Contributions of the Bone Marrow Microenvironment to Bone and Skeletal Metastasis.

The skeleton, a favored organ for prostate cancer is organized by a mineralized connective tissue, and a rich marrow where hematopoiesis replenishes a variety of blood cells and gives rise to many cell populations. Thus, when tumors metastasize to bones they encounter many cells, once considered bystanders such as hematopoietic stem cells and macrophages, which play key roles in tumor growth progression and metastasis. Macrophages are implicated in both skeletal homeostasis and tumorigenesis; yet their role in skeletal metastasis is unclear. Macrophage phagocytosis of apoptotic cells is referred to as efferocytosis, and is an integral process by which harmful by-products of dead and dying cells are removed to create a pro-resolving environment. The purpose of this study was to determine the role of macrophages and efferocytosis in prostate cancer skeletal metastasis. In vivo experimental approaches resulting in macrophage ablation showed significant reduction in tumor growth in tibiae after intratibial tumor inoculations. Efferocytosis of apoptotic tumor cells increased MFG-E8 expression and promoted macrophage polarization into the M2 macrophage phenotype. Conversely, efferocytosis inhibition with neutralizing MFG-E8 antibody resulted in reduced M2 polarization, suggesting that efferocytosis is important for macrophage polarization into tumor promoting M2 cells. The involvement of the STAT3/SOCS3 activation pathway in macrophage polarization was observed. Increased MFG-E8 levels when bone marrow macrophages were co-cultured with apoptotic cells was accompanied by SOCS3 downregulation. Inhibition of STAT3 phosphorylation resulted in decreased efferocytosis and M2 macrophage polarization with an associated increase in SOCS3 protein expression. This suggests that SOCS3 and phospho-STAT3 act in an inversely dependent manner when stimulated by MFG-E8 and efferocytosis. Therefore we report a novel mechanism by which MFG-E8, by mediating efferocytosis of prostate cancer cells, can support tumor growth through facilitation of M2 macrophage polarization and regulation of SOCS3/STAT3 activation. In conclusion, the bone microenvironment provides a dynamic and rich soil for tumors to thrive. Continued investigation on the role of bone marrow cells will provide a better understanding of the metastatic bone environment and aid in the advancement of new targets for the treatment and prevention of skeletal metastasis.PHDOral Health SciencesUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/111335/1/fabisoki_1.pd

Leptin Functions Peripherally to Regulate Differentiation of Mesenchymal Progenitor Cells

Leptin functions through a well-documented central neuroendocrine pathway to regulate bone mass. However, the ability of leptin to modulate bone mass through a peripheral mechanism has been debated due to conflicting in vitro results and lack of sufficient in vivo models. We utilized mice with LoxP sites introduced into the long-form leptin receptor (ObRb) gene to determine how leptin regulates mesenchymal progenitor cell (MPC) differentiation and osteoblast function in vitro and in vivo . Rapid phosphorylation of Stat3 after leptin treatment of bone marrow stromal cells (BMSCs) from mice with conditional deletion of ObRb in macrophages (LysM Cre+F/F ) confirmed expression of functional leptin receptors by BMSCs. Adenovirus-Cre mediated disruption of ObRb in primary stromal cells decreased mineralization and increased adipogenesis. In contrast, BMSCs harvested from leptin-signaling deficient Ob/Ob or Db/Db mice showed increased mineralization. To determine the physiologic relevance of these differences, mice with cell-specific deletion of ObRb in mesenchymal precursors (3.6 Cre+F/F ) or osteoblasts (2.3 Cre+F/F ) were generated. Although the 2.3 Cre+F/F mice were grossly normal, the 3.6 Cre+F/F mice displayed mild obesity that was not attributed to food intake. Femurs of 3.6 Cre+F/F animals showed a 58%–61.9% increase in trabecular bone volume and a 65.5%–74% increase in bone mineral density. Cortical volume and mineral content were also increased 18%–22%. Primary 3.6 Cre+F/F BMSCs recapitulated the high mineralization phenotype of Ob/Ob and Db/Db BMSCs. We conclude that leptin may have multiple peripheral roles depending on the differentiation state of MPC. Leptin (a) helps maintain MPCs in an undifferentiated state and (b) promotes mineralization of more differentiated osteoblasts. S TEM C ells 2010;28:1071–1080Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/77453/1/432_ftp.pd

Inhibitory effects of megakaryocytic cells in prostate cancer skeletal metastasis

Prostate cancer cells commonly spread through the circulation, but few successfully generate metastatic foci in bone. Osteoclastic cellular activity has been proposed as an initiating event for skeletal metastasis. Megakaryocytes (MKs) inhibit osteoclastogenesis, which could have an impact on tumor establishment in bone. Given the location of mature MKs at vascular sinusoids, they may be the first cells to physically encounter cancer cells as they enter the bone marrow. Identification of the interaction between MKs and prostate cancer cells was the focus of this study. K562 (human MK precursors) and primary MKs derived from mouse bone marrow hematopoietic precursor cells potently suppressed prostate carcinoma PC-3 cells in coculture. The inhibitory effects were specific to prostate carcinoma cells and were enhanced by direct cell-cell contact. Flow cytometry for propidium iodide (PI) and annexin V supported a proapoptotic role for K562 cells in limiting PC-3 cells. Gene expression analysis revealed reduced mRNA levels for cyclin D1, whereas mRNA levels of apoptosis-associated specklike protein containing a CARD (ASC) and death-associated protein kinase 1 (DAPK1) were increased in PC-3 cells after coculture with K562 cells. Recombinant thrombopoietin (TPO) was used to expand MKs in the marrow and resulted in decreased skeletal lesion development after intracardiac tumor inoculation. These novel findings suggest a potent inhibitory role of MKs in prostate carcinoma cell growth in vitro and in vivo. This new finding, of an interaction of metastatic tumors and hematopoietic cells during tumor colonization in bone, ultimately will lead to improved therapeutic interventions for prostate cancer patients. © 2011 American Society for Bone and Mineral Research.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/78486/1/204_ftp.pd

Stress intensity factor threshold in dental porcelains

The stress intensity factor threshold (K(IO)) is related to the stress level at which cracks start to grow stably, causing the weakening of porcelain prostheses during their use. The values of K(IO) of seven dental porcelains (with and without reinforcing leucite crystal, KAlSi(2)O(6)) stored in air (22 degrees C, 60% relative humidity) and artificial saliva (37 degrees C) were determined by measuring the crack growth velocity of radial cracks generated at the corner of Vickers indentations. The results of K(IO) were correlated with the leucite content, fracture toughness (K(Ic)), and chemical composition of the porcelains. It was observed that K(IO) increased with the increase of leucite content (only for the leucite-based porcelains) and with the increase of K(Ic). The increase in Al(2)O(3) content or the decrease in the alkali oxide (K(2)O and Na(2)O) content of the material`s glassy matrix tended to increase the K(IO) values. Storage media (air and saliva) did not significantly affect the K(IO) of porcelains tested, indicating that the control parameter of K(IO) value was not the water content of the storage media

Comparative analysis of two colorimetric assays in dental pulp cell density. International Endodontic Journal

Aim: To compare and contrast two colorimetric assays used for the measurement of proliferation using two dental pulp cell types: dental pulp stem cells (DPSC) and human dental pulp fibroblasts (HDPF). Methodology Dental pulp stem cells or HDPF were seeded at 0.25 × 104 cells per well in 96-well plates. Cell proliferation was evaluated after 24–72 h. At the end of the experimental period, the sulforhodamine B (SRB) assay or a water-soluble tetrazolium salt (WST-1) assay was performed. Optical densities were determined in a microplate reader (Genius; TECAN). Data were analysed by Student’s t-test (comparison between cell types) and one-way anova followed by Tukey test (time-point intervals). Pearson’ correlation tests were performed to compare the two assays for each cell line. Results Both assays showed that DPSC had higher proliferation rates than HDPF. A positive significant correlation between the two colorimetric assays tested for both cell types DPSC (Pearson’s correlation coefficient = 0.847; P \u3c 0.05) and HDPF (Pearson’s correlation coefficient = 0.775; P \u3c 0.05). Conclusion Both tests demonstrated similar trends of cell proliferation, and thus are both appropriate for the evaluation of DPSC and HDPF. The choice of assay is therefore one of the practical applications. SRB stained plates can be dried and stored so may have utility in laboratories where data may require review or when access to analytical equipment is limited. WST-1 assays have the benefit of both ease and speed and may have utility in laboratories requiring either high throughput or rapid analyses

Comparative analysis of two colorimetric assays in dental pulp cell density. International Endodontic Journal

Aim: To compare and contrast two colorimetric assays used for the measurement of proliferation using two dental pulp cell types: dental pulp stem cells (DPSC) and human dental pulp fibroblasts (HDPF). Methodology Dental pulp stem cells or HDPF were seeded at 0.25 × 104 cells per well in 96-well plates. Cell proliferation was evaluated after 24–72 h. At the end of the experimental period, the sulforhodamine B (SRB) assay or a water-soluble tetrazolium salt (WST-1) assay was performed. Optical densities were determined in a microplate reader (Genius; TECAN). Data were analysed by Student’s t-test (comparison between cell types) and one-way anova followed by Tukey test (time-point intervals). Pearson’ correlation tests were performed to compare the two assays for each cell line. Results Both assays showed that DPSC had higher proliferation rates than HDPF. A positive significant correlation between the two colorimetric assays tested for both cell types DPSC (Pearson’s correlation coefficient = 0.847; P \u3c 0.05) and HDPF (Pearson’s correlation coefficient = 0.775; P \u3c 0.05). Conclusion Both tests demonstrated similar trends of cell proliferation, and thus are both appropriate for the evaluation of DPSC and HDPF. The choice of assay is therefore one of the practical applications. SRB stained plates can be dried and stored so may have utility in laboratories where data may require review or when access to analytical equipment is limited. WST-1 assays have the benefit of both ease and speed and may have utility in laboratories requiring either high throughput or rapid analyses