Equine model for soft-tissue regeneration.

Soft-tissue regeneration methods currently yield suboptimal clinical outcomes due to loss of tissue volume and a lack of functional tissue regeneration. Grafted tissues and natural biomaterials often degrade or resorb too quickly, while most synthetic materials do not degrade. In previous research we demonstrated that soft-tissue regeneration can be supported using silk porous biomaterials for at least 18 months in vivo in a rodent model. In the present study, we scaled the system to a survival study using a large animal model and demonstrated the feasibility of these biomaterials for soft-tissue regeneration in adult horses. Both slow and rapidly degrading silk matrices were evaluated in subcutaneous pocket and intramuscular defect depots. We showed that we can effectively employ an equine model over 6 months to simultaneously evaluate many different implants, reducing the number of animals needed. Furthermore, we were able to tailor matrix degradation by varying the initial format of the implanted silk. Finally, we demonstrate ultrasound imaging of implants to be an effective means for tracking tissue regeneration and implant degradation.

Young developmental age cardiac extracellular matrix promotes the expansion of neonatal cardiomyocytes in vitro.

A major limitation to cardiac tissue engineering and regenerative medicine strategies is the lack of proliferation of postnatal cardiomyocytes. The extracellular matrix (ECM) is altered during heart development, and studies suggest that it plays an important role in regulating myocyte proliferation. Here, the effects of fetal, neonatal and adult cardiac ECM on the expansion of neonatal rat ventricular cells in vitro are studied. At 24h, overall cell attachment was lowest on fetal ECM; however, ~80% of the cells were cardiomyocytes, while many non-myocytes attached to older ECM and poly-l-lysine controls. After 5 days, the cardiomyocyte population remained highest on fetal ECM, with a 4-fold increase in number. Significantly more cardiomyocytes stained positively for the mitotic marker phospho-histone H3 on fetal ECM compared with other substrates at 5 days, suggesting that proliferation may be a major mechanism of cardiomyocyte expansion on young ECM. Further study of the beneficial properties of early developmental aged cardiac ECM could advance the design of novel biomaterials aimed at promoting cardiac regeneration.

In vivo flow cytometer for real-time detection and quantification of circulating cells.

An in vivo flow cytometer is developed that allows the real-time detection and quantification of circulating fluorescently labeled cells in live animals. A signal from a cell population of interest is recorded as the cells pass through a slit of light focused across a blood vessel. Confocal detection of the excited fluorescence allows continuous monitoring of labeled cells in the upper layers of scattering tissue, such as the skin. The device is used to characterize the in vivo kinetics of red and white blood cells circulating in the vasculatúre of the mouse ear. Potential applications in biology and medicine are discussed.

Instrumentation for multi-modal spectroscopic diagnosis of epithelial dysplasia.

Reflectance and fluorescence spectroscopies have shown great promise for early detection of epithelial dysplasia. We have developed a clinical reflectance spectrofluorimeter for multimodal spectroscopic diagnosis of epithelial dysplasia. This clinical instrument, the FastEEM, collects white light reflectance and fluorescence excitation-emission matrices (EEM's) within a fraction of a second. In this paper we describe the FastEEM instrumentation, designed for collection of multi-modal spectroscopic data. We illustrate its performance using tissue phantoms with well defined optical properties and biochemicals of known fluorescence properties. In addition, we discuss our plans to develop a system that combines a multi-spectral imaging device for wide area surveillance with this contact probe device.

Imaging human epithelial properties with polarized light-scattering spectroscopy.

Biomedical imaging with light-scattering spectroscopy (LSS) is a novel optical technology developed to probe the structure of living epithelial cells in situ without need for tissue removal. LSS makes it possible to distinguish between single backscattering from epithelial-cell nuclei and multiply scattered light. The spectrum of the single backscattering component is further analyzed to provide quantitative information about the epithelial-cell nuclei such as nuclear size, degree of pleomorphism, degree of hyperchromasia and amount of chromatin. LSS imaging allows mapping these histological properties over wide areas of epithelial lining. Because nuclear enlargement, pleomorphism and hyperchromasia are principal features of nuclear atypia associated with precancerous and cancerous changes in virtually all epithelia, LSS imaging can be used to detect precancerous lesions in optically accessible organs.

Fluorescence, reflectance, and light-scattering spectroscopy for evaluating dysplasia in patients with Barrett's esophagus.

BACKGROUND & AIMS: The aim of this study was to assess the potential of 3 spectroscopic techniques (fluorescence, reflectance, and light-scattering spectroscopy) individually and in combination, for evaluating low- and high-grade dysplasia in patients with Barrett's esophagus (BE). METHODS: Fluorescence spectra at 11 excitation wavelengths and a reflectance spectrum were acquired in approximately 1 second from each site before biopsy using an optical fiber probe. The measured fluorescence spectra were combined with the reflectance spectra to extract the intrinsic tissue fluorescence. The reflectance spectra provided morphologic information about the bulk tissue, whereas light-scattering spectroscopy was used to determine cell nuclear crowding and enlargement in Barrett's epithelium. RESULTS: Significant differences were observed between dysplastic and nondysplastic BE in terms of intrinsic fluorescence, bulk scattering properties, and levels of epithelial cell nuclear crowding and enlargement. The combination of all 3 techniques resulted in superior sensitivity and specificity for separating high-grade from non-high-grade and dysplastic from nondysplastic epithelium. CONCLUSIONS: Intrinsic fluorescence, reflectance, and light-scattering spectroscopies provide complementary information about biochemical and morphologic changes that occur during the development of dysplasia. The combination of these techniques (Tri-Modal Spectroscopy) can serve as an excellent tool for the evaluation of dysplasia in BE.

Effects of fluence rate on cell survival and photobleaching in meta-tetra-(hydroxyphenyl)chlorin-photosensitized Colo 26 multicell tumor spheroids.

We report the influence of fluence rate on the photobleaching and cell survival in Colo 26 multicell spheroids photosensitized by meta-tetra-(hydroxyphenyl)chlorin (mTHPC). Photosensitizer degradation and therapeutic efficacy increased dramatically and progressively when the fluence rate was reduced over the range from 90 to 5 mW cm-2. These experimental results were compared to a mathematical model of photobleaching based on self-sensitized singlet oxygen reactions with the photosensitizer ground state. This model incorporates photophysical parameters obtained from microelectrode measurements of oxygen depletion at the surface of mTHPC-sensitized spheroids and was refined by including the inhomogeneous distribution of mTHPC in spheroids and oxygen depletion in the bulk medium. Since the model is consistent with the experimental data we conclude that the fluence rate dependence of the cell survival and of mTHPC photobleaching is due to photochemical oxygen consumption and a predominantly singlet oxygen-mediated mechanism of mTHPC photobleaching. The threshold dose of reacting singlet oxygen was calculated to be 7.9 +/- 2.2 mM in this system.

Intrinsic fluorescence spectroscopy in turbid media: disentangling effects of scattering and absorption.

The fluorescence from a turbid medium such as biologic tissue contains information about scattering and absorption, as well as the intrinsic fluorescence, i.e., the fluorescence from an optically thin sample of pure fluorophores. The interplay of scattering and absorption can result in severe distortion of the intrinsic spectral features. These distortions can be removed by use of a photon-migration-based picture and information from simultaneously acquired fluorescence and reflectance spectra. We present experimental evidence demonstrating the validity of such an approach for extracting the intrinsic fluorescence for a wide range of scatterer and absorber concentrations in tissue models, ex vivo and in vivo tissues. We show that variations in line shape and intensity in intrinsic tissue fluorescence are significantly reduced compared with the corresponding measured fluorescence.

Interferometric phase-dispersion microscopy.

We describe a new scanning microscopy technique, phase-dispersion microscopy (PDM). The technique is based on measuring the phase difference between the fundamental and the second-harmonic light in a novel interferometer. PDM is highly sensitive to subtle refractive-index differences that are due to dispersion (differential optical path sensitivity, 5 nm). We apply PDM to measure minute amounts of DNA in solution and to study biological tissue sections. We demonstrate that PDM performs better than conventional phase-contrast microscopy in imaging dispersive and weakly scattering samples.

Hypoxia significantly reduces aminolaevulinic acid-induced protoporphyrin IX synthesis in EMT6 cells.

We have studied the effects of hypoxia on aminolaevulinic acid (ALA)-induced protoporphyrin IX (PpIX) synthesis in EMT6 monolayer cultures characterized by different cell densities and proliferation rates. Specifically, after ALA incubation under hypoxic or normoxic conditions, we detected spectrofluorometrically the PpIX content of the following populations: (a) low-density exponentially growing cells; (b) high-density fed-plateau cells; and (c) high-density unfed-plateau cells. These populations were selected either for the purpose of comparison with other in vitro studies (low-density exponentially growing cells) or as representatives of tumour regions adjacent to (high-density fed-plateau cells) and further away from (high-density unfed-plateau cells) capillaries. The amount of PpIX per cell produced by each one of these populations was higher after normoxic ALA incubation. The magnitude of the effect of hypoxia on PpIX synthesis was dependent on cell density and proliferation rate. A 42-fold decrease in PpIX fluorescence was observed for the high-density unfed-plateau cells. PpIX production by the low-density exponential cells was affected the least by ALA incubation under hypoxic conditions (1.4-fold decrease), whereas the effect on the high-density fed-plateau population was intermediate (20-fold decrease).

Effects of the subcellular redistribution of two nile blue derivatives on photodynamic oxygen consumption.

We present experimental evidence that demonstrates directly how the subcellular localization and redistribution of two nile blue derivatives, 5-ethylamino-9-diethyl-aminobenzo[a]phenothiazinium chloride (EtNBS) and 5-ethylamino-9-diethyl-aminobenzo[a]phenoselenazinium chloride (EtNBSe), affect oxygen consumption during irradiation of sensitized multicell EMT6 spheroids. Specifically, two well-defined phases of oxygen consumption are observed during treatment, with the onset of the second phase being a fluence-dependent event. Fluorescence microscopy during irradiation of EtNBS-sensitized EMT6 monolayer cultures indicates that sensitizer redistribution from intracellular organelles, presumably lysosomes, to the cytosol can explain the onset of the second oxygen consumption phase. This event requires eight times fewer photons for EtNBSe than for EtNBS, consistent with the higher singlet oxygen yield of the former dye. The existence of a second oxygen consumption phase suggests that the aggregated form of the dye is a less efficient photodynamic agent. Moreover, we present evidence suggesting that damage to the primary sites of localization might be less significant than damage incurred by the sites to which the sensitizer redistributes during irradiation.

Singlet oxygen- versus nonsinglet oxygen-mediated mechanisms of sensitizer photobleaching and their effects on photodynamic dosimetry.

We report the effects of singlet oxygen (1O2) and non-1O2-mediated sensitizer photobleaching on oxygen consumption and dosimetry during photodynamic therapy (PDT) of sensitized multicell tumor spheroids. We develop a theoretical model for the description of non-1O2-mediated photobleaching resulting from irreversible reactions of the excited singlet or triplet sensitizer populations with cell substrate. We show that the fluence-dependent simple exponential decay expression of sensitizer degradation is not consistent with these mechanisms and, therefore, with any reasonable mechanism that we consider, because we have shown previously that 1O2-mediated photobleaching cannot be described by a simple exponential with a constant photobleaching coefficient (I. Georgakoudi et al., Photochem. Photobiol. 65, 135-144, 1997). Analysis of oxygen microelectrode measurements performed at the edge of Nile blue selenium (EtNBSe)- and protoporphyrin IX (PpIX)-sensitized spheroids during PDT demonstrates that the former drug photobleaches via a non-1O2-mediated mechanism, while the latter is degraded via a 1O2-mediated mechanism. Comparisons of the cytotoxic effects of EtNBSe with those of Photofrin (a drug that is degraded via a 1O2-mediated mechanism) indicate that the lower threshold 1O2 dose and the higher extinction coefficient and 1O2 yield for EtNBSe do not necessarily result in improved photodynamic effects, thus emphasizing the importance of the sensitizer photobleaching mechanism for dosimetry.

The mechanism of Photofrin photobleaching and its consequences for photodynamic dosimetry.

We report experimental results that support a theory of self-sensitized singlet oxygen-mediated bleaching of the porphyrin photosensitizer Photofrin. Microelectrode measurements of photodynamic oxygen consumption were made near the surface of individual, Photofrin-sensitized EMT6 spheroids during laser irradiation. The progressive decrease in photochemical oxygen consumption with sustained irradiation is consistent with a theory in which bleaching occurs via self-sensitized singlet oxygen reaction with the photosensitizer ground state. A bleaching model based solely on absorbed optical energy density is inconsistent with the data. Photobleaching has a significant effect on calculated photodynamic dose distributions in 500 microns diameter spheroids. Dose distributions corrected for the effects of bleaching produce a new estimate (12.1 +/- 1.2 mM) for the threshold dose of reacting singlet oxygen in this system.