Raman microspectroscopy was used to determine biochemical markers during the differentiation of embryonic murine stem cells (mES) in vitro. Such markers are useful to determine the differentiation status of ES cells cultured on biomaterials. Raman spectra of mES cells as undifferentiated, spontaneously differentiated (4 days), and differentiated cells via formation of embryoid bodies (16, 20 days) were analyzed. Unsupervised hierarchical cluster analysis and principal component analysis were used to determine biochemical differences between mES cells in various states of differentiation. The undifferentiated cells were characterized by high scores of the first principal component (PC1, 49% variance). Similarity between the PC1 loading and the Raman spectrum of RNA indicated a high concentration of RNA in mES cells compared to differentiated cells. The ratio between the peak areas of RNA and proteins was used as a measure of mRNA translation. Using the same peak area ratio, it was possible to differentiate even between mES as undifferentiated and in early stages of differentiation (4 days). These findings were correlated with biological studies reporting high levels of nontranslated mRNA during early embryonic development. Therefore, the RNA translation obtained from the Raman spectra can be used as marker of differentiation state of mES cells.

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Prostate cancer cells with stem cell characteristics were identified in human prostate cancer cell lines by their ability to form from single cells self-renewing prostaspheres in non-adherent cultures. Prostaspheres exhibited heterogeneous expression of proliferation, differentiation and stem cell-associated makers CD44, ABCG2 and CD133. Treatment with WNT inhibitors reduced both prostasphere size and self-renewal. In contrast, addition of Wnt3a caused increased prostasphere size and self-renewal, which was associated with a significant increase in nuclear β-catenin, keratin 18, CD133 and CD44 expression. As a high proportion of LNCaP and C4-2B cancer cells express androgen receptor we determined the effect of the androgen receptor antagonist bicalutamide. Androgen receptor inhibition reduced prostasphere size and expression of PSA, but did not inhibit prostasphere formation. These effects are consistent with the androgen-independent self-renewal of cells with stem cell characteristics and the androgen-dependent proliferation of transit amplifying cells. As the canonical WNT signaling effector β-catenin can also associate with the androgen receptor, we propose a model for tumour propagation involving a balance between WNT and androgen receptor activity. That would affect the self-renewal of a cancer cell with stem cell characteristics and drive transit amplifying cell proliferation and differentiation. In conclusion, we provide evidence that WNT activity regulates the selfrenewal of prostate cancer cells with stem cell characteristics independently of androgen receptor activity. Inhibition of WNT signaling therefore has the potential to reduce the self-renewal of prostate cancer cells with stem cell characteristics and improve the therapeutic outcome.

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The graft polymerization of acrylic acid was carried out onto poly(ethylene terephthalate) films that had been pretreated with argon plasma and subsequently exposed to oxygen to create peroxides. The influence of synthesis conditions, such as plasma treatment time, plasma power, monomer concentration, temperature, and the presence of Mohr's salt, on the degree of grafting was investigated. The observed initial increase in grafting with monomer concentration accelerated at about 20% monomer. The grafting reached a maximum at 40% monomer and subsequently decreased with further increases in monomer concentration. The reaction temperature had a pronounced effect on the degree of grafting. The initial rate of grafting increased with increasing temperature, but the degree of grafting showed a maximum at 50°C. The activation energy of the grafting obtained from an Arrhenius plot was 29.1 kJ/mol. The addition of Mohr's salt to the reaction medium not only led to a homopolymer-free grafting reaction but also diminished the degree of grafting. The degree of grafting increased with increasing plasma power and plasma treatment time.

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Both Fourier Transform Infrared (FTIR) and Raman spectroscopy have been applied to thein vitrocharacterisation of biomaterials, mainly surface reactions leading to the formation of a biologically active hydroxycarbonate apatite (HCA) layer on the sample surface when immersed in simulated body fluids (SBF). The HCA layer indicates the degree of bioactivity of the sample, because it leads to a strong bond between the biomaterial and living tissue. Reflection measurements using FTIR allow quick, non-destructive detection of the HCA layer for solid and powder samples. Due to the low Raman scattering efficiency and low absorption of water in the visible-near infrared region, Raman micro-spectroscopy was successfully used for thein situcharacterisation of 20 and 40µm diameter 45S5 Bioglass®fibres. Thein situcapabilities of the Raman micro-spectrometer have also been extended to the characterisation of living cells attached on bioinert silica and bioactive 45S5 Bioglass®and 58S substrates. Using a high power 785 nm laser, living cells in physiological conditions can be real-time sampled over long periods of time without inducing cell damage and with good signal strength. Cell death can be monitored because it proved to induce strong changes in the Raman signature in the spectral regions 1000–1150 cm–1and 1550–1650 cm–1.