A Agnese Ravetto

Tuning the differentiation of periosteum-derived cartilage using biochemical and mechanical stimulations

OBJECTIVE In this study, we aim at tuning the differentiation of periosteum in an organ culture model towards cartilage, rich in collagen type II, using combinations of biochemical and mechanical stimuli. We hypothesize that addition of TGF-β will stimulate chondrogenesis, whereas sliding indentation will enhance collagen synthesis. DESIGN Periosteum was dissected from the tibiotarsus of 15-day-old chick embryos. Explants were embedded in between two agarose layers, and cultured without stimulation (control), with biochemical stimulation (10 ng/ml TGF-β1), with mechanical stimulation (sliding indentation), or both biochemical and mechanical stimulations. Sliding indentation was introduced as a method to induce tensile tissue strain. Analysis included quantification of DNA, collagen and GAG content, conventional histology, and immunohistochemistry for collagen type I and II at 1 or 2 weeks of culture. RESULTS Embedding the periosteal explants in between agarose layers induced cartilage formation, confirmed by synthesis of sGAG and collagen type II. Addition of TGF-β1 to the culture medium did not further enhance this chondrogenic response. Applying sliding indentation only to the periosteum in between agarose layers enhanced the production of collagen type I, leading to the formation of fibrous tissue without any evidence of cartilage formation. However, when stimulated by both TGF-β1 and sliding indentation, collagen production was still enhanced, but now collagen type II, while sGAG was found to be similar to TGF-β1 or unloaded samples. CONCLUSIONS The type of tissue produced by periosteal explants can be tuned by combining mechanical stimulation and soluble factors. TGF-β1 stimulated a chondrocyte phenotype and sliding indentation stimulated collagen synthesis. Such a combination may be valuable for improvement of the quality of tissue-engineered cartilage.

Cell types can be distinguished by measuring their viscoelastic recovery times using a micro-fluidic device

We introduce a simple micro-fluidic device containing an actuated flexible membrane, which allows the viscoelastic characterization of cells in small volumes of suspension by loading them in compression and observing the cell deformation in time. From this experiment, we can determine the characteristic time constant of recovery of the cell. To validate the device, two cell types known to have different cytoskeletal structures, 3T3 fibroblasts and HL60 cells, are tested. They show a substantially different response in the device and can be clearly distinguished on the basis of the measured characteristic recovery time constant. Also, the effect of breaking down the actin network, a main mechanical component of the cytoskeleton, by a treatment with Cytochalasin D, results in a substantial increase of the measured characteristic recovery time constant. Experimental variations in loading force, loading time, and surface treatment of the device also influence the measured characteristic recovery time constant significantly. The device can therefore be used to distinguish between cells with different mechanical structure in a quantitative way, and makes it possible to study changes in the mechanical response due to cell treatments, changes in the cell’s micro-environment, and mechanical loading conditions.

Monocytic Cells Become Less Compressible but More Deformable upon Activation

Aims Monocytes play a significant role in the development of atherosclerosis. During the process of inflammation, circulating monocytes become activated in the blood stream. The consequent interactions of the activated monocytes with the blood flow and endothelial cells result in reorganization of cytoskeletal proteins, in particular of the microfilament structure, and concomitant changes in cell shape and mechanical behavior. Here we investigate the full elastic behavior of activated monocytes in relation to their cytoskeletal structure to obtain a better understanding of cell behavior during the progression of inflammatory diseases such as atherosclerosis. Methods and Results The recently developed Capillary Micromechanics technique, based on exposing a cell to a pressure difference in a tapered glass microcapillary, was used to measure the deformation of activated and non-activated monocytic cells. Monitoring the elastic response of individual cells up to large deformations allowed us to obtain both the compressive and the shear modulus of a cell from a single experiment. Activation by inflammatory chemokines affected the cytoskeletal organization and increased the elastic compressive modulus of monocytes with 73–340%, while their resistance to shape deformation decreased, as indicated by a 25–88% drop in the cell’s shear modulus. This decrease in deformability is particularly pronounced at high strains, such as those that occur during diapedesis through the vascular wall. Conclusion Overall, monocytic cells become less compressible but more deformable upon activation. This change in mechanical response under different modes of deformation could be important in understanding the interplay between the mechanics and function of these cells. In addition, our data are of direct relevance for computational modeling and analysis of the distinct monocytic behavior in the circulation and the extravascular space. Lastly, an understanding of the changes of monocyte mechanical properties will be important in the development of diagnostic tools and therapies concentrating on circulating cells.

A membrane-based microfluidic device for mechano-chemical cell manipulation

We introduce a microfluidic device for chemical manipulation and mechanical investigation of circulating cells. The device consists of two crossing microfluidic channels separated by a porous membrane. A chemical compound is flown through the upper “stimulus channel”, which diffuses through the membrane into the lower “cell analysis channel”, in which cells are mechanically deformed in two sequential narrow constrictions, one before and one after crossing the stimulus channel. Thus, this system permits to measure cell deformability before and after chemical cues are delivered to the cells within one single chip. The validity of the device was tested with monocytic cells stimulated with an actin-disrupting agent (Cytochalasin-D). Furthermore, as proof of principle of the device application, the effect of an anti-inflammatory drug (Pentoxifylline) was tested on monocytic cells activated with Lipopolysaccharides and on monocytes from patients affected by atherosclerosis. The results show that the system can detect differences in cell mechanical deformation after chemical cues are delivered to the cells through the porous membrane. Diffusion of Cytochalasin-D resulted in a considerable decrease in entry time in the narrow constriction and an evident increase in the velocity within the constriction. Pentoxifylline showed to decrease the entry time but not to affect the transit time within the constriction for monocytic cells. Monocytes from patients affected by atherosclerosis were difficult to test in the device due to increased adhesion to the walls of the microfluidic channel. Overall, this analysis shows that the device has potential applications as a cellular assay for analyzing cell-drug interaction.