Functional and molecular characterisation of mesenchymal stem cells derived from bone marrow and dental tissues.

Mesenchymal stromal/stem cells (MSCs) are multipotent, progenitor cells with the ability to differentiate into cells of mesenchymal and non-mesenchymal tissues in vitro and in vivo. Characterisation of MSC-like cells residing in dental pulp (DPSC) and periodontal ligament (PDLSC) tissues has shown that these cells exhibit similar features to bone marrow stromal cells (BMSCs). The present project proposed that BMSCs, DPSCs and PDLSCs contain heterogeneous populations of progenitor cells including a minor subset of multipotential stem cells. Identification of the precise developmental stages and generation of gene expression profiles of these different cell populations will greatly improve our understanding of the fundamental cellular processes involved in MSC differentiation and proliferation pathways. Therefore, the aim of this PhD project was to assess the unique phenotype of BMSCs, DPSCs and PDLSCs and their differentiated progeny by studying their proliferative and differentiation potentials, and by assessing their gene expression profiles. Initial findings obtained from functional analyses of clonal populations present within BMSCs, DPSCs and PDLSCs support the hypothesis that within each of the populations, co-exist cells of varying levels of proliferation and differentiation potentials. Following on from these results, whole genome microarray analyses were used to determine gene expression patterns of clonal cell fractions exhibiting significant functional differences within each MSC population. The microarray data identified cohorts of differentially expressed genes in comparisons of cell clones of low proliferation potential and high/multi-differentiation potential within BMSC, DPSC and PDLSC populations. Further interrogation of the generated data identified 24 differentially up-regulated genes in long lived/multi-potential MSC clones, common to BMSC, DPSC and PDLSC populations. Specifically, E2F-2, LDB-2, PTTG-1 and TWIST-1, were identified as transcriptional regulators or co-regulators that may be involved in the proliferation and differentiation of these MSC populations. In light of the previously reported involvement of TWIST-1 in growth and development of BMSC, the effect of enforced TWIST-1 expression was investigated in DPSC and PDLSC populations. These findings demonstrated that TWIST-1 holds a stimulatory role in cell proliferation and is involved in the regulation of differentiation/commitment processes in MSCs. Overall, the identification of genes associated with MSCs of high growth and developmental potential lays the foundation for further definition of molecular mechanisms involved in MSC maintenance and survival. Elucidation of these fundamental processes is highly significant as it holds a critical role in the development of MSC-based tissue regeneration therapies.Thesis (Ph.D.) -- University of Adelaide, School of Dentistry, 201

Transport Parameters of Inkjet Printed Nanoparticle Silver on Polyimide Substrate Measured at Room and Liquid Nitrogen Temperatures

This brief presents the transport parameters of silver layers on a polyimide substrate fabricated by an inkjet printing technology using nanoparticle inks with 20 wt% and 40 wt% silver. All the electrical characteristics of the samples were measured by Hall effect measurement system at 0.37 T. The electron mobility in inkjet printed silver layer was 7.07 and 4.58 cm(2)/V s for 20 wt% silver, 29.6 and 21.3 cm(2)/V s for 40 wt% silver at 77 and 300 K, respectively. The resistivity in inkjet printed silver layer with 20 wt% is 20.6 x 10(-6) and 27.1 x 10(-6) Omega cm, and 4.81 x 10(-6) and 8.27 x 10(-6) Omega cm for 40 wt% silver at 77 and 300 K, respectively. The surface morphology and profiles of the samples were obtained with an atomic force and scanning electron microscope

Investigation of the Cell Surface Proteome of Human Periodontal Ligament Stem Cells

The present study examined the cell surface proteome of human periodontal ligament stem cells (PDLSC) compared to human fibroblasts. Cell surface proteins were prelabelled with CyDye before processing to extract the membrane lysates, which were separated using 2D electrophoresis. Selected differentially expressed protein “spots” were identified using Mass spectrometry. Four proteins were selected for validation: CD73, CD90, Annexin A2, and sphingosine kinase 1 previously associated with mesenchymal stem cells. Flow cytometric analysis found that CD73 and CD90 were highly expressed by human PDLSC and gingival fibroblasts but not by keratinocytes, indicating that these antigens could be used as potential markers for distinguishing between mesenchymal cells and epithelial cell populations. Annexin A2 was also found to be expressed at low copy number on the cell surface of human PDLSC and gingival fibroblasts, while human keratinocytes lacked any cell surface expression of Annexin A2. In contrast, sphingosine kinase 1 expression was detected in all the cell types examined using immunocytochemical analysis. These proteomic studies form the foundation to further define the cell surface protein expression profile of PDLSC in order to better characterise this cell population and help develop novel strategies for the purification of this stem cell population

Improved Performance of Multilayer CPW Inductors on Flexible Substrate

In this paper, we present the modeling, fabrication, measurement techniques, and characterization results of multilayer miniature coplanar waveguide (CPW) meander-type inductors for high-frequency applications. Detailed procedure of the fabrication process based on using ink-jet printing technology is given. Silver nanoparticle ink that contains 20 wt% of silver was printed on a flexible polyimide foil 50 mu m thick in one, two, and three layers. It is shown that multilayer CPW inductors have improved performance. Almost two times better Q-factor value was achieved. This is an encouraging result for multilayer printing in ink-jet technology

Fabrication of a novel triphasic and bioactive ceramic and evaluation of its in vitro and in vivo cytocompatibility and osteogenesis

We report, for the first time, the synthesis of a novel triphasic and crystalline bioactive ceramic (MSM-10) with the ability to simultaneously release three types of bioactive ions (strontium (Sr), silicon (Si) and magnesium (Mg)) to the surrounding microenvironment. An MSM-10 powder with a nominal composition (wt%) of 54 Mg2SiO4, 36 Si 3Sr5 and 10 MgO was prepared by the sol-gel method and fabricated as porous scaffolds using the foam replication method. The effects of the different amounts of the phases in the ceramics on the mechanical and physical properties of the scaffolds as well as their in vitro and in vivo behaviors were comprehensively investigated. Biphasic calcium phosphate (BCP, β-tricalcium phosphate (60 wt%)/hydroxyapatite (40 wt%)) scaffolds were used as the control material. The attachment, morphology, proliferation and differentiation of primary human osteoblasts (HOBs) were investigated after cell culturing on the various scaffolds. In vitro cytotoxicity (ISO/EN 10993-5) results not only indicated the biocompatibility of MSM-10, but also its positive effects on inducing the proliferation of HOBs. Our results showed significant enhancement in osteogenic gene expression levels (Runx2, osteocalcin, osteopontin and bone sialoprotein), when HOBs were cultured on MSM-10, compared to those for BCP and other generated ceramic scaffolds. For the in vivo studies, the different types of the materials were seeded with cultured human mesenchymal stem cells (hMSC) and then subcutaneously transplanted into the dorsal surface of eight-week-old immunocompromised (NOD/SCID) mice. MSM-10 demonstrated a significant amount of new bone formation compared to the other groups tested with no macroscopic signs of inflammation or toxicity in the tissue surrounding the implants. The novel MSM-10 ceramic presents promising potential for bone regeneration in orthopaedic and maxillofacial applications. © 2014 The Royal Society of Chemistry

Histology of CMSCs and DMSCs transplants.

<p>Cross sections are representative of CMSCs transplants (A-B) and DMSCs transplants (E-F) after 8 weeks stained with Haematoxylin and Eosin (H&E). In the transplant, the HA/TCP carrier surfaces (dashed lines) are lined with new bone formation (b), areas of immature bone (ob) together with the surrounding fibrous and hematopoietic tissue (a) and blood vessel (bv). Representative BrdU staining for localization of implanted CMSCs (C-D) and DMSCs (G-H). BrdU-stained implanted cells were found lining the mineralized matrix (black arrows) and surrounding fibrous tissue. Brown nuclear staining is indicative of DAB reactivity. There was no immunoreactivity present in sections stained with isotype-matched antibodies. HA/TCP: hydroxyapatite/tricalcium phosphate particles. Magnification is 100X and scalebar is 500 μm.</p

Antibodies used for characterizing CMSCs and DMSCs by flow cytometry.

<p>^a anti-human antibodies raised in mice.</p><p>Antibodies used for characterizing CMSCs and DMSCs by flow cytometry.</p

CMSC phenotypic characterization.

<p>A. (i) Bright field microscopy image of CMSCs at P0. Magnification is 100X and scalebar is 100 μm. (ii) CMSCs from placentae of male newborns were analyzed using interphase FISH on MSC nuclei. CMSCs showed one chromosome X (Spectrum Green) and one chromosome Y (Spectrum Orange) signals. Cell nuclei were stained blue with DAPI. Magnification is 630X. B. Primary CMSCs cell surface markers expression. Histograms of representative primary CMSC at P3 depicting the expression of CD90, CD146, CD166, CD44, CD73, CD105, CD45, HLA-DR, and CD19. The red histogram shows the MSC marker antibody staining while the white histogram shows the corresponding isotype control antibody staining. PE: phycoerythrin dye, APC: allophycocyanin dye, APC-Cy7: allophycocyanin-Cy7 dye. C. Representative photomicrographs showing CMSCs differentiation into mesenchymal lineages. (i) Osteogenic differentiation, Alizarin Red staining in cells after 5weeks growth in osteogenic induction medium. Arrows show calcium depositions. (ii) Adipogenic differentiation, Oil Red O staining in cells after 14 days growth in adipogenic induction medium. Arrows show fat droplets. (iii) Chondrogenic differentiation, Safranin O staining for proteoglycans depositions in cells after 21 days growth in chondrogenic induction medium. Inset shows control uninduced CMSCs. Scalebar is 100 μm.</p

Loss of Grem1-lineage chondrogenic progenitor cells causes osteoarthritis

Abstract Osteoarthritis (OA) is characterised by an irreversible degeneration of articular cartilage. Here we show that the BMP-antagonist Gremlin 1 (Grem1) marks a bipotent chondrogenic and osteogenic progenitor cell population within the articular surface. Notably, these progenitors are depleted by injury-induced OA and increasing age. OA is also caused by ablation of Grem1 cells in mice. Transcriptomic and functional analysis in mice found that articular surface Grem1-lineage cells are dependent on Foxo1 and ablation of Foxo1 in Grem1-lineage cells caused OA. FGFR3 signalling was confirmed as a promising therapeutic pathway by administration of pathway activator, FGF18, resulting in Grem1-lineage chondrocyte progenitor cell proliferation, increased cartilage thickness and reduced OA. These findings suggest that OA, in part, is caused by mechanical, developmental or age-related attrition of Grem1 expressing articular cartilage progenitor cells. These cells, and the FGFR3 signalling pathway that sustains them, may be effective future targets for biological management of OA