Evaluation of scaffold microstructure and comparison of cell seeding methods using micro-computed tomography-based tools

Micro-computed tomography (micro-CT) provides a means to analyse and model three-dimensional (3D) tissue engineering scaffolds. This study proposes a set of micro-CT-based tools firstly for evaluating the microstructure of scaffolds and secondly for comparing different cell seeding methods. The pore size, porosity and pore interconnectivity of supercritical CO 2 processed poly( l -lactide-co- ɛ -caprolactone) (PLCL) and PLCL/β-tricalcium phosphate scaffolds were analysed using computational micro-CT models. The models were supplemented with an experimental method, where iron-labelled microspheres were seeded into the scaffolds and micro-CT imaged to assess their infiltration into the scaffolds. After examining the scaffold architecture, human adipose-derived stem cells (hASCs) were seeded into the scaffolds using five different cell seeding methods. Cell viability, number and 3D distribution were evaluated. The distribution of the cells was analysed using micro-CT by labelling the hASCs with ultrasmall paramagnetic iron oxide nanoparticles. Among the tested seeding methods, a forced fluid flow-based technique resulted in an enhanced cell infiltration throughout the scaffolds compared with static seeding. The current study provides an excellent set of tools for the development of scaffolds and for the design of 3D cell culture experiments.

Tissue Engineered Esophageal Patch by Mesenchymal Stromal Cells: Optimization of Electrospun Patch Engineering

Aim of work was to locate a simple, reproducible protocol for uniform seeding and optimal cellularization of biodegradable patch minimizing the risk of structural damages of patch and its contamination in long-term culture. Two seeding procedures are exploited, namely static seeding procedures on biodegradable and biocompatible patches incubated as free floating (floating conditions) or supported by CellCrownTM insert (fixed conditions) and engineered by porcine bone marrow MSCs (p-MSCs). Scaffold prototypes having specific structural features with regard to pore size, pore orientation, porosity, and pore distribution were produced using two different techniques, such as temperature-induced precipitation method and electrospinning technology. The investigation on different prototypes allowed achieving several implementations in terms of cell distribution uniformity, seeding efficiency, and cellularization timing. The cell seeding protocol in stating conditions demonstrated to be the most suitable method, as these conditions successfully improved the cellularization of polymeric patches. Furthermore, the investigation provided interesting information on patches’ stability in physiological simulating experimental conditions. Considering the in vitro results, it can be stated that the in vitro protocol proposed for patches cellularization is suitable to achieve homogeneous and complete cellularizations of patch. Moreover, the protocol turned out to be simple, repeatable, and reproducible.

Improving cell distribution on 3D additive manufactured scaffolds through engineered seeding media density and viscosity

In order to ensure the long-term in vitro and in vivo functionality of cell-seeded 3D scaffolds, an effective and reliable method to control cell seeding efficiency and distribution is crucial. Static seeding on 3D additive manufactured scaffolds made of synthetic polymers still remains challenging, as it often results in poor cell attachment, high cell sedimentation and non-uniform cell distribution, due to gravity and to the intrinsic macroporosity and surface chemical properties of the scaffolds. In this study, the bio-inert macromolecules dextran and Ficoll were used for the first time as temporary supplements to alter the viscosity and density of the seeding media, respectively, and improve the static seeding output. The addition of these macromolecules drastically reduced the cell sedimentation velocities, allowing for homogeneous cell attachment to the scaffold filaments. Both dextran- and Ficoll-based seeding methods supported human mesenchymal stromal cells viability and osteogenic differentiation post-seeding. Interestingly, the improved cell distribution led to increased matrix production and mineralization compared to scaffolds seeded by conventional static method. These results suggest a simple and universal method for an efficient seeding of 3D additive manufactured scaffolds, independent of their material and geometrical properties, and applicable for bone and various other tissue regeneration.

A Confirmatory Snowfall Enhancement Project in the Snowy Mountains of Australia. Part I: Project Design and Response Variables

The Snowy Precipitation Enhancement Research Project (SPERP) was undertaken from May 2005 to June 2009 in the Snowy Mountains of southeastern Australia with the aim of enhancing snowfall in westerly flows associated with winter cold fronts. Building on earlier field studies in the region, SPERP was developed as a confirmatory experiment of glaciogenic static seeding using a silver-chloroiodide material dispersed from ground-based generators. Seeding of 5-h experimental units (EUs) was randomized with a seeding ratio of 2:1. A total of 107 EUs were undertaken at suitable times, based on surface and upper-air observations. Indium (III) oxide was released during all EUs for comparison of indium and silver concentrations in snow in seeded and unseeded EUs to test the targeting of seeding material. A network of gauges was deployed at 44 sites across the region to detect whether precipitation was enhanced in a fixed target area of 832 km2, using observations from a fixed control area to estimate the natural precipitation in the target. Additional measurements included integrated supercooled liquid water at a site in the target area and upper-air data from a site upwind of the target.

Processing and Characterization of Apatite-Wollastonite Porous Scaffolds for Bone Tissue Engineering

There is a clinical and socio-economic need to produce synthetic alternatives to autologous or allogenic bone grafts. Bioactive glasses and glass-ceramics offer great potential in this area. The aims of this study were to optimise production of apatite-wollastonite (A-W) glassceramic scaffolds produced by selective laser sintering, in terms of their physical and biological properties and to look at how human Mesenchymal Stem Cells (MSCs) responded to these 3-D scaffolds in vitro. An indirect selective laser sintering process successfully produced strong, porous scaffolds. Depending upon particle size(s) and infiltration of the porous structure, flexural strengths between 35 MPa and 100 MPa were obtained. Following static seeding of A-W scaffolds with MSCs, fluoresecent actin and nuclei staining, as observed by confocal microscopy, showed that these scaffolds supported the adherence of human MSC’s at time periods of up to 21 days. As such these seeded scaffolds show great potential for use in bone regenerative medicine.

Testing, Implementation, and Evolution of Seeding Concepts—A Review

In this paper, testing, implementation, and evolution of both static and dynamic seeding concepts are reviewed. A brief review of both waterspray and hygroscopic seeding is first presented. This is followed by reviews of static seeding of stable orographic clouds and supercooled cumuli. We conclude with a review of dynamic seeding concepts with particular focus on the Florida studies. It is concluded that it is encouraging that our testing procedures have evolved from single-response-variable “blackbox” experiments to randomized experiments that attempt to test a number of components in the hypothesized chain of physical responses to seeding. It is cautioned, however, that changes in the seeding strategy to optimize detection of a physical response (in any of the intermediate links in the hypothesized chain of responses) can have an adverse effect upon rainfall on the ground.

Static Mode Seeding of Summer Cumuli—A Review

A review of the state of knowledge of the physics of the static mode seeding hypothesis for convective clouds is presented. The central thesis of the review is that the results of past experimental work are diverse but valid and that credibility of the science depends on understanding the physical reasons for the diverse results. Areas of uncertainty and conflicts in evidence associated with the statement of physical hypothesis, the concept of seedability, the seeding operation, and the chain of physical events following seeding are highlighted to identify what issues need to be resolved to further progress in precipitation enhancement research and application. It is concluded that the only aspect of static seeding that meets scientific standards of cause-and-effect relationships and repeatability is that glaciogenic seeding agents can produce distinct “seeding signatures” in clouds. However, the reviewer argues that a body of inferential physical evidence has been amassed that provides a better understanding of which clouds are seedable (susceptible to precipitation enhancement by artificial seeding) and which are not, even though the tools for recognizing and properly treating them are imperfect. In particular, the inferred evidence appears to support the claims of physical plausibility for the positive statistical results of the Israeli experiments. It is suggested that future work continue to be designed for physical understanding and evaluation through comprehensive field studies and numerical modeling. Duplicating the Israeli experiments in another location should receive high priority but, in general, future experiments should move upscale from cumulus congestus to convective complexes. In doing so, a new, more complex physical hypothesis that accounts for cloud–environment and microphysical–dynamical interactions and their response to seeding will have to be developed.