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Mesenchymal stem cells (MSCs) have attracted great interest in recent years for tissue engineering and regenerative medicine applications due to their ease of isolation and multipotent differentiation capacity. In the past, MSC research has focussed on the effects of soluble cues, such as growth factors and cytokines; however, there is now increasing interest in understanding how parameters such as substrate modulus, specific extracellular matrix (ECM) components and the ways in which these are presented to the cell can influence MSC properties. Here we use surfaces of self-assembled maleimide-functionalized polystyrene-block-poly(ethylene oxide) copolymers (PS-PEO-Ma) to investigate how the spatial arrangement of cell adhesion ligands affects MSC behaviour. By changing the ratio of PS-PEO-Ma in mixtures of block copolymer and polystyrene homopolymer, we can create surfaces with lateral spacing of the PEO-Ma domains ranging from 34 to 62 nm. Through subsequent binding of cysteine–GRGDS peptides to the maleimide-terminated end of the PEO chains in each of these domains, we are able to present tailored surfaces of controlled lateral spacing of RGD (arginine-glycine-aspartic acid) peptides to MSCs. We demonstrate that adhesion of MSCs to the RGD-functionalized block-copolymer surfaces is through specific attachment to the presented RGD motif and that this is mediated by α5, αV, β1 and β3 integrins. We show that as the lateral spacing of the peptides is increased, the ability of the MSCs to spread is diminished and that the morphology changes from well-spread cells with normal fibroblastic morphology and defined stress-fibres, to less-spread cells with numerous cell protrusions and few stress fibres. In addition, the ability of MSCs to form mature focal adhesions is reduced on substrates with increased lateral spacing. Finally, we investigate differentiation and use qRT-PCR determination of gene expression levels and a quantitative alkaline phosphatase assay to show that MSC osteogenesis is reduced on surfaces with increased lateral spacing while adipogenic differentiation is increased. We show here, for the first time, that the lateral spacing of adhesion peptides affects human MSC (hMSC) properties and might therefore be a useful parameter with which to modify hMSC behaviour in future tissue engineering strategies.

Mesenchymal stromal cells (MSCs) are of clinical interest due to their ability to self-renew and differentiate into multiple tissue types. Unfortunately, both the number of MSCs within tissues and their reparative potential decreases with age, resulting in a decreased capability for tissue repair. The ability to isolate MSCs from a multitude of tissue sources and expand and differentiate them in vitro has made them excellent candidates for a range of therapeutic applications. However, a number of existing limitations in terms of their culture must first be addressed prior to them being widely available for use in the clinic. In this chapter we discuss potential strategies to produce therapeutically-relevant numbers of high-quality MSCs for use in clinical applications. The relative merits of MSCs derived from different tissue sources and the development of defined culture conditions, in terms of oxygen levels, soluble media and culture substrates from which high-quality MSCs may, in future be derived for clinical applications, are described.

As strategies for manipulating cellular behaviour in vitro and in vivo become more sophisticated, synthetic biomaterial substrates capable of reproducing critical biochemical and biophysical properties (or cues) of tissue micro-environments will be required. Cytoskeletal tension has been shown to be highly deterministic of cell fate decisions, yet few synthetic biomaterials are capable of modulating cytoskeletal tension of adhered cells through variations in stiffness, at least in the ranges applicable to tissue properties (e.g., 1-100 kPa), whilst also possessing other required properties, such as biodegradability, biocompatibility and processability. In this paper we describe a non-cytotoxic polymer system based on acrylated polypropylene glycol triol (aPPGT). This new elastomer system has tunable elastic moduli, is degradable, can be easily surface modified and can be manufactured into porous three dimensional scaffolds or micropatterned substrates. We demonstrate that the PPGT substrates can modulate hMSC morphology, growth, and differentiation, and that they can produce similar outcomes as observed for a non-degradable polyacrylamide substrate, confirming their utility as a degradable elastomer for tissue engineering and other biomedical applications.

Studying the rate of cell migration provides insight into fundamental cell biology as well as a tool to assess the functionality of synthetic surfaces and soluble environments used in tissue engineering. The traditional tools used to study cell migration include the fence and wound healing assays. In this paper we describe the development of a microchannel based device for the study of cell migration on defined surfaces. We demonstrate that this device provides a superior tool, relative to the previously mentioned assays, for assessing the propagation rate of cell wave fronts. The significant advantage provided by this technology is the ability to maintain a virgin surface prior to the commencement of the cell migration assay. Here, the device is used to assess rates of mouse fibroblasts (NIH 3T3) and human osteosarcoma (SaOS2) cell migration on surfaces functionalized with various extracellular matrix proteins as a demonstration that confining cell migration within a microchannel produces consistent and robust data. The device design enables rapid and simplistic assessment of multiple repeats on a single chip, where surfaces have not been previously exposed to cells or cellular secretions.

A 29 year old woman, who was an intermittent contact lens wearer, is referred from her local ophthalmologist with a diagnosis of microbial keratitis. There is a large central corneal ulcer with stromal white cell recruitment and low grade anterior chamber (AC) activity. The patient has no other eye problems and is in good health. Would you perform a corneal scraping? Which antibiotics would you use to begin empirical treatment for her microbial keratitis?

The effect that synthetic steroid components of different oral contraceptives have on the assay system for measurement of sex hormones binding globulin (SHBG)-binding capacity, has been examined. Interference with the assay was not demonstrable at therapeutic levels of the drugs. d-Norgestrel was the only steroid studied which bound to SHBG with an affinity comparable to the endogenous sex hormones; it also tended to decrease SHBG-finding capacity, whereas preparations containing low levels of norethisterone increased the binding capacity. We suggest that measurement of changes in SHBG-binding capacity in subjects taking oral contraceptives may prove useful in indicating changes in sex hormone balance, particularly in view of the well-recognised side-effect of these drugs.This study examines the effect of synthetic steroid components of 7 commonly prescribed oral contraceptives on the assay of sex hormone binding globulin (SHBG). The 7 oral contraceptives and the number of patients taking each preparation are: 1) Minovlar (n=10); 2) Ortho-Novum 1/50 (n=2); 3) Gynovlar 21 (n=8); 4) Minilyn (n=5); 5) Eugynon-50 (n=2); 6) Eugynon-30 (n=10) and 7) Microgynon (n=3). The blood SHBG-binding capacity of these 40 women was measured. The method of Rosner was used in the SHBG-binding capacity assay. Serum samples from each woman were treated with charcoal to remove the endogenous and exogenous steroids; the samples were assayed for SHBG-binding capacity before and after the charcoal treatment. Binding studies were also done. The results show that the synthetic compounds do not cause significant interference with the assay system when used at therapeutic levels. Of the synthetic steroids studied, d-Norgestrel was the only 1 which bound to SHBG with an affinity comparable to the endogenous sex hormones. D-Norgestrel also tended to decrease SHBG-binding capacity. This finding supports the observation that d-Norgestrel has androgenic activity due to its structure, as there is an apparent relation between the in vivo androgenic activity of steroids and their ability to bind to SHBG; the more potent the androgen, the greater its binding to SHBG. It is probable that d-Norgestrel exerts a direct inhibiting effect on SHBG synthesis, as do endogenous androgens. The ideal oral contraceptive preparation is one which maintains the existing sex hormone balance, avoiding the undesirable effects of excess estrogen administration on the one hand and excess androgenicity on the other. Measurement of SHBG-binding capacity would be a useful tool for determining changes in sex hormone balance. As women vary in their requirement, measurement of SHBG-binding capacity before and during contraceptive therapy may be a better indicator of hormonal balance in an individual woman.

Transient stability problems become much more severe where the system design criteria is based on "stuck-breaker" fault clearing times, particularly where three-phase faults are used. Such a criterion is difficult and frequently impossible to meet with normal system design. Results of studies exploring the relative effect on transient stability limits of several options available to the system planner, including transmission strength, excitation system response, and early valving, are presented. Early valving appears to offer attractive stability improvement in those cases where such improvement is needed.