This PDF file contains the front matter associated with SPIE Proceedings Volume 7179, including the Title Page, Copyright information, Table of Contents, and the Conference Committee listing.

Using high speed ultrahigh resolution optical coherence tomography (OCT) at 800nm, non-invasive 3D cellular imaging has been accomplished. Cellular resolution imaging on and within these types of substrates is not possible with conventional microscopy techniques such as interference contrast microscopy, and requires the use of fluorescent staining. It is possible to achieve data acquisition rates of 20,000 samples per second with OCT which, in combination with its high axial and transverse resolution (>2-3μm), allows it to be used as a non-invasive technique to analyze cell migration in 3D with time. Comparatively high penetration depth also makes OCT a uniquely suited imaging technique for visualization of cells within a 3D construct. In this paper it is demonstrated that it is possible to resolve ~10μm Dictyostelium discoideum cells, a well established and useful model for investigation of cell motility and chemotaxis, in 3D and follow them in time lapse using an 800nm ultrahigh resolution high speed frequency domain based OCT microscope. Ultimately, these visualization techniques could enable monitoring of cell behavior in regenerative medicine, for example tracking of individual cells within a cell scaffold.

As the repair of injured or degenerated tendon is often compromised by the shortage of suitable donor tissue, other procedures need to be developed. The application of a functional tissue engineered tendon could prove to be a promising alternative therapy. Due to their good biocompatibility, collagen hydrogel based scaffolds have been considered to be potentially suitable for engineering tendon tissue in vitro. One of the major limitations of collagen hydrogels for engineering tissues is the difficulty in controlling their architecture and collagen concentration which results in poor mechanical strength. This study aims to overcome these limitations by creating a highly biocompatible scaffold that is both mechanically robust and aligned. Collagen fibers were pre-aligned under a high magnetic field then concentrated using plastic compression. Primary tenocytes cultured from rats were seeded on the aligned scaffolds. Following a protocol in public domain, thick cultured collagen constructs were rolled up into a spiral after undergoing plastic compressed. Both a light microscopy and a polarization sensitive optical coherence tomography (PSOCT) with central beam at 1300 nm were used to monitor the birefringence in the constructs. Conventional light microscopy showed that the tenocytes aligned along the pre-organized collagen bundles in contrast to the random distributed observed on unaligned scaffolds. PSOCT only revealed weak birefringence from aligned but uncompressed constructs. However, PSOCT images showed contrast band structures in the spiral constructs which suggests that the birefringence signal depends on the density of aligned collagen fibers. The effect of aligned cells, neo-formed matrix and the plastic compression on the birefringence signals are discussed in this paper briefly.

The ability of optical imaging techniques such as optical coherence tomography (OCT) to non-destructively characterize tissue-engineered constructs has generated enormous interest recently. Collagen gels are 3D structures that represent a simple common model of many engineered tissues that contain 2 primary scatterers: collagen and cells. We are testing the ability of OCT data to characterize the remodeling of such collagen-based constructs by 3 different types of cells: vascular smooth muscle cells (SMCs), endothelial cells (ECs), and osteoblasts (OBs). Collagen gels were prepared with SMCs, ECs, and OBs with a seeding density of 1×10⁶ cells/ml; additionally, acellular controls were also prepared. The disk-shaped constructs were allowed to remodel in the incubator for 5 days, with OCT imaging occurring on days 1 and 5. From the OCT data, the attenuation and reflectivity were evaluated by fitting the data to a theoretical model that relates the tissue optical properties (scattering coefficient and anisotropy factor) and imaging conditions to the OCT signal. The degree of gel compaction was determined from the volume of the culture medium that feeds the constructs. We found that gel compaction (relative to the acellular control) occurred in the SMC constructs, but not in the OB or EC constructs. The optical property data showed that at day 5 the SMC constructs had an overall higher reflectivity (lower g) relative to day 1, whereas there was no obvious change in reflectivity of the EC, OB constructs and acellular controls relative to day 1. Moreover, there was a difference in the attenuation of the OB constructs on day 5 relative to day 1, but not in the other constructs. The apparent decrease in anisotropy observed in the SMC constructs, but not in the OB and EC constructs and acellular controls, suggests that OCT is sensitive to the remodeling of the collagen matrix that accompanies gel compaction, and can offer highly localized information on the construct microstructure. The apparent increase in the scattering coefficient of the OB constructs is believed to be caused by a higher rate of proliferation by these cell types relative to the others. Overall, these results suggest that the optical properties of collagen gels contain information on both cell number and collagen gel microstructure.

Optical imaging modalities such as confocal microscopy and optical coherence tomography (OCT) are emerging as appealing methods for non-destructive evaluation of engineered tissues. The information offered by such optical imaging methods depends on the wavelength vis-á-vis the optical scattering properties of the sample. These properties affect many factors critical to image analysis in a nonlinear and nontrivial manner. Thus, we sought to characterize the effect wavelength has on the optical properties collagen remodeled by cells at 3 common imaging wavelengths: 488, 633, and 1310 nm. To do this, we seeded smooth muscle cells (SMCs) in soluble collagen gels at a density of 1×106 cells/ml; similar acellular control constructs were also prepared. The constructs were allowed to remodel in the incubator for 5 days, and were examined at 24 and 120 hours by confocal imaging at 488 and 633 nm, and by OCT imaging at 1310 nm. From the confocal and OCT data, the attenuation and reflectivity were evaluated by fitting the data to a theoretical model that relates the tissue optical properties (scattering coefficient and anisotropy factor) and imaging conditions to the signal. In general, we found that at 1310 nm, the optical properties of the acellular control constructs had a lower reflectivity (higher anisotropy) than the SMC constructs. The difference in reflectivity between the SMC construct and acellular controls tended to decrease with wavelength, owing to a relative increase in reflectivity of acellular controls at lower wavelengths relative to the cellular constructs. Overall, the largest difference in optical properties occurred at 1310 nm. Taken together, the data show that the shift in optical properties of soluble collagen gels caused by cellular remodeling is nonlinearly wavelength dependent, and that this information should be considered when devising how to optimally characterize engineered tissues using optical imaging methods.

The lung is an organ heavily involved in cancer as both the origin of lung cancer and where metastases from tumours originating in other organs develop. Therefore, it is obvious that the interaction between cancer cells and lung tissue plays a role in cancer development, invasion, and/or growth. Out of all the components of the lung's extracellular matrix, elastin fragmentation products, the so-called elastin peptides, have been associated with tumour growth and invasion. Therefore, we studied using a 3D model whether elastin peptides could increase the proliferative activity of lung cancer cells. To this purpose, we grew lung cancer cells in collagen type I 3D models and used Optical Coherence Tomography to study lung cancer cells growth in the absence and presence of elastin peptides in real-time and at different time-points for each specimen. Our work shows that the addition of elastin peptides to lung cancer cells increased not only the size of the cancer cell clusters in 3D models but also the number of these clusters. This work demonstrates that, using OCT, the effects of extracellular matrix components on cancer cells can be characterised in 3D models. The biomedical applications of this methodology can be extended to other systems.

Myocardial infarction leads to remodeling of the myocardium, resulting in a deterioration of cardiac function. This remodeling involves changes in the extracellular matrix, particularly an increase in collagen. Recently developed stem cell based regenerative treatments have been shown to reduce myocardial remodeling and collagen formation after infarction leading to an improvement in overall cardiac function. However, this emerging field is in dire need of biomarkers to monitor the progress and success of these treatments. Collagen is a fibrous protein and exhibits birefringence due to different refractive indices parallel and perpendicular to the direction of the fibers. As a result, changes in the collagen content and organization in the myocardium should lead to changes in birefringence. Birefringence measurements were made through ex vivo myocardial tissues from rats with induced myocardial infarctions including a number that had undergone regenerative treatment with mesenchymal stem cells. Results show a decrease in birefringence from normal to infracted myocardium, indicating a decrease in tissue organization associated with scar formation, however, an increase in birefringence was seen in those myocardial tissues that had undergone regenerative treatment indicating reorganization of tissue structure. These results demonstrate promise for this technique and are motivating further work towards performing measurements in vivo.

Successful development of cultured tissues is heavily influenced by cell alignment within the tissue scaffold. Proper cell alignment leads to optimum tissue strength. It has been demonstrated that proper alignment is engendered by application of physiologically realistic stresses during the cell proliferation process. In situ monitoring of cell alignment during development thus can provide important feedback information in determining the optimum stresses. T-matrix calculations suggest that cell alignment characteristics (cell aspect and orientation) can be inferred from the spectral polarization of light scattered by the cells. Therefore, a spectral polarimetry system has been created to measure these effects to provide feedback for proper cell alignment. In order to properly use the system, a calibration procedure was first established. The calibration procedure entailed making mathematical predictions for the system performance based on the system components, and then empirically validating these predictions. Upon system calibration, measurements were made on a biologically relevant sample. We present results of experimental measurements on the sample and discuss structural inferences made from these measurements. In addition, we compare our results with structural information obtained from histological analysis.

To ensure the sustainability of tissue engineered products there is a need to consider the engineering and manufacturing issues related to them particularly for the purposes of process optimization and product quality assurance. This work describes the application of Raman spectroscopy for in process monitoring of a skin substitute and rotating orthogonal polarization imaging to track collagen alignment in a tissue engineered tendon. The skin substitute studied is produced from culturing fibroblasts in a fibrin matrix. Throughout the production process the fibroblasts secrete extracellular matrix and in doing so deposit collagen in the matrix. Key to optimization of the skin substitute production process is development of strategies to track the collagen and fibrin content. The work presented here discusses the feasibility of Raman spectroscopy to resolve fibrin and collagen components in the skin substitutes. Collagen alignment is also important in the engineering of many tissues, in particular tendons. Thus, this work will also investigate the ability of rotating orthogonal polarization imaging to track collagen alignment in a tissue engineered tendon.

The effect of laser radiation on the generation of hyaline cartilage in spine disc and joints has been demonstrated. The paper considers physical processes and mechanisms of laser regeneration, presents results of investigations aimed to optimize laser settings and to develop feedback control system for laser reconstruction of spine discs. Possible mechanisms of laser-induced regeneration include: (1) Space and temporary modulated laser beam induces nonhomogeneous and pulse repetitive thermal expansion and stress in the irradiated zone of cartilage. Mechanical effect due to controllable thermal expansion of the tissue and micro and nano gas bubbles formation in the course of the moderate (up to 45-50 oC) heating of the NP activate biological cells (chondrocytes) and promote cartilage regeneration. (2) Nondestructive laser radiation leads to the formation of nano and micro-pores in cartilage matrix. That promotes water permeability and increases the feeding of biological cells. Results provide the scientific and engineering basis for the novel low-invasive laser procedures to be used in orthopedics for the treatment cartilages of spine and joints. The technology and equipment for laser reconstruction of spine discs have been tested first on animals, and then in a clinical trial. Since 2001 the laser reconstruction of intervertebral discs have been performed for 340 patients with chronic symptoms of low back or neck pain who failed to improve with non-operative care. Substantial relief of back pain was obtained in 90% of patients treated who returned to their daily activities. The experiments on reparation of the defects in articular cartilage of the porcine joints under temporal and spase modulated laser radiation have shown promising results.

Destructive fat tissue engineering could be realized using the optical method, which provides reduction of regional or site-specific accumulations of subcutaneous adipose tissue on the cell level. We hypothesize that light irradiation due to photodynamic and selective photothermal effects may lead to fat cell lypolytic activity (the enhancement of lipolysis of cell triglycerides due to expression of lipase activity and cell release of free fat acids (FFAs) due to temporal cell membrane porosity), and cell delayed killing due to apoptosis caused by the induced fat cell stress and/or limited cell necrosis.

Laser technique has been applied to measure cyclic contractile movement of cultured myotubes in vitro. The designed measurement system includes light source (helium neon, 632.8 nm wave length), charge-coupled devise cameras and detectors. Cyclic contraction of myotubes cultured from C2C12 (mouse myoblast) was generated by cyclic electric pulses (amplitude < 60 V, 1 ms width) through electrodes of platinum wire dipped in the medium. The spectrum of fluctuating intensity of the transmitted laser beam through the myotubes, which periodically repeated contraction and relaxation, was analyzed. The results show that the designed laser system is effective to detect frequency of cyclic contractile movement of myotubes between 0.5 Hz and 5 Hz.

Hyperspectral imaging devices are common in remote sensing reconnaissance technology. It captures both spatial and spectral information in a single pixel. Hyperspectral imaging collects the same picture on many bands of the spectrum to generate a "datacube" that can identify the materials that make up an imaging target. Hyperspectral imaging technique has an advance in molecular imaging for high-throughput evaluation using target-specific optically imaging probes and/or using intrinsic optical property. The purpose of this study is to investigate the potential of the hyperspectral imaging technology for cellular functional evaluation to determine the effectiveness and validity of the regenerative medicine. Hyperspectral imaging system was developed for minute scale imaging with high resolution using relay lens, combination of lenses to magnify the image. Various cultured cells in monolayer were used as imaging target. Hyperspectral imaging has been finally achieved by improvements in spatial resolution up to 9 μm with the spectral resolution of 1.2 nm. In order to apply this developed hyperspectral imaging system for validation of regenerative medicine, Hyperspectral imaging system performed during the process of differentiation and dedifferentiation and the process of spheroid formation as 3D culture model. Changes of cellular condition enabled to produce significant changes in the observed spectrum. Therefore, hyperspectral imaging is revealed to have a significant potential to evaluate cellular function.

Quantitative and qualitative monitoring of neovascular growth is required in many vascular tissue engineering applications. For example, the contribution of progenitor cells in growing microvasculature has been demonstrated; however, the process of vascularization from progenitor cells is not well understood. Therefore, there is a need for an imaging technique that is consistent, easy to use, and can quantitatively assess the dynamics of vascular growth or regression in a three-dimensional environment. In this study, we evaluate the ability of combined ultrasound and photoacoustic imaging to assess the dynamics of vascular growth. The experiments were performed using hydrogels that spontaneously promote tube formation from implanted mesenchymal stem cells (MSCs). Specifically, PEGylated fibrin gels, supporting the development of capillary growth were implanted in a Lewis rat. After one week, the rat was euthanized and the gel implants were excised and positioned in water cuvettes for imaging. Simultaneous ultrasound and photoacoustic images were obtained using single-element, focused ultrasound transducers interfaced with a nanosecond pulsed laser source. To image samples, ultrasound transducers operating at either 25 MHz or 48 MHz and interfaced with laser sources operating at either 532 nm or within 680-800 nm wavelengths were used. The 3-D ultrasound and photoacoustic images were acquired by mechanically scanning the transducer over the region of interest and capturing spatially co-registered and temporally consecutive photoacoustic transients and ultrasound pulse-echo signals. The ultrasound and photoacoustic images agree well with the overall anatomy and vascular structure in the gel samples. The results suggest that the photoacoustic and ultrasound imaging could be used to sequentially monitor the growth of neovasculature in-vivo.

Current efforts in tissue engineering target the growth of 3D volumes of tissue cultures in bioreactor conditions. Fluorescence optical tomography has the potential to monitor cells viability and tissue growth non-destructively directly within the bioreactor via bio-molecular fluorescent labelling strategies. We currently work on developing the imaging instrumentation for tissue cultures in bioreactor conditions. Previously, we localized in 3D thin fluorescent-labelled capillaries in a cylindrically shaped bioreactor phantom containing a diffusive medium with our time-of-flight localization technique. Here, we present our first reconstruction results of the spatial distribution of fluorophore concentrations for labelled capillaries embedded in a bioreactor phantom.