Implementation of a High-Throughput Pilot Screen in Peptide Hydrogel-Based Three-Dimensional Cell Cultures.

Cell-based high-throughput drug screening (HTS) is a common starting point for the drug discovery and development process. Currently, there is a push to combine complex cell culture systems with HTS to provide more clinically applicable results. However, there are mechanistic requirements inherent to HTS as well as material limitations that make this integration challenging. Here, we used the peptide-based shear-thinning hydrogel MAX8 tagged with the RGDS sequence to create a synthetic extracellular scaffold to culture cells in three dimensions and showed a preliminary implementation of the scaffold within an automated HTS setup using a pilot drug screen targeting medulloblastoma, a pediatric brain cancer. A total of 2202 compounds were screened in the 384-well format against cells encapsulated in the hydrogel as well as cells growing on traditional two-dimensional (2D) plastic. Eighty-two compounds passed the first round of screening at a single point of concentration. Sixteen-point dose-response was done on those 82 compounds, of which 17 compounds were validated. Three-dimensional (3D) cell-based HTS could be a powerful screening tool that allows researchers to finely tune the cell microenvironment, getting more clinically applicable data as a result. Here, we have shown the successful integration of a peptide-based hydrogel into the high-throughput format.

Beta-hairpin hydrogels as scaffolds for high-throughput drug discovery in three-dimensional cell culture.

Automated cell-based high-throughput screening (HTS) is a powerful tool in drug discovery, and it is increasingly being recognized that three-dimensional (3D) models, which more closely mimic in vivo-like conditions, are desirable screening platforms. One limitation hampering the development of 3D HTS is the lack of suitable 3D culture scaffolds that can readily be incorporated into existing HTS infrastructure. We now show that ß-hairpin peptide hydrogels can serve as a 3D cell culture platform that is compatible with HTS. MAX8 ß-hairpin peptides can physically assemble into a hydrogel with defined porosity, permeability and mechanical stability with encapsulated cells. Most importantly, the hydrogels can then be injected under shear-flow and immediately reheal into a hydrogel with the same properties exhibited prior to injection. The post-injection hydrogels are cell culture compatible at physiological conditions. Using standard HTS equipment and medulloblastoma pediatric brain tumor cells as a model system, we show that automatic distribution of cell-peptide mixtures into 384-well assay plates results in evenly dispensed, viable MAX8-cell constructs suitable for commercially available cell viability assays. Since MAX8 peptides can be functionalized to mimic the microenvironment of cells from a variety of origins, MAX8 peptide gels should have broad applicability for 3D HTS drug discovery.

ß-hairpin peptide hydrogels for package delivery.

The underlying challenge of drug delivery is the safe, controlled transport of a supply of therapeutic agent to its intended location at its effective dose. New and expanding solutions to payload delivery are being discovered in the field of hydrogels. Hydrogels are highly hydrated polymer networks that vary greatly depending on the underlying molecular structure. The subgroup of hydrogels that will be the focus of this chapter is the ß-hairpin peptide hydrogel. These peptide-based materials are formed through a molecular self-assembly mechanism that only occurs after desired triggering of intramolecular peptide folding. Once folded, the ß-hairpins assemble intermolecularly into a nanofibrillar network. The physical properties of the hydrogel network and its peptide foundation result in advantageous material properties which can be used for multiple biomedical applications including drug delivery. As a shear thinning solid that is easily injectable, cytocompatible, customizable, and well characterized, ß-hairpin hydrogels are an exciting candidate as a drug delivery vehicle.

Beta Hairpin Peptide Hydrogels as an Injectable Solid Vehicle for Neurotrophic Growth Factor Delivery.

There is intense interest in developing novel methods for the sustained delivery of low levels of clinical therapeutics. MAX8 is a peptide-based beta-hairpin hydrogel that has unique shear thinning properties that allow for immediate rehealing after the removal of shear forces, making MAX8 an excellent candidate for injectable drug delivery at a localized injury site. The current studies examined the feasibility of using MAX8 as a delivery system for nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF), two neurotrophic growth factors currently used in experimental treatments of spinal cord injuries. Experiments determined that encapsulation of NGF and BDNF within MAX8 did not negatively impact gel formation or rehealing and that shear thinning did not result in immediate growth factor release. ELISA, microscopy, rheology, and Western blotting experiments collectively demonstrate the functional capabilities of the therapeutic-loaded hydrogels to (i) maintain a protective environment against in vitro degradation of encapsulated therapeutics for at least 28 days; and (ii) allow for sustained release of NGF and BDGF capable of initiating neurite-like extensions of PC12 cells, most likely due to NGF/BDGF signaling pathways. Importantly, while the 21 day release profiles could be tuned by adjusting the MAX8 hydrogel concentration, the initial shear thinning of the hydrogel (e.g., during injection) does not induce significant premature loss of the encapsulated therapeutic, most likely due to effective trapping of growth factors within structurally robust domains that are maintained during the application of shear forces. Together, our data suggests that MAX8 allows for greater dosage control and sustained therapeutic growth factor delivery, potentially alleviating side effects and improving the efficacy of current therapies.

Peptide Hydrogels - Versatile Matrices for 3D Cell Culture in Cancer Medicine.

Traditional two-dimensional (2D) cell culture systems have contributed tremendously to our understanding of cancer biology but have significant limitations in mimicking in vivo conditions such as the tumor microenvironment. In vitro, three-dimensional (3D) cell culture models represent a more accurate, intermediate platform between simplified 2D culture models and complex and expensive in vivo models. 3D in vitro models can overcome 2D in vitro limitations caused by the oversupply of nutrients, and unphysiological cell-cell and cell-material interactions, and allow for dynamic interactions between cells, stroma, and extracellular matrix. In addition, 3D cultures allow for the development of concentration gradients, including oxygen, metabolites, and growth factors, with chemical gradients playing an integral role in many cellular functions ranging from development to signaling in normal epithelia and cancer environments in vivo. Currently, the most common matrices used for 3D culture are biologically derived materials such as matrigel and collagen. However, in recent years, more defined, synthetic materials have become available as scaffolds for 3D culture with the advantage of forming well-defined, designed, tunable materials to control matrix charge, stiffness, porosity, nanostructure, degradability, and adhesion properties, in addition to other material and biological properties. One important area of synthetic materials currently available for 3D cell culture is short sequence, self-assembling peptide hydrogels. In addition to the review of recent work toward the control of material, structure, and mechanical properties, we will also discuss the biochemical functionalization of peptide hydrogels and how this functionalization, coupled with desired hydrogel material characteristics, affects tumor cell behavior in 3D culture.

Why traditional statistical process control charts for attribute data should be viewed alongside an xmr-chart.

The use of statistical process control (SPC) charts in healthcare is increasing. The general advice when plotting SPC charts is to begin by selecting the right chart. This advice, in the case of attribute data, may be limiting our insights into the underlying process and consequently be potentially misleading. Given the general lack of awareness that additional insights may be obtained by using more than one SPC chart, there is a need to review this issue and make some recommendations. Under purely common cause variation the control limits on the xmr-chart and traditional attribute charts (eg, p-chart, c-chart, u-chart) will be in close agreement, indicating that the observed variation (xmr-chart) is consistent with the underlying Binomial model (p-chart) or Poisson model (c-chart, u-chart). However, when there is a material difference between the limits from the xmr-chart and the attribute chart then this also constitutes a signal of an underlying systematic special cause of variation. We use one simulation and two case studies to demonstrate these ideas and show the utility of plotting the SPC chart for attribute data alongside an xmr-chart. We conclude that the combined use of attribute charts and xmr-charts, which requires little additional effort, is a useful strategy because it is less likely to mislead us and more likely to give us the insight to do the right thing.

Relationships between fast bowling technique and ball release speed in cricket.

The aim of this study was to identify the key aspects of technique that characterize the fastest bowlers. Kinematic data were collected for 20 elite male fast bowlers with 11 kinematic parameters calculated, describing elements of fast bowling technique that have previously been linked to ball release speed. Four technique variables were identified as being the best predictors of ball release speed, explaining 74% of the observed variation in ball release speed. The results indicate that the fastest bowlers have a quicker run-up and maintain a straighter knee throughout the front foot contact phase. The fastest bowlers were also observed to exhibit larger amounts of upper trunk flexion up to ball release and to delay the onset of arm circumduction. This study identifies those technique variables that best explain the differences in release speeds among fast bowlers. These results are likely to be useful in both the coaching and talent identification of fast bowlers.

The influence of cricket fast bowlers' front leg technique on peak ground reaction forces.

High ground reaction forces during the front foot contact phase of the bowling action are believed to be a major contributor to the high prevalence of lumbar stress fractures in fast bowlers. This study aimed to investigate the influence of front leg technique on peak ground reaction forces during the delivery stride. Three-dimensional kinematic data and ground reaction forces during the front foot contact phase were captured for 20 elite male fast bowlers. Eight kinematic parameters were determined for each performance, describing run-up speed and front leg technique, in addition to peak force and time to peak force in the vertical and horizontal directions. There were substantial variations between bowlers in both peak forces (vertical 6.7 ± 1.4 body weights; horizontal (braking) 4.5 ± 0.8 body weights) and times to peak force (vertical 0.03 ± 0.01 s; horizontal 0.03 ± 0.01 s). These differences were found to be linked to the orientation of the front leg at the instant of front foot contact. In particular, a larger plant angle and a heel strike technique were associated with lower peak forces and longer times to peak force during the front foot contact phase, which may help reduce the likelihood of lower back injuries.

The effect of coaching intervention on elite fast bowling technique over a two year period.

Fast bowling in cricket is an activity that is well recognised as having high injury prevalence and there has been debate regarding the most effective fast bowling technique. The aim of this study was to determine whether two-year coaching interventions conducted in a group of elite young fast bowlers resulted in fast bowling technique alteration. Selected kinematics of the bowling action of 14 elite young fast bowlers were measured using an 18 camera Vicon Motion Analysis system before and after two-year coaching interventions that addressed specific elements of fast bowling technique. Mann-Whitney tests were used to determine whether any changes in kinematic variables occurred pre- and post-intervention between those who had the coaching interventions and those who didn't. The coaching interventions, when applied, resulted in a more side-on shoulder alignment at back foot contact (BFC) (p = 0.002) and decreased shoulder counter-rotation (p = 0.001) however, there was no difference in the degree of change in back and front knee flexion angles or lower trunk side-flexion. This study has clearly shown that specific aspects of fast bowling technique are changeable over a two-year period in elite level fast bowlers and this may be attributed to coaching intervention.