Novel burn device for rapid, reproducible burn wound generation.

INTRODUCTION: Scarring following full thickness burns leads to significant reductions in range of motion and quality of life for burn patients. To effectively study scar development and the efficacy of anti-scarring treatments in a large animal model (female red Duroc pigs), reproducible, uniform, full-thickness, burn wounds are needed to reduce variability in observed results that occur with burn depth. Prior studies have proposed that initial temperature of the burner, contact time with skin, thermal capacity of burner material, and the amount of pressure applied to the skin need to be strictly controlled to ensure reproducibility. The purpose of this study was to develop a new burner that enables temperature and pressure to be digitally controlled and monitored in real-time throughout burn wound creation and compare it to a standard burn device. METHODS: A custom burn device was manufactured with an electrically heated burn stylus and a temperature control feedback loop via an electronic microstat. Pressure monitoring was controlled by incorporation of a digital scale into the device, which measured downward force. The standard device was comprised of a heat resistant handle with a long rod connected to the burn stylus, which was heated using a hot plate. To quantify skin surface temperature and internal stylus temperature as a function of contact time, the burners were heated to the target temperature (200±5°C) and pressed into the skin for 40s to create the thermal injuries. Time to reach target temperature and elapsed time between burns were recorded. In addition, each unit was evaluated for reproducibility within and across three independent users by generating burn wounds at contact times spanning from 5 to 40s at a constant pressure and at pressures of 1 or 3lbs with a constant contact time of 40s. Biopsies were collected for histological analysis and burn depth quantification using digital image analysis (ImageJ). RESULTS: The custom burn device maintained both its internal temperature and the skin surface temperature near target temperature throughout contact time. In contrast, the standard burner required more than 20s of contact time to raise the skin surface temperature to target due to its quickly decreasing internal temperature. The custom burner was able to create four consecutive burns in less than half the time of the standard burner. Average burn depth scaled positively with time and pressure in both burn units. However, the distribution of burn depth within each time-pressure combination in the custom device was significantly smaller than with the standard device and independent of user. CONCLUSIONS: The custom burn device's ability to continually heat the burn stylus and actively control pressure and temperature allowed for more rapid and reproducible burn wounds. Burns of tailored and repeatable depths, independent of user, provide a platform for the study of anti-scar and other wound healing therapies without the added variable of non-uniform starting injury.

Myoferlin depletion elevates focal adhesion kinase and paxillin phosphorylation and enhances cell-matrix adhesion in breast cancer cells.

Breast cancer is the second leading cause of malignant death among women. A crucial feature of metastatic cancers is their propensity to lose adhesion to the underlying basement membrane as they transition to a motile phenotype and invade surrounding tissue. Attachment to the extracellular matrix is mediated by a complex of adhesion proteins, including integrins, signaling molecules, actin and actin-binding proteins, and scaffolding proteins. Focal adhesion kinase (FAK) is pivotal for the organization of focal contacts and maturation into focal adhesions, and disruption of this process is a hallmark of early cancer invasive potential. Our recent work has revealed that myoferlin (MYOF) mediates breast tumor cell motility and invasive phenotype. In this study we demonstrate that noninvasive breast cancer cell lines exhibit increased cell-substrate adhesion and that silencing of MYOF using RNAi in the highly invasive human breast cancer cell line MDA-MB-231 also enhances cell-substrate adhesion. In addition, we detected elevated tyrosine phosphorylation of FAK (FAK(Y397)) and paxillin (PAX(Y118)), markers of focal adhesion protein activation. Morphometric analysis of PAX expression revealed that RNAi-mediated depletion of MYOF resulted in larger, more elongated focal adhesions, in contrast to cells transduced with a control virus (MDA-231(LVC) cells), which exhibited smaller focal contacts. Finally, MYOF silencing in MDA-MB-231 cells exhibited a more elaborate ventral cytoskeletal structure near focal adhesions, typified by pronounced actin stress fibers. These data support the hypothesis that MYOF regulates cell adhesions and cell-substrate adhesion strength and may account for the high degree of motility in invasive breast cancer cells.

Influence of hydration on fiber geometry in electrospun scaffolds.

Finite element models of tissue engineering scaffolds are powerful tools to understand scaffold function, including how external mechanical signals deform the scaffold at the meso- and microscales. Fiber geometry is needed to inform finite element models of fiber-based tissue engineering scaffolds; however, the accuracy and utility of these models may be limited if they are informed by non-hydrated geometries. Scanning electron microscopy and confocal microscopy, coupled with Fourier analysis of the resulting images, were used to quantify how hydration alters fiber geometry in electrospun collagen and polycaprolactone (PCL) scaffolds. The results also quantify how image size affects fiber geometry. Hydration is demonstrated to increase fiber tortuosity, defined as the ratio of actual fiber length:end-to-end fiber length. For collagen scaffolds, hydration increased the mean tortuosity from 1.05 to 1.21, primarily from large ∼2- to 10-fold) increases in smaller (<40µm) wavelength amplitudes. For PCL fibers, the mean tortuosity increased from 1.01 to only 1.04, primarily from modest ∼2-fold) increases in larger (>100µm) wavelength amplitudes. The results demonstrate that mechanical simulations of electrospun scaffolds should be informed with hydrated scaffold geometries of at least 200µm scale, in order to capture geometrical effects associated with fiber straightening.

Plasma surface modification of electrospun fibers for adhesion-based cancer cell sorting.

Personalized cancer therapies drive the need for devices that rapidly and accurately segregate cancer cells from solid tumors. One potential sorting strategy is to segregate populations of cells based on their relative strength of adhesion. To investigate the effect of surface hydrophilicity and cell phenotype on adhesion, primary human breast skin fibroblasts and keratinocytes and MCF-7 breast cancer cells were seeded onto air and CF(4) plasma-treated nanofibers followed by exposure to three shear stresses (200, 275 and 350 dynes per cm(2)) 1 hour after inoculation. No difference in strength of adhesion was measured in either fibroblasts or keratinocytes on either plasma treated-surface: all exhibited >60% of the initial cell count after a 5 minute exposure to 350 dynes per cm(2) of shear stress. In contrast, a significant difference between relative strength of adhesion on air versus CF(4) plasma-treated surfaces was observed for MCF-7 cells: 26% and 6.6% of cells remained on the air and CF(4) plasma-treated surfaces, respectively. The ability to sort this cancer cell line from two non-cancerous primary human cells was evaluated by inoculating a mixture of all three cell types simultaneously onto CF(4) treated nanofibers followed by 1 hour of culture and exposure to 350 dynes per cm(2) shear stress. The majority of MCF-7 cells were removed (0.7% remained) while a majority of fibroblasts and keratinocytes remained adhered (74 and 57%). Post-sorted MCF-7 viability and morphology remained unchanged, preserving the possibility of post-separation and analysis. These data suggest that the plasma treatment of electrospun scaffolds provides a tool useful in sorting cancer cells from a mixed cell population based on adhesion strength.

Regulation of electrospun scaffold stiffness via coaxial core diameter.

Scaffold mechanics influence cellular behavior, including migration, phenotype and viability. Scaffold stiffness is commonly modulated through cross-linking, polymer density, or bioactive coatings on stiff substrates. These approaches provide useful information about cellular response to substrate stiffness; however, they are not ideal as the processing can change substrate morphology, density or chemistry. Coaxial electrospinning was investigated as a fabrication method to produce scaffolds with tunable stiffness and strength without changing architecture or surface chemistry. Core solution concentration, solvent and feed rate were utilized to control core diameter with higher solution concentration and feed rate positively correlating with increased fiber diameter and stiffness. Coaxial scaffolds electrospun with an 8 wt./vol.% polycaprolactone (PCL)-HFP solution at 1 ml h(-1) formed scaffolds with an average core diameter of 1.1±0.2 µm and stiffness of 0.027±3.3×10(-3) N mm(-1). In contrast, fibers which were 2.6±0.1 µm in core diameter yielded scaffolds with a stiffness of 0.065±4.7×10(-3) N mm(-1). Strength and stiffness positively correlated with core diameter with no significant difference in total fiber diameter and interfiber distance observed in as-spun scaffolds. These data indicate that coaxial core diameter can be utilized to tailor mechanical properties of three-dimensional scaffolds and would provide an ideal scaffold for assessing the effect of scaffold mechanics on cell behavior.

Epidermal differentiation governs engineered skin biomechanics.

Engineered skin must be mechanically strong to facilitate surgical application and prevent damage during the early stages of engraftment. However, the evolution of structural properties during culture, the relative contributions of the epidermis and dermis, and any correlation with tissue morphogenesis are not well known. These aspects are investigated by assessing the mechanical properties of engineered skin (ES) and engineered dermis (ED) during a 21-day culture period, including correlations with cellular metabolism, cellular organization and epidermal differentiation. During culture, the epidermis differentiates and begins to cornify, as evidenced by immunostaining and surface electrical capacitance. Tensile testing reveals that the ultimate tensile strength and linear stiffness increase linearly with time for ES, but are relatively unchanged for ED. ES strength correlates significantly with epidermal differentiation (p < 0.001) and a composite strength model indicates that strength is largely determined by the epidermis. These data suggest that strategies to improve ES biomechanics should target the dermis. Additionally, time-dependant changes in average ES strength and percent elongation can be used to set upper bound limits on mechanical stimulation profiles to avoid tissue damage.

Fiber density of electrospun gelatin scaffolds regulates morphogenesis of dermal-epidermal skin substitutes.

Porous, nowoven fibrous gelatin scaffolds were prepared using electrospinning. Electrospun scaffolds with varying fiber diameter, interfiber distance, and porosity were fabricated by altering the concentration of the electrospinning solution. Solution concentration was a significant predictor of fiber diameter, interfiber distance, and porosity with higher solution concentration correlated with larger fiber diameters and interfiber distances. The potential of electrospun gelatin as a scaffolding material for dermal and epidermal tissue regeneration was also evaluated. Interfiber distances >5.5 microm allowed deeper penetration of human dermal fibroblasts into the scaffold, whereas cells in scaffolds with more densely packed fibers were able to infiltrate only into the upper regions. Scaffolds with interfiber distances

Design guidelines for interactive multimedia learning environments to promote social inclusion.

PURPOSE: There is a continuing need for guidelines to aid in the design of Interactive Multimedia Learning Environments (IMLE) to promote effective learning. The project introduced in this paper looks at an important subset of this problem, the design of interactive learning environments to promote social inclusion. METHOD: A consortium of six partners contributed toward defining learning material to develop a range of work based skills, including horticulture, IT and catering. These were then developed into IMLE prototypes. Formative evaluation of these prototypes then revealed a range of usability problems, which were grouped into generic types and frequency of occurrence. RESULTS: The most important and frequently occurring problems were used to distil a set of design guidelines for the development of effective IMLE. The results from this usability content analysis were also used to refine the initial prototypes to improve their usability and effectiveness. CONCLUSIONS: These guidelines, termed the Greenhat Design Guidelines, can be adopted for use by all multimedia developers aiming to promote the social inclusion of vulnerable or socially disadvantaged groups of people. The refined IMLE can be accessed via the Greenhat Server to improve the employment-related skills of socially excluded people.

Do knee injuries have long-term consequences for isometric and dynamic muscle strength?

To ascertain whether decrements in knee extensor muscle strength persist years after a traumatic ligamentous or meniscal knee injury, with reference to (1) the type of muscle activity, (2) the dominance of injury, and (3) the time since injury, 36 subjects with previous unilateral knee injuries were assessed. Peak voluntary quadriceps muscle strength was measured using the KinCom 500H dynamometer during isometric, concentric (30 degrees.s-1 and 120 degrees.s-1) and eccentric contractions (30.s-1), and the findings for each type of contraction were compared. Significant differences in quadriceps muscle strength were seen between (1) the injured and uninjured limbs during maximal voluntary isometric (P = 0.0003), concentric (P < 0.0001) and eccentric (P < 0.0001) contractions, and (2) the isometric and concentric decrements (P = 0.004), and the isometric and eccentric decrements (P = 0.012) within the same injured limb. The decrements in eccentric strength were significantly greater if they affected the dominant rather than the non-dominant limb (P = 0.0186). No relationship was seen between the time since injury and the degree of isometric or dynamic decrement. Deficits in quadriceps muscle strength remained for a long time after traumatic knee injury, with exercise levels rarely returning to the previously uninjured state. The degree of decrement in muscle strength was dependent upon the type of muscle activity, with concentric and eccentric activity showing greater decrements than those seen with isometric activity. Deficits in the type of activity varied widely in the same individual, and eccentric decrements were significantly worse following dominant knee injuries. Possible explanations for these findings and the implications for rehabilitation following knee injury are discussed.

Unilateral lower-limb musculoskeletal injury: its long-term effect on balance.

OBJECTIVE: To assess if any long-term decrements in balance occur after unilateral musculoskeletal injury. The relation between the size of decrement and the dominance, the type, and the time since injury were also considered. DESIGN: With eyes open and closed, postural sway in one-legged standing was recorded for 10 seconds in 48 subjects who sustained a unilateral musculoskeletal injury 6 months to 42 years earlier. Comparative data were also collected in 108 healthy subjects with no previous injury. SETTING: A university physiologic laboratory. PATIENTS: Injured subjects were recruited locally via the district general hospital, sports injury clinic, and the university, and had not received any treatment within the past 6 months. MAIN OUTCOME MEASURE: Postural sway of the injured and uninjured limb (or dominant and nondominant limb in the uninjured subjects). RESULTS: Postural sway was significantly greater in the injured limb compared with the uninjured limb (p = .0118). The ratio of the postural sway of the injured limb compared with the uninjured limb (I/UI%) was significantly lower in the group with nondominant injuries (p = .0085). Subjects with nondominant injuries performed significantly better than those with dominant injuries (p = .0085). No relation was found between the decrements in balance performance and the type of injury and time since injury. CONCLUSIONS: Full recovery is frequently not achieved and perhaps recovery does not continue to improve once the formal rehabilitation period is over.

Reduction in range of movement can increase maximum voluntary eccentric forces for the human knee extensor muscles.

Using the KinCom 500H isokinetic dynamometer the first part of this study measured the characteristics of the force velocity relationship curve for the human knee extensors between -1.57 (eccentric) and 3.67 (concentric) rads x s(-1) (-90 and 210 degrees s(1)) for both legs in 4 subjects. A significant increase in force generation was seen in eccentric activity at 0.52 rads x s(-1) (30 degrees s(-1)) but not at 1.57 rads x s(-1) (90 degrees s(-1)) compared to maximum voluntary isometric force (P < 0.005). This increase was, however, lower than would be expected from the classical force-velocity relationship. The second part of the study examined whether restricting the range of movement was able to further increase the eccentric forces. In a further 6 subjects, the eccentric contractions were repeated during either an 80 degrees (15-95 degrees flexion) and a 50 degrees (45-95 degrees flexion) range of movement. Significant increases in force were seen over the shorter range of movement at 0.52 rads x s(-1) (30 degrees s(-1)) (P = 0.006) and 1.57 rads x s(-1) (90 degrees s(-1)) (P < 0.001).

Unilateral lower limb injury: its long-term effects on quadriceps, hamstring, and plantarflexor muscle strength.

OBJECTIVE: To ascertain if long-term deficits in quadriceps, hamstring, and plantarflexor muscle strength remain after unilateral lower-limb musculoskeletal injury and to quantify whether improvements in performance continue once a subject concludes rehabilitation and returns to everyday activities. The relation between the size of decrement and limb dominance, type of injury, and time since injury was also considered. DESIGN: Isometric and/or dynamic muscle strength of both legs was measured (using the KinCom 500H isokinetic dynamometer) in 48 subjects. SETTING: A physiological laboratory at Brunel University. PATIENTS: Patients were recruited locally via a district general hospital, sports injury clinic, and university. MAIN OUTCOME MEASURES: Muscle strength in the injured limb, reported as a percentage of muscle strength in the uninjured limb. It was assumed that the preinjury state of the injured limb was similar to that of the uninjured limb. RESULTS: Decrements were seen in mean isometric and peak isometric, concentric, and eccentric quadriceps activity (p < .0001) and isometric plantarflexor activity (p < .05) in the injured limb, with the type of injury influencing the size of the decrement. Minimal difference was found in the hamstring muscles. CONCLUSIONS: The decrements in performance in the quadriceps muscle imply that full recovery (as defined by the preinjury state) is frequently not achieved and stress the need for accurate, objective assessment of muscle strength and further investigation into the nature and duration of rehabilitation after musculoskeletal injury.

Impedance imaging using linear electrode arrays.

The characteristics of using a linear array of electrodes for impedance imaging are examined. The advantages of a linear array include known electrode position, localised resolution, and mobility, but there are some disadvantages. The problems of the choice of data set, and of the range of measurements are examined. The results of investigations into resolution and sensitivity are presented, and also of some preliminary in vivo measurements. It is concluded that a linear array may have possible clinical applications.