilage in order to establish the optimal enzyme blend to ensure the best outcomes of the overall procedure. We show, for the first time, that collagenase H activity alone is required for effective cartilage digestion, resulting in an improvement in the yield of viable cells. The extracted chondrocytes proved able to grow and activate differentiation/dedifferentiation programs, as assessed by morphological and gene expression analyses.
- Published
- 2020
- Full Text
- View/download PDF
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