Indocyanine Green (ICG) Lymphography Is Superior to Lymphoscintigraphy for Diagnostic Imaging of Early Lymphedema of the Upper Limbs

BACKGROUND: Secondary lymphedema causes swelling in limbs due to lymph retention following lymph node dissection in cancer therapy. Initiation of treatment soon after appearance of edema is very important, but there is no method for early diagnosis of lymphedema. In this study, we compared the utility of four diagnostic imaging methods: magnetic resonance imaging (MRI), computed tomography (CT), lymphoscintigraphy, and Indocyanine Green (ICG) lymphography. PATIENTS AND METHODS: Between April 2010 and November 2011, we examined 21 female patients (42 arms) with unilateral mild upper limb lymphedema using the four methods. The mean age of the patients was 60.4 years old (35-81 years old). Biopsies of skin and collecting lymphatic vessels were performed in 7 patients who underwent lymphaticovenous anastomosis. RESULTS: The specificity was 1 for all four methods. The sensitivity was 1 in ICG lymphography and MRI, 0.62 in lymphoscintigraphy, and 0.33 in CT. These results show that MRI and ICG lymphography are superior to lymphoscintigraphy or CT for diagnosis of lymphedema. In some cases, biopsy findings suggested abnormalities in skin and lymphatic vessels for which lymphoscintigraphy showed no abnormal findings. ICG lymphography showed a dermal backflow pattern in these cases. CONCLUSIONS: Our findings suggest the importance of dual diagnosis by examination of the lymphatic system using ICG lymphography and evaluation of edema in subcutaneous fat tissue using MRI

Smooth Muscle-Like Tissue Constructs with Circumferentially Oriented Cells Formed by the Cell Fiber Technology

<div><p>The proper functioning of many organs and tissues containing smooth muscles greatly depends on the intricate organization of the smooth muscle cells oriented in appropriate directions. Consequently controlling the cellular orientation in three-dimensional (3D) cellular constructs is an important issue in engineering tissues of smooth muscles. However, the ability to precisely control the cellular orientation at the microscale cannot be achieved by various commonly used 3D tissue engineering building blocks such as spheroids. This paper presents the formation of coiled spring-shaped 3D cellular constructs containing circumferentially oriented smooth muscle-like cells differentiated from dedifferentiated fat (DFAT) cells. By using the cell fiber technology, DFAT cells suspended in a mixture of extracellular proteins possessing an optimized stiffness were encapsulated in the core region of alginate shell microfibers and uniformly aligned to the longitudinal direction. Upon differentiation induction to the smooth muscle lineage, DFAT cell fibers self-assembled to coiled spring structures where the cells became circumferentially oriented. By changing the initial core-shell microfiber diameter, we demonstrated that the spring pitch and diameter could be controlled. 21 days after differentiation induction, the cell fibers contained high percentages of ASMA-positive and calponin-positive cells. Our technology to create these smooth muscle-like spring constructs enabled precise control of cellular alignment and orientation in 3D. These constructs can further serve as tissue engineering building blocks for larger organs and cellular implants used in clinical treatments.</p></div

Core ECM protein optimization.

<p>(a) Bar graph of the average Young’s modulus of various types of ECM protein, organized in increasing stiffness. n = 50, and data are expressed as the mean ± standard deviation. (b) PCol and ACol-L failed to support fiber formation as shown in the day 1 images where DFAT cells mainly aggregated into spheroids as opposed to cell fibers. (c) ACol-H is stiff enough to support initial formation of DFAT cells into fiber structures, but failed to maintain the fiber structures as almost half of the cells already aggregated into spheroids by day 10. All concentrations of fibrin tested successfully supported initial DFAT cell fiber formation (day 1), but by day 33, almost all cells in Fibrin-L (d) and about half of the cells in Fibrin-M (e) aggregated to spheroids, and only Fibrin-H (f) still maintained the fiber structures. (g) Mixture of collagen and fibrin, ACol-Fib, is also able to support initial DFAT cell fiber formation as well as maintain the fiber structures for at least 33 days. Scale bar = 1 mm.</p

Formation of DFAT cell fiber.

<p>(a) Schematic drawing of the microfluidic double co-axial device used to encapsulate cells into the core of alginate fibers with the default flow rates. The inner core stream consist of DFAT cells suspended in the appropriate ECM proteins. The outer shell stream is sodium alginate solution that solidifies when it comes in contact with the sheath stream of calcium chloride solution. (b) Cell fiber formation overnight. A day 0 image captured immediately following fiber fabrication with single DFAT cells dispersed in ECM proteins encapsulated in the center core of the alginate fiber, and a day 1 image showing a section of the formed DFAT cell fiber inside alginate shell. (c) Images comparing DFAT cell alignment when cultured in conventional 2D dish and as 3D fiber (day 2) structure. DFAT cells are randomly oriented when attached on 2D dishes, whereas DFAT cells are highly aligned along the longitudinal fiber direction when cultured as 3D fiber structure.</p

Overall concept of the study.

<p>DFAT cells derived from adipocytes are encapsulated into the center core of alginate fibers and allowed to form into cell fiber. DFAT cell fiber is subsequently induced for differentiation into smooth muscle cell lineage in induction media containing TGF-β. As cells’ inherent cell traction force increases during this process, linear cell fiber self-assembles to coiled spring structure. Such smooth muscle-like cell springs may serve as tissue engineering building blocks.</p

Cell spring characterization.

<p>(a) Schematic image defining various parameters of a spring. (b) Graph showing the spring pitch over 30 days of culture. The spring pitch reached about 300 μm by day 2 or 3 of culture and remains constant. n = 9, and data are expressed as the mean ± standard deviation. (c) Graph showing the spring diameter over 30 days of culture. The spring diameter decreased to about 200 μm by day 5 and stays constant for the rest of the culture period. n = 10, and data are expressed as the mean ± standard deviation. (d) Spring pitch and (e) spring diameter as functions of fiber diameter D_f. As the fiber diameter increases, both spring pitch and diameter also increase. n ≥ 3, and data are expressed as the mean ± standard deviation. (f), (g), (h) Images of cell springs with D_f = 300, 400, and 500 μm, respectively. Scale bar = 200 μm.</p