Integration of Cyanine, Merocyanine and Styryl Dye Motifs with Synthetic Bacteriochlorins.

Understanding the effects of substituents on spectral properties is essential for the rational design of tailored bacteriochlorins for light-harvesting and other applications. Toward this goal, three new bacteriochlorins containing previously unexplored conjugating substituents have been prepared and characterized. The conjugating substituents include two positively charged species, 2-(N-ethyl 2-quinolinium)vinyl- (B-1) and 2-(N-ethyl 4-pyridinium)vinyl- (B-2), and a neutral group, acroleinyl- (B-3); the charged species resemble cyanine (or styryl) dye motifs whereas the neutral unit resembles a merocyanine dye motif. The three bacteriochlorins are examined by static and time-resolved absorption and emission spectroscopy and density functional theoretical calculations. B-1 and B-2 have Qy absorption bathochromically shifted well into the NIR region (822 and 852 nm), farther than B-3 (793 nm) and other 3,13-disubstituted bacteriochlorins studied previously. B-1 and B-2 have broad Qy absorption and fluorescence features with large peak separation (Stokes shift), low fluorescence yields, and shortened S1 (Qy ) excited-state lifetimes (~700 ps and ~100 ps). More typical spectra and S1 lifetime (~2.3 ns) are found for B-3. The combined photophysical and molecular-orbital characteristics suggest the altered spectra and enhanced nonradiative S1 decay of B-1 and B-2 derive from excited-state configurations in which electron density is shifted between the macrocycle and the substituents.

Extending the short and long wavelength limits of bacteriochlorin near-infrared absorption via dioxo- and bisimide-functionalization.

Six new bacteriochlorins expanding the range of the strong near-infrared (NIR) absorption (Qy band) to both shorter and longer wavelengths (∼690 to ∼900 nm) have been synthesized and characterized. The architectures include bacteriochlorin-bisimides that have six-membered imide rings spanning the 3,5- and 13,15-macrocycle positions or five-membered imide rings spanning the ß-pyrrolic 2,3- and 12,13-positions. Both bisimide types absorb at significantly longer wavelength than the bacteriochlorin precursors (no fused rings), whereas oxo-groups at the 7- or 7,17-positions shift the Qy band to a new short wavelength limit. Surprisingly, bacteriochlorin-bisimides with five-membered ß-pyrrolic-centered imide rings have a Qy band closer to that of six-membered bacteriochlorin-monoimides. However, the five-membered bisimides (versus the six-membered bacteriochlorin-monoimides) have significantly enhanced absorption intensity that is paralleled by an ∼2-fold higher fluorescence yield (∼0.16 vs ∼0.07) and longer singlet excited-state lifetime (∼4 ns vs ∼2 ns). The photophysical enhancements derive in part from mixing of the lowest unoccupied frontier molecular orbitals of the five-membered imide ring with those of the bacteriochlorin framework. In general, all of the new bacteriochlorins have excited-state lifetimes (1-4 ns) that are sufficiently long for use in molecular-based systems for photochemical applications.

Photophysical Properties and Electronic Structure of Chlorin-Imides: Bridging the Gap between Chlorins and Bacteriochlorins.

Efficient light harvesting for molecular-based solar-conversion systems requires absorbers that span the photon-rich red and near-infrared (NIR) regions of the solar spectrum. Reported herein are the photophysical properties of a set of six chlorin-imides and nine synthetic chlorin analogues that extend the absorption deeper (624-714 nm) into these key spectral regions. These absorbers help bridge the gap between typical chlorins and bacteriochlorins. The new compounds have high fluorescence quantum yields (0.15-0.34) and long singlet excited-state lifetimes (4.2-10.9 ns). The bathochromic shift in Qy absorption is driven by substituent-based stabilization of the lowest unoccupied molecular orbital, with the largest shifts for chlorins that bear an electron-withdrawing, conjugative group at the 3-position in combination with a 13,15-imide ring.

Probing electronic communication for efficient light-harvesting functionality: dyads containing a common perylene and a porphyrin, chlorin, or bacteriochlorin.

The synthesis, photophysical, redox, and molecular-orbital characteristics of three perylene-tetrapyrrole dyads were investigated to probe the efficacy of the arrays for use as light-harvesting constituents. Each dyad contains a common perylene-monoimide that is linked at the N-imide position via an arylethynyl group to the meso-position of the tetrapyrrole. The tetrapyrroles include a porphyrin, chlorin, and bacteriochlorin, which have zero, one, and two reduced pyrrole rings, respectively. The increased pyrrole-ring reduction results in a progressive red shift and intensification of the lowest-energy absorption band, as exemplified by benchmark monomers. The arylethyne linkage affords moderate perylene-tetrapyrrole electronic coupling in the dyads as evidenced by the optical, molecular-orbital, and redox properties of the components of the dyads versus the constituent parts. All three dyads in nonpolar solvents exhibit relatively fast (subpicosecond) energy transfer from the perylene to the tetrapyrrole. Competing charge-transfer processes are also absent in nonpolar solvents, but become active for both the chlorin and bacteriochlorin-containing dyads in polar solvents. Calculations of energy-transfer rates via the Förster, through-space mechanism reveal that these rates are, on average, 3-fold slower than the observed rates. Thus, the Dexter through-bond mechanism contributes more substantially than the through-space mechanism to energy transfer in the dyads. The electronic communication between the perylene and tetrapyrrole falls in a regime intermediate between those operative in other classes of perylene-tetrapyrrole dyads that have previously been studied.

Distinct photophysical and electronic characteristics of strongly coupled dyads containing a perylene accessory pigment and a porphyrin, chlorin, or bacteriochlorin.

The synthesis, photophysical, redox, and molecular-orbital characteristics of three perylene-tetrapyrrole dyads were investigated to elucidate characteristics favorable for use in next-generation light-harvesting assemblies. Each dyad contains a common perylene-monoimide that is linked at the 9-position via an ethynyl group to the meso-position of the tetrapyrrole. The tetrapyrroles include a porphyrin, chlorin, and bacteriochlorin, which have zero, one, and two reduced pyrrole rings, respectively. The increased pyrrole-ring reduction results in a progressive red shift and intensification of the lowest-energy absorption band, as exemplified by benchmark monomers. The direct ethyne linkage and accompanying strong perylene-tetrapyrrole electronic coupling in the dyads is evident by significant differences in optical absorption versus the sum of the features of the constituents. The perturbations decrease for the tetrapyrrole constituent along the series porphyrin > chlorin > bacteriochlorin. This trend is explained by the relative configurational mixing in the tetrapyrrole excited states and how the configuration-interaction energy (and not simply the energies of the configurations) is affected by coupling to the perylene. The perylene-tetrapyrrole electronic coupling is further evidenced in the redox and MO characteristics of the three dyads. All three dyads in nonpolar solvents exhibit relatively long singlet excited-state lifetimes (3.3-6.5 ns) and relatively large fluorescence quantum yields (0.14-0.40). Collectively, the physicochemical characteristics of the strongly coupled perylene-tetrapyrrole dyads render these architectures excellent candidates for light-harvesting materials with significant, even panchromatic, near-ultraviolet to near-infrared absorption.

Photophysical properties and electronic structure of bacteriochlorin-chalcones with extended near-infrared absorption.

Synthetic bacteriochlorins enable systematic tailoring of substituents about the bacteriochlorin chromophore and thereby provide insights concerning the native bacteriochlorophylls of bacterial photosynthesis. Nine free-base bacteriochlorins (eight prepared previously and one prepared here) have been examined that bear diverse substituents at the 13- or 3,13-positions. The substituents include chalcone (3-phenylprop-2-en-1-onyl) derivatives with groups attached to the phenyl moiety, a "reverse chalcone" (3-phenyl-3-oxo-1-enyl), and extended chalcones (5-phenylpenta-2,4-dien-1-onyl, retinylidenonyl). The spectral and photophysical properties (τs, Φf, Φ(ic), Φ(isc), τT, k(f), k(ic), k(isc)) of the bacteriochlorins have been characterized. The bacteriochlorins absorb strongly in the 780-800 nm region and have fluorescence quantum yields (Φf) in the range 0.05-0.11 in toluene and dimethylsulfoxide. Light-induced electron promotions between orbitals with predominantly substituent or macrocycle character or both may give rise to some net macrocycle ↔ substituent charge-transfer character in the lowest and higher singlet excited states as indicated by density functional theory (DFT) and time-dependent DFT calculations. Such calculations indicated significant participation of molecular orbitals beyond those (HOMO - 1 to LUMO + 1) in the Gouterman four-orbital model. Taken together, the studies provide insight into the fundamental properties of bacteriochlorins and illustrate designs for tuning the spectral and photophysical features of these near-infrared-absorbing tetrapyrrole chromophores.

Molecular electronic tuning of photosensitizers to enhance photodynamic therapy: synthetic dicyanobacteriochlorins as a case study.

Photophysical, photostability, electrochemical and molecular-orbital characteristics are analyzed for a set of stable dicyanobacteriochlorins that are promising photosensitizers for photodynamic therapy (PDT). The bacteriochlorins are the parent compound (BC), dicyano derivative (NC)2BC and corresponding zinc (NC)2BC-Zn and palladium chelate (NC)2BC-Pd. The order of PDT activity against HeLa human cancer cells in vitro is (NC)2BC-Pd > (NC)2BC > (NC)2BC-Zn ≈ BC. The near-infrared absorption feature of each dicyanobacteriochlorin is bathochromically shifted 35-50 nm (748-763 nm) from that for BC (713 nm). Intersystem crossing to the PDT-active triplet excited state is essentially quantitative for (NC)2BC-Pd. Phosphorescence from (NC)2BC-Pd occurs at 1122 nm (1.1 eV). This value and the measured ground-state redox potentials fix the triplet excited-state redox properties, which underpin PDT activity via Type-1 (electron transfer) pathways. A perhaps counterintuitive (but readily explicable) result is that of the three dicyanobacteriochlorins, the photosensitizer with the shortest triplet lifetime (7 µs), (NC)2BC-Pd has the highest activity. Photostabilities of the dicyanobacteriochlorins and other bacteriochlorins studied recently are investigated and discussed in terms of four phenomena: aggregation, reduction, oxidation and chemical reaction. Collectively, the results and analysis provide fundamental insights concerning the molecular design of PDT agents.

Stable synthetic bacteriochlorins for photodynamic therapy: role of dicyano peripheral groups, central metal substitution (2H, Zn, Pd), and Cremophor EL delivery.

A series of four stable synthetic bacteriochlorins was tested in vitro in HeLa cells for their potential in photodynamic therapy (PDT). The parent bacteriochlorin (BC), dicyano derivative (NC)(2)BC and corresponding zinc chelate (NC)(2)BC-Zn and palladium chelate (NC)(2)BC-Pd were studied. Direct dilution of a solution of bacteriochlorin in an organic solvent (N,N-dimethylacetamide) into serum-containing medium was compared with the dilution of bacteriochlorin in Cremophor EL (CrEL; polyoxyethylene glycerol triricinoleate) micelles into the same medium. CrEL generally reduced aggregation (as indicated by absorption and fluorescence) and increased activity up to tenfold (depending on bacteriochlorin), although it decreased cellular uptake. The order of PDT activity against HeLa human cancer cells after 24 h incubation and illumination with 10 J cm(-2) of near-infrared (NIR) light is (NC)(2)BC-Pd (LD(50)=25 nM) > (NC)(2)BC > (NC)(2)BC-Zn ≈ BC. Subcellular localization was determined to be in the endoplasmic reticulum, mitochondria and lysosomes, depending on the bacteriochlorin. (NC)(2)BC-Pd showed PDT-mediated damage to mitochondria and lysosomes, and the greatest production of hydroxyl radicals as determined using a hydroxyphenylfluorescein probe. The incorporation of cyano substituents provides an excellent motif for the enhancement of the photoactivity and photostability of bacteriochlorins as PDT photosensitizers.

Synthesis and physicochemical properties of metallobacteriochlorins.

Access to metallobacteriochlorins is essential for investigation of a wide variety of fundamental photochemical processes, yet relatively few synthetic metallobacteriochlorins have been prepared. Members of a set of synthetic bacteriochlorins bearing 0-4 carbonyl groups (1, 2, or 4 carboethoxy substituents, or an annulated imide moiety) were examined under two conditions: (i) standard conditions for zincation of porphyrins [Zn(OAc)(2)·2H(2)O in N,N-dimethylformamide (DMF) at 60-80 °C], and (ii) treatment in tetrahydrofuran (THF) with a strong base [e.g., NaH or lithium diisopropylamide (LDA)] followed by a metal reagent MX(n). Zincation of bacteriochlorins that bear 2-4 carbonyl groups proceeded under the former method whereas those with 0-2 carbonyl groups proceeded with NaH or LDA/THF followed by Zn(OTf)(2). The scope of metalation (via NaH or LDA in THF) is as follows: (a) for bacteriochlorins that bear two electron-releasing aryl groups, M = Cu, Zn, Pd, and InCl (but not Mg, Al, Ni, Sn, or Au); (b) for bacteriochlorins that bear two carboethoxy groups, M = Ni, Cu, Zn, Pd, Cd, InCl, and Sn (but not Mg, Al, or Au); and (c) a bacteriochlorin with four carboethoxy groups was metalated with Mg (other metals were not examined). Altogether, 15 metallobacteriochlorins were isolated and characterized. Single-crystal X-ray analysis of 8,8,18,18-tetramethylbacteriochlorin reveals the core geometry provided by the four nitrogen atoms is rectangular; the difference in length of the two sides is ∼0.08 Å. Electronic characteristics of (metal-free) bacteriochlorins were probed through electrochemical measurements along with density functional theory calculation of the energies of the frontier molecular orbitals. The photophysical properties (fluorescence yields, triplet yields, singlet and triplet excited-state lifetimes) of the zinc bacteriochlorins are generally similar to those of the metal-free analogues, and to those of the native chromophores bacteriochlorophyll a and bacteriopheophytin a. The availability of diverse metallobacteriochlorins should prove useful in a variety of fundamental photochemical studies and applications.

Photophysical properties and electronic structure of stable, tunable synthetic bacteriochlorins: extending the features of native photosynthetic pigments.

Bacteriochlorins, which are tetrapyrrole macrocycles with two reduced pyrrole rings, are Nature's near-infrared (NIR) absorbers (700-900 nm). The strong absorption in the NIR region renders bacteriochlorins excellent candidates for a variety of applications including solar light harvesting, flow cytometry, molecular imaging, and photodynamic therapy. Natural bacteriochlorins are inherently unstable due to oxidative conversion to the chlorin (one reduced pyrrole ring) or the porphyrin. The natural pigments are also only modestly amenable to synthetic manipulation, owing to a nearly full complement of substituents on the macrocycle. Recently, a new synthetic methodology has afforded access to stable synthetic bacteriochlorins wherein a wide variety of substituents can be appended to the macrocycle at preselected locations. Herein, the spectroscopic and photophysical properties of 33 synthetic bacteriochlorins are investigated. The NIR absorption bands of the chromophores range from ∼700 to ∼820 nm; the lifetimes of the lowest excited singlet state range from ∼2 to ∼6 ns; the fluorescence quantum yields range from ∼0.05 to ∼0.25; and the yield of the lowest triplet excited state is ∼0.5. The spectroscopic/photophysical studies of the bacteriochlorins are accompanied by density functional theory (DFT) calculations that probe the characteristics of the frontier molecular orbitals. The DFT calculations indicate that the impact of substituents on the spectral properties of the molecules derives primarily from effects on the lowest unoccupied molecular orbital. Collectively, the studies show how the palette of synthetic bacteriochlorins extends the properties of the native photosynthetic pigments (bacteriochlorophylls). The studies have also elucidated design principles for tuning the spectral and photophysical characteristics as required for a wide variety of photochemical applications.

Synthesis and photophysical characterization of stable indium bacteriochlorins.

Bacteriochlorins have wide potential in photochemistry because of their strong absorption of near-infrared light, yet metallobacteriochlorins traditionally have been accessed with difficulty. Established acid-catalysis conditions [BF(3)·OEt(2) in CH(3)CN or TMSOTf/2,6-di-tert-butylpyridine in CH(2)Cl(2)] for the self-condensation of dihydrodipyrrin-acetals (bearing a geminal dimethyl group in the pyrroline ring) afford stable free base bacteriochlorins. Here, InBr(3) in CH(3)CN at room temperature was found to give directly the corresponding indium bacteriochlorin. Application of the new acid catalysis conditions has afforded four indium bacteriochlorins bearing aryl, alkyl/ester, or no substituents at the ß-pyrrolic positions. The indium bacteriochlorins exhibit (i) a long-wavelength absorption band in the 741-782 nm range, which is shifted bathochromically by 22-32 nm versus the analogous free base species, (ii) fluorescence quantum yields (0.011-0.026) and average singlet lifetime (270 ps) diminished by an order of magnitude versus that (0.13-0.25; 4.0 ns) for the free base analogues, and (iii) higher average yield (0.9 versus 0.5) yet shorter average lifetime (30 vs 105 µs) of the lowest triplet excited state compared to the free base compounds. The differences in the excited-state properties of the indium chelates versus free base bacteriochlorins derive primarily from a 30-fold greater rate constant for S(1) → T(1) intersystem crossing, which stems from the heavy-atom effect on spin-orbit coupling. The trends in optical properties of the indium bacteriochlorins versus free base analogues, and the effects of 5-OMe versus 5-H substituents, correlate well with frontier molecular-orbital energies and energy gaps derived from density functional theory calculations. Collectively the synthesis, photophysical properties, and electronic characteristics of the indium bacteriochlorins and free base analogues reported herein should aid in the further design of such chromophores for diverse applications.

Stable synthetic cationic bacteriochlorins as selective antimicrobial photosensitizers.

Photodynamic inactivation is a rapidly developing antimicrobial treatment that employs a nontoxic photoactivatable dye or photosensitizer in combination with harmless visible light to generate reactive oxygen species that are toxic to cells. Tetrapyrroles (e.g., porphyrins, chlorins, bacteriochlorins) are a class of photosensitizers that exhibit promising characteristics to serve as broad-spectrum antimicrobials. In order to bind to and efficiently penetrate into all classes of microbial cells, tetrapyrroles should have structures that contain (i) one or more cationic charge(s) or (ii) a basic group. In this report, we investigate the use of new stable synthetic bacteriochlorins that have a strong absorption band in the range 720 to 740 nm, which is in the near-infrared spectral region. Four bacteriochlorins with 2, 4, or 6 quaternized ammonium groups or 2 basic amine groups were compared for light-mediated killing against a gram-positive bacterium (Staphylococcus aureus), a gram-negative bacterium (Escherichia coli), and a dimorphic fungal yeast (Candida albicans). Selectivity was assessed by determining phototoxicity against human HeLa cancer cells under the same conditions. All four compounds were highly active (6 logs of killing at 1 microM or less) against S. aureus and showed selectivity for bacteria over human cells. Increasing the cationic charge increased activity against E. coli. Only the compound with basic groups was highly active against C. albicans. Supporting photochemical and theoretical characterization studies indicate that (i) the four bacteriochlorins have comparable photophysical features in homogeneous solution and (ii) the anticipated redox characteristics do not correlate with cell-killing ability. These results support the interpretation that the disparate biological activities observed stem from cellular binding and localization effects rather than intrinsic electronic properties. These findings further establish cationic bacteriochlorins as extremely active and selective near-infrared activated antimicrobial photosensitizers, and the results provide fundamental information on structure-activity relationships for antimicrobial photosensitizers.

In vitro photodynamic therapy and quantitative structure-activity relationship studies with stable synthetic near-infrared-absorbing bacteriochlorin photosensitizers.

Photodynamic therapy (PDT) is a rapidly developing approach to treating cancer that combines harmless visible and near-infrared light with a nontoxic photoactivatable dye, which upon encounter with molecular oxygen generates the reactive oxygen species that are toxic to cancer cells. Bacteriochlorins are tetrapyrrole compounds with two reduced pyrrole rings in the macrocycle. These molecules are characterized by strong absorption features from 700 to >800 nm, which enable deep penetration into tissue. This report describes testing of 12 new stable synthetic bacteriochlorins for PDT activity. The 12 compounds possess a variety of peripheral substituents and are very potent in killing cancer cells in vitro after illumination. Quantitative structure-activity relationships were derived, and subcellular localization was determined. The most active compounds have both low dark toxicity and high phototoxicity. This combination together with near-infrared absorption gives these bacteriochlorins great potential as photosensitizers for treatment of cancer.

Stable synthetic bacteriochlorins overcome the resistance of melanoma to photodynamic therapy.

Cutaneous malignant melanoma remains a therapeutic challenge, and patients with advanced disease have limited survival. Photodynamic therapy (PDT) has been successfully used to treat many malignancies, and it may show promise as an antimelanoma modality. However, high melanin levels in melanomas can adversely affect PDT effectiveness. Herein the extent of melanin contribution to melanoma resistance to PDT was investigated in a set of melanoma cell lines that markedly differ in the levels of pigmentation; 3 new bacteriochlorins successfully overcame the resistance. Cell killing studies determined that bacteriochlorins are superior at (LD(50) approximately 0.1 microM) when compared with controls such as the FDA-approved Photofrin (LD(50) approximately 10 microM) and clinically tested LuTex (LD(50) approximately 1 microM). The melanin content affects PDT effectiveness, but the degree of reduction is significantly lower for bacteriochlorins than for Photofrin. Microscopy reveals that the least effective bacteriochlorin localizes predominantly in lysosomes, while the most effective one preferentially accumulates in mitochondria. Interestingly all bacteriochlorins accumulate in melanosomes, and subsequent illumination leads to melanosomal damage shown by electron microscopy. Fluorescent probes show that the most effective bacteriochlorin produces significantly higher levels of hydroxyl radicals, and this is consistent with the redox properties suggested by molecular-orbital calculations. The best in vitro performing bacteriochlorin was tested in vivo in a mouse melanoma model using spectrally resolved fluorescence imaging and provided significant survival advantage with 20% of cures (P<0.01).

Excited-state photodynamics of perylene-porphyrin dyads. 5. Tuning light-harvesting characteristics via perylene substituents, connection motif, and three-dimensional architecture.

Seven perylene-porphyrin dyads were examined with the goal of identifying those most suitable for components of light-harvesting systems. The ideal dyad should exhibit strong absorption by the perylene in the green, undergo rapid and efficient excited-state energy transfer from perylene to porphyrin, and avoid electron-transfer quenching of the porphyrin excited state by the perylene in the medium of interest. Four dyads have different perylenes at the p-position of the meso-aryl group on the zinc porphyrin. The most suitable perylene identified in that set was then incorporated at the m- or o-position of the zinc porphyrin, affording two other dyads. An analogue of the o-substituted architecture was prepared in which the zinc porphyrin was replaced with the free base porphyrin. The perylene in each dyad is a monoimide derivative; the perylenes differ in attachment of the linker (either via a diphenylethyne linker at the N-imide or an ethynylphenyl linker at the C9 position) and the number (0-3) of 4-tert-butylphenoxy groups (which increase solubility and slightly alter the electrochemical potentials). In the p-linked dyad, the monophenoxy perylene with an N-imide diphenylethyne linker is superior in providing rapid and essentially quantitative energy transfer from excited perylene to zinc porphyrin with minimal electron-transfer quenching in both toluene and benzonitrile. The dyads with the same perylene at the m- or o-position exhibited similar results except for one case, the o-linked dyad bearing the zinc porphyrin in benzonitrile, where significant excited-state quenching is observed; this phenomenon is facilitated by close spatial approach of the perylene and porphyrin and the associated thermodynamic/kinetic enhancement of the electron-transfer process. Such quenching does not occur with the free base porphyrin because electron transfer is thermodynamically unfavorable even in the polar medium. The p-linked dyad containing a zinc porphyrin attached to a bis(4-tert-butylphenoxy)perylene via an ethynylphenyl linker at the C9 position exhibits ultrafast and quantitative energy transfer in toluene; the same dyad in benzonitrile exhibits ultrafast (<0.5 ps) perylene-to-porphyrin energy transfer, rapid (∼5 ps) porphyrin-to-perylene electron transfer, and fast (∼25 ps) charge recombination to the ground state. Collectively, this study has identified suitable perylene-porphyrin constructs for use in light-harvesting applications.

Effects of amniotic membrane suspension in human corneal wound healing in vitro.

PURPOSE: To investigate the biochemical mechanism of amniotic membrane (AM) suspension on corneal wound healing, particularly on epithelial proliferation and migration. METHODS: Human corneal epithelial cells (HCECs) were cultured in media with different concentrations of AM suspension (5% and 30%), Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12 (negative control), and serum containing Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12 (positive control). In an effort to evaluate the migratory potential of AM, migration assays were conducted via the manual scraping of HCECs and immunocytochemical staining of cell adhesion molecules (E-cadherin). The relative expression of matrix metallopeptidase 9 (MMP9) and adhesion molecules (E-cadherin, fibronectin) was determined via reverse transcription-polymerase chain reaction (RT-PCR) and western blot analysis. The proliferative potential of AM was evaluated via a proliferation assay using 5-Bromo-2-deoxyuridine (BrdU) incorporation and western blot analysis for proliferating cell nuclear antigen (PCNA). In addition, enzyme-linked immunosorbent assay (ELISA) was used to measure the protein concentrations of mitogenic growth factors (epidermal growth factor [EGF],keratinocyte growth factor [KGF], hepatocyte growth factor [HGF], and basic fibroblast growth factor [bFGF]) in AM suspensions. RESULTS: Migration assay rates were enhanced as AM concentrations increased, with statistically significant changes seen in 30% AM-treated and positive control cells, compared to negative control cells (p<0.05). RT-PCRs revealed that the expression of the MMP9 gene was upregulated by AM, and the expressions of E-cadherin and fibronectin genes were downregulated by AM. Western blot analysis demonstrated significantly higher MMP9 expression in AM-treated groups, versus significantly lower levels of E-cadherin and fibronectin expression in AM-treated groups. Immunocytochemistry showed large quantities of E-cadherin near the wound edges after 24 h of injury in the AM-treated groups. The proliferation assay showed that the BrdU positive cell counts/total cell counts (labeling index) were augmented by AM to a statistically significant degree (p<0.05 in the 30% AM and positive control groups). Western blot analysis showed that the expression cell cycle-associated protein, PCNA, increased gradually as a result of AM treatment. ELISA showed that our AM suspension contained 4 growth factors (HGF, EGF, KGF, and FGF). The amount of HGF was especially large, followed by that of EGF. CONCLUSIONS: These results demonstrate that the suspension form of AM maintains its beneficial effect on corneal epithelial wound healing in vitro, and that AM suspension leads to significant increases in corneal epithelial migration and proliferation with increasing AM concentrations.

Reconstruction of rabbit corneal epithelium on lyophilized amniotic membrane using the tilting dynamic culture method.

Rabbit corneal epithelium was reconstructed using tilting dynamic culture with a self-manufactured, amniotic membrane (AM) supporter and a lyophilized amniotic membrane (LAM). Rabbit corneal epithelial (RCE) cells were cultured and cryopreserved after isolation from the limbus. The second- and third-passage RCE cells were plated onto the epithelial side of the LAM of Ahn's AM supporter. Two days later, the air-liquid interface culture was maintained with third-passage RCE cells for 6 days and second-passage corneal epithelial cells for 9 days. The average viability of thawed RCE cells, assessed using trypan blue dye exclusion, was 77.42%. The reconstructed corneal epithelium was characterized by histological (hematoxylin and eosin) and immunohistochemical staining (proliferating cell nuclear antigen) for light microscopy, and by reverse transcriptase-polymerase chain reaction, glucose assay, and transmission electron microscopy. The basal layer of the reconstructed corneal epithelium was well formed, and the epithelium was tightly constructed due to the increase in cell proliferation and differentiation caused by the tilting dynamic culture, as opposed to static culture. Tilting dynamic culture was useful for the reconstruction of the epithelium using easily damaged epithelial cells and resulted in more stratum cell layers. Moreover, cytokeratin (CK3) mRNA expression in tilting dynamic cultured third-passage RCE cells seeded onto AM was greater than in static cultured third-passage RCE cells. The morphology of the reconstructed corneal epithelium on LAM by tilting dynamic culture for 9 days resembled that of the skin epidermis. This was thought to be because the tilting dynamic culture not only accelerated the proliferation and differentiation of cells by physical or mechanical stimulation, but also ensured that the supply of medium was delivered to the basal cells more efficiently. Thus, the reconstruction of the corneal epithelium using LAM and tilting dynamic culture was considered to be a good in vitro model for autologous or allogeneic transplantation of corneal epithelium and skin epidermis in patients with damaged epithelia.

Fabrication of myomucosal flap using tissue-engineered bioartificial mucosa constructed with oral keratinocytes cultured on amniotic membrane.

The purpose of this study was to fabricate bioartificial mucosa using cultured oral keratinocytes (OKCs) on an amniotic membrane (AM), and to evaluate the possibility of developing a prelaminated myomucosal flap using the fabricated bioartificial mucosa and local muscle flap. Buccal mucosa was harvested from male New Zealand rabbits (n = 40, 2.5-3.0 kg) and primary cultivation was performed. The cultured OKCs were seeded on the AM and a submerged culture was performed. Prelamination of the bioartificial mucosa was performed on the latissimus dorsi (LD) muscle of rabbits. Survival rate, layer of OKCs, and Cinamon's score (CS) based on macroscopic and microscopic examinations were evaluated 7, 10, 14, and 21 days after prelamination (n = 10 per day). The OKCs cultured on AM showed multiple layers (3.85 +/- 1.32) and cells were tightly adhered with desmosomes. Basal layer cells adhered to the AM with hemidesmosomes. In addition, the AM played an excellent role as a substrate for the OKCs and simplified handling during prelamination. A myomucosal flap with OKCs cultured on AM was fabricated within 2 weeks (CS: 11.05 +/- 2.63). The basement component of laminin was observed 2 weeks after prelamination and showed enough strength to adhere to the underlying fascia. A myomucosal flap was successfully developed using prelamination of bioartificial mucosa on the LD muscle between 10 and 14 days.

Transplantation of reconstructed corneal layer composed of corneal epithelium and fibroblasts on a lyophilized amniotic membrane to severely alkali-burned cornea.

The purpose of this article was to evaluate the graft efficacy of reconstructed corneal layer, composed of autologous corneal epithelium and fibroblasts on a lyophilized amniotic membrane (LAM), in a severely alkali-burned corneal model. After biopsy specimens were obtained from the left eyes of 24 rabbits, the corneal epithelial cells and fibroblasts were expanded in vitro and the corneal layer was reconstructed on LAM. Thirty-six eyes of rabbits underwent alkali burn (1 N NaOH, 30 s) to create a limbal deficiency and a deeply damaged corneal stroma. Four weeks later, group 1 underwent a graft of the reconstructed corneal layer composed of autologous corneal epithelium and fibroblasts on LAM. Group 2 was transplanted with a graft of the reconstructed autologous corneal epithelium, and group 3 served as a control without surgery. Wound healing and stabilization of the ocular surfaces occurred much faster in group 1 than in groups 2 and 3. The eyes in group 3 revealed typical limbal deficiencies with conjuctivalization and persistent corneal epithelial defects. However, the corneas in group 1 developed only mild peripheral neovascularization. Immunohistochemical staining in group 1 demonstrated that p63, cytokeratin 3, E-cadherin, transforming growth factor (TGF)-beta1, and collagen IV were expressed strongly in the corneal epithelium and basement membrane. On the basis of these results, transplantation of the reconstructed corneal layer, composed of autologous corneal epithelium and fibroblasts on LAM, partially accelerated the recovery of the alkali-injured rabbit ocular surface, and might be useful therapeutically for the treatment of patients with severely damaged cornea.