Contributions of the integrin ß1 tail to cell adhesive forces.

Integrin receptors connect the extracellular matrix to the cell cytoskeleton to provide essential forces and signals. To examine the contributions of the ß1 integrin cytoplasmic tail to adhesive forces, we generated cell lines expressing wild-type and tail mutant ß1 integrins in ß1-null fibroblasts. Deletion of ß1 significantly reduced cell spreading, focal adhesion assembly, and adhesive forces, and expression of human ß1 (hß1) integrin in these cells restored adhesive functions. Cells expressing a truncated tail mutant had impaired spreading, fewer and smaller focal adhesions, reduced integrin binding to fibronectin, and lower adhesion strength and traction forces compared to hß1-expressing cells. All these metrics were equivalent to those for ß1-null cells, demonstrating that the ß1 tail is essential to these adhesive functions. Expression of the constitutively-active D759A hß1 mutant restored many of these adhesive functions in ß1-null cells, although with important differences when compared to wild-type ß1. Even though there were no differences in integrin-fibronectin binding and adhesion strength between hß1- and hß1-D759A-expressing cells, hß1-D759A-expressing cells assembled more but smaller adhesions than hß1-expressing cells. Importantly, hß1-D759A-expressing cells generated lower traction forces compared to hß1-expressing cells. These differences between hß1- and hß1-D759A-expressing cells suggest that regulation of integrin activation is important for fine-tuning cell spreading, focal adhesion assembly, and traction force generation.

Gradient biomaterials for soft-to-hard interface tissue engineering.

Interface tissue engineering (ITE) is a rapidly developing field that aims to fabricate biological tissue alternates with the goal of repairing or regenerating the functions of diseased or damaged zones at the interface of different tissue types (also called "interface tissues"). Notable examples of the interface tissues in the human body include ligament-to-bone, tendon-to-bone and cartilage-to-bone. Engineering interface tissues is a complex process, which requires a combination of specialized biomaterials with spatially organized material composition, cell types and signaling molecules. Therefore, the use of conventional biomaterials (monophasic or composites) for ITE has certain limitations to help stimulate the tissue integration or recreating the structural organization at the junction of different tissue types. The advancement of micro- and nanotechnologies enable us to develop systems with gradients in biomaterials properties that encourage the differentiation of multiple cell phenotypes and subsequent tissue development. In this review we discuss recent developments in the fabrication of gradient biomaterials for controlling cellular behavior such as migration, differentiation and heterotypic interactions. Moreover, we give an overview of potential uses of gradient biomaterials in engineering interface tissues such as soft tissues (e.g. cartilage) to hard tissues (e.g. bone), with illustrated experimental examples. We also address fundamentals of interface tissue organization, various gradient biomaterials used in ITE, micro- and nanotechnologies employed for the fabrication of those gradients, and certain challenges that must be met in order for ITE to reach its full potential.

Portable microcontact printing device for cell culture.

In this work, we developed a portable device to perform microcontact printing in a safety cabinet for cell culture. The device was designed to be small and non-bulky, easy to sterilize, while not requiring the use of electricity, and which requires very little manual handling. Moreover, the portable microcontact printer is reproducibly fabricated with a rapid prototyping system, and allows for the easy micropatterning of biomolecules with a resolution ranging from 20 to 500 µm. This opens new horizons in the direct and simple micropatterning of culture dishes and the mimicking and biofabrication of complex architectures of tissues.

Variability in the structure of epiphytic assemblages of Posidonia oceanica in relation to human interferences in the Gulf of Gabes, Tunisia.

In this study we evaluate whether the pattern of spatial variability of the macro-epiphyte assemblages of leaves of Posidonia oceanica differed in relation to anthropogenic interference in the Gulf of Gabes (southern coast of Tunisia). A hierarchical sampling design was used to compare epiphytic assemblages at 5 m depth in terms of abundance and spatial variability at disturbed and control locations. The results indicate that the biomass and mean percentage cover decreased at locations near the point of sewage outlet in comparison to control locations. These losses were related to the distance from the source of disturbance. This study revealed that the diversity is reduced in disturbed locations by the loss of biomass and the mean percentage cover, explained by means of a multiple-stressor model which plays an important role in the macro-epiphytes' setting. It is urgent to propose the best management plans to save the remaining P. oceanica meadow in the Gulf of Gabes and its associated epiphytes.

Fabrication of transferable micropatterned-co-cultured cell sheets with microcontact printing.

The purpose of the present study is to develop a novel method for the fabrication of transferable micropatterned cell sheets for tissue engineering. To achieve this development, microcontact printing of fibronectin on commercially available temperature-responsive dishes was employed. Primary rat hepatocytes were seeded on the dish surfaces printed with fibronectin. Under serum-free conditions, hepatocytes were attached onto fibronectin domains selectively. Then, a second cell type of endothelial cells was seeded in the presence of serum. Double fluorescent staining revealed that endothelial cells successfully adhered to the intervals of hepatocyte domains. Finally, all the cells were harvested as a single contiguous micropatterned cell sheet upon temperature-reduction. With a cell sheet manipulator having a gelatin layer for the support of harvested cell sheets, harvested micropatterned cell sheets were transferred to new dish surfaces. This technique would be useful for the fabrication of thick tissue constructs having a complex microarchitecture.

Construction of multifunctional proteins for tissue engineering: epidermal growth factor with collagen binding and cell adhesive activities.

The development of different techniques based on natural and polymeric scaffolds are useful for the design of different biomimetic materials. These approaches, however, require supplementary steps for the chemical or physical modification of the biomaterial. To avoid such steps, in the present study, we constructed a new multifunctional protein that can be easily immobilized onto hydrophobic surfaces, and at the same time helps enhance specific cell adhesion and proliferation onto collagen substrates. A collagen binding domain was fused to a previously constructed protein, which had an epidermal growth factor fused to a hydrophobic peptide that allows for cell adhesion. The new fusion protein, designated fnCBD-ERE-EGF is produced in Escherichia coli, and its abilities to bind to collagen and promote cell proliferation were investigated. fnCBD-ERE-EGF was shown to keep both collagen binding and cell growth-promoting activities comparable to those of the corresponding unfused proteins. The results obtained in this study also suggest the use of a fnCBD-ERE-EGF as an alternative for the design of multifunctional ECM-bound growth factor based materials.

Cell sheet technology and cell patterning for biofabrication.

We have developed cell sheet technology as a modern method for the fabrication of functional tissue-like and organ-like structures. This technology allows for a sheet of interconnected cells and cells in full contact with their natural extracellular environment to be obtained. A cell sheet can be patterned and composed according to more than one cell type. The key technology of cell sheet engineering is that a fabricated cell sheet can be harvested and transplanted utilizing temperature-responsive surfaces. In this review, we summarize different aspects of cell sheet engineering and provide a survey of the application of cell sheets as a suitable material for biofabrication and clinics. Moreover, since cell micropatterning is a key tool for cell sheet engineering, in this review we focus on the introduction of our approaches to cell micropatterning and cell co-culture to the principles of automation and how they can be subjected to easy robotics programming. Finally, efforts towards making cell sheet technology suitable for biofabrication and robotic biofabrication are also summarized.