Nanomechanical Analysis of Extracellular Matrix and Cells in Multicellular Spheroids.

INTRODUCTION: Over the last decade, atomic force microscopy (AFM) has played an important role in understanding nanomechanical properties of various cancer cell lines. This study is focused on Lewis lung carcinoma cell tumours as 3D multicellular spheroid (MS). Not much is know about the mechanical properties of the cells and the surrounding extracellular matrix (ECM) in rapidly growing tumours. METHODS: Depth-dependent indentation measurements were conducted with the AFM. Force-vs.-indentation curves were used to create stiffness profiles as a function of depth. Here studies were focused on the outer most layer, i.e., proliferation zone of the spheroid. RESULTS: Both surface and sub-surface stiffness profiles of MS were created. This study revealed three nanomechanical topographies, Type A-high modulus due to collagen fibers, Type B-high stiffness at cell membrane and ECM interface and Type C-increased modulus due to cell lying deep inside matrix at a depth of 1.35 µm. Both Type and Type-B topographies result from collagen-based structures in ECM. CONCLUSION: This study has first time revealed mechanical constitution of an MS. Depth-dependent indentation studies have the revealed role of various molecular and cellular components responsible for providing mechanical stability to MS. Nanomechanical heterogeneities revealed in this investigation can shed new light in developing correct dosage regime for collagenase treatment of tumours and designing better controlled artificial extracellular matrix systems for replicating tissue growth in-vitro.

Dynamic and Depth Dependent Nanomechanical Properties of Dorsal Ruffles in Live Cells and Biopolymeric Hydrogels.

The nanomechanical properties of various biological and cellular surfaces are increasingly investigated with Scanning Probe Microscopy. Surface stiffness measurements are currently being used to define metastatic properties of various cancerous cell lines and other related biological tissues. Here we present a unique methodology to understand depth dependent nanomechanical variations in stiffness in biopolymers and live cells. In this study we have used A2780 and NIH3T3 cell lines and 0.5% and 1% Agarose to investigate depth dependent stiffness and porosity on nanomechanical properties in different biological systems. This analytical methodology can circumvent the issue associated with the contribution of substrates on cell stiffness. Here we demonstrate that by calculating 'continuous-step-wise-modulus' on force versus distance curves one can observe minute variation as function of depth. Due to the presence of different kinds of cytoskeletal filament, dissipation of contact force might vary from one portion of a cell to another. On NIH3T3 cell lines, stiffness profile of Circular Dorsal Ruffles could be observed in form of large parabolic feature with changes in stiffness at different depth. In biopolymers like agarose, depending upon the extent of polymerization in there can be increase or decrease in stiffness due variations in pore size and extent to which crosslinking is taking place at different depths. 0.5% agarose showed gradual decrease in stiffness whereas with 1% agarose there was slight increase in stiffness as one indents deeper into its surface.

Mechanically Loading Cell/Hydrogel Constructs with Low-Intensity Pulsed Ultrasound for Bone Repair.

Low-intensity pulsed ultrasound (LIPUS) has been shown to be effective for orthopedic fracture repair and nonunion defects, but the specific mechanism behind its efficacy is still unknown. Previously, we have shown a measurable acoustic radiation force at LIPUS intensities traditionally used for clinical treatment and have applied this force to osteoblastic cells encapsulated in type I collagen hydrogels. Our goal in this study is to provide insight and inform the appropriate design of a cell therapy approach to bone repair in which osteoblasts are embedded in collagen hydrogels, implanted into a bony defect, and then transdermally stimulated using LIPUS-derived acoustic radiation force to enhance bone formation at the earliest time points after bone defect repair. To this end, in this study, we demonstrate the ability to measure local hydrogel deformations in response to LIPUS-induced acoustic radiation force and reveal that hydrogel deformation varies with both LIPUS intensity and hydrogel stiffness. Specifically, hydrogel deformation is positively correlated with LIPUS intensity and this deformation is further increased by reducing the stiffness of the hydrogel. We have also shown that encapsulated osteoblastic cells respond to increases in LIPUS intensity by upregulating both cyclooxygenase 2 and prostaglandin E2 (PGE2), both implicated in new bone formation and well-established responses to the application of fluid forces on osteoblast cells. Finally, we demonstrate that combining an increase in LIPUS with a three-dimensional culture environment upregulates both markers beyond their expression noted from either experimental condition alone, suggesting that both LIPUS and hydrogel encapsulation, when combined and modulated appropriately, can enhance osteoblastic response considerably. These studies provide important information toward a clinically relevant cell therapy treatment for bone defects that allows the transdermal application of mechanical loading to bone defects without physically destabilizing the defect site.

The effect of acoustic radiation force on osteoblasts in cell/hydrogel constructs for bone repair.

Ultrasound, or the application of acoustic energy, is a minimally invasive technique that has been used in diagnostic, surgical, imaging, and therapeutic applications. Low-intensity pulsed ultrasound (LIPUS) has been used to accelerate bone fracture repair and to heal non-union defects. While shown to be effective the precise mechanism behind its utility is still poorly understood. In this study, we considered the possibility that LIPUS may be providing a physical stimulus to cells within bony defects. We have also evaluated ultrasound as a means of producing a transdermal physical force that could stimulate osteoblasts that had been encapsulated within collagen hydrogels and delivered to bony defects. Here we show that ultrasound does indeed produce a measurable physical force and when applied to hydrogels causes their deformation, more so as ultrasound intensity was increased or hydrogel stiffness decreased. MC3T3 mouse osteoblast cells were then encapsulated within hydrogels to measure the response to this force. Statistically significant elevated gene expression for alkaline phosphatase and osteocalcin, both well-established markers of osteoblast differentiation, was noted in encapsulated osteoblasts (p < 0.05), suggesting that the physical force provided by ultrasound may induce bone formation in part through physically stimulating cells. We have also shown that this osteoblastic response is dependent in part on the stiffness of the encapsulating hydrogel, as stiffer hydrogels resulted in reducing or reversing this response. Taken together this approach, encapsulating cells for implantation into a bony defect that can potentially be transdermally loaded using ultrasound presents a novel regenerative engineering approach to enhanced fracture repair.

Investigation of in vitro cytotoxicity of the redox state of ionic iron in neuroblastoma cells.

BACKGROUND: there is an intimate relation between transition metals and cell homeostasis due to the physiological necessity of metals in vivo. Particularly, iron (ferrous and ferric state) is utilized in many physiological processes of the cell but in excess has been linked with negative role contributing in many neurodegenerative processes. OBJECTIVE: the aim of this study was to investigate which oxidation state of ionic iron (Ferrous (II) versus Ferric (III)) is more toxic to neuronal cells (SHSY(5)Y). MATERIALS AND METHODS: The neuroblastoma (SHSY(5)Y) cells were exposed to varying concentration of ferric and ferrous iron. Morphological studies using immunofluorescence staining and microscopic analysis as confirmed by intracellular glutathione (GSH) test demonstrated oxidative stress to cells in iron microenvironment. In addition, MTT assay was performed to evaluate the viability and metabolic state of the cells. RESULTS: the results showed that ferrous form has significantly higher toxicity compared to the ferric ionic state of higher concentration. In addition, microscopic analysis shows cell fenestration at higher concentrations and swelling at intermediate ferric dosages as demonstrated by atomic force microscopy (AFM). Interestingly, the addition of a differentiation inducing factor, trans-retinoic rcid (RA) retains significant viability and morphological features of the cells irrespective of the ionic state of the iron. AFM images revealed clustered aggregates arising from iron chelation with RA. CONCLUSIONS: the results indicate that Fe (II) has more toxic effects on cells. In addition, it could be an interesting finding with respect to the antioxidant properties of RA as a chelating agent for the neurodegenerative therapeutics.