Substrate Strain and Proliferation & Differentiation of Bone Marrow Mesenchymal Stem Cells

New methods of surface modification of transparent silicone substrate were developed, and a new set of cell culture devices that provide homogeneous substrate strain was designed. Using the new device, effects of cyclic substrate strain on bone marrow mesenchymal stem cells(MSCs) were studied. It was found that cyclic strain influenced proliferation and differentiation of bone marrow MSCs in different ways.

Vascular endothelial growth factor regulates melanoma cell adhesion and growth in the bone marrow microenvironment via tumor cyclooxygenase-2

Background: Human melanoma frequently colonizes bone marrow (BM) since its earliest stage of systemic dissemination, prior to clinical metastasis occurrence. However, how melanoma cell adhesion and proliferation mechanisms are regulated within bone marrow stromal cell (BMSC) microenvironment remain unclear. Consistent with the prometastatic role of inflammatory and angiogenic factors, several studies have reported elevated levels of cyclooxygenase-2 (COX-2) in melanoma although its pathogenic role in bone marrow melanoma metastasis is unknown. Methods: Herein we analyzed the effect of cyclooxygenase-2 (COX-2) inhibitor celecoxib in a model of generalized BM dissemination of left cardiac ventricle-injected B16 melanoma (B16M) cells into healthy and bacterial endotoxin lipopolysaccharide (LPS)-pretreated mice to induce inflammation. In addition, B16M and human A375 melanoma (A375M) cells were exposed to conditioned media from basal and LPS-treated primary cultured murine and human BMSCs, and the contribution of COX-2 to the adhesion and proliferation of melanoma cells was also studied. Results: Mice given one single intravenous injection of LPS 6 hour prior to cancer cells significantly increased B16M metastasis in BM compared to untreated mice; however, administration of oral celecoxib reduced BM metastasis incidence and volume in healthy mice, and almost completely abrogated LPS-dependent melanoma metastases. In vitro, untreated and LPS-treated murine and human BMSC-conditioned medium (CM) increased VCAM-1-dependent BMSC adherence and proliferation of B16M and A375M cells, respectively, as compared to basal medium-treated melanoma cells. Addition of celecoxib to both B16M and A375M cells abolished adhesion and proliferation increments induced by BMSC-CM. TNF alpha and VEGF secretion increased in the supernatant of LPS-treated BMSCs; however, anti-VEGF neutralizing antibodies added to B16M and A375M cells prior to LPS-treated BMSC-CM resulted in a complete abrogation of both adhesion-and proliferation-stimulating effect of BMSC on melanoma cells. Conversely, recombinant VEGF increased adherence to BMSC and proliferation of both B16M and A375M cells, compared to basal medium-treated cells, while addition of celecoxib neutralized VEGF effects on melanoma. Recombinant TNFa induced B16M production of VEGF via COX-2-dependent mechanism. Moreover, exogenous PGE2 also increased B16M cell adhesion to immobilized recombinant VCAM-1. Conclusions: We demonstrate the contribution of VEGF-induced tumor COX-2 to the regulation of adhesion-and proliferation-stimulating effects of TNFa, from endotoxin-activated bone marrow stromal cells, on VLA-4-expressing

A Trabecular Bone Explant Model of Osteocyte–Osteoblast Co-Culture for Bone Mechanobiology

The osteocyte network is recognized as the major mechanical sensor in the bone remodeling process, and osteocyte-osteoblast communication acts as an important mediator in the coordination of bone formation and turnover. In this study, we developed a novel 3D trabecular bone explant co-culture model that allows live osteocytes situated in their native extracellular matrix environment to be interconnected with seeded osteoblasts on the bone surface. Using a low-level medium perfusion system, the viability of in situ osteocytes in bone explants was maintained for up to 4 weeks, and functional gap junction intercellular communication (GJIC) was successfully established between osteocytes and seeded primary osteoblasts. Using this novel co-culture model, the effects of dynamic deformational loading, GJIC, and prostaglandin E-2 (PGE(2)) release on functional bone adaptation were further investigated. The results showed that dynamical deformational loading can significantly increase the PGE(2) release by bone cells, bone formation, and the apparent elastic modulus of bone explants. However, the inhibition of gap junctions or the PGE(2) pathway dramatically attenuated the effects of mechanical loading. This 3D trabecular bone explant co-culture model has great potential to fill in the critical gap in knowledge regarding the role of osteocytes as a mechano-sensor and how osteocytes transmit signals to regulate osteoblasts function and skeletal integrity as reflected in its mechanical properties.

Fluid flow induced calcium response in bone cell network

In our previous work, bone cell networks with controlled spacing and functional intercellular gap junctions had been successfully established by using microcontact printing and self assembled monolayers technologies [Guo, X. E., E. Takai, X. Jiang, Q. Xu, G. M. Whitesides, J. T. Yardley, C. T. Hung, E. M. Chow, T. Hantschel, and K. D. Costa. Mol. Cell. Biomech. 3:95-107, 2006]. The present study investigated the calcium response and the underlying signaling pathways in patterned bone cell networks exposed to a steady fluid flow. The glass slides with cell networks were separated into eight groups for treatment with specific pharmacological agents that inhibit pathways significant in bone cell calcium signaling. The calcium transients of the network were recorded and quantitatively evaluated with a set of network parameters. The results showed that 18 alpha-GA (gap junction blocker), suramin (ATP inhibitor), and thapsigargin (depleting intracellular calcium stores) significantly reduced the occurrence of multiple calcium peaks, which were visually obvious in the untreated group. The number of responsive peaks also decreased slightly yet significantly when either the COX-2/PGE(2) or the NOS/nitric oxide pathway was disrupted. Different from all other groups, cells treated with 18 alpha-GA maintained a high concentration of intracellular calcium following the first peak. In the absence of calcium in the culture medium, the intracellular calcium concentration decreased slowly with fluid flow without any calcium transients observed. These findings have identified important factors in the flow mediated calcium signaling of bone cells within a patterned network.

Ring-opening metathesis of bulky norbornene monomers and the radical-mediated hydrophosphonation of olefins

This thesis discusses two major topics: the ring-opening metathesis polymerization (ROMP) of bulky monomers and the radical-mediated hydrophosphonation of olefins. The research into the ROMP of bulky monomers is further divided into three chapters: wedge-shaped monomers, the alternating copolymerization of 1-methyloxanorbornene derivatives with cyclooctene, and the kinetic resolution polymerization of 1-methyloxanorbornene derivatives. The wedge-shaped monomers can be polymerized into diblock copolymers that possess photonic crystal properties. The alternating copolymerization of 1-methyloxanorbornene derivatives with cyclooctene is performed with > 90% alternation via two different routes: typical alternating copolymerization and a sequence editing approach. The kinetic resolution polymerization of these same 1-methyloxanorbornene monomers achieves only modest selectivity (S=4), but there is evidence that the growing polymer chain forms a helix that influences the selectivity of the resolution. The last topic is the radical-mediated hydrophosphonation of olefins. This synthetic method provides access to Wittig reagents that are capable of highly cis-selective olefinations of aldehydes.

Aqueous ring-opening metathesis polymerizations

Past workers in this group as well as in others have made considerable progress in the understanding and development of the ring-opening metathesis polymerization (ROMP) technique. Through these efforts, ROMP chemistry has become something of an organometallic success story. Extensive work was devoted to trying to identify the catalytically active species in classical reaction mixtures of early metal halides and alkyl aluminum compounds. Through this work, a mechanism involving the interconversion of metal carbenes and metallacyclobutanes was proposed. This preliminary work finally led to the isolation and characterization of stable metal carbene and metallacyclobutane complexes. As anticipated, these well-characterized complexes were shown to be active catalysts. In a select number of cases, these catalysts have been shown to catalyze the living polymerization of strained rings such as norbornene. The synthetic control offered by these living systems places them in a unique category of metal catalyzed reactions. To take full advantage of these new catalysts, two approaches should be explored. The first takes advantage of the unusual fact that all of the unsaturation present in the monomer is conserved in the polymer product. This makes ROMP techniques ideal for the synthesis of highly unsaturated, and fully conjugated polymers, which find uses in a variety of applications. This area is currently under intense investigation. The second aspect, which should lend itself to fruitful investigations, is expanding the utility of these catalysts through the living polymerization of monomers containing interesting functional groups. Polymer properties can be dramatically altered by the incorporation of functional groups. It is this latter aspect which will be addressed in this work.

After a general introduction to both the ring-opening metathesis reaction (Chapter 1) and the polymerization of fuctionalized monomers by transition metal catalysts (Chapter 2), the limits of the existing living ROMP catalysts with functionalized monomers are examined in Chapter 3. Because of the stringent limitations of these early metal catalysts, efforts were focused on catalysts based on ruthenium complexes. Although not living, and displaying unusually long induction periods, these catalysts show high promise for future investigations directed at the development of catalysts for the living polymerization of functionalized monomers. In an attempt to develop useful catalysts based on these ruthenium complexes, efforts to increase their initiation rates are presented in Chapter 4. This work eventually led to the discovery that these catalysts are highly active in aqueous solution, providing the opportunity to develop aqueous emulsion ROMP systems. Recycling the aqueous catalysts led to the discovery that the ruthenium complexes become more activated with use. Investigations of these recycled solutions uncovered new ruthenium-olefin complexes, which are implicated in the activation process. Although our original goal of developing living ROMP catalysts for the polymerization of fuctionalized monomers is yet to be realized, it is hoped that this work provides a foundation from which future investigations can be launched.

In the last chapter, the ionophoric properties of the poly(7-oxanobornene) materials is briefly discussed. Their limited use as acyclic host polymers led to investigations into the fabrication of ion-permeable membranes fashioned from these materials.

Ultrafast transient ring-shaped population grating

We demonstrate an ultrafast transient, ring-shaped population grating induced by an ultrashort hollow Gaussian laser bullet by solving the three-dimensional full-wave Maxwell-Bloch equations. Through adjusting the beam waist and the area of the pulse, we can control the number of lines and the period of the grating. Based on this coherent control scheme, a door to produce gratings with complex transverse structure is opened.

Characteristics of an add-drop filter composed of a Mach-Zehnder interferometer and double ring resonators

A planar lightwave circuit (PLC) add-drop filter is proposed and analyzed, which consists of a symmetric Mach-Zehnder interferometer (MZI) combined with double microring resonators. A critical coupling condition is derived for a better box-like drop spectrum. Comparisons of its characteristics with other schemes, such as a MZI with a single ring resonator, arepresented, and some of the issues about device design and fabrication are also discussed.

Analysis of a second-order polarization-independent filter made of a single ring resonator and a Sagnac interferometer

A novel second-order polarization-independent filter made of a single ring resonator and a Sagnac interferometer (SRRSI) is proposed, and its filtering characteristics are investigated. By using birefringence in waveguide, a single ring resonator can be used to synthesize a filter with second-order response. Analytical formulas are derived for characteristics of the SRRSI varied with waveguide parameters.. such as the coupling coefficient; and the critical condition of a second-order Butterworth filter is given. The influence of loss in the ring resonator is also analyzed. (c) 2005 Elsevier B.V. All rights reserved.