This PDF file contains the front matter associated with SPIE Proceedings Volume 6439, including the Title Page, Copyright information, Table of Contents, Introduction (if any), and the Conference Committee listing.

Tissue engineering aims to create in vitro functional tissues that could ultimately be used as autologous implants. Considering the large number of parameters that have to be tested to optimize the tissue growth and to achieve a better understanding of tissue formation, relevant biological in vitro models are needed which can be monitored and characterized all along the different stages of tissue engineering: cell seeding, cell growth, extra-cellular matrix (ECM) deposition, matrix turn-over and tissue organization. We developed porous chitosan scaffolds (&fgr;1.5mm) that enclose a 300&mgr;m microchannel to encourage fluid shear-stress stimulation and more specifically to support bundle formation for the specific case of tendon tissue engineering. These scaffolds were loaded in perfusion bioreactors and monitored during several days by optical coherence tomography (OCT). The fiber based time domain OCT employed a 1300nm superluminescent diode with a bandwith of 52 nm and a xyz resolution of 16*16*14 in free space. This set up allowed us to assess the volume fraction of cell seeded in the microchannels, and thus to optimize the seeding procedure. The cell growth and ECM deposition were successfully monitored at different time point as the channels were filled by newly formed material. Different scattering behaviors have been observed during cell growth and ECM production. The possibility to monitor continuously the scaffolds under perfusion will allow an easy discrimination of the parameters affecting the pre-tissue formation rate growth.

The purpose of this work is to investigate the microstructure of blended materials using non-invasive, optical imaging modality. Multiphoton autofluorescence and second harmonic generation signals will be used for characterizing and quantifying individual non-linear optical properties of each polymer in pure polymeric thin films. In addition, reflected confocal signals will used to outline the interface of refractive index mismatch. And the phase separation phenomenon of immiscible blended membranes composed of different ratio of nylon and chitosan are analyzed and differentiated using the non-invasive optical information including autofluerescence, second harmonic generation, and reflected signals. We therefore propose the potentiality of using multiphoton autofluorescence and second harmonic generation microscopy complemented with reflected confocal microscopy for studying the synthetic blended polymeric scaffolds and also, in the future, the dynamic, in vivo, cell-matrix interaction in the field of tissue engineering.

The development of atherosclerotic plaques includes changes in the cellular and extracellular composition of the arterial wall. Although these changes in composition affect the manner in which light scatters in the vessel wall and thus affect any optical signal, experimentally determining how features of atherosclerosis affect optical signals has remained elusive. Using current tissue-engineering methods, we developed a 3D tissue construct model for assessing how certain features of atherosclerosis (the increased concentrations of lipids and macrophages) affect optical signals. The model is based on vascular tissue constructs made of smooth muscle cells (SMCs) and macrophages (M&Fgr;s) that are co-cultured inside a 3D scaffold matrix of collagen fibers with interspersed lipids. To make the scaffold matrix, powdered collagen was dissolved in acetic acid, homogenized, and neutralized by sequential dialyses to yield a soft gel of 2 μm thick collagen fibers in which cells were seeded. In "normal" constructs, only SMCs were seeded in the collagen gel; in "athero-like" constructs, both SMCs and M&Fgr;s (loaded or unloaded with lipid) were seeded in the gel. To demonstrate the use of this model, sets of slab-shaped normal and athero-like constructs were imaged by optical coherence tomography (OCT) and qualitatively analyzed. 2D frames from 3D OCT image cubes were compared to 2D histology sections. Our results indicate that the cellular composition of the construct affects morphological features of the OCT image.

The depth-sensing micro-indentation has been well recognized as a powerful tool for characterizing mechanical properties of solid materials due to its non-destructive approach. Based on the depth-sensing principle, we have developed a new indentation method combined with a high resolution imaging technique, Optical Coherence Tomography (OCT), which can accurately measure the deformation of soft tissues or hydrogels under a spherical indenter of constant force. The Hertz contact theory has been applied for quantitatively correlating the indentation force and the deformation with the mechanical properties of the materials. The Young's modulus of hydrogels estimated by the new method are comparable with those measured by conventional depth-sensing micro-indentation. The advantages of this new method include its capability to characterize mechanical properties of bulk soft materials and amenability to perform creeping tests. More importantly, the measurement can be performed under sterile condition allowing non-destructive, in-situ and real-time investigations on the changes in mechanical properties of soft materials (e.g. hydrogel). This unique character can be applied for various biomechanical investigations such as monitoring reconstruction of engineered tissue.

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.

Tendon and ligament are the transition tissues from a hard tissue to a soft tissue. The regenerative medicine of tendons needs reasonable biomaterials to regenerate precisely from the view point of composition and adhesion properties. In regenerative medicine of hard tissues, it has been reported that calcifications are influenced by phosphorylated proteins (phosphate groups) and the biomaterial possessing phosphate groups promote or inhibit the formation of HAP. We have studied to develop and evaluate the phosphorylated soft biomaterials, which is possible to control a calcification by the introduction ratio of phosphate groups, as biomaterials for tendon regeneration. In addition, we have studied measurement technologies. In the present study, we studied a FT-IR analysis of gelatins with different introduction ratio of phosphate groups, an evaluation of calcifications by the difference of introduction ratio of phosphate groups, and a fundamental survey on OCT imaging for calcifications of a gelatin and a phosphorylated gelatin. We use phosphorylated gelatins with different introduction ratios of phosphate group linked by ester bonds. The introduction ratios are measured by the FT-IR calibrated by a molybdenum blue method. Phosphorylated gelatin sheets were calcified using 1.5SBF soaking process and alternative soaking process. These gelatin sheets with different calcification conditions were measured using SD-OCT systems with 843nm centered wavelength SLD. As a result, we demonstrated that it was possible to measure the calcification on/in the gelatin sheets and sponges and phosphorylated using OCT. The main mechanism is the strong back scattering and the high scattering of deposited calcium particles.

Previous results of tissue optical clearing have qualitatively indicated that optical properties of bio-tissue can be changed by the use of biocompatible hyperosmotic agents. In this talk, it is our aim to try to quantitatively evaluate how relative between changes of optical properties and hyperosmotic agents. Tissue-like turbid media, i.e. intralipid-10% suspension and porcine skin tissues, medicated without and with biocompatible hyperosmotic agents, have been investigated within NIR range. Optical parameters (absorption coefficient, scattering coefficient and anisotropy factor) of samples have been calculated by Inverse Adding-Doubling (IAD) modelling method. The results have demonstrated that optical property parameters (absorption coefficient, scattering coefficient and anisotropy factor) of tissue samples have changed with the course of the administration of biocompatible hyperosmotic agents into intralipid-10% suspension and porcine skin tissues respectively. This reveals better refractive index matching environment has been gradually created within tissue. Further, the results have showed the potential capabilities to quantitatively control optical properties of bio-tissue in this way. Therefore, this would facilitate explanation of the mechanism of tissue optical clearing by the use of biocompatible hyperosmotic agents.

Cells, scaffold and culture environment are the three essential elements in engineering tissue constructs. Among these elements, the scaffold plays a critical role in converting cells into tissue since it provides a template and space for cells to grow and produce the desired matrix. Scaffolds are usually fabricated into three-dimensional blocks from biodegradable polymers with different internal architectures, for instance they are with fibrous or porous structures. The mechanical properties and nutrient diffusion ability of scaffolds are highly dependent on their internal structure. The biodegradable feature of scaffolds leads to a dramatic change in their microstructure during in vitro culture or after implantation. In this study, we explore optical coherence tomography (OCT) as a potential tool to characterize architecture of scaffolds including porosity, pore distribution and interconnectivity. This instrument is a fibre based time domain OCT equipped with a 1300 nm superluminescent diode, with a bandwidth of 52 nm and a free space resolution of 16x16x14 μm. Two model scaffold systems have been investigated. One was porous poly(lactide) scaffold fabricated by solvent-evaporation and salt leaching technique with dual poregens. Another was fibrous chitosan scaffold produced by wet spinning. Variations of scaffolds architecture, in term of porosity and interconnectivity, with different fabrication conditions could be quantified with the help of a commercial software (Volocity, Improvision). This study demonstrated that OCT can be used as a tool to guide scaffold fabrication and optimise their internal structure. Moreover, it can be used as on-line monitoring for scaffold degradation in various culture conditions.

In order to achieve functional tissue with the correct biomechanical properties it is critical to stimulate mechanically the cells. Perfusion bioreactor induces fluid shear stress that has been well characterized for two-dimensional culture where both simulation and experimental data are available. However these results can't be directly translated to tissue engineering that makes use of complex three-dimensional porous scaffold. Moreover, stimulated cells produce extensive extra-cellular matrix (ECM) that alter dramatically the micro-architecture of the constructs, changing the local flow dynamic. In this study a Fourier domain Doppler optical coherent tomography (FD-DOCT) system working at 1300nm with a bandwidth of 50nm has been used to determine the local flow rate inside different types of porous scaffolds used in tissue engineering. Local flow rates can then be linearly related, for Newtonian fluid, to the fluid shear stress occurring on the pores wall. Porous chitosan scaffolds (&fgr;1.5mm x 3mm) with and without a central 250 &mgr;m microchannel have been produced by a freeze-drying technique. This techniques allow us to determine the actual shear stress applied to the cells and to optimise the input flow rate consequently, but also to relate the change of the flow distribution to the amount of ECM production allowing the monitoring of tissue formation.

Stem cells and its differentiations have got a lot of attentions in regenerative medicine. The process of differentiations, the formation of tissues, has become better understood by the study using a lot of cell types progressively. These studies of cells and tissue dynamics at molecular levels are carried out through various approaches like histochemical methods, application of molecular biology and immunology. However, in case of using regenerative sources (cells, tissues and biomaterials etc.) clinically, they are measured and quality-controlled by non-invasive methods from the view point of safety. Recently, the use of Fourier Transform Infrared spectroscopy (FT-IR) has been used to monitor biochemical changes in cells, and has gained considerable importance. The objective of this study is to establish the infrared spectroscopy of cell differentiation as a quality control of cell sources for regenerative medicine. In the present study, as a basic study, we examined the adipose differentiation kinetics of preadipocyte (3T3-L1) and the osteoblast differentiation kinetics of bone marrow mesenchymal stem cells (Kusa-A1) to analyze the infrared absorption spectra. As a result, we achieved to analyze the adipose differentiation kinetics using the infrared absorption peak at 1739 cm-1 derived from ester bonds of triglyceride and osteoblast differentiation kinetics using the infrared absorption peak at 1030 cm-1 derived from phosphate groups of calcium phosphate.

A spectral domain Polarization sensitive optical coherence tomography (SDPS-OCT) system has been developed to acquire depth images of biological tissues such as porcine tendon, rabbit eye. The Stocks vectors (I, Q, U, and V) of the backscattered light from the biological tissues have been reconstructed. Further, the phase retardation and polarization degree between the two orthogonal polarizing states have been computed. Reconstructed images, i.e. birefringence images, from Stokes parameters, retardation and polarization degree of biological tissues show significant local variations in the polarization state. And the birefringence contrast of biological tissue possibly changes by some outside force. In addition, the local thickness of the birefringence layer determined with our system is significant. The results presented show SDPS-OCT is a potentially powerful technique to investigate tissue structural properties on the basis of the fact that any fibrous structure with biological tissues can influence the polarization state of light.

In this talk, a spectral domain polarization sensitive optical coherence tomography (SDPS-OCT) system has been developed so as to obtain high scan speed, high dynamic range and high sensitivity, and simultaneously get birefringence contrast of some biological tissue. To reduce corruption of the DC and autocorrelation terms to images, we introduce the two phase method. The stocks vectors (I, Q, U, and V) of the backscattered light from the specimen have been reconstructed by processing the signals from the two channels which are responsible for detecting the vertical and horizontal polarization state light separately. Further, the phase retardation between the two orthogonal polarization states has been acquired. The results from rabbit eye show that SDPS-OCT system based on the two phase method has great potential to imaging biological tissue.

In order to improve the imaging contrast and resolution in photoacoustic tomography(PAT), the deconvolution between the transducer impulse response and the recorded photoacoustic(PA) signal of the tissue phantom is often used. The suppression of noise is critical in the deconvolution. Compared with the traditional band-pass filter in Fourier domain, wiener filter is more appropriate for the wide band PA signal. The scaling parameter in wiener filter is hard to determine using the traditional Fourier domain method. To solve the problem, the deconvolution algorithm with wiener filter based on the wavelet transform is presented. The scaling parameter is estimated using discrete wavelet transform(DWT) by its multi-resolution analysis(MRA) ability. The white noise had been effectively suppressed. Both numerical simulation and experimental results demonstrated that the contrast and resolution of PA images had been improved.

In photoacoustic (PA) tomography, a piezoelectrical signal of inner characteristic of interesting object is mainly acquired by a hydrophone. Every piezoelectrical signal as output signal is the convolution of the original input signal that denotes the ultrasonic signal emitting from the substance and the system transfer function. The undistorted input signal is the very physical quantity that we want actually. Therefore an original input signal is computed with the deconvolution of the system transfer function and the output signal. While most practical deconvolution problems are called as blind deconvolution because the system transfer function and the input signal are both unknown and estimated from the output signal in the same time. In common, the deconvolution problem has an important property that it is called ill-condition, which is a special and intractable difficulty that both the theoretic analysis and the numerical computation would meet. For the sake of getting the solution of the deconvolution problem reasonable in physics and responsible for the gained data continually, a package of theory method called regularization to cure the ill-conditioned problems is applied in the PA signal processing.

A highly accurate, fast three-dimensional in vivo temperature mapping method is developed using MRI water photon chemical shift. It is important to have the precise temperature distribution information during laser-tissue thermal treatment. Several methods can be used for temperature measurement including thermal couple, optical fiber sensor, and MRI (magnetic resonance imaging) methods. MRI is the only feasible method for 3D in vivo, non-invasive temperature distribution measurement for laser-tissue interaction. The water proton chemical shift method is used in 3D MRI mapping. Varies MRI parameters, such as flip angle, TE, TR, spatial resolution, and temporal repetition, were optimized for the temperature mapping. The laser radiation of 805nm wavelength and a light-absorbing dye, indocyanine green (ICG) was used for temperature elevation. The measurement was conducted using gel phantom, chicken tissue and rats. The phantom system was constructed with a dye-enhanced spherical gel embedded in uniform gel phantom, simulating a tumor within normal tissue. The normal temperature elevation within ex vivo tissue such as chicken breast can reach up to 45-50 degree C with a power density of 1.3W/cm2 (with laser power of 3W and 1.7cm beam size). The temperature resolution is 0.37 degree C with a 0.2-mm spatial resolution and repetition rate of around 40 seconds. The external magnetic field drift effect is also evaluated.

Propagation of ultrashort pulses of intense, infrared light through transparent medium gives rise to a visually spectacular phenomenon known as supercontinuum (white light) generation wherein the spectrum of transmitted light is very considerably broader than that of the incident light. We have studied the propagation of ultrafast (<45 fs) pulses of intense infrared light through biological media (water, and water doped with salivary proteins) which reveal that white light generation is severely suppressed in the presence of a major salivary protein, &agr;-amylase.