Optical study of chemotherapy efficiency in cancer treatment via intracellular structural disorder analysis using partial wave spectroscopy.

As cancer progresses, macromolecules, such as DNA, RNA or lipids, inside cells undergo spatial structural rearrangements and alterations. Mesoscopic light transport-based optical partial wave spectroscopy (PWS) was recently introduced to quantify changes in the nanoscale structural disorder in biological cells. The PWS measurement is performed using a parameter termed as "disorder strength" (L d ), which represents the degree of nanoscale structural disorder inside the cells. It was shown that cancerous cells have higher disorder strength than normal cells. In this work, we first used the PWS to analyze the hierarchy of different types of prostate cancer cells, namely, C4-2, DU-145 and PC-3, by quantifying their average disorder strengths. Results expectedly showed that L d values increases in accordance with the increasing aggressiveness/tumorigenicity levels of these cells. Using the L d parameter, we then analyzed the chemoresistance properties of these prostate cancer cells to docetaxel drug compared to their chemosensitivity. Results show that chemoresistant cancer cells have increased L d values, that is, higher disorder strength, relative to chemosensitive cancer cells. Thus, use of the L d metric can be effective in determining the efficacy of particular chemotherapy.

Novel naturally crosslinked electrospun nanofibrous chitosan mats for guided bone regeneration membranes: material characterization and cytocompatibility.

Guided bone regeneration (GBR) barrier membranes are used to prevent soft tissue infiltration into the graft space during dental procedures that involve bone grafting. Chitosan materials have shown promise as GBR barrier membranes, due to their biocompatibility and predictable biodegradability, but degradation rates may still be too high for clinical applications. In this study, chitosan GBR membranes were electrospun using chitosan (70% deacetylated, 312 kDa, 5.5 w/v%), with or without the addition of 5 or 10 mm genipin, a natural crosslinking agent, in order to extend the degradation to meet the clinical target time frame of 4-6 months. Membranes were evaluated for fibre diameter, tensile strength, biodegradation rate, bond structure and cytocompatibility. Genipin addition, at 5 or 10 mm, resulted in median fibre diameters 184, 144 and 154 nm for uncrosslinked, 5 mm and 10 mm crosslinked, respectively. Crosslinking, examined by Fourier transform infrared spectroscopy, showed a decrease in N-H stretch as genipin levels were increased. Genipin-crosslinked mats exhibited only 22% degradation based on mass loss, as compared to 34% for uncrosslinked mats at 16 weeks in vitro. The ultimate tensile strength of the mats was increased by 165% to 32 MPa with 10 mm crosslinking as compared to the uncrosslinked mats. Finally, genipin-crosslinked mats supported the proliferation of SAOS-2 cells in a 5 day growth study, similar to uncrosslinked mats. These results suggest that electrospun chitosan mats may benefit from genipin crosslinking and have the potential to meet clinical degradation time frames for GBR applications.

Quantitative trait loci for tibial bone strength in C57BL/6J and C3H/HeJ inbred strains of mice.

Three-point bending technology has been widely used in the measurement of bone strength. Quantitative trait loci (QTLs) for bone strength have been identified using mouse femurs. In this study, we investigate the use of mouse tibiae in identification of QTLs that regulate bone strength. Mouse tibiae were from a F(2) population derived from C57BL/6J (B6) and C3H/HeJ (C3H). Three-point bending was measured using ISO 4049, with the support width adjustable to accommodate specimen sizes outside the scope of ISO 4049. The strain rate is selectable from 0.05 to 500 mm per min. All stress strain diagrams are recorded and retrieved in digital electronic form. Genome scan was performed in The Jackson Laboratory (TJL). QTL mapping was conducted using Map Manager QTX software. Data show that (i) both elastic modulus (stiffness) and maximum loading (strength) value appear as normal distributions, suggesting that multiple genetic factors control the bone strength; (ii) 11 QTLs, accounting for 90% of variation for strength, have been detected. More than half QTLs of three-point bending are located on the same locations of bone density earlier identified from mouse femurs; (iii) a major QTL of femoral and vertebral bone mineral density (BMD) was not detected for bone strength of tibiae; (iv) the QTL on chromosome 4 has extremely high LOD score of 31.8 and represents 60% of the variation of bone strength; and (v) four QTLs of stiffness (chromosomes 2, 11, 15 and 19) have been identified.

Computation of transient flow rates in passive pumping micro-fluidic systems.

Motion in micro-channels of passive flow micro-fluidic systems can be controlled by proper design and estimated by careful modeling. We report on methods to describe the flow rate as function of time in a passive pump driven micro-fluidic system. The model considers the surface energy present in small droplets, which prompts their shrinkage and induces flow. The droplet geometries are controlled by the micro-fluidic system geometry and hydrophilicity of the droplet channel contact area so that the chord of the droplet's cross section is restrained as the fluid is pumped. The model uses interfacial thermodynamics and the Hagen-Poiseuille equation for calculating the flow rate in micro-channels. Existing analyses consider the theoretical relationships among sample volume and induced flow rate, surface energy of the drops at the entrance and exit ports, and the resistance to flow. This model provides more specific information on the influence of the experimental conditions in computations of the flow rate. The model was validated in four sets of experiments. Passive pumps with 1.8 mm diameter, hydrophobic or hydrophilic entry ports, 5.0 or 10.0 mm channel length, and 2.5 or 3.3 mm diameter reservoir ports provided initial flow rates between 85 nL s(-1) and 196 nL s(-1).

Localized elasticity measured in epithelial cells migrating at a wound edge using atomic force microscopy.

Restoration of lung homeostasis following injury requires efficient wound healing by the epithelium. The mechanisms of lung epithelial wound healing include cell spreading and migration into the wounded area and later cell proliferation. We hypothesized that mechanical properties of cells vary near the wound edge, and this may provide cues to direct cell migration. To investigate this hypothesis, we measured variations in the stiffness of migrating human bronchial epithelial cells (16HBE cells) approximately 2 h after applying a scratch wound. We used atomic force microscopy (AFM) in contact mode to measure the cell stiffness in 1.5-microm square regions at different locations relative to the wound edge. In regions far from the wound edge (>2.75 mm), there was substantial variation in the elastic modulus in specific cellular regions, but the median values measured from multiple fields were consistently lower than 5 kPa. At the wound edge, cell stiffness was significantly lower within the first 5 microm but increased significantly between 10 and 15 microm before decreasing again below the median values away from the wound edge. When cells were infected with an adenovirus expressing a dominant negative form of RhoA, cell stiffness was significantly decreased compared with cells infected with a control adenovirus. In addition, expression of dominant negative RhoA abrogated the peak increase in stiffness near the wound edge. These results suggest that cells near the wound edge undergo localized changes in cellular stiffness that may provide signals for cell spreading and migration.

Quantitative trait loci that determine mouse tibial nanoindentation properties in an F2 population derived from C57BL/6J x C3H/HeJ.

Use of nanoindentation technology to identify quantitative trait loci (QTL) that regulate bone properties represents a novel approach to improving our understanding of molecular mechanisms that control bone matrix properties. Tibiae for QTL mapping were from an F2 population derived from C57BL/6J and C3H/HeJ. A nanoindenter (Triboindenter; Hysitron, Minneapolis, MN) was used to conduct indentation tests on transverse sections. Genotyping was performed in The Jackson Laboratory. QTL mapping was conducted using software. We found that (1) tibiae from mice at 16 weeks of age were mature and suitable for measurement by a nanoindentor; (2) both stiffness modulus and hardness modulus in the F2 population appeared to have normal distributions, which suggested that multiple genetic factors control the bone properties; and (3) QTL for hardness were identified from five chromosomes (Chr 8, 12, 13, 17, and 19) and for stiffness, from four chromosomes (Chr 3, 8, 12, and 13). Among all detected QTL, one at the same location on Chr 12 was detected for both hardness and stiffness data. It explained the highest percentage of phenotypic variation in bone properties. Using nanoindentation technology to identify QTL that regulate bone properties yielded as many as six different chromosomal regions. Although the actual genes remain to be identified, nanoindentation will contribute to our understanding of molecular mechanisms and normal development processes that control the matrix properties of bone.

Mechanical properties of OI type III bone tissue measured by nanoindentation.

Nanoindentation was used to characterize the intrinsic mechanical properties of bone tissue from eight (8) children with type III Osteogenesis Imperfecta (OI). The bone samples were harvested from the cortex portion at the site of bowing (the mid 2/3 of the shaft of the tibia/femur). Unlike normal bone tissue, OI type III cortical bone exhibited more isotropic material properties. Young's modulus and hardness values measured in the longitudinal direction did not show significant differences from the transverse measurements. No differences were observed in modulus or hardness in an analysis of the cortical and trabecular samples. The deformation patterns of the OI type III bone during nanoindentation were found to be similar to those of normal adult bone in an analysis of the ratio of modulus to hardness. No correlation was found between nanoindentation measurement and age in an analysis of regression.

Matrix remodeling expression in anulus cells subjected to increased compressive load.

STUDY DESIGN: Mechanobiology study of gene expression changes as a result of compressive overload of anular fibrochondrocytes. OBJECTIVE: To test hypotheses regarding phenotype shift in genes coding for representative extracellular matrix (ECM) proteins and matrix modulators. SUMMARY OF THE BACKGROUND DATA: In degenerative disc disease, the transfer of compressive load through the disc shifts largely from the nucleus onto the anulus. In vivo models simulating this condition have shown derangement of the collagenous ultrastructure in the anulus. In vitro models of cultured anulus cells subjected to static compressive stress generally suggest a down-regulation of synthesis. This study evaluated the expression of specific isomers of genes responsible for mechanical viability and metabolism of the disc under cyclic compressive loads. METHODS: Fibrochondrocytes were digested from the anuli of 3, 2-week-old pigs, embedded in 1.5% alginate gel, and hydrostatically compressed at 0.5 Hz for 3 hours to amplitudes of 10 and 30 atm. These levels represented nominal load transfer through the healthy disc and high load transfer through the degenerative disc. Ribonucleic acid was isolated, reverse transcribed, and evaluated by real-time polymerase chain reaction for expression of type I (C-I) and type II (C-II) collagen, aggrecan, the matrix metalloproteinase (MMP-1), and the transforming growth factor beta (TGFbeta-1). Results were expressed at percentages of uncompressed controls. RESULTS: The lower pressure of 10 atm resulted in up-regulation of all ECM protein genes. C-I and C-II both averaged 141%, and aggrecan 121% of controls (P < 0.05). MMP-1 and TGFbeta-1 were essentially unchanged. With the pressure increased to 30 atm, C-II remained approximately at the level expressed under lower pressure, but C-I was reduced to 42% of controls (P < 0.05), indicating a phenotype shift. MMP-1 and TGFbeta-1 also were down-regulated to 71% and 54% of controls, respectively (P < 0.05). CONCLUSIONS: The up-regulation of the ECM genes with nominal pressure highlights the mechanobiological importance of common activity in fibrocartilage homeostasis. Differential regulation of the 2 primary collagen types with high pressure indicates a capacity of the anulus to remodel according to pathomechanical conditions.