Richard M. Baker

Automated, contour-based tracking and analysis of cell behaviour over long time scales in environments of varying complexity and cell density

Understanding single and collective cell motility in model environments is foundational to many current research efforts in biology and bioengineering. To elucidate subtle differences in cell behaviour despite cell-to-cell variability, we introduce an algorithm for tracking large numbers of cells for long time periods and present a set of physics-based metrics that quantify differences in cell trajectories. Our algorithm, termed automated contour-based tracking for in vitro environments (ACTIVE), was designed for adherent cell populations subject to nuclear staining or transfection. ACTIVE is distinct from existing tracking software because it accommodates both variability in image intensity and multi-cell interactions, such as divisions and occlusions. When applied to low-contrast images from live-cell experiments, ACTIVE reduced error in analysing cell occlusion events by as much as 43% compared with a benchmark-tracking program while simultaneously tracking cell divisions and resulting daughter–daughter cell relationships. The large dataset generated by ACTIVE allowed us to develop metrics that capture subtle differences between cell trajectories on different substrates. We present cell motility data for thousands of cells studied at varying densities on shape-memory-polymer-based nanotopographies and identify several quantitative differences, including an unanticipated difference between two ‘control’ substrates. We expect that ACTIVE will be immediately useful to researchers who require accurate, long-time-scale motility data for many cells.

Contour-Based Algorithm for Tracking Cells and Cell-Material Analyses

Cellular tracking has been employed to investigate complex cell-cell and cell-material interactions that play critical roles in tissue development and disease progression. Tracking is often performed manually, however limitations associated with manual tracking make it impractical for tracking dense populations of cells. To address these limitations, several automated tracking algorithms have been developed, buy most of these algorithms are incapable of tracking cells after occlusion events or cell divisions. Here we have developed a custom algorithm in MATLAB that employs a contour-based segmentation approach to identify and track cell divisions and occlusion events. The algorithm further analyzes cell tracks during occlusion events using a cost analysis to detect and relabel mislabeled cells.

Poly(caprolactone) shape memory scaffold for bone tissue engineering

Using synthetic scaffolds as a vessel for bone tissue engineering is a promising technique that could address current limitations associated with autologous grafts, such as donor site scarcity and morbidity. In this study, we aimed to synthesize a biocompatible scaffold with controllable pore morphology that could be used to fill critical size defects through the shape memory effect. To achieve this goal we used a porogen-leaching technique in which a shape memory poly(caprolactone) (PCL) was cured in the presence of salt particles, which were subsequently removed to create high porosity in the scaffold. The overall shape memory behavior of the scaffold was investigated and it was found that the material exhibited greater than 99 % shape fixing and 93 % shape recovery. Preliminary cell culture studies on a non-triggered scaffold showed that cells readily attached to the material and remained viable within the scaffold.

Shape memory activation can affect cell seeding of shape memory polymer scaffolds designed for tissue engineering and regenerative medicine

The ability of a three-dimensional scaffold to support cell seeding prior to implantation is a critical criterion for many scaffold-based tissue engineering and regenerative medicine strategies. Shape memory polymer functionality may present important new opportunities and challenges in cell seeding, but the extent to which shape memory activation can positively or negatively affect cell seeding has yet to be reported. The goal of this study was to determine whether shape memory activation can affect cell seeding. The hypothesis was that shape memory activation of porous scaffolds during cell seeding can affect both the number of cells seeded in a scaffold and the distribution (in terms of average infiltration distance) of cells following seeding. Here, we used a porous shape memory foam scaffold programmed to expand when triggered to study cell number and average cell infiltration distance following shape memory activation. We found that shape memory activation can affect both the number of cells and the average cell infiltration distance. The effect was found to be a function of rate of shape change and scaffold pore interconnectivity. Magnitude of shape change had no effect. Only reductions in cell number and infiltration distance (relative to control and benchmark) were observed. The findings suggest that strategies for tissue engineering and regenerative medicine that involve shape memory activation in the presence of a cell-containing medium in vitro or in vivo should consider how recovery rate and scaffold pore interconnectivity may ultimately impact cell seeding.Graphical abstract

Wrinkle formation on a biocompatible shape memory polymer

Ordered wrinkles have been used for a variety of applications and have found much interest in the field of mechanobiology, as topography has been shown to influence cell behavior. In this study we aimed to synthesize a biocompatible material that could be triggered to form wrinkles under physiological conditions. To achieve this aim, we used a shape memory polymer (SMP) substrate to induce surface buckling of a thin gold film resting atop the SMP. The SMP composition was tuned to allow for surface buckling to occur under cell culture compatible conditions. The SMP was strained to different values prior to buckling and the resulting wavelength and amplitude for each strain was characterized. Increasing the strain allowed for smaller wavelengths and larger wrinkle amplitudes. Preliminary cell culture experiments showed high cell viability and cell alignment atop the wrinkled topography.

Preservation of osteogenic capacity following shape memory triggering of foam and e-spun scaffolds

Shape memory polymers (SMPs) have received significant attention for their potential to be applied in the development of dynamic, functional scaffolds for tissue engineering and regenerative medicine. Recent work has employed shape memory polymers in the development of topography-changing substrates, and these studies have shown that changes in topography can direct cell alignment, cell migration, and stem cell lineage commitment. Additional efforts have focused on expanding these strategies to 3D scaffolds that are capable of undergoing architecture changes under physiological conditions. Such shape changing scaffolds show promise for regenerative medicine applications, but it remains unknown whether shape memory actuated changes in 3D architecture have detrimental effect on the differentiation capacity of stem cells resident in the scaffold during the change. In this study, we investigated the effect of architectural changes in 3D foams and fiber mats on the osteogenic capacity of human adipose-derived stem cells. The results demonstrate osteogenic capacity to be preserved following shape memory triggering, with no reduction in osteogenesis compared to a static control.

Shape Memory Scaffold with a Tunable Recovery Temperature for Filling Critical-Size Bone Defects

Traditionally, critical-size defects have been treated using autologous bone grafts which, while being effective, have limitations that include donor site scarcity, additional pain, and donor site morbidity. Synthetic scaffolds show promise as alternate graft materials, but current scaffolds have limitations associated with filling and conforming to the defect site. In this study, we aimed to synthesize a cytocompatible scaffold with shape memory functionality that could address limitations associated with filling and conforming to the defect site. To achieve this goal we employed a porogen-leaching technique to fabricate a shape memory poly(epsilon-caprolactone) (PCL) foam capable of expanding to fill space under physiological temperatures. Tuning of the recovery temperature to a physiological temperature was achieved by copolymerizing with a second, hydrophilic polymer, as well as by varying the deformation temperature. The scaffold showed excellent shape fixing and shape recovery, and the transition temperature was tuned to a physiological range. Preliminary cell studies showed qualitatively that cells remain viable and proliferate on the scaffold.

A contour-based particle tracking system for the study of cell migratory behavior

Due to a recent focus on cell based therapies in biomedical applications, the ability to accurately track cellular behavior has become an increasingly crucial research tool. Cell motility is of particular importance as it influences basic cellular interactions, effecting macroscale processes such as general tissue development, wound healing, and disease progression. Traditional particle tracking systems focus on intensity thresholds as a means of identifying particles and removing noise. This work presents a contour-based particle tracking algorithm capable of identifying and sorting cells based on nuclear stained images. The two functions presented act cohesively to accurately identify individual cells as particles, capitalizing on a new technique for more accurate depiction of variably stained cells. Based on full-width half maximum theorem fitting, preliminary results demonstrate that this tool has strong potential for use in cell based biomedical applications.