Faculty Bibliography

PURPOSE: To evaluate the fixation integrity at time zero of a type I/III collagen patch secured to a chondral defect in the porcine knee using methods typically employed in autologous chondrocyte implantation (ACI) and matrix-assisted chondrocyte implantation. METHODS: Twenty-four porcine knee specimens underwent a medial parapatellar arthrotomy. A prefabricated template was used to create cartilage defects of 2 cm2 in the medial femoral condyle. A size-matched collagen patch was fashioned. Four methods of fixation to the chondral defect were analyzed: group 1-saline, group 2-fibrin glue around the periphery of the patch, group 3-fibrin glue applied to the base of the defect and around the periphery of the patch, group 4-6-0 vicryl suture and fibrin glue around the periphery of the patch. Collagen patch fixation was assessed at intervals of 60, 300, 600, 900, and 1,200 cycles from full extension to 90 degrees of flexion, performed manually without application of axial force. Patch fixation was evaluated by 2 independent observers using a customized scoring scale. RESULTS: Mean peripheral detachment of the patch and chondral defect uncovering remained less than 25% for all groups. Area of defect uncovering was significantly increased in group 2 compared with group 4 after 900 and 1,200 cycles (P = .0014 and P = .0025, respectively). Fibrin glue applied to the base of the defect, or suturing of the patch, reduced deformation significantly after 900 cycles. CONCLUSIONS: Suture increases the stability of fixation of a type I/III collagen patch to a chondral defect better than fibrin glue alone in the porcine knee after repetitive cycling, with respect to patch detachment and chondral defect uncovering. Application of fibrin glue to the base of the defect, or securing the patch with suture, decreases collagen patch deformation. CLINICAL RELEVANCE: In cases where minimally invasive techniques do not allow suture fixation of the collagen patch, scaffold fixation may be compromised during articular motion protocols typically used after second- and third-generation ACI procedures.

Flow cytometry provides a high throughput, multi-dimensional analysis of cells flowing in suspension. In order to combine this feature with the ability to resolve detailed structures in 3D, we developed an optofluidic device that combines a microfluidic system with a dual beam trap. This allows for the rotation of single cells in a continuous flow, around an axis perpendicular to the imaging plane. The combination of both techniques enables the tomographic reconstruction of the 3D structure of the cell. In addition this method is capable to provide detailed 3D structural data for flow cytometry, as it improves the reconstructed z-resolution of a standard microscopy system to produce images with isotropic resolution in all three axes.

The mechanical properties of biological cells have long been considered as inherent markers of biological function and disease. However, the screening and active sorting of heterogeneous populations based on serial single-cell mechanical measurements has not been demonstrated. Here we present a novel monolithic glass chip for combined fluorescence detection and mechanical phenotyping using an optical stretcher. A new design and manufacturing process, involving the bonding of two asymmetrically etched glass plates, combines exact optical fiber alignment, low laser damage threshold and high imaging quality with the possibility of several microfluidic inlet and outlet channels. We show the utility of such a custom-built optical stretcher glass chip by measuring and sorting single cells in a heterogeneous population based on their different mechanical properties and verify sorting accuracy by simultaneous fluorescence detection. This offers new possibilities of exact characterization and sorting of small populations based on rheological properties for biological and biomedical applications.

We describe a method for quantifying the mechanical properties of cells in suspension with a microfluidic device consisting of a parallel array of micron-sized constrictions. Using a high-speed charge-coupled device camera, we measure the flow speed, cell deformation, and entry time into the constrictions of several hundred cells per minute during their passage through the device. From the flow speed and the occupation state of the microconstriction array with cells, the driving pressure across each constriction is continuously computed. Cell entry times into microconstrictions decrease with increased driving pressure and decreased cell size according to a power law. From this power-law relationship, the cell elasticity and fluidity can be estimated. When cells are treated with drugs that depolymerize or stabilize the cytoskeleton or the nucleus, elasticity and fluidity data from all treatments collapse onto a master curve. Power-law rheology and collapse onto a master curve are predicted by the theory of soft glassy materials and have been previously shown to describe the mechanical behavior of cells adhering to a substrate. Our finding that this theory also applies to cells in suspension provides the foundation for a quantitative high-throughput measurement of cell mechanical properties with microfluidic devices.

Phospholipid vesicles are common model systems for cell membranes. Important aspects of the membrane function relate to its mechanical properties. Here we have investigated the deformation behaviour of phospholipid vesicles in a dual-beam laser trap, also called an optical stretcher. This study explicitly makes use of the inherent heating present in such traps to investigate the dependence of vesicle deformation on temperature. By using lasers with different wavelengths, optically induced mechanical stresses and temperature increase can be tuned fairly independently with a single setup. The phase transition temperature of vesicles can be clearly identified by an increase in deformation. In the case of no heating effects, a minimal model for drop deformation in an optical stretcher and a more specific model for vesicle deformation that takes explicitly into account the angular dependence of the optical stress are presented to account for the experimental results. Elastic constants are extracted from the fitting procedures, which agree with literature data. This study demonstrates the utility of optical stretching, which is easily combined with microfluidic delivery, for the future serial, high-throughput study of the mechanical and thermodynamic properties of phospholipid vesicles.

Determining cell mechanical properties is increasingly recognized as a marker-free way to characterize and separate biological cells. This emerging realization has led to the development of a plethora of appropriate measurement techniques. Here, we use a fairly novel approach, deterministic lateral displacement (DLD), to separate blood cells based on their mechanical phenotype with high throughput. Human red blood cells were treated chemically to alter their membrane deformability and the effect of this alteration on the hydrodynamic behaviour of the cells in a DLD device was investigated. Cells of defined stiffness (glutaraldehyde cross-linked erythrocytes) were used to test the performance of the DLD device across a range of cell stiffness and applied shear rates. Optical stretching was used as an independent method for quantifying the variation in stiffness of the cells. Lateral displacement of cells flowing within the device, and their subsequent exit position from the device were shown to correlate with cell stiffness. Data showing how the isolation of leucocytes from whole blood varies with applied shear rate are also presented. The ability to sort leucocyte sub-populations (T-lymphocytes and neutrophils), based on a combination of cell size and deformability, demonstrates the potential for using DLD devices to perform continuous fractionation and/or enrichment of leucocyte sub-populations from whole blood.

The classical purpose of optical fibres is delivery of either optical power, as for welding, or temporal information, as for telecommunication. Maximum performance in both cases is provided by the use of single-mode optical fibres. However, transmitting spatial information, which necessitates higher-order modes, is difficult because their dispersion relation leads to dephasing and a deterioration of the intensity distribution with propagation distance. Here we consciously exploit the fundamental cause of the beam deterioration-the dispersion relation of the underlying vectorial electromagnetic modes-by their selective excitation using adaptive optics. This allows us to produce output beams of high modal purity, which are well defined in three dimensions. The output beam distribution is even robust against significant bending of the fibre. The utility of this approach is exemplified by the controlled rotational manipulation of live cells in a dual-beam fibre-optical trap integrated into a modular lab-on-chip system.

OBJECTIVE:Exercise-induced bronchoconstriction (EIB) is more prevalent in elite athletes than in the general population. Many of these athletes provide a positive eucapnic voluntary hyperpnoea (EVH) challenge without previous diagnosis of EIB. It is unknown whether this is specific to elite athletes or whether the same risk applies to recreationally active individuals. The purpose of this study was to investigate the prevalence of a positive EVH challenge in a population of recreationally active individuals. METHODS:136 recreationally active individuals (Age: 21.9 ± 3.7 years; Height: 175 ± 9 cm; Weight: 70.9 ± 10.0 kg) without previous history of asthma or EIB, volunteered to take part in the study. All participants completed an EVH challenge, which was deemed positive if FEV1 fell ≥10% from baseline at two consecutive time points, and was reversible following inhalation of a short acting β2-agonist. RESULTS:18 of 136 (13.2%) participants had a positive EVH challenge. Of the 18 individuals, the fall in FEV1 from baseline ranged from -12% to -50%. At baseline, percentage predicted FEV1 (97.5 ± 12.5% versus 104.9 ± 10%; p < 0.01), FEV1/FVC ratio (79.5 ± 6.9% versus 87.8 ± 5.5%; p < 0.01), FEF25-75 (3.73 ± 1.00 versus 4.73 ± 1.00 l/s; p < 0.01) and predicted PEF (89.4 ± 8.8% versus 97.5 ± 13.6%; p < 0.05) values for EVH positive participants were significantly lower than EVH negative participants respectively. CONCLUSIONS:Overall, 13.2% of recreationally active individuals with no previous history of asthma presented with a positive EVH challenge. Individuals who are recreationally active may benefit from an objective bronchial provocation challenge, given that self-reported symptoms alone only provide a supportive role towards a valid EIB diagnosis.

In this paper we describe a pneumatically actuated fibre-optic spanner integrated into a microfluidic Lab-on-a-Chip device for the controlled trapping and rotation of living cells. The dynamic nature of the system allows interactive control over the rotation speed with the same optical power. The use of a multi-layer device makes it possible to rotate a cell both in the imaging plane and also in a perpendicular plane allowing tomographic imaging of the trapped living cell. The integrated device allows easy operation and by combining it with high-resolution confocal microscopy we show for the first time that the pattern of rotation can give information regarding the sub-cellular composition of a rotated cell.