The clinical applications of human amnion in plastic surgery.

Since the early 1900s, human amnion has been applied to a wide variety of clinical scenarios including burns, chronic ulcers, dural defects, intra-abdominal adhesions, peritoneal reconstruction, genital reconstruction, hip arthroplasty, tendon repair, nerve repair, microvascular reconstruction, corneal repair, intra-oral reconstruction and reconstruction of the nasal lining and tympanic membrane. Amnion epithelial and mesenchymal cells have been shown to contain a variety of regulatory mediators that result in the promotion of cellular proliferation, differentiation and epithelialisation and the inhibition of fibrosis, immune rejection, inflammation and bacterial invasion. The full repertoire of biological factors that these cells synthesise, store and release and the mechanisms by which these factors exert their beneficial effects are only now being fully appreciated. Although many commercially available biological and synthetic alternatives to amnion exist, ethical, religious, and financial constraints may limit the widespread utilisation of these products. Amnion is widely available, economical and is easy to manipulate, process and store. Although many clinical applications are of historical interest only, amnion offers an alternative source of multi-potent or pluripotent stem cells and therefore may yet have a great deal to offer the plastic surgery and regenerative medicine community. It is the purpose of this article to review the clinical applications of human amnion relevant to plastic surgery.

Light-activated tissue bonding for excisional wound closure: a split-lesion clinical trial.

BACKGROUND: Apposition of wound edges by sutures provides a temporary scaffold and tension support for healing. We have developed a novel tissue-sealing technology, photoactivated tissue bonding (PTB), which immediately crosslinks proteins between tissue planes, thereby sealing on a molecular scale. OBJECTIVES: To determine the effectiveness of PTB for superficial closure of skin excisions and to compare the results with standard epidermal suturing. METHODS: A split-lesion, paired comparison study of 31 skin excisions was performed. Following deep closure with absorbable sutures, one-half of each wound was superficially closed with nonabsorbable nylon sutures while the other half was stained with Rose Bengal dye and treated with green light. Overall appearance and scar characteristics were rated at 2weeks and 6months in a blinded manner by three dermatologists viewing photographs, by two onsite physicians and by patients. RESULTS: At 2weeks, neither sutured nor PTB-treated segments showed dehiscence; however, PTB-sealed segments showed less erythema than sutured segments as determined by photographic (P=0·001) and onsite evaluations (P=0·005). Overall appearance after PTB was judged better than after sutures (P=0·002). At 6months, scars produced by PTB were deemed superior to scars resulting from sutures in terms of appearance (P<0·001), width (P=0·002) and healing (P=0·003). Patients were more satisfied with the appearance of the PTB-sealed wound half after 2weeks and 6months (P=0·013 and P=0·003, respectively). CONCLUSIONS: A novel molecular suturing technique produces effective wound sealing and less scarring than closure with nylon interrupted epidermal sutures. Comparisons with better suturing techniques are warranted.

A photobiological and photophysical-based study of phototoxicity of two chlorins.

To understand the fundamental determinants of phototoxic efficacy and absorbed photodynamic dose, the triplet state and photobleaching quantum yields in living cells, cellular uptake, intracellular localization, and correlation with cell viability were studied for the two purpurins tin ethyl etiopurpurin 1 (SnET2) and tin octaethylbenzochlorin (SnOEBC) in ovarian cancer cells (OVCAR5). Although the triplet yields of these two photosensitizers were not significantly affected by cellular incorporation, the photobleaching yields were shown to be 3 orders of magnitude higher for cellular-bound sensitizer than for free or albumin-bound photosensitizer and higher for SnET2 than for SnOEBC for all of the cases. The intracellular concentration of SnOEBC was half that of SnET2 after 3 h- and 24 h-incubation times for both 0.1 microM and 1.0 microM incubation concentrations. Despite the lower concentrations of SnOEBC, the phototoxicity of the two photosensitizers was comparable at 1-microM incubation concentration and was up to 10-fold higher for SnOEBC at the lower concentration. The subcellular localization established using confocal microscopy and molecular probes showed that both photosensitizers were primarily lysosomally localized. SnOEBC, however, had an extra-lysosomal, mitochondrial localization component. The photophysical measurements allowed calculation of the intracellular singlet oxygen production, which indicated that the photosensitizer-light dose reciprocity was limited by photobleaching for SnET2 but only minimally for SnOEBC, and this was confirmed through cell-survival studies. Taken together, these data indicate that the critical determinant of differences in phototoxicity between the two molecules was their relative rates of photobleaching and their subcellular localization. The study points to the importance of combining photosensitizer uptake and localization with photophysical measurements in the relevant biological milieu to reasonably interpret and/or predict photosensitization efficacies.

Photochemical keratodesmos for repair of lamellar corneal incisions.

PURPOSE: To determine the efficacy of photochemical keratodesmos (PKD) for closing surgical incisions in the cornea of enucleated rabbit eyes compared with that achieved using sutures and self-sealing incisions. METHODS: A 3.5-mm incision, at an angle parallel to the iris, was made in the cornea of enucleated New Zealand White rabbit eyes. The intraocular pressure required to cause leakage (IOP(L)) from the untreated incision was then recorded. Photochemical keratodesmos treatment was then performed by application of a dye, Rose Bengal (RB), in saline solution to the surfaces of the incision wound, followed by laser irradiation at 514 nm from an argon ion laser. Immediately after treatment, the IOP(L) was measured. Both dose and laser irradiance dependencies were studied in five or more eyes for each condition and appropriate control eyes. The IOP(L)s were compared with those obtained using conventional interrupted 10-0 nylon sutures. Other dyes were tested in a similar fashion. RESULTS: The IOP(L) of 300 mm Hg was obtained using a fluence of 1270 J/cm(2) with an irradiance of 1.27 W/cm(2) (laser exposure time, 16 minutes 40 seconds). No sealing was observed using dye or light alone where control pressures of approximately 30 mm Hg were found. At higher dose (1524 J/cm(2)) and irradiance (3.82 W/cm(2); 6 minutes 35 seconds), PKD was less effective, which may be attributable to thermal effects. PKD produced IOP(L)s similar to those in closure by sutures. Other dyes such as riboflavin-5-phosphate and N:-hydroxy-pyridine thione also produced efficient bonding after PKD. Nonphotochemically active dyes did not produce significant increases in the IOP(L) at which leakage occurred. CONCLUSIONS: The increase in IOP(L) after PKD treatment, comparable with that with sutures, in enucleated rabbit eyes demonstrates the feasibility of this technique ex vivo.

Photosensitized production of singlet oxygen.

Photosensitization is a simple and controllable method for the generation of singlet oxygen in solution and in cells. Methods are described for determining the yield of singlet oxygen in solution, for measurement of the rate of reaction between singlet oxygen and a substrate, and for comparing the effectiveness of singlet oxygen generated by different photosensitizers in cells. These quantitative measurements can lead to better understanding of the interaction of singlet oxygen with biomolecules.

Evidence for peroxynitrite formation during S-nitrosoglutathione photolysis in air saturated solutions.

Flash photolysis of S-nitrosoglutathione (GSNO) in aerated solutions at pH 10 gave rise to an absorption with a maximum around 310-320 nm. This peak is spectrally similar to that displayed by ONOO-. The decay kinetics of this absorption was compared to that of authentic ONOO-, generated independently. An excellent correlation was obtained. Further proof of ONOO- generation was provided by HPLC studies showing the production of 3-nitrotyrosine on irradiation of GSNO in the presence of tyrosine at pH 7.4. In addition, the nitration yield was increased approximately 5-fold in the presence of bicarbonate and totally eliminated with DMPO, indicating the requirement of a radical intermediate for peroxynitrite production during S-nitrosothiol photolysis.

Can cellular phototoxicity be accurately predicted on the basis of sensitizer photophysics?

The phototoxicity of three structurally related photosensitizers (PS), deuteroporphyrin IX (DP) and monobromo (Br-DP) and dibromo (Br2-DP) derivatives, was studied in murine L1210 leukemia cells. These compounds were chosen on the basis of heavy-atom-induced differences in triplet yield, phi T, and lifetime, tau T, and used as tools to test a model for phototoxicity based on photophysical parameters. All three porphyrins were found to localize preferentially in the plasma membrane of L1210 cells by confocal fluorescence microscopy. A poor correlation was observed between the measured photodynamic efficacies of these PS and a model using photophysical parameters determined by laser flash photolysis in homogeneous solution. However, an excellent correlation was obtained when the same parameters measured directly in the cells were used. The biological microenvironment of the porphyrins in cells induces significant changes in the photophysics of the PS. Reduction in fluorescence yield, phi F, and phi T observed for Br2-DP in cell suspensions arises from self association of the molecule due to increased hydrophobicity and high local concentrations. The photophysical model was also tested for its ability to handle variations in the oxygen dependence of cellular phototoxicity of these PS. The good correlation achieved between laser flash photolysis data determined in cells and the measured phototoxicity under air, 1.5% and 0.5% O2-saturated conditions, proves the intermediacy of singlet oxygen. This study gives further credence to the direct use of photophysical techniques to elucidate photochemical mechanisms in biological media while highlighting the potential pitfalls of using solution data to predict photosensitizing potential.

Photophysical properties of tin ethyl etiopurpurin I (SnET2) and tin octaethylbenzochlorin (SnOEBC) in solution and bound to albumin.

The photophysical characteristics of two second-generation PDT photosensitizers, tin ethyl etiopurpurin I (SnET2) and tin octaethylbenzochlorin (SnOEBC), have been measured in homogeneous solution and when bound to bovine serum albumin (BSA). The ground state and triplet state absorption spectra have been characterized, as have triplet lifetimes and quantum yields for intersystem crossing, singlet oxygen formation and photobleaching. In total, these parameters provide a complete set of data that can be used to quantitatively compare the photosensitizing efficiencies of these molecules. The photo-bleaching quantum yield of SnET2 is increased dramatically when it is bound to BSA, thus limiting the production of singlet oxygen at incident fluences above 1 J/cm2. In contrast, the quantum yield of photobleaching of SnOEBC is at least an order of magnitude lower than that of SnET2 under these conditions and does not significantly limit the photosensitization process for typical in vivo or in vitro fluences. This difference is expected to play a significant role in determining the relative photo-sensitizing ability of these compounds in vivo.

Exclusive free radical mechanisms of cellular photosensitization.

In order to determine the specific effects of radical-induced reactions in the absence of complicating excited-state pathways, four different thiohydroxamic esters and their parent molecule, N-hydroxypyridine-2(1H)-thione, have been studied in murine L1210 leukemia cells for their ability to produce photobiological damage. Irradiation (lambda exc = 355 nm) of cells in the presence of thiopyridone esters, specific photolytic precursors of sulfur-, carbon- and oxygen-centered radicals, caused toxicity that was unambiguously demonstrated to result from radical photosensitization mechanisms. Cellular morphological changes were observed following irradiation but apoptosis was not found to take place. A good correlation was evident between lipid peroxidation, measured by the thiobarbituric acid method, and phototoxicity, assessed by the trypan blue exclusion assay, indicating that the ester derivatives exert their effects mainly in plasma and/or subcellular membranes. Irradiation performed under deaerated conditions also induced significant phototoxicity but the effects of deaeration were dependent on the ester used and are discussed in terms of the nature of the primary radical species generated in each case. Irradiation of L1210 cells in the presence of N-hydroxypyridine-2(1H)-thione, a nonspecific, photochemical source of hydroxyl radical, was also found to trigger phototoxicity and lipid peroxidation. However in this case, photodamage cannot yet be definitely attributed to a radical or type II mechanism although the apparent oxygen independence of phototoxicity would indicate that type II contribution is not significant.

Environmental effects on cellular photosensitization: correlation of phototoxicity mechanism with transient absorption spectroscopy measurements.

Little is directly known about the influence of the local environment experienced by a photosensitizer in a biological system on its photophysics and photochemistry. In this paper, we have addressed this issue by correlating mechanistic studies using laser flash photolysis with cellular phototoxicity data, obtained under the same experimental conditions. In particular, we have focused on the interaction between local concentrations of photosensitizer (deuteroporphyrin) and oxygen in determining the mechanism of phototoxicity in L1210 cells. In cells, as well as in models such as liposomes and red blood cell ghosts, hypochromicity and a reduction in fluorescence and intersystem crossing yields are observed on increasing the photosensitizer concentration between 0.5 and 20 microM, which illustrates the onset of a self-association. In aerated cellular preparations, the phototoxicity is predominantly type II (singlet oxygen) for all concentrations studied but an oxygen-independent mechanism occurs at the higher concentrations in deaerated samples. These observations are readily explained by consideration of triplet state kinetics as a function of oxygen and photosensitizer concentrations in cells. The rate constant for quenching of the photosensitizer triplet state by oxygen in cells was measured as 6.6 x 10(8) M-1 s-1 and by photosensitizer ground state as approximately 10(6) M-1 s-1 (in terms of local concentration). The latter reaction gave rise to a long-lived species that is presumably responsible for the oxygen-independent phototoxicity observed at the higher photosensitizer concentrations used. This self-quenching of the triplet state is postulated to arise from electron transfer resulting in radical ion formation. Under conditions where no self-quenching contributes, the phototoxicity measured as a function of oxygen concentration correlates well with a model based on the determined kinetic parameters, thus, unambiguously proving the intermediacy of singlet oxygen. These effects should be borne in mind when interpreting phototoxicity mechanisms from in vitro cell studies. The excellent correlation achieved between laser flash photolysis data and measured phototoxicity gives credence to the direct use of photophysical techniques to elucidate photochemical mechanisms in biological media.

A photo-activated, protein-based, NO/H2O2 generating system with tumoricidal activity composed of the nitric oxide derivative of apo-metallothionein (thionein-NO) and glucose oxidase.

The S-nitroso derivative of apo-metallothionein (thionein) was prepared by transnitrosation with S-nitrosoglutathione. The thionein-NO thus formed has an absorption maximum at 334 nm. Light-induced NO release from thionein-NO was demonstrated by flash photolysis. This system produces peroxynitrite at neutral pH as evidenced by nitrotyrosine formation. The cytotoxic potential of this protein-based, light-activated NO/H2O2 generating system was demonstrated by exposing human colon adenocarcinoma cells (SW 948) in culture to thionein-NO and glucose oxidase in the presence and absence of light. The cell density of the samples, 72 h subsequent to receiving 1 h of light exposure, decreased by approximately 98%, relative to controls. In comparison, cell density of the samples that were incubated in the presence of catalase and did not receive light treatment, decreased by only approximately 22% after 72 h.

The effects of aggregation, protein binding and cellular incorporation on the photophysical properties of benzoporphyrin derivative monoacid ring A (BPDMA).

Absorption, fluorescence and laser flash photolysis spectroscopies were used to investigate the effects of self-aggregation, binding to human serum albumin and incorporation in cancer cells on the photophysics of benzoporphyrin derivative monoacid ring A (BPDMA). Aggregation of BPDMA has been studied in mixtures of methanol and phosphate-buffered saline (PBS). The extent of aggregation was dependent on dye concentration and solvent composition, becoming particularly marked in mixtures containing less than 30% methanol. A dimerization constant Kd or 9 x 10(6) M-1 was determined by fluorescence experiments for BPDMA in pure PBS. In addition to spectral modifications, aggregation induces a lowering of the fluorescence and intersystem crossing quantum yields. Human serum albumin binds BPDMA with an association constant Kb of 5.2 x 10(5) M-1 in PBS. When bound to HSA, BPDMA displays photophysical properties very similar to the monomer in organic solvents. The molar ratio [HSA]/[BPDMA] corresponding to complete binding of the dye was determined to be approximately 10. Efficient generation of the triplet state of BPDMA was also observed from aqueous cellular suspensions containing incorporated photosensitizer.

Merocyanine dyes: effect of structural modifications on photophysical properties and biological activity.

Merocyanine derivatives were prepared by structural alterations at the barbituric acid or chalcogenazole moieties. The photophysical properties of the dyes were markedly influenced by the presence of selenium rather than sulfur as a substituent at position 2 of the barbiturate. In methanol, quantum yields of both triplet state (phi T) and singlet oxygen sensitization (phi delta) were increased by over an order of magnitude, with a concomitant decrease in fluorescence, when selenium was present in the molecule. Photoisomerization, one of the dominant deactivation pathways in the sulfur- or oxygen-containing analogues, was completely absent in the selenium-containing derivatives. Efficient triplet state formation was observed for selenium-containing derivatives incorporated into L1210 cells by diffuse reflectance laser flash photolysis. Cytotoxicity studies, carried out using clonogenic assays on L1210 leukemia cells, showed a good correlation with phi T and phi delta, measured in solution. Experimental evidence provided by this paper supports a triplet state-, and probably singlet oxygen-, mediated phototoxic mechanism. Photoisomerization or singlet state mechanisms can be discounted.

Photophysical and photosensitizing properties of benzoporphyrin derivative monoacid ring A (BPD-MA).

The photophysical properties of benzoporphyrin derivative monoacid ring A (BPD-MA), a second-generation photosensitizer currently in phase II clinical trials, were investigated in homogeneous solution. Absorption, fluorescence, triplet-state, singlet oxygen (O2 (1 delta g)) sensitization studies and photobleaching experiments are reported. The ground state of this chlorin-type molecule shows a strong absorbance in the red (lambda approximately 688 nm, epsilon approximately 33,000 M-1 cm-1 in organic solvents). For the singlet excited state the following data were determined in methanol: energy level, Es = 42.1 kcal mol-1, lifetime, tau f = 5.2 ns and fluorescence quantum yield, phi f = 0.05 in air-saturated solution. The triplet state of BPD-MA has a lifetime, tau T > or = 25 microseconds, an energy level, ET = 26.9 kcal mol-1 and the molar absorption coefficient is epsilon T = 26,650 M-1 cm-1 at 720 nm. A dramatic effect of oxygen on the fluorescence (phi f) and intersystem crossing (phi T) quantum yields has been observed. The BPD-MA presents rather high triplet (phi T = 0.68 under N2-saturated conditions) and singlet oxygen (phi delta = 0.78) quantum yields. On the other hand, the presence of oxygen does not significantly modify the photobleaching of this photostable compound, the photodegradation quantum yield (phi Pb) of which was found to be on the order of 5 x 10(-5) in organic solvents.

Effects of structural modifications on the photosensitizing properties of dialkylcarbocyanine dyes in homogeneous and heterogeneous solutions.

The photophysical characterization of structurally modified symmetric dialkylthiacarbocyanine dyes in homogeneous and biomimetic media is reported. The aim of the two specific structural modifications was to increase singlet oxygen production, hence enhancing the photosensitizing properties of these cyanine dyes. Specifically, (1) the sulfur was exchanged with selenium in order to enhance intersystem crossing via an internal heavy atom effect and (2) substituents of differing size were introduced into the meso-position of the polymethine chain to reduce photoisomerization. The result of incorporation of an internal heavy atom (selenium) into the structure of the dye yields the expected effect: this modification results in a 22-fold increase in the rate of intersystem crossing, but does not change the remaining competing deactivation rates of the first excited singlet state. As a consequence, singlet oxygen quantum yields increase from 0.001 to 0.014 in ethanol and from 0.006 to 0.08 in unilamellar liposomes. In the case of the meso-substituted thiacarbocyanine dyes, a significant reduction in photoisomerization is indeed observed. However, this modification drastically enhances internal conversion which then becomes the main deactivation pathway of the first excited singlet state. As a result, very small fluorescence and singlet oxygen quantum yields are obtained, e.g. 0.006 and 0.001, respectively, in ethanol.

An efficient oxygen independent two-photon photosensitization mechanism.

A novel oxygen-independent photosensitization mechanism from the upper triplet state (Tn) of rose bengal has been demonstrated by selectively populating Tn by sequential two-color laser excitation. Products formed from Tn inhibit red blood cell acetylcholinesterase and decrease viability of P388D1 mouse macrophage monocyte cells as measured by trypan blue exclusion assay. Laser flash photolysis studies indicate that Tn reacts efficiently, as evidenced by permanent photobleaching of T1 absorption, with chemical yields approaching unit efficiency. This mechanism may have application for oxygen deficient photosensitization under high intensity, pulsed laser irradiation.

Photophysical properties of 3,3'-dialkylthiacarbocyanine dyes in organized media: unilamellar liposomes and thin polymer films.

All symmetrical dialkylthiacarbocyanine dyes, with the exception of the diethyl derivatives, are incorporated into liposomes. Absorption and fluorescence data indicate a solubilization site close to the bilayer surface with the alkyl chains penetrating into the lipid bilayer. Incorporation into organized assemblies affects the photophysical parameters of these dyes. Photoisomerization occurring from the first excited state becomes more difficult as the restrictive effect of the solubilization site increases. As a consequence, competing deactivation processes, such as fluorescence and triplet formation, become more efficient with the result that fluorescence quantum yields, triplet yields and singlet oxygen quantum yields are larger in liposomes than in homogeneous solution. Dihexylthiacarbocyanine iodide has a fluorescence quantum yield of 0.27 and 0.10 (25 degrees C) in dimyristoylphosphatidyl-choline liposomes and ethanol, respectively, and the singlet oxygen yield increases by a factor three to 0.006 on going from ethanol to liposomes. The effect of a highly organized environment is even more pronounced in thin polymer films. In these systems, photoisomerization is completely inhibited and only triplet formation is observed in the transient absorption spectrum.

Photophysical properties of 3,3'-dialkylthiacarbocyanine dyes in homogeneous solution.

The photophysical properties of 3,3'-dialkylthiacarbocyanine iodides and chlorides were measured in various solvents. It was found that photoisomerization and fluorescence are the major contributors to the deactivation of the excited singlet state; intersystem crossing occurs with only a very low efficiency. In ethanol, a triplet yield of 0.004 and a singlet oxygen quantum yield of 0.002 were determined. The photophysical parameters of these dyes are not substantially influenced by the length of the alkyl chain or the size of the halide counterion. The substitution of an ethyl with an octadecyl-chain only slightly hinders photoisomerization, and the replacement of the chloride with an iodide reduces only marginally the fluorescence lifetimes and fluorescence quantum yields in chloroform. A significant external heavy-atom effect is observed using dibromoethane as a solvent: triplet and singlet oxygen yields increase 7-10-fold, and the triplet lifetime decreases from 55 microseconds to 15 microseconds.

Phototoxic and photochemical properties of sanguinarine.

Sanguinarine, a commercial drug exhibiting antimicrobial and antitumor properties, was studied with respect to its basic photochemical characteristics and also with regard to its phototoxicity to mosquito larvae (Aedes atropalpus). Sanguinarine proved to be clearly phototoxic to larvae, with an LD50 of 0.096 mg/mL with near UV exposure as compared with 23.3 mg/mL without. Flash photolysis experiments enabled the study of the triplet state of sanguinarine to be undertaken. Quenching by oxygen occurs with a rate constant of 6 x 10(9) M-1s-1 and time-resolved emission studies indicate that sanguinarine produces a significant amount of singlet oxygen (phi delta = 0.16) as does the isoquinoline alkaloid, berberine (phi delta = 0.25). These values represent the first direct quantitative measurements of photosensitization parameters of these compounds. Additionally, sanguinarine exhibits efficient electron donation properties, undergoing reaction with methyl viologen with a rate constant greater than 10(10) M-1s-1, but is a poor electron acceptor. Phototoxicity of sanguinarine can thus be explained in terms of its photosensitization properties.