Orofacial prosthesis design and fabrication using stereolithography.

The use of stereolithography for the manufacture of implantable prostheses is relatively new. Until now, its use with regard to mandibular resection has been to produce pre-operative models that allow more sophisticated planning of the contour and better preparation of the metallic framework to be implanted. This framework rejoins the sections of the mandible, returning some function, but providing no soft tissue support. Hence, the aesthetic results are not optimal. The aim of this study was to investigate the accuracy with which a stereolithographic model could be made to completely reunite both surfaces of the resected mandible and restore full contour. The design and fabrication of a mandibular prosthesis utilizing CAD/CAM technology and stereolithography is still in the early stages, but initial results indicate that with further research and better tools this could provide a new approach to mandibular resections, providing better aesthetic results.

The topography of acetylcholinesterase in dimyristoylphosphatidylcholine liposomes.

Unilamellar liposomes prepared from sn-3-(dimyristoyl)phosphatidylcholine (DMPC) in the presence and absence of acetylcholinesterase were examined by ESR for lipid/protein interactions. Using 5-, 12-, and 16-doxyl stearic acid probes incorporated into the phospholipid bilayers, no measurable differences in the gel to liquid-crystalline phase transition temperature of DMPC (as determined by ESR spectroscopy) were observed when the enzyme was present. These results have established that no significant incorporation of acetylcholinesterase into the hydrophobic region of the phospholipid bilayer is detectable at the protein: lipid ratios used in these experiments. Confirmation of these results was also obtained by differential scanning calorimetry. Interaction of the enzyme with the outermost region of the bilayer was established by trypsin digestion which indicated that as much as 25% of liposome-associated enzyme was removable and was, therefore, exposed to the outer surface. The results of this study have established that acetylcholinesterase associated with unilamellar DMPC liposomes was primarily entrapped within the aqueous compartment of the vesicles and was not present in the phospholipid bilayer.

Liposomes are effective carriers for the ocular delivery of prophylactics.

Liposomes containing acetylcholinesterase were prepared by the freeze-drying method. The multilamellar morphology of the vesicles was revealed by freeze-fracture electron microscopy and their size distribution was determined by quasi-elastic light scattering. The vesicle diameters were in the range of about 0.2-4.0 micron. The liposome preparations were tested for their ocular delivery of an entrapped cholinesterase enzyme in counteracting the miotic effect of diisopropylfluorophosphate (DFP), a prototype of a family of organophosphate poisons. The topical application of the enzyme-containing liposomes to the rabbit eye was found to confer a significant level of protection against DFP-induced miosis. In comparing the prophylactic effectiveness of different enzyme-bearing liposomes, positively charged vesicles were found to be more effective than either neutral or negatively charged vesicles. Although the precise protective mechanism is not clear, our in vitro studies indicate that DFP molecules freely associate with liposomes and tear fluid promotes the release of liposome-entrapped enzymes. Thus, it is conceivable that the enzyme-liposome complex may act somewhat like a sponge by sequestering DFP molecules which diffuse into the vesicle, and also by releasing the entrapped enzyme to combine with DFP, thereby neutralizing its in vivo toxic effect.

THE ROLE OF FREE RADICALS IN SENESCENCE AND WOUNDING.

Reactions involving free radicals are an inherent feature of plant senescence and appear to contribute to a process of oxidative deterioration that leads ultimately to cell death. Radical species derived from molecular oxygen are the primary mediators of this oxidative damage, but non-radical excited states of oxygen, specifically singlet oxygen, may also be involved. Several lines of evidence suggest that degradation of lipids in senescing membranes and the ensuing release of free fatty acids initiate oxidative deterioration by providing substrate for lipoxygenase. In some tissues, lipoxygenase activity increases with advancing senescence in a pattern that is consistent with its putative role in promoting oxidative damage. However, there are important exceptions to this which may be explained by the fact that the timing and extent of peroxidative reactions initiated by lipoxygenase are likely to be determined more by the availability of substrate for the enzyme than by changes in its activity. There are both membranous and cytosolic forms of lipoxygenase in senescing tissues, and peroxidation of membrane lipids appears to be initiated by the membranous enzyme once the appropriate fatty acid substrates, linoleic acid and linolenic acid, become available. Since lipid peroxidation is known to form alkoxy and peroxy radicals as well as singlet oxygen, these reactions in membrane bilayers are probably a major source of activated oxygen species in senescing tissues. Further-more, there are indications that activated oxygen from the lipoxygenase reaction can become substrate for the cytosolic form of the enzyme which, in turn, may raise the titre of activated oxygen during senescence. Additional possible sources of increased free radical production in senescing tissues include peroxidase, which shows greatly increased activity with advancing age, leakage of electrons from electron transport systems to oxygen, in particular from the photosynthetic electron transport system, and decompartmentalization of iron, which would facilitate formation of the highly reactive hydroxyl radical from the less reactive superoxide anion. A variety of macromolecules can be damaged by activated oxygen. Unsaturated fatty acids are especially prone to attack, and this implies that membranes are primary targets of free radical damage. The manifestations of this damage in senescing tissues range from altered membrane fluidity and phase properties to leakiness that can be attributed to a destabilized and highly perturbed membrane bilayer. There is also a progressive breakdown of cellular protein with advancing senescence. Free radicals can inactivate proteins by reacting with specific amino acid residues, and a number of in zitro studies have indicated that such alteration renders the proteins more prone to hydrolysis by proteases. Thus, although there is no direct evidence linking enhanced proteolysis during senescence to free radical damage, there is reason to believe that this may be a contributing factor. Wounding of certain plant tissues also initiates a series of reactions that revolve around the breakdown of membrane lipids and their peroxidation. Indeed, as in the case of senescence, membrane deterioration follokving wounding appears to be facilitated by a self-perpetuating wave of free radical production emanating from peroxidation within the lipid bilayer. There is also recent evidence for activation of an O2 - -producing NADPH oxidase in plant tissues following fungal infection that may be analogous to the well-characterized O2 - -generating NADPH oxidase associated with the plasma membrane of polymorphonuclear leukocytes. This raises the interesting possibility that plants and animals share a common defence response to invading organisms. Contents Summary 317 I. Introduction 318 II. Species of activated oxygen 319 III. Sites of activated oxygen production 319 IV. Free radical production during senescence 323 V. Targets of free radical damage in senescing tissues 330 VI. The role of free radicals in seed ageing 336 VII. The role of free radicals in wounding 337 VIII. Concluding remarks 338 Acknowledgement 338 References 338.

Liposomes and tear fluid. I. Release of vesicle-entrapped carboxyfluorescein.

The successful application of liposomes as a topical ophthalmic drug delivery device requires knowledge of vesicle stability in the presence of tear fluid. The release of 5-carboxyfluorescein from large unilamellar liposomes in the presence of rabbit tear fluid was studied in vitro as a function of bilayer cholesterol content. Reverse evaporation vesicles were prepared from egg phosphatidylcholine, stearylamine and varying amounts of cholesterol. Both the rate and the extent of fluorescent dye release were significantly increased in the presence of rabbit tear fluid at all cholesterol levels. However, by incorporating increasing amounts of cholesterol in the vesicle bilayers, tear-induced leakage was reduced. The release kinetics reported in this study are similar to those observed in the presence of human serum. While serum-induced leakage is attributed to high-density lipoprotein-mediated destabilization, reported differences in tear protein composition suggest some other, as yet unidentified, factor.