List of Plates

Figure 1.3 Cell migration into a synthetic three-dimensional (3D) scaffold. A composite 3D scaffold composed of poly(methacrylic acid) (PMMA) and poly(hydroxyethyl methacrylate) (PHEMA) was developed for cornea tissue engineering. Confocal microscopy was used to monitor migration of corneal fibroblasts into the acellular scaffold. Using a viability assay, the live or dead cells fluoresce different in different wavelengths. Because the confocal images were collected at different focal depths, reconstructed 3D images can be produced with detailed information in the direction of cell migration into the scaffold. * indicates p

Figure 1.4 Four-color imaging of osteogenesis. Human MSCs were induced to differentiate to osteoblasts. At day 14, the cells were labeled and visualized to quantify the extent of osteogenesis. Using a multiphotom microscope (Bio-Rad, Radiance 2000), a set of four fluorophores was selected to label and image simultaneously the nuclei (blue), osteocalcin expression (green), microtubule (yellow), and microfilament (red) organization. These four fluorophores were carefully chosen to minimize potential spectroscopic overlaps.

Figure 2.7 The calculated T1 and T2 as functions of the water molecule tumbling rate and magnetic field strength (100-700?MHz) calculated using the BPP theory of relaxation [50]. As the tissue matures, both T1 and T2 decrease, as shown by the red arrow. Most human tissues fall within the range shown by the blue ellipse. T2 is commonly used in the assessment of cartilage regeneration, as can be seen in a recent clinical trial [2, 6]. The figure shows that relaxation times are field dependent for soft tissues. The inset shows an example of how T2/T1 can be used as a unit-free biomarker for the assessment. As shown in the inset, the control gel has the highest T2/T1, but the ratio is lowest for osteogenic constructs with chondrogenic constructs falling between the two. Data in the inset are adapted from [25, 51].

Figure 3.6 (a) Schematic of a sample preparation for MRI measurement (the black arrow indicates the sample inside a 5?mm tube). (b) A representative T2-weighted proton MRI of an acellular scaffold. (c-e) Representative sodium MRI of chondrogenic constructs at day 7, day 14, and day 28 along with representative ROIs of the construct (bottom box) and reference media (top box). The average number of voxels in sodium images is 231?±?22. Majumdar [23]. Reproduced with the permission of Springer.

Figure 3.8 (a) TQ signal intensity as a function of creation time for tissue-engineered cartilage and their best fit with Equation 3.1 for 1-day-old engineered cartilage constructs. (b) The TQ signal of human marrow stromal cells (HMSCs) seeded in biomimetic scaffolds at week 2 and week 4. The week 4 spectrum is narrow compared to the week 2 spectrum, indicating faster motion or lower ?0tc at week 4. Reproduced with the permission from Kotecha et al. [26].

Figure 5.6 Material properties are calculated from each filtered dataset and averaged with a weight corresponding to the amplitude of the motion at each pixel.

Figure 5.7 A circular low-pass Butterworth filter is applied on every map of material properties so that they appear smoother.

Figure 5.9 Construct development map over 4-week period. Adipogenic (A) and osteogenic (O) constructs are shown from left to right with corresponding shear wave image, elastogram, and average shear stiffness. The colormap for the elastogram corresponds with the color scheme of the bar chart.

Figure 5.11 Shear wave images (top) and corresponding stiffness maps (bottom) in engineered constructs after 4 weeks of implantation. The displacement map shows the propagation of shear waves through constructs. Notice that, multiple waves are visible in adipose construct, indicating a lower stiffness and softer tissue structure, while for stiffer tissues-both osteogenic and chondrogenic-a full shear wave is not attained. Reconstructed elastogram on the bottom shows estimated stiffness of 2, 9, 15?kPa for adipogenic, chondrogenic, and osteogenic constructs, respectively.

Figure 5.12 Silk construct development map over 8-week study. Shown from left to right are the magnitude image, T2 relaxation map, shear wave image, and stiffness map of the constructs. Average T2 relaxation times decreased from 91.2 67.6?±?3.1 at week 8. Average stiffness values increased from 7.6?±?2.0?kPa 17.2?±?3.1 at week 8.

Figure 5.13 Collagen construct development map over 8-week study. Shown from left to right are the magnitude image, T2 relaxation map, and stiffness map of the constructs. Average T2 relaxation times decreased from 75.2?±?18.4?ms at week 2 to 58.4?±?4.2 at week 8. Average stiffness values increased from 4.6?±?1.7?kPa at week 2 to 14.7?±?3.8?kPa at week 8.

Figure 6.1 Example of using FEA to verify the inversion algorithm in MRE. (a) The geometry of the model is that of a fluid-filled spherical shell embedded in a stiffer medium, and a solid spherical medium with the same density as the shell embedded in the medium for comparison. (b) The wave pattern of the model under a horizontal 80-Hz harmonic excitation. (c) The stiffness map obtained from the regular Helmholtz inversion algorithm. (d) The stiffness map obtained from an effective stiffness estimation algorithm.

Figure 6.3 Displacement amplitude results for three simulations. The top row is the displacement amplitude shown on the 3D models of (a) harmonic excitation on a 3-mm diameter area in the vertical direction, (b) harmonic excitation on a 3-mm diameter area in the direction normal to the excitation plane, and (c) harmonic excitation on a 6-mm diameter area in the direction normal to the excitation plane. The bottom row is the displacement amplitude map on the short-axis slice plane of (d) harmonic excitation on a 3-mm diameter area in the vertical direction, (e) harmonic excitation on a 3-mm area in the direction normal to the excitation plane, and (f) harmonic excitation on a 6-mm are in the direction normal to the excitation plane.

Figure 7.1 Three-dimensional oxygen map of fibrosarcoma tumor and tumor bearing leg. The tumor outline, determined from a registered MR image, is shown in red. The image was acquired using 250-MHz pulse EPR oxygen imager.

Figure 7.7 Example of MR and EPR image registration. The tumor area (brighter than the leg) is determined in MR image. After image registration, the tumor area is transferred from MR to EPR image for oxygen analysis.

Figure 7.8 (a) MRI and EPR spin probe distribution in bulky acellular PLGA scaffold in PBS (sample courtesy of Dr. Syam Nukavarapu). (b) 1-mm-thick acellular collagen gel (2?mg/ml) (The gel was provided by Dr. Michael Cho.) Registered CT, EPR spin probe distribution, and mock-up oxygen image of the deoxygenated sample.

Figure 8.3 Femur example of image registration, 2D segmentation, and 3D reconstruction process. (a) CT images are loaded into and properly registered. (b) ROI is identified as appropriate differentiating color mask. (c) 3D voxel-based femur model. Sun et al. [1]. Reproduced with the permission of Elsevier.

Figure 8.5 Anatomy of the knee joint: anterior view. The knee meniscus is situated between the femur and the tibia. Crossing the meniscus are various ligaments, which aid in stabilizing the knee joint. Kohn and Moreno [82]. Reproduced with the permission of Elsevier.

Figure 8.7 Spatiotemporally released rhCTGF- and rhTGFß3-induced fibrocartilage-like matrix formation in 3D-printed porous scaffolds. (a) Anatomic reconstruction of human meniscus. Human meniscus scaffolds were 3D-printed with layer-by-layer deposition of PCL fibers (100?µm diameter), forming 100-200?µm channels. (b) Poly(lactic-co-glycolic acid) (PLGA) microspheres (mS) encapsulating rhCTGF and rhTGFß3 were in physical contact with PCL microfibers. (c) Fluorescent dextrans simulating CTGF (green, 40?kDa) and TGFß3 (red, 10 kDa) were delivered into the outer and inner zones, respectively, of human meniscus scaffolds to show scaffold loading. Distribution of dextrans was maintained from day 1 to day 8. (d) rhCTGF and rhTGFß3 release from the PCL scaffolds over time in vitro. (e) When the scaffolds were incubated at top human synovium MSC monolayers for 6 weeks, spatiotemporally delivered rhCTGF and rhTGFß3 induced cells to form zone-specific collagen type I and II matrices, similar to the native rat meniscus. (f) Scaffold with empty mS showed little matrix formation after 6 weeks of coculture with 1:1 mixture of fibrogenic and chondrogenic supplements (no growth factors in medium). Spatiotemporal delivery of rhCTGF- and rhTGFß3-induced fibrocartilaginous matrix formation, consisting of alcian blue-positive, collagen II-rich cartilaginous matrix in the inner zone and...