Delphine Dean

Cell damage evaluation of thermal inkjet printed Chinese hamster ovary cells.

Thermal inkjet printing technology has been applied successfully to cell printing. However, there are concerns that printing process may cause cell damages or death. We conducted a comprehensive study of thermal inkjet printed Chinese hamster ovary (CHO) cells by evaluating cell viability and apoptosis, and possible cell membrane damages. Additionally, we studied the cell concentration of bio‐ink and found optimum printing of concentrations around 8 million cells per mL. Printed cell viability was 89% and only 3.5% apoptotic cells were observed after printing. Transient pores were developed in the cell membrane of printed cells. Cells were able to repair these pores within 2 h after printing. Green fluorescent protein (GFP) DNA plasmids were delivered to CHO‐S cells by co‐printing. The transfection efficiency is above 30%. We conclude that thermal inkjet printing technology can be used for precise cell seeding with minor effects and damages to the printed mammalian cells. The printing process causes transient pores in cell membranes, a process which has promising applications for gene and macroparticles delivery to induce the biocompatibility or growth of engineered tissues. Biotechnol. Bioeng. 2010;106: 963–969.

Green synthesis of robust, biocompatible silver nanoparticles using garlic extract

This paper details a facile approach for the synthesis of stable and monodisperse silver nanoparticles performed at ambient/low temperature where Allium sativum (garlic) extract functions as the silver salt reducing agent during nanoparticle synthesis as well as the post-synthesis stabilizing ligands. Varying the synthesis conditions provides control of particle size, size-distribution, and kinetics of particle formation. Infrared spectroscopy, energy dispersive x-ray chemical analysis, and high performance liquid chromatography indicated that the carbohydrates present in the garlic extract are the most likely nanoparticle stabilizing chemistry. The synthesized silver nanoparticles also demonstrate potential for biomeical applications, owing to the 1) enhanced stability in biological media, 2) resistance to oxidation by the addition of H2O2, 3) ease and scalability of synthesis, and 4) lack of harsh chemicals required for synthesis. Cytotoxicity assays indicated no decrease in cellular proliferation for vascular smooth muscle cells and 3T3 fibroblasts at a concentration of 25 μg/ml, confirming that garlic extract prepared silver nanoparticles are ideal candidates for future experimentation and implementation into biomedical applications.

Fluid flow forces and rhoA regulate fibrous development of the atrioventricular valves

Fibrous development of the extracellular matrix (ECM) of cardiac valves is necessary for proper heart function. Pathological remodeling of valve ECM is observed in both pediatric and adult cardiac disorders. It is well established that intracardiac hemodynamics play a significant role in the morphogenesis of cardiovascular tissues. However, the mechanisms that transduce mechanical forces into morphogenetic processes are not well understood. Here, we report the development of a three-dimensional, in vitro culture system that allows for culture of embryonic valve tissue under specific pulsatile flow conditions. This system was used to investigate the role that fluid flow plays in fibrous ECM expression during valve formation and to test the underlying cellular mechanisms that regulate this mechanotransduction. When cultured under pulsatile flow, developing valve tissues upregulated fibrous ECM expression at both the transcript and protein levels in comparison to no-flow controls. Flow-cultured valve tissues also underwent morphological development, as cushions elongated into leaflet-like structures that were absent in no-flow controls. Furthermore, rhoA, a member of the cytoskeletal actin-regulating GTPase family of proteins, was upregulated and activated by flow culture. Inhibition of the downstream rhoA effector kinase, ROCK, blocked flow-driven fibrous ECM accumulation and tissue stiffening, while the addition of lysophosphatidic acid (LPA), a rhoA activator, stimulated fibrous ECM deposition and tissue stiffening. These results support a prominent role for the rhoA pathway in the mechanotransduction of hemodynamic forces during fibrous remodeling of developing valve tissue. Our results also point to a potential link between regulation of the actinomyosin cytoskeleton and fibrous ECM synthesis in cardiovascular tissues.

Alteration of Dentin-Enamel Mechanical Properties Due to Dental Whitening Treatments

The mechanical properties of dentin and enamel affect the reliability and wear properties of a tooth. This study investigated the influence of clinical dental treatments and procedures, such as whitening treatments or etching prior to restorative procedures. Both autoclaved and non-autoclaved teeth were studied in order to allow for both comparison with published values and improved clinical relevance. Nanoindentation analysis with the Oliver-Pharr model provided elastic modulus and hardness across the dentin-enamel junction (DEJ). Large increases were observed in the elastic modulus of enamel in teeth that had been autoclaved (52.0 GPa versus 113.4 GPa), while smaller increases were observed in the dentin (17.9 GPa versus 27.9 GPa). Likewise, there was an increase in the hardness of enamel (2.0 GPa versus 4.3 GPa) and dentin (0.5 GPa versus 0.7 GPa) with autoclaving. These changes suggested that the range of elastic modulus and hardness values previously reported in the literature may be partially due to the sterilization procedures. Treatment of the exterior of non-autoclaved teeth with Crest Whitestrips, Opalescence or UltraEtch caused changes in the mechanical properties of both the enamel and dentin. Those treated with Crest Whitestrips showed a reduction in the elastic modulus of enamel (55.3 GPa to 32.7 GPa) and increase in the elastic modulus of dentin (17.2 GPa to 24.3 GPa). Opalescence treatments did not significantly affect the enamel properties, but did result in a decrease in the modulus of dentin (18.5 GPa to 15.1 GPa). Additionally, as expected, UltraEtch treatment decreased the modulus and hardness of enamel (48.7 GPa to 38.0 GPa and 1.9 GPa to 1.5 GPa, respectively) and dentin (21.4 GPa to 15.0 GPa and 1.9 GPa to 1.5 GPa, respectively). Changes in the mechanical properties were linked to altered protein concentration within the tooth, as evidenced by fluorescence microscopy and Fourier transform infrared spectroscopy.

Cartilage Aggrecan Can Undergo Self-Adhesion

Here it is reported that aggrecan, the highly negatively charged macromolecule in the cartilage extracellular matrix, undergoes Ca(2+)-mediated self-adhesion after static compression even in the presence of strong electrostatic repulsion in physiological-like solution conditions. Aggrecan was chemically end-attached onto gold-coated planar silicon substrates and gold-coated microspherical atomic force microscope probe tips (end radius R approximately 2.5 mum) at a density ( approximately 40 mg/mL) that simulates physiological conditions in the tissue ( approximately 20-80 mg/mL). Colloidal force spectroscopy was employed to measure the adhesion between opposing aggrecan monolayers in NaCl (0.001-1.0 M) and NaCl + CaCl(2) ([Cl(-)] = 0.15 M, [Ca(2+)] = 0 - 75 mM) aqueous electrolyte solutions. Aggrecan self-adhesion was found to increase with increasing surface equilibration time upon compression (0-30 s). Hydrogen bonding and physical entanglements between the chondroitin sulfate-glycosaminoglycan side chains are proposed as important factors contributing to aggrecan self-adhesion. Self-adhesion was found to significantly increase with decreasing bath ionic strength (and hence, electrostatic double-layer repulsion), as well as increasing Ca(2+) concentration due to the additional ion-bridging effects. It is hypothesized that aggrecan self-adhesion, and the macromolecular energy dissipation that results from this self-adhesion, could be important factors contributing to the self-assembled architecture and integrity of the cartilage extracellular matrix in vivo.

Increased extracellular matrix density decreases MCF10A breast cell acinus formation in 3D culture conditions

The extracellular matrix (ECM) contributes to the generation and dynamic of normal breast tissue, in particular to the generation of polarized acinar and ductal structures. In vitro 3D culture conditions, including variations in the composition of the ECM, have been shown to directly influence the formation and organization of acinus‐like and duct‐like structures. Furthermore, the density of the ECM appears to also play a role in the normal mammary tissue and tumour formation. Here we show that the density of the ECM directly influences the number, organization and function of breast acini. Briefly, non‐malignant human breast MCF10A cells were incubated in increasing densities of a Matrigel®–collagen I matrix. Elastic moduli near and distant to the acinus structures were measured by atomic force microscopy, and the number of acinus structures was determined. Immunochemistry was used to investigate the expression levels of E‐cadherin, laminin, matrix metalloproteinase‐14 and ß‐casein in MCF10A cells. The modulus of the ECM was significantly increased near the acinus structures and the number of acinus structures decreased with the increase in Matrigel–collagen I density. As evaluated by the expression of laminin, the organization of the acinus structures present was altered as the density of the ECM increased. Increases in both E‐cadherin and MMP14 expression by MCF10A cells as ECM density increased were also observed. In contrast, MCF10A cells expressed lower ß‐casein levels as the ECM density increased. Taken together, these observations highlight the key role of ECM density in modulating the number, organization and function of breast acini. Copyright

Role of cytoskeletal components in stress-relaxation behavior of adherent vascular smooth muscle cells.

A number of recent studies have demonstrated the effectiveness of atomic force microscopy (AFM) for characterization of cellular stress-relaxation behavior. However, this techniques recent development creates considerable need for exploration of appropriate mechanical models for analysis of the resultant data and of the roles of various cytoskeletal components responsible for governing stress-relaxation behavior. The viscoelastic properties of vascular smooth muscle cells (VSMCs) are of particular interest due to their role in the development of vascular diseases, including atherosclerosis and restenosis. Various cytoskeletal agents, including cytochalasin D, jasplakinolide, paclitaxel, and nocodazole, were used to alter the cytoskeletal architecture of the VSMCs. Stress-relaxation experiments were performed on the VSMCs using AFM. The quasilinear viscoelastic (QLV) reduced-relaxation function, as well as a simple power-law model, and the standard linear solid (SLS) model, were fitted to the resultant stress-relaxation data. Actin depolymerization via cytochalasin D resulted in significant increases in both rate of relaxation and percentage of relaxation; actin stabilization via jasplakinolide did not affect stress-relaxation behavior. Microtubule depolymerization via nocodazole resulted in nonsignificant increases in rate and percentage of relaxation, while microtubule stabilization via paclitaxel caused significant decreases in both rate and percentage of relaxation. Both the QLV reduced-relaxation function and the power-law model provided excellent fits to the data (R(2)=0.98), while the SLS model was less adequate (R(2)=0.91). Data from the current study indicate the important role of not only actin, but also microtubules, in governing VSMC viscoelastic behavior. Excellent fits to the data show potential for future use of both the QLV reduced-relaxation function and power-law models in conjunction with AFM stress-relaxation experiments.

Effects of serum deprivation on the mechanical properties of adherent vascular smooth muscle cells

Vascular smooth muscle cell (VSMC) function plays a key role in regulating the development and progression of vascular lesions. Among the more significant phenomena that occur during the development of these lesions is the phenotypic switching of VSMCs from a contractile to a synthetic state. A better understanding of the concurrent changes to VSMC mechanical properties that occur with phenotypic shifts can help to elucidate the role of VSMC mechanics in the development of vascular diseases. In the current study, the mechanical properties of adherent cultured rat aortic VSMCs were assessed by atomic force microscopy. Serum starvation was used to induce a phenotypic shift in vitro. It was concluded that serum starvation led to a statistically significant increase in apparent elastic modulus after 5 days, as well as a statistically significant decrease in hysteresis after culture for 3 days. If this trend of VSMC mechanical properties changing concurrently with phenotypic shifts were to hold true in vivo, such changes could affect the processes of mechanotransduction and/or arterial mechanical properties, thereby contributing to the progression of vascular disease.

Molecular Adhesion between Cartilage Extracellular Matrix Macromolecules

In this study, we investigated the molecular adhesion between the major constituents of cartilage extracellular matrix, namely, the highly negatively charged proteoglycan aggrecan and the type II/IX/XI fibrillar collagen network, in simulated physiological conditions. Colloidal force spectroscopy was applied to measure the maximum adhesion force and total adhesion energy between aggrecan end-attached spherical tips (end radius R ≈ 2.5 μm) and trypsin-treated cartilage disks with undamaged collagen networks. Studies were carried out in various aqueous solutions to reveal the physical factors that govern aggrecan–collagen adhesion. Increasing both ionic strength and [Ca2+] significantly increased adhesion, highlighting the importance of electrostatic repulsion and Ca2+-mediated ion bridging effects. In addition, we probed how partial enzymatic degradation of the collagen network, which simulates osteoarthritic conditions, affects the aggrecan–collagen interactions. Interestingly, we found a significant increase in aggrecan–collagen adhesion even when there were no detectable changes at the macro- or microscales. It is hypothesized that the aggrecan–collagen adhesion, together with aggrecan–aggrecan self-adhesion, works synergistically to determine the local molecular deformability and energy dissipation of the cartilage matrix, in turn, affecting its macroscopic tissue properties.

Collagen Matrix Alignment Using Inkjet Printer Technology

Collagen fiber orientation plays an important role in many cell properties and actions in vivo. While it is easy to replicate randomly oriented collagen in vitro, it is much more difficult to create aligned collagen matrices for cell culture. In this work, a novel inkjet printer-based collagen alignment technique was established. A collagen type I solution was printed in a line pattern onto glass substrates using a modified inkjet printer. Staining studies indicated that the heat involved in the printing process is not great enough to denature the collagen. The extent of alignment was observed using light microscopy, atomic force microscopy (AFM), and polarized light microscopy. Additionally, cardiomyocytes, which require extracellular matrix alignment to maintain their in vivo phenotype, were cultured on the aligned matrices. The cells grew along the lines of collagen and coordinated beating, indicating the success of the aligned matrix. This collagen alignment technique is cheap, fast, precise, and easy to use in comparison to other current techniques. It may be used to align other extracellular matrix proteins and could even be used to create a three dimensional construct with varying fiber orientations.