Rami Mhanna

Artificial Bacterial Flagella for Remote‐Controlled Targeted Single‐Cell Drug Delivery

We hypothesized that ABFs can be functionalized with liposomes, thus allowing these micromachines to per-form biological or biomedical tasks in a remotely con-trolled fashion. To test this hypothesis fl uorescently labeled liposomes and calcein-loaded liposomes were adsorbed on the surface of ABFs. The adsorption of liposomes on the ABFs was confi rmed by quartz crystal microbalance with dissipation monitoring (QCM-D) and fl uorescence recovery after photobleaching (FRAP). The liposome-functionalized ABFs were then placed in contact with cells in vitro and the uptake of calcein (a model water soluble drug) by cells was monitored using fl uorescence microscopy. The fabrication of ABFs was followed as described ear-lier by Tottori et al.

Layer-by-layer films made from extracellular matrix macromolecules on silicone substrates.

The layer-by-layer (LbL) technique has been widely used to produce nanofilms for biomedical applications. Naturally occurring polymers such as ECM macromolecules are attractive candidates for LbL film preparation. In this study, we assessed the build-up of type I collagen (Col1)/chondroitin sulfate (CS) or Col1/Heparin (HN) on polydimethylsiloxane (PDMS) substrates. The build-up was assessed by quartz crystal microbalance with dissipation (QCM-D) and atomic force microscopy (AFM). Integrin-mediated cell adhesion was assessed by studying the cytoskeletal organization of mammalian primary cells (chondrocytes) seeded on different end layers and number of layers. Data generated from the QCM-D observations showed a consistent build-up of films with more adsorption in the case of Col1/HN. Col1/CS films were stable in media, whereas Col1/HN films were not. AFM analysis showed that the layers were fibrillar in structure for both systems and between 20 and 30 nm thick. The films promoted cell adhesion when compared with tissue culture plastic in serum-free media with cycloheximide. Crosslinking of the films resulted in constrained cell spreading and a ruffled morphology. Finally, beta1 integrin blocking antibodies prevented cell spreading, suggesting that cell adhesion and spreading were mediated mainly by interaction with the collagen fibrils. The ability to construct stable ECM-based films on PDMS has particular relevance in mechanobiology, microfluidics, and other biomedical applications.

Interference with the contractile machinery of the fibroblastic chondrocyte cytoskeleton induces re-expression of the cartilage phenotype through involvement of PI3K, PKC and MAPKs

Chondrocytes rapidly lose their phenotypic expression of collagen II and aggrecan when grown on 2D substrates. It has generally been observed that a fibroblastic morphology with strong actin-myosin contractility inhibits chondrogenesis, whereas chondrogenesis may be promoted by depolymerization of the stress fibers and/or disruption of the physical link between the actin stress fibers and the ECM, as is the case in 3D hydrogels. Here we studied the relationship between the actin-myosin cytoskeleton and expression of chondrogenic markers by culturing fibroblastic chondrocytes in the presence of cytochalasin D and staurosporine. Both drugs induced collagen II re-expression; however, renewed glycosaminoglycan synthesis could only be observed upon treatment with staurosporine. The chondrogenic effect of staurosporine was augmented when blebbistatin, an inhibitor of myosin/actin contractility, was added to the staurosporine-stimulated cultures. Furthermore, in 3D alginate cultures, the amount of staurosporine required to induce chondrogenesis was much lower compared to 2D cultures (0.625 nM vs. 2.5 nM). Using a selection of specific signaling pathway inhibitors, it was found that PI3K-, PKC- and p38-MAPK pathways positively regulated chondrogenesis while the ERK-pathway was found to be a negative regulator in staurosporine-induced re-differentiation, whereas down-regulation of ILK by siRNA indicated that ILK is not determining for chondrocyte re-differentiation. Furthermore, staurosporine analog midostaurin displayed only a limited chondrogenic effect, suggesting that activation/deactivation of a specific set of key signaling molecules can control the expression of the chondrogenic phenotype. This study demonstrates the critical importance of mechanobiological factors in chondrogenesis suggesting that the architecture of the actin cytoskeleton and its contractility control key signaling molecules that determine whether the chondrocyte phenotype will be directed along a fibroblastic or chondrogenic path.

Use of biomimetic microtissue spheroids and specific growth factor supplementation to improve tenocyte differentiation and adaptation to a collagen-based scaffold in vitro.

Tenocytes represent a valuable source of cells for the purposes of tendon tissue engineering and regenerative medicine and as such, should possess a high degree of tenogenic differentiation prior to their use in vivo in order to achieve maximal efficacy. In the current report, we identify an efficient means by which to maintain differentiated tenocytes in vitro by employing the hanging drop technique in combination with defined growth media supplements. Equine tenocytes retained a more differentiated state when cultured as scaffold-free microtissue spheroids in low serum-containing medium supplemented with L-ascorbic acid 2-phosphate, insulin and transforming growth factor (TGF)-β1. This was made evident by significant increases in the expression levels of pro-tenogenic markers collagen type I (COL1A2), collagen type III (COL3A1), scleraxis (SCX) and tenomodulin (TNMD), as well as by enhanced levels of collagen type I and tenomodulin protein. Furthermore, tenocytes cultured under these conditions demonstrated a typical spindle-like morphology and when embedded in collagen gels, became highly aligned with respect to the orientation of the collagen structure following their migration out from the microtissue spheroids. Our findings therefore provide evidence to support the use of a biomimetic microtissue approach to culturing tenocytes and that in combination with the defined growth media described, can improve their differentiation status and functional repopulation of collagen matrix.

Probing the microenvironmental conditions for induction of superficial zone protein expression

OBJECTIVE To determine the in vitro conditions which promote expression of superficial zone protein (SZP). METHODS Chondrocytes from 6-month-old calves were expanded in monolayer culture and the expression of SZP in alginate bead and monolayer culture was quantified with quantitative real time-polymerase chain reaction (qRT-PCR) and immunostaining. The effect of oxygen tension on SZP expression was determined by qRT-PRC analysis of cells cultured in two dimension (2D) and three dimension (3D) under hypoxic (1% pO2) or normoxic (21% pO2) conditions. Finally, to examine the effect of cyclic tensile strain on expression of SZP in 2D and 3D cultures, chondrocytes encapsulated in alginate beams or seeded on type I collagen coated polydimethylsiloxane (PDMS) chambers were subjected to 5% strain at 1 Hz, 2 h/day for 4 days or 2 h at the fourth day of culture and mRNA levels were quantified. RESULTS Bovine chondrocytes in monolayer showed a drastic decrease in SZP expression, similar in trend to the commonly reported downregulation of type II collagen (Col2). Chondrocytes embedded in alginate beads for 4 days re-expressed SZP but not Col2. SZP expression was higher under normoxic conditions whereas Col2 was upregulated only in alginate beads under hypoxic conditions. Cyclic mechanical strain showed a tendency to upregulate mRNA levels of SZP. CONCLUSIONS A microenvironment encompassing a soft encapsulation material and 21% oxygen is sufficient for fibroblastic chondrocytes to re-express SZP. These results serve as a guideline for the design of stratified engineered articular cartilage and suggest that microenvironmental cues (oxygen tension level) strongly influence the pattern of SZP expression in vivo.

Sulfated Alginate as a Mimic of Sulfated Glycosaminoglycans: Binding of Growth Factors and Effect on Stem Cell Behavior

Sulfated glycosaminoglycans (GAGs) are principal elements of the extracellular matrix, where they are involved in a plethora of signaling pathways mainly via interactions with diverse proteins such as growth factors and cytokines. However, the mechanisms that drive these interactions are not yet clear, mostly because of the difficulty to access large quantities of homogeneously sulfated natural GAGs. In this work, GAG mimics are synthesized from readily available alginate with different degrees of sulfation (DS, from 0.8 to 2.6) by simple process. The effect of the DS is determined on the binding of basic fibroblast growth factor (FGF‐2). The enzyme‐linked immunosorbent assay demonstrates that the binding of FGF‐2 is significantly greater for alginates with high DS as compared to unmodified and low sulfated analogs. These results are further applied to engineer FGF‐2‐loaded substrates for stem cell culturing using the layer‐by‐layer approach. All films support the attachment and growth of adipose derived stem cells (ADSCs). Noteworthy, highly sulfated alginates maintain the stemness of the ADSCs that exhibit remarkably long filopodia. These results can be exploited in the engineering of novel substrates that induce targeted cell behavior via controlled protein delivery and for tissue engineering constructs applicable in various regenerative approaches.

Development of Microplatforms to Mimic the In Vivo Architecture of CNS and PNS Physiology and Their Diseases

Understanding the mechanisms that govern nervous tissues function remains a challenge. In vitro two-dimensional (2D) cell culture systems provide a simplistic platform to evaluate systematic investigations but often result in unreliable responses that cannot be translated to pathophysiological settings. Recently, microplatforms have emerged to provide a better approximation of the in vivo scenario with better control over the microenvironment, stimuli and structure. Advances in biomaterials enable the construction of three-dimensional (3D) scaffolds, which combined with microfabrication, allow enhanced biomimicry through precise control of the architecture, cell positioning, fluid flows and electrochemical stimuli. This manuscript reviews, compares and contrasts advances in nervous tissues-on-a-chip models and their applications in neural physiology and disease. Microplatforms used for neuro-glia interactions, neuromuscular junctions (NMJs), blood-brain barrier (BBB) and studies on brain cancer, metastasis and neurodegenerative diseases are addressed. Finally, we highlight challenges that can be addressed with interdisciplinary efforts to achieve a higher degree of biomimicry. Nervous tissue microplatforms provide a powerful tool that is destined to provide a better understanding of neural health and disease.

Cell-Free Approaches in Synthetic Biology Utilizing Microfluidics

Synthetic biology is a rapidly growing multidisciplinary branch of science which aims to mimic complex biological systems by creating similar forms. Constructing an artificial system requires optimization at the gene and protein levels to allow the formation of entire biological pathways. Advances in cell-free synthetic biology have helped in discovering new genes, proteins, and pathways bypassing the complexity of the complex pathway interactions in living cells. Furthermore, this method is cost- and time-effective with access to the cellular protein factory without the membrane boundaries. The freedom of design, full automation, and mimicking of in vivo systems reveal advantages of synthetic biology that can improve the molecular understanding of processes, relevant for life science applications. In parallel, in vitro approaches have enhanced our understanding of the living system. This review highlights the recent evolution of cell-free gene design, proteins, and cells integrated with microfluidic platforms as a promising technology, which has allowed for the transformation of the concept of bioprocesses. Although several challenges remain, the manipulation of biological synthetic machinery in microfluidic devices as suitable ‘homes’ for in vitro protein synthesis has been proposed as a pioneering approach for the development of new platforms, relevant in biomedical and diagnostic contexts towards even the sensing and monitoring of environmental issues.

Multi-variant bioreactor for cartilage tissue engineering

Articular cartilage is a stratified tissue with distinct layers expressing different protein types/amounts and having different cell morphologies. Research on articular cartilage has shown that compression, hydrostatic pressure, and hypoxic conditions tend to give the articular cartilage the properties of the middle and bottom layers of the natural tissue, while surface motion and normoxic conditions induce a superficial zone cartilage phenotype. Our objective is to build a bioreactor that can control all of these parameters in order to test for different values and find optimal ranges to create an engineered cartilage tissue with ideal characteristics. Thus we built a four-chamber bioreactor that can apply hydrostatic pressure, compression, shear and torsion, in addition to controlling oxygen tension supplied to the cartilage. The mechanical simulation is applied using a gear-rack mechanism having a frequency of 0.5Hz. The oxygen tension is controlled by electric valves connected to O2 and N2 bottles coming to the bioreactors chambers, oxygen and pressure sensors are used in the process. The bioreactor is controlled and coded using Arduino software. After building the bioreactor, a Matlab computer vision test was done to check for the precision of the mechanism, and finally injurious and proliferation tests were performed to check for the effectiveness of the bioreactor. Results of the precision testing showed a 4% error for a 1mm displacement. Results of the injurious tests showed significant numbers of dead cells for compressive forces larger than 10 MPa. In conclusion, our newly developed system is capable of delivering a variety of mechanical stimuli and oxygen tension simulating those in native cartilage. The importance of this system lies in its applicability to cartilage but also to other mechanoresponsive and oxygen sensitive tissues such as bone, muscle, tendons, ligaments, and blood vessels. In the future, we plan to improve our bioreactor by using a cam-follower mechanism for higher precision.