Francesca Boccafoschi

Scratch wound closure of C2C12 mouse myoblasts is enhanced by human platelet lysate

The effect of a platelet lysate (PL) on muscle wound healing, based on in vitro scratch wound of C2C12 mouse myoblasts, has been investigated. Cell viability assays show that PL induced an increase in cell proliferation at concentrations of 1–20%, but was slightly cytotoxic at 100%. PL promoted wound closure after scratch wounding of cell monolayers. The p38 inhibitor SB203580 and the PI3K inhibitor, wortmannin, decreased the PL effect, whereas the ERK inhibitor, PD98059, did not. Transwell migration of cells was also increased by PL, and although SB203580 abrogated this effect, wortmannin reduced it, whereas PD98059 was ineffective. Western blot analyses of scratch wounded cells showed activation of AKT and p38, while in the presence of PL there was a faster and sustained activation of AKT and p38 (up to 6 h), and a transient activation of ERK1/2. Taken together, the data show that PL promotes C2C12 wound healing by enhancing cell proliferation and motility.

Properties of Zinc Releasing Surfaces for Clinical Applications

Two series of glasses of general formula (2-p) SiO2·1.1Na 2O·CaO·pP2O5·xZnO (p=0.10, 0.20; x=0.0, 0.16, 0.35, and 0.78) have been analyzed for physico-chemical surface features before and after contact with simulated body fluid, morphological characteristics, and osteoblast-like cells behavior when cultured on them. The resulted good cell adhesion and growth, along with nonsignificant changes of the focal contacts, allow the authors to indicate HZ5 and HP5Z5 glasses as the ones having optimal ratio of Zn/P to maintain acceptable cell behavior, comparable to the bioactive glass (Bioglass®) used as a control; results are also rationalized by means of three-dimensional models derived by molecular dynamic simulations, with decomposition and conversion rates optimized with respect to the parent Henchs Bioglass®.

Composite Films of Gelatin and Hydroxyapatite/Bioactive Glass for Tissue-Engineering Applications

Cross-linked gelatin/hydroxyapatite/bioactive glass (G/HA/CEL2) films with different compositions (100:0:0 (G1); 30:70:0 (G2); 30:0:70 (G3); 30:35:35 (G4) (%, w/w/w)) were prepared as scaffold materials for tissue-engineering applications, particularly in the field of bone repair. A bioactive glass with 45% SiO2, 3% P2O5, 26% CaO, 7% MgO, 15% Na2O and 4% K2O molar composition was selected (CEL2). Genipin was used as a cross-linker for the gelatin component. Samples were characterized in terms of their bioactivity, thermal properties, mechanical behaviour and cell compatibility. After only 3 days of incubation in simulated body fluid (SBF) at 37°C, calcium phosphate crystals precipitated on G3 and G4 surfaces, due to the high CEL2 bioactivity. Cross-linking increased the thermal stability of the gelatine component as indicated by thermal analysis (denaturation temperature was 92.3°C and 97.6°C for not cross-linked and cross-linked gelatin, respectively). Furthermore, tensile modulus of samples increased with increasing the inorganic phase amount (from 4.72 ± 0.23 MPa for G1 to 6.46 ± 0.05 MPa for G4). The adhesion and proliferation of human primary osteoblasts on composite films was evaluated. Cell viability was high with respect to the control for all samples and the presence of hydroxyapatite exerted an important role in the ability of mineralization.

Photoactive chitosan switching on bone-like apatite deposition.

The work was focused on the synthesis and characterization of the chitosan-g-fluorescein (CHFL) conjugate polymer as a biocompatible amphiphilic water-soluble photosensitizer, able to stimulate hydroxyapatite deposition upon visible light irradiation. Fluorescein (FL) grafting to chitosan (CH) chains was confirmed by UV-vis analysis of water solutions of FL and CHFL and by Fourier transform infrared spectroscopy (FTIR-ATR) analysis of CHFL and CH. Smooth CHFL cast films with 4 microm thickness were obtained by solvent casting. Continuous exposure to visible light for 7 days was found to activate the deposition of calcium phosphate crystals from a conventional simulated body fluid (SBF 1.0x) on the surface of CHFL cast films. EDX and FTIR-ATR analyses confirmed the apatite nature of the deposited calcium phosphate crystals. CHFL films preincubated in SBF (1.0x) solution under visible light irradiation and in the dark for 7 days were found to support the in vitro adhesion and proliferation of MG63 osteoblast-like cells (MTT viability test; 1-3 days culture time). On the other hand, the mineralization ability of MG63 osteoblast-like cells was significantly improved on CHFL films preincubated under visible light exposure (alkaline phosphatase activity (ALP) test for 1, 3, 7, and 14 days). The use of photoactive biocompatible conjugate polymer, such as CHFL, may lead to new therapeutic options in the field of bone/dental repair, exploiting the photoexcitation mechanism as a tool for biomineralization.

Dynamic fibroblast cultures: response to mechanical stretching.

Mechanical forces play an important role in the organization, growth and function of tissues. Dynamic extracellular environment affects cellular behavior modifying their orientation and their cytoskeleton. In this work, human fibroblasts have been subjected for three hours to increasing substrate deformations (1-25%) applied as cyclic uniaxial stretching at different frequencies (from 0.25Hz to 3Hz). Our objective was to identify whether and in which ranges the different deformations magnitude and rate were the factors responsible of the cell alignment and if actin cytoskeleton modification was involved in these responses. After three hours of cyclically stretched substrate, results evidenced that fibroblasts aligned perpendicularly to the stretch direction at 1% substrate deformation and reached statistically higher orientation at 2% substrate deformation with unmodified values at 5-20%, while 25% substrate deformation induced cellular death. It was also shown that a percentage of cells oriented perpendicularly to the deformation were not influenced by increased frequency of cyclical three hours deformations (0.25-3Hz). Cyclic substrate deformation was shown also to involve actin fibers which orient perpendicularly to the stress direction as well. Thus, we argue that a substrate deformation induces a dynamic change in cytoskeleton able to modify the entire morphology of the cells.

The induction of MMP-9 release from granulocytes by Vitamin E in UHMWPE

Ultra-high molecular weight polyethylene (UHMWPE) is a biopolymer widely used in orthopaedic implants and its oxidation is considered as major responsible for inflammation and the prosthesis failure. We have studied the effect on the activation of resting human granulocytes of the addition of Vitamin E (Vit.E, alpha-tocopherol), a natural biological antioxidant and antiinflammatory agent, to UHMWPE. We have measured changes in granulocytes morphology and respiratory burst by flow cytometry using Dihydrorhodamine 123 and matrix metalloproteinase 9 (MMP-9, gelatinase B) release and activity in the growth medium using substrate zymography following contact (60 min at 37 degrees C) with cell grade polystyrene (PS), normal UHMWPE (PE) and Vit.E added UHMWPE (PE Vit.E). FTIR analyses showed that the surfaces of PE and PE-Vit.E were not significantly different. PS, PE and PE Vit.E did not alter granulocytes morphology and respiratory burst as showed by the mean fluorescence emitted (PS=12.0+/-0.1, PE=13.0+/-0.4, PE Vit.E=14.5+/-0.1). PE Vit.E was able to increase MMP-9 release compared to PS and normal PE (215+/-16% of the control, p<0.001). The PE Vit.E-induced MMP-9 release was abolished by okadaic acid (0.5 nM), suggesting a direct role of Vit.E in the phenomenon.

Biomimetic myocardial patches fabricated with poly(ɛ-caprolactone) and polyethylene glycol-based polyurethanes.

The production of efficient heart patches for myocardium repair requires the use of biomaterials with high elastomeric properties and controllable biodegradability. To fulfil these design criteria we propose biodegradable poly(ester urethanes) and poly(ether ester urethanes) from poly(ɛ-caprolactone) (PCL) and poly(ethylene glycol) (PEG) as macrodiols, 1,4-diisocyanatobutane as diisocyanate, l-Lysine Ethyl Ester and Alanine-Alanine-Lysine (AAK) as chain extenders. This peptide was used to tune biodegradability properties, since the Alanine-Alanine sequence is a target for the elastase enzyme. Enzymatic degradation tests demonstrated the feasibility of tuning biodegradability properties due to the introduction of AAK peptide in polyurethane backbone. Two formulations have been processed into porous scaffolds by Thermally-Induced Phase Separation (TIPS). Scanning Electron Microscopy micrographs revealed promising microstructures, which were characterized by stretched and unidirectional pores and mimicked the striated muscle tissue. Tensile tests showed that, although scaffolds are characterized by lower mechanical properties than films, these substrates have suitable elastomeric behaviors and elastic moduli for contractile and soft tissue regeneration. Viability tests on cardiomyocytes revealed the best cell response for dense film and porous scaffold obtained from PCL and Lysine Ethyl Ester-based polyurethane, with an increased viability for the porous substrate, which is ascribable to the morphological features of its microstructure. Future works will be addressed to study the in vivo behavior of these constructs and to confirm their applicability for myocardial tissue engineering.

Chondrogenic induction of human mesenchymal stem cells using combined growth factors for cartilage tissue engineering.

The objective of this study was to evaluate whether growth factors (FGF‐2, FGF‐4 and FGF‐6) used alone or in combination with TGFβ2 are able to increase the proliferation and induce the differentiation of human bone marrow mesenchymal stem cells (hMSCs) to chondrocytes, with a view to using them in cartilage tissue engineering. Cells cultured in monolayer, used to test the activity of the growth factors on cell proliferation, showed that a combination of FGFs with TGFβ2 increases cell proliferation compared to cells cultured in control medium or in the presence of growth factors alone. The chondrogenic potential, evaluated in three‐dimensional (3D) cell aggregates, showed that FGF‐2 and FGF‐6, when used in combination with TGFβ2 increased the size and glycosaminoglycan content of the cell aggregates without increasing cell number. Extracellular matrix (ECM) also showed higher collagen type II immunoreactivity, which was particularly evident in an area similar to a germinative pole that was observed only in pellets cultured with FGF‐2 and FGF‐6 combined with TGFβ2, or in pellets cultured with FGF‐2 alone. Moreover, the RT‐PCR assay has highlighted an increased expression of collagen type II and Sox9, used as gene markers for chondrogenesis. We can conclude that combinations of FGF‐2 or FGF‐6 with TGFβ2 may provide a novel tool to induce the differentiation of adult human mesenchymal stem cells for applications in cartilage tissue engineering. Copyright

Cardiovascular biomaterials: when the inflammatory response helps to efficiently restore tissue functionality?

The evaluation of biological host response to implanted materials permits the determination of the safety and biocompatibility of biomedical devices, prostheses and biomaterials. Once a biomaterial is introduced into the body to a corresponding implant site, a sequence of events occurs promoting the activation of inflammatory mediators such as leukocytes and the release of signaling molecules such as cytokines and growth factors, evoking an inflammatory and wound healing process. This review examines the cellular and molecular mechanisms involved in the foreign body reaction, especially how cytokines impact the overall inflammatory response to devices. It also reviews how these events can be modulated by the physical and chemical properties of the biomaterials such as wettability, chemistry and geometry of surface. Particular attention is dedicated to the cardiovascular field, where the use of synthetic polymers has several limitations such as thrombogenicity and risk of infection. New materials and strategies to improve biomaterial characteristics are discussed. Copyright

The Role of Mechanical Stretching in the Activation and Localization of Adhesion Proteins and Related Intracellular Molecules

The molecular complexity of the processes which lead to cell adhesion includes membrane and cytoskeletal proteins, involved in the focal adhesion formation, as well as signaling molecules tightly associated with the main intracellular regulatory cascades (Akt/PKB and MAPK/Erk). Dynamic environments, which create substrate deformations at determined frequencies and timing, have significant influences on adhesion mechanisms and in general in cellular behavior. In this work, we investigated the role of mechanical stretching (10% substrate deformation, 1 Hz frequency applied up to 60 min) on adhesion proteins (vinculin and focal adhesion kinase—FAK), related RhoGTPases (Rac1 and RhoA), and intracellular pathways (Akt/PKB and MAPK/Erk) in terms of activation and membrane recruitment in relation with cytoskeletal changes observed (membrane ruffling and filopodia formation). These changes are due to intracellular molecular rearrangements, acting with sequential concerted dynamics, able to modify the cytoskeletal conformation. The observed cellular response adds some important issues for better understanding the cellular behavior in environment which mimic as close as possible the physiological conditions. J. Cell. Biochem. 112: 1403–1409, 2011.