Regenerative medicine by the transplantation of differentiated cells or tissue stem cells has been clinically
performed, particularly in the form of cell sheets. To ensure the safety and effectiveness of cell therapy, the
efficient selection of desired cells with high quality is a critical issue, which requires the development of a new
evaluation method to discriminate cells non-invasively with high throughput. There were many ways to
characterize cells and their components, among which the optical spectral analysis has a powerful potential for this
purpose. We developed a cellular hyperspectral imaging system, which captured both spatial and spectral
information in a single pixel. Hyperspectral data are composed of continual spectral bands, whereas multispectral
data are usually composed of about 5 to 10 discrete bands of large bandwidths. The hyperspectral imaging system
which we developed was set up by a commonly-used inverted light microscope for cell culture experiments, and
the time-lapse imaging system with automatic focus correction. Spectral line imaging device with EMCCD was
employed for spectral imaging. The system finally enabled to acquire 5 dimensional (x, y, z, time, wavelength)
data sets and cell-by-cell evaluation. In this study, we optimized the protocol for the creation of cellular spectral
database under biological understanding. We enabled to confirm spectrum of autofluorescence of collagen,
absorption of specific molecules in the cultural sample and increase of scattering signal due to cell components
although detail spectral analyses have not been performed.
There is a demand in the field of regenerative medicine for measurement technology that enables determination of
functions and components of engineered tissue. To meet this demand, we developed a method for extracellular matrix
characterization using time-resolved autofluorescence spectroscopy, which enabled simultaneous measurements with
mechanical properties using relaxation of laser-induced stress wave. In this study, in addition to time-resolved
fluorescent spectroscopy, hyperspectral sensor, which enables to capture both spectral and spatial information, was used
for evaluation of biochemical characterization of tissue-engineered cartilage. Hyperspectral imaging system provides
spectral resolution of 1.2 nm and image rate of 100 images/sec. The imaging system consisted of the hyperspectral
sensor, a scanner for x-y plane imaging, magnifying optics and Xenon lamp for transmmissive lighting. Cellular
imaging using the hyperspectral image system has been achieved by improvement in spatial resolution up to 9
micrometer. The spectroscopic cellular imaging could be observed using cultured chondrocytes as sample. At early
stage of culture, the hyperspectral imaging offered information about cellular function associated with endogeneous
fluorescent biomolecules.
There is a demand in the field of regenerative medicine for measurement technology that enables determination of functions and characterizations of engineered tissue. Regenerative medicine involving the articular cartilage in particular requires measurement of viscoelastic properties and characterization of the extracellular matrix, which plays a major role in articular cartilage. To meet this demand, we previously proposed a noninvasive method for determination of the viscoelasticity using laser-induced thermoelastic wave (1,2). We also proposed a method for characterization of the extracellular matrix using time-resolved autofluorescence spectroscopy, which could be performed simultaneously with laser-induced thermoelastic wave measurement(3). The purpose of this study was to verify the usefulness and limitation of these methods for evaluation of actual engineered cartilage. 3rd Q-SW Nd:YAG laser pulses, which are delivered through optical fiber, were used for the light source. Laser-induced thermoelastic waves were detected by a sensor consisting of a piezoelectric transducer, which was designed for use in arthroscopy(4). The time-resolved fluorescence spectroscopy was measured by a photonic multichannel analyzer with 4ch digital signal generator. Various tissue-engineered cartilages were developed as samples. Only a limited range of sample thickness could be measured, however, the measured viscoelastic parameters had a positive correlation with culture time, that is, the degree of formation of extracellular matrix(5,6). There were significant differences in the fluorescent parameters among the phenotypic expressions of cartilage because chondrocyte produces specific extracellular matrix as in collagen types depending on its phenotype.
We demonstrated the capability of a photoacoustic method for viscoelastic measurement. The measurement method has already proved to be useful for evaluation of regenerative medicine of articular cartilage. However, characterization of the extracellular matrix as well as determination of the viscoelastic property should be carried out for evaluation of regenerative medicine because the extracellular matrix plays an important role. We therefore developed a method for characterization of the extracellular matrix that can be performed simultaneously with the photoacoustic measurement. Since collagen molecules, which are the major contents of the cartilage extracellular matrix, are well known as endogenous fluorescent molecules, it is possible that fluorescence measurement will enable characterization of the extracellular matrix. Third harmonic Q-switched Nd:YAG laser pulses were used as an excitation light source. The time-resolved fluorescence spectroscopy was obtained by using a photonic multi-channel analyzer. Tissue-engineered cartilages cultured under different conditions for various periods were used as samples. Different culture conditions resulted in different extracellular matrix formations. There were significant differences in the measured fluorescent parameters among the culture conditions of cartilage because chondrocytes produce a specific extracellular matrix depending on its culture condition. The specific extracellular matrix contained a specific type of collagen such as collagen type I or type II, which each have specific fluorescent features. Thus, the fluorescent parameters enabled characterization of synthesis of cartilage-associated extracellular matrix. Therefore, the combination of fluorescence and photoacoustic measurement is expected to become a useful evaluation method in regenerative medicine.
We demonstrated the capability of photoacoustic measurement for viscoelastic characterization. Since tissue viscoelasticity affects the propagation and attenuation of photoacoustic waves generated in the tissue, the relaxation times of the photoacoustic waves give the viscosity-elasticity ratio of the tissue. The relaxation times of photoacoustic waves of articular cartilage tissues engineered under various culture conditions were closely correlated with intrinsic viscosity-elasticity ratios measured by using a conventional viscoelastic analyzer (R > 0.98). In order to apply the photoacoustic measurement method to evaluation of the regeneration of articular cartilage as a method to validate the surgery, the method should enable not only evaluation of engineered tissue during cultivation in vitro but also evaluation after transplantation of engineered tissue in vivo. The aim of this study was to verify the usefulness of the photoacoustic method for repeated measurement of viscoelastic properties in order to evaluate the process of regeneration of a full-thickness defect in rabbit articular cartilage using allografted tissue-engineered cartilage. Photoacoustic waves were induced by 266- and 355-nm, 5-7 ns, light pulses delivered through an optical silica fiber from an Q-switched Nd:YAG laser and were detected by a piezoelectric transducer, which we had designed. About a 40% difference between the viscosity-elasticity ratio of allografted cartilage that of tissue surrounding the defect was shown just after surgery. The difference was significantly reduced at 4 and 12 postoperative weeks. Therefore, since the photoacoustic measurement method enables assessment of the progress of restoration of the viscoelasticity of articular cartilage, its main function, this method would be useful as an evaluation method in regenerative medicine.
We demonstrated a capability of biomechanical characterization by photoacoustic measurement for the purpose of non-invasive functional evaluation of articular cartilage. In this study, the scheme of photoacoustic measurement was improved. For in vivo application, the measurement scheme was changed from a transmittance mode to a reflectance mode in which an optical fiber was coaxially arranged with a piezoelectric transducer. In order to verify the applicability of this measurement for diagnosis of cartilage degeneration, photoacoustic measurements in a reflectance mode were performed using various degenerated cartilages. As a model of degenerated cartilage, cartilage-bone plugs were punched out from a porcine knee joint and treated with trypsin (1 mg/ml). Stress waves were induced by 250-355 nm, 7-ns light pulses delivered through an optical silica fiber from an OPO and were detected by a piezoelectric transducer. The change in relaxation time, which was correlated with the viscosity-elasticity ratio, had a positive correlation with time of trypsin treatment. Our results revealed the applicability of photoacoustic measurement to in vivo preoperative diagnosis of cartilage degeneration.
We have demonstrated a capability of biomechanical characterization by photoacoustic measurement using various concentraiton gelatins as tissue pahntom. We have also evaluated the viscoelasticity of the cartilages tissue-engineered under the different culture conditions. Structural tissues, such as cartilage, bone, tendon, and muscle require time-dependent mechanical responses (viscoelastic properties) to describe their mechanical behavior. However, non-invasive measurement of tissue viscoelastic has not been developed; such measurement is necessary for tissue engineering applications on weight-bearing tissues. As tissue viscoelasticity affects the propagation and attenuation of the stress waves generated in the tissue, their relaxation times which are defined as the time for the stress wave amplitude to decrease by a factor of 1/e, give the viscosity-elasticity ratio of the tissue. In this study, stress waves (photoacoustic waves) which were induced by 250-nm, 6-ns, light pulses from an OPO were detected by a piezoelectric transducer. The relaxation time of the photoacoustic wave was measured for various concentrations of gelatins which had been measured their viscoelastic properties by a conventional method. Consequently, the relaxation time corresponded to the known viscosity-elasticity ratio of the gelatins. For the tissue-engineered cartileges, photoacoustic measurements were performed under the different cultured conditions. The relaxation time of the cartilages closely correlated with the viscosity-elasticity ratio measured by a convetional method. Therefore, the photoacoustic measurement is one of the qualified candidates for a non-invasive viscoelastic measurement of tissue.
The purpose of this study is to evaluate the influence on the intervertebral disc cells after laser irradiation using three- dimensional culture system and to clarify the optimum Ho:YAG laser irradiation condition on percutaneous laser disc decompression (PLDD) for lumbar disc herniation. Since the Ho:YAG laser ablation is characterized by water-vapor bubble dynamics, not only thermal effect but also acoustic effect on cell metabolism might occur in the intervertebral disc. We studied the disc cell reaction from the metabolic point of view to investigate photothermal and photoacoustic effects on three-dimensional cultured disc cell. Intervertebral discs were obtained from female 30 Japanese white rabbits weighing about 1 kg. A pulsed Ho:YAG laser (wavelength: 2.1 micrometer, pulse width: about 200 microseconds) was delivered through a 200 micrometer-core diameter single silica glass fiber. We used the Ho:YAG laser irradiation fluence ranging from 60 to approximately 800 J/cm2 at the fiber end. To investigate acoustic effect, the acoustic transducer constructed with polyvinylidene fluoride (PVdF) film and acoustic absorber was used to detect the stress wave. Thermocouple and thermography were used to investigate thermal effect. Concerning damage of plasma membrane and ability of matrix synthesis, thermal effect might mainly affect cell reaction in total energy of 54 J (closed to practically used condition), but in 27 J, acoustic effect might contribute to it. We found that total energy was key parameter among the optimum condition, so that temperature and/or stress wave may influence Ho:YAG laser-disc cell interactions.
The purpose of this study is to evaluate the influence on disc cells after laser irradiation using three-dimensional (3-D) culture system and to clarify the optimum Ho:YAG laser irradiation condition on percutaneous laser disc decompression (PLDD) therapy. Since the Ho:YAG laser ablation is characterized by water-vapor bubble dynamics with pressure wave, thermal effect on cell metabolism might occur in the intervertebral disc. We studied the disc cell damage on the metabolic point of view to investigate the optimum irradiation parameter of the Ho:YAG laser. We have developed the 3-D cultured disc cell system using agarose gel to investigate laser - disc cell interaction. This culture system provides a highly in vivo-like environment for disc cells in which cell- extracellular matrix interactions appear to be more important than contacts among cells. Intervertebral discs were obtained from Japanese white. The isolated disc cells were seeded in 96-well culture plates at the cell densities of 1 X 106 cells/ml, and incubated for 12 days. A pulsed Ho:YAG laser was delivered through a 200 micrometer-core diameter single silica glass fiber. On the agarose gel including the 3-D cultured disc cells, we used the Ho:YAG laser irradiation energy ranging from 40 to 180 mJ/pulse at the fiber end. Cytotoxicity and matrix synthesis after the laser irradiations were evaluated in time course to determine the optimum condition of laser irradiations. It was confirmed that laser irradiation causes necrosis of the cells and additionally produces apoptosis depending on the condition. The ability of matrix synthesis was maintained even after the irradiation, which differed depending on the irradiation conditions. The optimum irradiation conditions seemed related to the preservation of intact area and the acceleration of matrix synthesis in reactive area.
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