Chondrogenesis and mineralization during in vitro culture of human mesenchymal stem cells on three-dimensional woven scaffolds.

Human mesenchymal stem cells (hMSCs) and three-dimensional (3D) woven poly(ɛ-caprolactone) (PCL) scaffolds are promising tools for skeletal tissue engineering. We hypothesized that in vitro culture duration and medium additives can individually and interactively influence the structure, composition, mechanical, and molecular properties of engineered tissues based on hMSCs and 3D poly(ɛ-caprolactone). Bone marrow hMSCs were suspended in collagen gel, seeded on scaffolds, and cultured for 1, 21, or 45 days under chondrogenic and/or osteogenic conditions. Structure, composition, biomechanics, and gene expression were analyzed. In chondrogenic medium, cartilaginous tissue formed by day 21, and hypertrophic mineralization was observed in the newly formed extracellular matrix at the interface with underlying scaffold by day 45. Glycosaminoglycan, hydroxyproline, and calcium contents, and alkaline phosphatase activity depended on culture duration and medium additives, with significant interactive effects (all p < 0.0001). The 45-day constructs exhibited mechanical properties on the order of magnitude of native articular cartilage (aggregate, Young's, and shear moduli of 0.15, 0.12, and 0.033 MPa, respectively). Gene expression was characteristic of chondrogenesis and endochondral bone formation, with sequential regulation of Sox-9, collagen type II, aggrecan, core binding factor alpha 1 (Cbfα1)/Runx2, bone sialoprotein, bone morphogenetic protein-2, and osteocalcin. In contrast, osteogenic medium produced limited osteogenesis. Long-term culture of hMSC on 3D scaffolds resulted in chondrogenesis and regional mineralization at the interface between soft, newly formed engineered cartilage, and stiffer underlying scaffold. These findings merit consideration when developing grafts for osteochondral defect repair.

Hand-held spectroscopic device for in vivo and intraoperative tumor detection: contrast enhancement, detection sensitivity, and tissue penetration.

Surgery is one of the most effective and widely used procedures in treating human cancers, but a major problem is that the surgeon often fails to remove the entire tumor, leaving behind tumor-positive margins, metastatic lymph nodes, and/or satellite tumor nodules. Here we report the use of a hand-held spectroscopic pen device (termed SpectroPen) and near-infrared contrast agents for intraoperative detection of malignant tumors, based on wavelength-resolved measurements of fluorescence and surface-enhanced Raman scattering (SERS) signals. The SpectroPen utilizes a near-infrared diode laser (emitting at 785 nm) coupled to a compact head unit for light excitation and collection. This pen-shaped device effectively removes silica Raman peaks from the fiber optics and attenuates the reflected excitation light, allowing sensitive analysis of both fluorescence and Raman signals. Its overall performance has been evaluated by using a fluorescent contrast agent (indocyanine green, or ICG) as well as a surface-enhanced Raman scattering (SERS) contrast agent (pegylated colloidal gold). Under in vitro conditions, the detection limits are approximately 2-5 × 10(-11) M for the indocyanine dye and 0.5-1 × 10(-13) M for the SERS contrast agent. Ex vivo tissue penetration data show attenuated but resolvable fluorescence and Raman signals when the contrast agents are buried 5-10 mm deep in fresh animal tissues. In vivo studies using mice bearing bioluminescent 4T1 breast tumors further demonstrate that the tumor borders can be precisely detected preoperatively and intraoperatively, and that the contrast signals are strongly correlated with tumor bioluminescence. After surgery, the SpectroPen device permits further evaluation of both positive and negative tumor margins around the surgical cavity, raising new possibilities for real-time tumor detection and image-guided surgery.

Molecular characterization of chordoma xenografts generated from a novel primary chordoma cell source and two chordoma cell lines.

OBJECT: Chordoma cells can generate solid-like tumors in xenograft models that express some molecular characteristics of the parent tumor, including positivity for brachyury and cytokeratins. However, there is a dearth of molecular markers that relate to chordoma tumor growth, as well as the cell lines needed to advance treatment. The objective in this study was to isolate a novel primary chordoma cell source and analyze the characteristics of tumor growth in a mouse xenograft model for comparison with the established U-CH1 and U-CH2b cell lines. METHODS: Primary cells from a sacral chordoma, called "DVC-4," were cultured alongside U-CH1 and U-CH2b cells for more than 20 passages and characterized for expression of CD24 and brachyury. While brachyury is believed essential for driving tumor formation, CD24 is associated with healthy nucleus pulposus cells. Each cell type was subcutaneously implanted in NOD/SCID/IL2Rγ(null) mice. The percentage of solid tumors formed, time to maximum tumor size, and immunostaining scores for CD24 and brachyury (intensity scores of 0-3, heterogeneity scores of 0-1) were reported and evaluated to test differences across groups. RESULTS: The DVC-4 cells retained chordoma-like morphology in culture and exhibited CD24 and brachyury expression profiles in vitro that were similar to those for U-CH1 and U-CH2b. Both U-CH1 and DVC-4 cells grew tumors at rates that were faster than those for U-CH2b cells. Gross tumor developed at nearly every site (95%) injected with U-CH1 and at most sites (75%) injected with DVC-4. In contrast, U-CH2b cells produced grossly visible tumors in less than 50% of injected sites. Brachyury staining was similar among tumors derived from all 3 cell types and was intensely positive (scores of 2-3) in a majority of tissue sections. In contrast, differences in the pattern and intensity of staining for CD24 were noted among the 3 types of cell-derived tumors (p < 0.05, chi-square test), with evidence of intense and uniform staining in a majority of U-CH1 tumor sections (score of 3) and more than half of the DVC-4 tumor sections (scores of 2-3). In contrast, a majority of sections from U-CH2b cells stained modestly for CD24 (scores of 1-2) with a predominantly heterogeneous staining pattern. CONCLUSIONS: This is the first report on xenografts generated from U-CH2b cells in which a low tumorigenicity was discovered despite evidence of chordoma-like characteristics in vitro. For tumors derived from a primary chordoma cell and U-CH1 cell line, similarly intense staining for CD24 was observed, which may correspond to their similar potential to grow tumors. In contrast, U-CH2b tumors stained less intensely for CD24. These results emphasize that many markers, including CD24, may be useful in distinguishing among chordoma cell types and their tumorigenicity in vivo.

Contrast in intracardiac acoustic radiation force impulse images of radiofrequency ablation lesions.

We have previously shown that intracardiac acoustic radiation force impulse (ARFI) imaging visualizes tissue stiffness changes caused by radiofrequency ablation (RFA). The objectives of this in vivo study were to (1) quantify measured ARFI-induced displacements in RFA lesion and unablated myocardium and (2) calculate the lesion contrast (C) and contrast-to-noise ratio (CNR) in two-dimensional ARFI and conventional intracardiac echo images. In eight canine subjects, an ARFI imaging-electroanatomical mapping system was used to map right atrial ablation lesion sites and guide the acquisition of ARFI images at these sites before and after ablation. Readers of the ARFI images identified lesion sites with high sensitivity (90.2%) and specificity (94.3%) and the average measured ARFI-induced displacements were higher at unablated sites (11.23 ± 1.71 µm) than at ablated sites (6.06 ± 0.94 µm). The average lesion C (0.29 ± 0.33) and CNR (1.83 ± 1.75) were significantly higher for ARFI images than for spatially registered conventional B-mode images (C = -0.03 ± 0.28, CNR = 0.74 ± 0.68).

Signal Improvement and Contrast Enhancement in Magnetic Resonance Imaging

This thesis reports advances in magnetic resonance imaging (MRI), with the ultimate goal of improving signal and contrast in biomedical applications. More specifically, novel MRI pulse sequences have been designed to characterize microstructure, enhance signal and contrast in tissue, and image functional processes. In this thesis, rat brain and red bone marrow images are acquired using iMQCs (intermolecular multiple quantum coherences) between spins that are 10 μm to 500 μm apart. As an important application, iMQCs images in different directions can be used for anisotropy mapping. We investigate tissue microstructure by analyzing anisotropy mapping. At the same time, we simulated images expected from rat brain without microstructure. We compare those with experimental results to prove that the dipolar field from the overall shape only has small contributions to the experimental iMQC signal. Besides magnitude of iMQCs, phase of iMQCs should be studied as well. The phase anisotropy maps built by our method can clearly show susceptibility information in kidneys. It may provide meaningful diagnostic information. To deeply study susceptibility, the modified-crazed sequence is developed. Combining phase data of modified-crazed images and phase data of iMQCs images is very promising to construct microstructure maps. Obviously, the phase image in all above techniques needs to be highly-contrasted and clear. To achieve the goal, algorithm tools from Susceptibility-Weighted Imaging (SWI) and Susceptibility Tensor Imaging (STI) stands out superb useful and creative in our system.

In vivo small animal micro-CT using nanoparticle contrast agents.

Computed tomography (CT) is one of the most valuable modalities for in vivo imaging because it is fast, high-resolution, cost-effective, and non-invasive. Moreover, CT is heavily used not only in the clinic (for both diagnostics and treatment planning) but also in preclinical research as micro-CT. Although CT is inherently effective for lung and bone imaging, soft tissue imaging requires the use of contrast agents. For small animal micro-CT, nanoparticle contrast agents are used in order to avoid rapid renal clearance. A variety of nanoparticles have been used for micro-CT imaging, but the majority of research has focused on the use of iodine-containing nanoparticles and gold nanoparticles. Both nanoparticle types can act as highly effective blood pool contrast agents or can be targeted using a wide variety of targeting mechanisms. CT imaging can be further enhanced by adding spectral capabilities to separate multiple co-injected nanoparticles in vivo. Spectral CT, using both energy-integrating and energy-resolving detectors, has been used with multiple contrast agents to enable functional and molecular imaging. This review focuses on new developments for in vivo small animal micro-CT using novel nanoparticle probes applied in preclinical research.