Yvonne Reinwald

Cryogels: Freezing unveiled by thawing

Cryogels are interconnected supermacroporous gels prepared at sub-zero temperatures having applications in various research fields. The process of cryogelation is ideally thought to take place via following steps: phase separation with ice-crystal formation, cross-linking and polymerization followed by thawing of ice-crystals to form an interconnected porous cryogel network. This phenomenon mostly thought as a theoretical concept has now been revealed here in practical terms via data generated by micro-computed tomography (Micro CT). Micro CT is mainly used for characterizing the gel materials in terms of their physical properties like pore size, porosity, strut size, etc., whereas this work has pioneered its role in elucidating the process of cryogel formation.

Analysis of sintered polymer scaffolds using concomitant synchrotron computed tomography and in situ mechanical testing

The mechanical behaviour of polymer scaffolds plays a vital role in their successful use in bone tissue engineering. The present study utilised novel sintered polymer scaffolds prepared using temperature-sensitive poly(dl-lactic acid-co-glycolic acid)/poly(ethylene glycol) particles. The microstructure of these scaffolds was monitored under compressive strain by image-guided failure assessment (IGFA), which combined synchrotron radiation computed tomography (SR CT) and in situ micro-compression. Three-dimensional CT data sets of scaffolds subjected to a strain rate of 0.01%/s illustrated particle movement within the scaffolds with no deformation or cracking. When compressed using a higher strain rate of 0.02%/s particle movement was more pronounced and cracks between sintered particles were observed. The results from this study demonstrate that IGFA based on simultaneous SR CT imaging and micro-compression testing is a useful tool for assessing structural and mechanical scaffold properties, leading to further insight into structure–function relationships in scaffolds for bone tissue engineering applications.

Dynamic 3D culture: Models of chondrogenesis and endochondral ossification

The formation of cartilage from stem cells during development is a complex process which is regulated by both local growth factors and biomechanical cues, and results in the differentiation of chondrocytes into a range of subtypes in specific regions of the tissue. In fetal development cartilage also acts as a precursor scaffold for many bones, and mineralization of this cartilaginous bone precursor occurs through the process of endochondral ossification. In the endochondral formation of bones during fetal development the interplay between cell signalling, growth factors, and biomechanics regulates the formation of load bearing bone, in addition to the joint capsule containing articular cartilage and synovium, generating complex, functional joints from a single precursor anlagen. These joint tissues are subsequently prone to degeneration in adult life and have poor regenerative capabilities, and so understanding how they are created during development may provide useful insights into therapies for diseases, such as osteoarthritis, and restoring bone and cartilage lost in adulthood. Of particular interest is how these tissues regenerate in the mechanically dynamic environment of a living joint, and so experiments performed using 3D models of cartilage development and endochondral ossification are proving insightful. In this review, we discuss some of the interesting models of cartilage development, such as the chick femur which can be observed in ovo, or isolated at a specific developmental stage and cultured organotypically in vitro. Biomaterial and hydrogel-based strategies which have emerged from regenerative medicine are also covered, allowing researchers to make informed choices on the characteristics of the materials used for both original research and clinical translation. In all of these models, we illustrate the essential importance of mechanical forces and mechanotransduction as a regulator of cell behavior and ultimate structural function in cartilage.

Interconnectivity analysis of supercritical CO2-foamed scaffolds

This paper describes a computer algorithm for the determination of the interconnectivity of the pore space inside scaffolds used for tissue engineering. To validate the algorithm and its computer implementation, the algorithm was applied to a computer-generated scaffold consisting of a set of overlapping spherical pores, for which the interconnectivity was calculated exactly. The algorithm was then applied to micro-computed X-ray tomography images of supercritical CO(2)-foamed scaffolds made from poly(lactic-co-glycolic acid) (PLGA), whereby the effect of using different weight average molecular weight polymer on the interconnectivity was investigated.

Post-processing of polymer foam tissue scaffolds with high power ultrasound: a route to increased pore interconnectivity, pore size and fluid transport.

The aim of this work is to demonstrate that the structural and fluidic properties of polymer foam tissue scaffolds, post-fabrication but prior to the introduction of cells, can be engineered via exposure to high power ultrasound. Our analysis is supported by measurements of fluid uptake during insonification and imaging of the scaffold microstructure via X-ray computed tomography, scanning electron microscopy and acoustic microscopy. The ultrasonic treatment is performed with a frequency of 30 kHz, average intensities up to 80,000 Wm(-2) and exposure times up to 20 h. The treatment is found to increase the mean pore size by over 10%. More striking is the improvement in fluid uptake: for scaffolds with only 40% water uptake via standard immersion techniques, we can routinely achieve full saturation of the scaffold over approximately one hour of exposure. These desirable modifications occur with negligible loss of scaffold integrity and mass, and are optimized when the ultrasound treatment is coupled to a pre-wetting stage with ethanol. Our findings suggest that high power ultrasound is highly targeted towards flow obstructions in the scaffold architecture, thereby providing an efficient means to promote pore interconnectivity and fluid transport in thick foam tissue scaffolds.

Comparability : Manufacturing, characterization and controls, report of a UK Regenerative Medicine Platform Pluripotent Stem Cell Platform Workshop, Trinity Hall, Cambridge, 14-15 September 2015

This paper summarizes the proceedings of a workshop held at Trinity Hall, Cambridge to discuss comparability and includes additional information and references to related information added subsequently to the workshop. Comparability is the need to demonstrate equivalence of product after a process change; a recent publication states that this ‘may be difficult for cell-based medicinal products’. Therefore a well-managed change process is required which needs access to good science and regulatory advice and developers are encouraged to seek help early. The workshop shared current thinking and best practice and allowed the definition of key research questions. The intent of this report is to summarize the key issues and the consensus reached on each of these by the expert delegates.

Electrospun PLGA fibre sheets incorporating fluorescent nanosensors: self-reporting scaffolds for application in tissue engineering

Ratiometric analyte responsive nanosensors have been incorporated into electrospun poly(lactic-co-glycolic) acid (PLGA) fibres to create self-reporting scaffolds. It has been demonstrated that the self-reporting scaffolds could be utilised to monitor microenvironment conditions without damaging the fabricated scaffold or the cells being cultured upon the construct. This presents opportunities to fully understand, monitor and optimise the growth of 3D model tissue constructs in vitro.

Real-time and non-invasive measurements of cell mechanical behaviour with optical coherence phase microscopy

Cell mechanical behaviour is increasingly recognised as a central biophysical parameter in cancer and stem cell research, and methods of investigating their mechanical behaviour are therefore needed. We have developed a novel qualitative method based on quantitative phase imaging which is capable of investigating cell mechanical behaviour in real-time at cellular resolution using optical coherence phase microscopy (OCPM), and stimulating the cells non-invasively using hydrostatic pressure. The method was exemplified to distinguish between cells with distinct mechanical properties, and transient change induced by Cytochalasin D. We showed the potential of quantitative phase imaging to detect nanoscale intracellular displacement induced by varying hydrostatic pressure in microfluidic channels, reflecting cell mechanical behaviour. Further physical modelling is required to yield quantitative mechanical properties.

Pluripotent Stem Cells and Their Dynamic Niche

Cell-seeded implants are a regenerative medicine strategy that aims to replace injured tissue and restore tissue function. Pluripotent stem cells are promising cell candidates for the development of regenerative medicine therapies as they have the ability to self-renew and commit towards numerous cell types. In vivo, stem cells reside in a dynamic niche, a stem cell-specific microenvironment that possesses chemical, biological and mechanical cues, which drive the stem cell fate and renewal. The connection between stem cells and their niche is a two-way relationship consisting of both cell–cell interac‐tion and cell–extracellular matrix (ECM) interactions. An alternative regenerative medicine approach is the manipulation of the stem cell microenvironment. Hence, novel strategies have been developed including 3D biomaterials and bioreactor technologies providing topographical, chemical and mechanical cues to recreate the stem cell niche. Understanding the mechanisms controlling stem cell fate and the dynamic nature of thestem cell niche will enable researchers to replicate this stem cell-specific microenvironment, and therefore, harness and control the valuable attributes which stem cells possess. This chapter elucidates the importance of pluripotent stem cells and their dynamic niche in regenerative medicine. It further presents novel strategies to replicate chemical, topographical and mechanical stimuli which are essential for the regulation of stem cell fate and hence tissue regeneration.

Online monitoring of mechanical properties of three-dimensional tissue engineered constructs for quality assessment

Mechanical preconditioning and mechanical properties of tissue engineered constructs are essential for their capability to regenerate damaged tissues. To online monitor the mechanical properties a hydrostatic pressure bioreactor was coupled with optical coherence tomography into a new image modality termed hydrostatic pressure optical coherence elastography (HP-OCE). HP-OCE was utilised to assess the properties of three-dimensional (3D) tissue constructs while being physically stimulated within the hydrostatic force bioreactor. Hydrogels have been infiltrated into porous rapid prototyped or salt-leached scaffolds to mimic heterogeneous mechanical properties of cell-seeded constructs. Variations of mechanical properties in the solid scaffolds and agarose gels with different gel concentrations as well as the presences of cells have been clearly delineated by HP-OCE. Results indicate that HP-OCE allows contactless real-time non-invasive monitoring of the mechanical properties of tissue constructs and the effect of physical stimulation on cellular activities.