Organic and Third Phase in HNO3/TBP/n-Dodecane System: No Reverse Micelles

The composition and speciation of the organic and third phases in the system HNO3/TBP (tri-n-butyl phosphate)/n-dodecane have been examined by a combination of gravimetric, Karl Fischer analysis, chemical analysis, FTIR, and 31P NMR spectroscopy, with particular emphasis on the transition from the two-phase to the three-phase region. Phase densities indicate that third-phase formation takes place for initial aqueous HNO3 concentrations above 15 M, while the results from the stoichiometric analysis imply that the organic and third phases are characterized by two distinct species, namely the mono-solvate TBP⋅HNO3 and the hemi-solvate TBP⋅2HNO3, respectively. Furthermore, the 31P NMR spectra of organic and third phase show no significant chemical differences at the phosphorus centers, suggesting that the second HNO3 molecule in the third phase is bound to HNO3 rather than TBP. The third-phase FTIR spectra reveal stronger vibrational absorption bands at 1028, 1310, 1653, and 3200–3500 cm−1, reflecting higher concentrations of H2O, HNO3, and TBP. The molecular dynamics simulation data predict structures in accord with the spectroscopically identified speciation, indicating inequivalent HNO3 molecules in the third phase. The predicted structures of the organic and third phases are more akin to microemulsion networks rather than the distinct, reverse micelles assumed in previous studies. H2O appears to be present as a disordered hydrogen-bonded solvate stabilizing the polar TBP/HNO3 aggregates in the organic matrix, and not as a strongly bound hydrate species in aggregates with defined stoichiometry

What more do we want out of hair and from whom?

At the age of non-invasive approach to regenerative as well as other treatments, harmless yield of starting cell material plays a crucial role in the development of a therapy. The small sample size, absence of pain, bleeding, wound or any kind of postoperative complications can make all the difference in acceptance of the progressing therapy by regulatory bodies, medical practitioners and patients. Within such concept – particularly when it comes to autologous treatments – the importance of the mentioned benign sources grows and gains focus. Perhaps paradoxical in the venture-gain sense, such source appears within a tiny hair follicle, or more precise its outer root sheath (ORS). This is, to the best of our knowledge, the smallest source of adult stem cells, among which are, at that, the naivest cells of the adult human body, able to give raise to many cell types. Those ‘mother of all stem cells’ as they are called, present the closest match to embryonic stem cells in terms of their developmental potency. Potential of this compact stem cell pool to differentiate into neurons, glia, melanocytes, keratinocytes, fibroblasts, chondrocytes, osteocytes, cardiomyocites, and endothelial cells has already been shown. Mainly, our group at the Translational Centre for Regenerative Medizin / Dermatological Clinic At Leipzig University deals with the ectodermal and mesenchymal developmental potential of the ORS stem cell pool. We are currently focused on development of melanocytes and keratinocytes out of ORS for the purposes of non-invasive, autologous, transplantation-based therapies for wound healing and depigmentation, in particular Vitiligo. Standardized procedures have already been developed for quick, reliable and high yield cultivation of well defined, uniform primary cells available in antibiotic free sterile cultures based on explant culture of harmlessly plucked hair follicles. These procedures involve outgrowth on nylon meshes, favorable cultivation conditions for melanocytes, selection into pure culture and end characterization on protein and gene expression level. Those cells have been tested within a number of biocompatible scaffolds and the procedures for the cultivation and application have been patented. In cooperation with Fraunhofer Institute for Cell Therapy and Immunology, enzymatic modifications of scaffold materials by lacase have brought about more adhesive and friendlier conditions for the melanocytes and keratinocytes and with use of cafeic acid and L-DOPA as substrates granted us very promising candidates for graft carrier. Using similar procedures, we have cultivated cells that closely match mesenchymal stem cells out of human hair follicle ORS for the purposes of chondrogenic and osteogenic differentiation, aiming at regenerative therapies of cartilage and bone. Further on, the procedures have been adjusted to physiologically fit cultivation conditions for explant cultures other than human – horse, chimpanzee and rabbit. This abstract outlines not only our work in the field of ORS stem cells, but also that of others and comments on the entire vast regenerative potential of this elegant, nevertheless putative source