X-ray scanning microscopies of microcalcifications in abdominal aortic and popliteal artery aneurysms.

Abdominal aortic and popliteal artery aneurysms are vascular diseases which show massive degeneration, weakening of the vascular wall and loss of the vascular tissue functionality. They are driven by inflammatory, hemodynamical factors and biological alterations that may lead, in the case of an abdominal aortic aneurysm, to sudden and dangerous ruptures of the arteries. Here, human aortic and popliteal aneurysm tissues were obtained during surgical repair, and studied by synchrotron radiation X-ray scanning microdiffraction and small-angle scattering, to investigate the microcalcifications present in the tissues. Data collected during the experiments were transformed into quantitative microscopy images through the combination of statistical approaches and crystallographic methods. As a result of this multi-step analysis, microcalcifications, which are markers of the pathology, were classified in terms of chemical and structural content. This analysis helped to identify the presence of nanocrystalline hy-droxy-apatite and microcrystalline cholesterol, embedded in myofilament, and elastin-containing tissue with low collagen content in predominantly nanocrystalline areas. The generality of the approach allows it to be transferred to other types of tissue and other pathologies affected by microcalcifications, such as thyroid carcinoma, breast cancer, testicular microli-thia-sis or glioblastoma.

An optimized table-top small-angle X-ray scattering set-up for the nanoscale structural analysis of soft matter.

The paper shows how a table top superbright microfocus laboratory X-ray source and an innovative restoring-data algorithm, used in combination, allow to analyze the super molecular structure of soft matter by means of Small Angle X-ray Scattering ex-situ experiments. The proposed theoretical approach is aimed to restore diffraction features from SAXS profiles collected from low scattering biomaterials or soft tissues, and therefore to deal with extremely noisy diffraction SAXS profiles/maps. As biological test cases we inspected: i) residues of exosomes' drops from healthy epithelial colon cell line and colorectal cancer cells; ii) collagen/human elastin artificial scaffolds developed for vascular tissue engineering applications; iii) apoferritin protein in solution. Our results show how this combination can provide morphological/structural nanoscale information to characterize new artificial biomaterials and/or to get insight into the transition between healthy and pathological tissues during the progression of a disease, or to morphologically characterize nanoscale proteins, based on SAXS data collected in a room-sized laboratory.

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.

Biological evaluation of materials for cardiovascular application: the role of the short-term inflammatory response in endothelial regeneration.

Because of their suitable bio-mechanical properties, polymeric materials, such as Poly(L-lactic acid) (PLLA), and poly (lactic-co-glycolic acid) (PLGA), are often used in the biomedical field, in particular for cardiovascular applications. Implanted materials induce several events related to the inflammatory reaction, such as macrophage adhesion and activation with following cytokine release. This work considered the effect of macrophage adhesion and related cytokine release on endothelial cells (PAOEC) proliferation and migration. Slight differences have been shown by the macrophages reaction when in contact with PLLA, PLGA, or PLLA/PLGA blend. However, these differences showed to differently enhance endothelial cells behavior in terms of wound healing. These data suggest the inflammatory reaction as a useful way to consider concerning materials biocompatibility, in order to optimize the endothelial regeneration following vascular prosthetic implants.

Study on the interaction between gelatin and polyurethanes derived from fatty acids.

In this study, gelatin was blended to proprietary noncytotoxic polyurethanes (PU) derived from vegetable oils with different weight ratios, as material for the preparation of novel biomedical products. The PU/gelatin blends were characterized for their morphology through scanning electron microscopy. Mechanical and thermal properties, chemical interactions between components, degradation behavior, surface properties, cell adhesion, and bioactivity were investigated as a function of the protein content. Higher blend miscibility was observed for the amorphous PUs, derived from oleic acid. Properties of PU/gelatin films were strongly influenced by the concentration of gelatin in the films. Gelatin enhanced the hydrophilicity, bioactivity, and cell adhesion of PUs.

The biological response of poly(L-lactide) films modified by different biomolecules: role of the coating strategy.

The interactions between the surface of synthetic scaffolds and cells play an important role in tissue engineering applications. To improve these interactions, two strategies are generally followed: surface coating with large proteins and surface grafting with small peptides. The proteins and peptides more often used and derived from the extracellular matrix, are fibronectin, laminin, and their active peptides, RGD and SIKVAV, respectively. The aim of this work was to compare the effects of coating and grafting of poly(L-lactide) (PLLA) films on MRC5 fibroblast cells. Grafting reactions were verified by X-ray photoelectron spectroscopy. Cell adhesion and proliferation on coated and grafted PLLA surfaces were measured by cell counting. Vinculin localization and distribution were performed on cell cultured on PLLA samples using a fluorescence microscopy technique. Finally, western blot was performed to compare signals of cell adhesion proteins, such as vinculin, Rac1, and RhoA, as well as cell proliferation, such as PCNA. These tests showed similar results for fibronectin and laminin coated PLLA, while RGD grafting is more effective compared with SIKVAV grafting. Considering the overall view of these results, although coating and grafting can both be regarded as effective methods for surface modification to enhance cell adhesion and proliferation on a biomaterial, RGD grafted PLLA show better cell adhesion and proliferation than coated PLLA, while SIKVAV grafted PLLA show similar adhesion but worse proliferation. These data verified different biological effects depending on the surface modification method used.

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.

Overstressed mechanical stretching activates survival and apoptotic signals in fibroblasts.

The interest of scientists in the effects of mechanical stresses on cells is growing, in order to reproduce and understand cell behaviour in an environment closely reproducing physiological conditions. There have been many studies showing that mechanical stimulations are involved in regulating the proliferation, apoptosis and synthesis of proteins and cell morphology. In this study, we have considered the effects of a 20% stretching mechanical stress on MRC5 lung fibroblast cells in order to verify the role of survival/apoptotic pathways. As a survival pathway, the activation of Akt has been studied in association with pro-apoptotic or anti-apoptotic signals such as the Bax/Bcl-2 ratio and cleavage of caspases 3 and 9. Findings have shown the effects of overstressed cellular stretching to be a balance of a cause-and-effect reaction between survival and apoptosis.

Effects of mechanical stress on cell adhesion: a possible mechanism for morphological changes.

The transmission of mechanical forces to cells is followed among all by biological signals related to changes in the assembly or disassembly of integrins associated linker proteins, such as vinculin. We applied for 3 hours 2% cyclic mechanical strain at the frequency of 1 Hz to human fibroblasts cultured on a deformable substrate; substrate deformation resulted to modify the number, length and area of vinculin positive focal adhesion contacts when compared to not stretched cells. The mechanism behind these morphological changes is related to Akt and RhoA roles in focal adhesion assembly. In the case of Akt and Rho inhibition, focal contacts disassembled only in presence of stretching mechanical stress, highlighting the role of mechanical stress on focal adhesion maturation in terms of multimolecolar assembly which from focal complexes leads to fibrillar adhesion.

Behaviour of human mesenchymal stem cells on a polyelectrolyte-modified HEMA hydrogel for silk-based ligament tissue engineering.

The aim of this study was to design a functional bio-engineered material to be used as scaffold for autologous mesenchymal stem cells in ligament tissue engineering. Polyelectrolyte modified HEMA hydrogel (HEMA-co-METAC), applied as coating on silk fibroin fibres, has been formulated in order to take advantage of the biocompatibility of the polyelectrolyte by increasing its mechanical properties with silk fibres. Human bone marrow mesenchymal stem cells behaviour on such reinforced polyelectrolyte has been studied by evaluating cell morphology, cell number, attachment, spreading and proliferation together with collagen matrix production and its mRNA expression. Silk fibroin fibres matrices with HEMA-co-METAC coating exhibited acceptable mechanical behaviour compared to the natural ligament, good human mesenchymal stem cell adhesion and with mRNA expression studies higher levels of collagen types I and III expression when compared to control cells on polystyrene. These data indicate high expression of mRNA for proteins responsible for the functional characteristics of the ligaments and suggest a potential for use of this biomaterial in ligament tissue-engineering applications.

Properties of zinc releasing surfaces for clinical applications.

Two series of glasses of general formula (2-p) SiO2.1.1Na2O.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 Hench's Bioglass.

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 0.25 Hz to 3 Hz). 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%3 Hz). 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.

Biological performances of collagen-based scaffolds for vascular tissue engineering.

Collagen is widely used for biomedical applications and it could represent a valid alternative scaffold material for vascular tissue engineering. In this work, reconstituted collagen films were prepared from neutralized acid-soluble solutions for subsequent haemocompatibility and cell viability performance assays. First, haemoglobin-free, thrombelastography and platelet adhesion tests were performed in order to investigate the blood contact performance. Secondly, specimens were seeded with endothelial cells and smooth muscle cells, and cell viability tests were carried out by MTT and SEM. Results show that neutralized acid-soluble type I collagen films do not enhance blood coagulation, do not alter normal viscoelastic properties of blood and slightly activate platelet adhesion and aggregation. Cell culture shows that the samples are adequate substrates to support the adhesion and proliferation of endothelial and smooth muscle cells.

Evaluation of bioresorbable implants from bovine bone: In vitro preliminary observations.

Biocompatibility evaluation is a fundamental step in developing new biomaterials. Implants derived from bovine tibial compact bone were analyzed with in vitro tests using fibroblast and osteoblast-like cells. Initially, cell attachment and proliferation were quantified. Results indicated that the pins did not interfere with normal cell adhesion and proliferation; more-over, cell morphology was observed using scanning electron microscopy (SEM) and confocal fluorescence microscopy. In vivo experiments to evaluate material osteointegration are currently in progress. (Journal of Applied Biomaterials and Biomechanics 2005; 3: 35-41).

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.