Application of a human bone engineering platform to an in vitro and in vivo breast cancer metastasis model

Application of a Human Bone Engineering Platform to an In Vitro and In Vivo Breast Cancer Metastasis Model Verena Maria Charlotte Reichert, MD Faculty of Built Environment and Engineering, School of Engineering Systems, Queensland University of Technology Thesis submitted for: Doctor of Philosophy (PhD) 2011 Keywords Breast cancer, bone metastasis, human osteoblast matrix, breast cancer related bone disease, adhesion, single cell force spectroscopy, migration, invasion, tissue engineering, scaffold, polycaprolactone, osteoblasts, human mesenchymal stem cells I II Abstract Breast cancer in its advanced stage has a high predilection to the skeleton Currently, treatment options of breast cancer-related bone metastasis are restricted to only palliative therapeutic modalities This is due to the fact that mechanisms regarding the breast cancer celI-bone colonisation as well as the interactions of breast cancer cells with the bone microenvironment are not fully understood, yet This might be explained through a lack of appropriate in vitro and in vivo models that are currently addressing the above mentioned issue Hence the hypothesis that the translation of a bone tissue engineering platform could lead to improved and more physiological in vitro and in vivo model systems in order to investigate breast cancer related bone colonisation was embraced in this PhD thesis Therefore the first objective was to develop an in vitro model system that mimics human mineralised bone matrix to the highest possible extent to examine the specific biological question, how the human bone matrix influences breast cancer cell behaviour Thus, primary human osteoblasts were isolated from human bone and cultured under osteogenic conditions Upon ammonium hydroxide treatment, a cell-free intact mineralised human bone matrix was left behind Analyses revealed a similar protein and mineral composition of the decellularised osteoblast matrix to human bone Seeding of a panel of breast cancer cells onto the bone mimicking matrix as well as reference substrates like standard tissue culture plastic and collagen coated tissue culture plastic revealed substrate specific differences of cellular behaviour Analyses of attachment, alignment, migration, proliferation, invasion, as well as downstream signalling pathways showed that these cellular properties were influenced through the osteoblast matrix The second objective of this PhD project was the development of a human ectopic bone polycaprolactone model in NOD/SCID tricalcium phosphate mice using medical grade (mPCL-TCP) scaffold Human osteoblasts and mesenchymal stem cells were seeded onto an mPCL-TCP III scaffold, fabricated using a fused deposition modelling technique After subcutaneous implantation in conjunction with the bone morphogenetic protein 7, limited bone formation was observed due to the mechanical properties of the applied scaffold and restricted integration into the soft tissue of flank of NOD/SCID mice Thus, a different scaffold fabrication technique was chosen using the same polymer Electrospun tubular scaffolds were seeded with human osteoblasts, as they showed previously the highest amount of bone formation and implanted into the flanks of NOD/SCID mice Ectopic bone formation with sufficient vascularisation could be observed After implantation of breast cancer cells using a polyethylene glycol hydrogel in close proximity to the newly formed bone, macroscopic communication between the newly formed bone and the tumour could be observed Taken together, this PhD project showed that bone tissue engineering platforms could be used to develop an in vitro and in vivo model system to study cancer cell colonisation in the bone microenvironment IV Table of contents Keywords I Abstract III Table of contents V List of illustrations and diagrams XIII List of abbreviations XIX Statement of original authorship XXV Acknowledgements XXVII Chapter - Overview 1 Overview Chapter - Literature Review - Mechanisms of breast cancer-related bone metastasis – overview of currently used in vitro and in vivo models 2.1 Epidemiology – facts about breast cancer 2.2 Clinical presentation of bone metastases and treatment options 2.3 Tumour cell metastasis – a multistep process – from the primary site to the skeleton 11 2.4 Characteristics of the “the congenial soil” – bone 13 2.5 How the “seeds” find their “congenial” soil 18 2.6 Attachment at the metastatic site - interaction of integrins with bone matrix constituents 19 2.7 More factors involved in the promotion of bone metastasis 23 2.8 Osteomimicry of breast cancer cells 26 2.9 Osteolytic bone metastasis 27 2.10 Models investigating bone-cancer cell interaction in vitro 29 2.11 Models investigating bone-cancer cell interaction in vivo 37 2.12 Summary 46 Chapter - Establishment of a human primary osteoblast matrix model 49 3.1 Introduction 51 V 3.2 Material and Methods 53 3.2.1 Characterisation of type I collagen coated tissue culture plastic 53 3.2.1.1 Collagen type I coating of tissue culture polystyrene or thermanox coverslips 53 3.2.1.2 Anti-col I staining of col I coated coverlips 53 3.2.1.3 Scanning electron microscopy analysis of col I coated thermanox coverslips 54 3.2.2 Generation and characterisation of primary human osteoblast matrices 54 3.2.2.1 Isolation of human primary osteoblasts and matrix production 54 3.2.2.2 Confocal laser microscopy to confirm completeness of decellularisation 55 3.2.2.3 Morphology - SEM 56 3.2.2.4 Morphology - Transmission Electron microscopy 56 3.2.2.5 Mineralisation - Alizarin red S 56 3.2.2.6 Mineralisation - Wako HRII calcium assay 56 3.2.2.7 Mineralisation - X-ray photoelectron spectroscopy (XPS) 57 3.2.2.8 Mineralisation - RAMAN analysis 57 3.2.2.9 Composition - Immunohistochemistry 58 3.2.2.10 Composition - Growth factor analysis 59 3.2.2.11 Composition - 2D PAGE with MALDI or LC/MS/MS 59 3.2.2.12 Composition - Western blot analysis 59 3.2.3 Image analysis 60 3.2.4 Statistical analysis 61 3.3 Results 62 VI 3.3.1 Presence and Morphology of collagen I on coated thermanox coverslips 62 3.3.2 Characterisation of the composition and morphology of the human decellularised osteoblast matrix 63 3.4 Discussion 75 3.5 Conclusion 79 Chapter - Interactions of human breast cancer cells with the OBM 81 4.1 Introduction 83 4.2 Materials and Methods 85 4.2.1 Characteristics and growth conditions for cell lines used in this study 85 4.2.1.1 MCF10A 85 4.2.1.2 T47D 86 4.2.1.3 SUM1315 86 4.2.1.4 MDA-MB-231 86 4.2.1.5 MDA-MB-231SA 87 4.2.1.6 Gene expression profile 88 4.2.2 Morphology 89 4.2.2.1 Flow cytometry 89 4.2.2.2 Morphology - Transmitted light microscopy 90 4.2.2.3 Morphology – Fluorescent staining 90 4.2.2.4 Morphology - SEM 91 4.2.3 Cell proliferation 92 4.2.4 Cell adhesion 92 4.2.5 Cell migration 95 4.2.6 Cell Invasion 96 4.2.6.1 SEM image analysis 96 4.2.6.2 Confocal microscopy image analysis 98 VII 4.2.7 Cell signalling pathways 98 4.2.8 Gene expression 100 4.2.8.1 RNA isolation 100 4.2.8.2 cDNA synthesis 100 4.2.8.3 qRT-PCR 101 4.2.9 4.3 Statistical analysis 102 Results 103 4.3.1 Characteristics of utilised cell lines 103 4.3.1.1 MCF10A 103 4.3.1.2 T47D 104 4.3.1.3 SUM1315 105 4.3.1.4 MDA-MB-231 106 4.3.1.5 MDA-MB-231-SA 107 4.3.2 Cell morphology of different cell lines on TCP, col I and OBM 113 4.3.2.1 Morphology on TCP, col I and OBM assessed with transmitted light and confocal laser scanning microscopy 113 4.3.2.2 4.3.3 Image analysis 116 Proliferation of MCF10A, T47D, SUM1315, MDA-MB231 and MDA-MB-231SA on TCP, col I and OBM 120 4.3.4 Assessment of established detachment forces of each cell line to disconnect from col I and OBM 121 4.3.5 Migratory features of the BC cell lines on the three investigated substrates 123 4.3.6 Invasive potential of MCF10A, T47D, SUM1315, MDA-MB-231 and MDA-MB-231SA cells 127 4.3.6.1 SEM image analysis 127 4.3.6.2 Confocal microscopy image analysis 127 VIII 100 101 102 103 104 105 106 107 108 109 110 111 112 Ruppender, N.S., A.R Merkel, T.J Martin, G.R Mundy, J.A Sterling, and S.A Guelcher, Matrix rigidity induces osteolytic gene expression of metastatic breast cancer cells PLoS One 5(11): p e15451 Kim, S.S., M Sun Park, O Jeon, C Yong Choi, and B.S Kim, Poly(lactide-co-glycolide)/hydroxyapatite composite scaffolds for bone tissue engineering Biomaterials, 2006 27(8): p 1399-409 Hutmacher, D.W., D Loessner, S Rizzi, D.L Kaplan, D.J Mooney, and J.A Clements, Can tissue engineering concepts advance tumor biology research? Trends Biotechnol, 2010 28(3): p 125-33 Ingber, D.E., Can cancer be reversed by engineering the tumor microenvironment? Semin Cancer Biol, 2008 18(5): p 356-64 Goldstein, R.H., R.A Weinberg, and M Rosenblatt, Of mice and (wo)men: mouse models of breast cancer metastasis to bone J Bone Miner Res, 2010 25(3): p 431-6 Burga, L.N., H Hu, A Juvekar, N.M Tung, S.L Troyan, E.W Hofstatter, and G.M Wulf, Loss of BRCA1 leads to an increase in epidermal growth factor receptor expression in mammary epithelial cells, and epidermal growth factor receptor-inhibition prevents estrogen receptor-negative cancers in BRCA1-mutant mice Breast Cancer Res, 2011 13(2): p R30 Bist, P., S.C Leow, Q.H Phua, S Shu, Q Zhuang, W.T Loh, T.H Nguyen, J.B Zhou, S.C Hooi, and L.H Lim, Annexin-1 interacts with NEMO and RIP1 to constitutively activate IKK complex and NFkappaB: implication in breast cancer metastasis Oncogene, 2011 Fang, W.B., I Jokar, A Chytil, H.L Moses, T Abel, and N Cheng, Loss of one Tgfbr2 allele in fibroblasts promotes metastasis in MMTV: polyoma middle T transgenic and transplant mouse models of mammary tumor progression Clin Exp Metastasis, 2011 Ciampricotti, M., K Vrijland, C.S Hau, T Pemovska, C.W Doornebal, E.N Speksnijder, K Wartha, J Jonkers, and K.E de Visser, Development of metastatic HER2(+) breast cancer is independent of the adaptive immune system J Pathol, 2010 Mohammadnejad, J., M.J Rasaee, M.H Babaei, M Paknejad, M.H Zahir, M Salouti, A.B Rajabi, and M Mazidi, A new radiopharmaceutical compound (131I-PR81) for radioimmunotherapy of breast cancer: labeling of antibody and its quality control Hum Antibodies, 2010 19(4): p 79-88 Nannuru, K.C., M Futakuchi, M.L Varney, T.M Vincent, E.G Marcusson, and R.K Singh, Matrix metalloproteinase (MMP)-13 regulates mammary tumor-induced osteolysis by activating MMP9 and transforming growth factor-beta signaling at the tumor-bone interface Cancer Res, 2010 70(9): p 3494-504 Bendre, M.S., A.G Margulies, B Walser, N.S Akel, S Bhattacharrya, R.A Skinner, F Swain, V Ramani, K.S Mohammad, L.L Wessner, A Martinez, T.A Guise, J.M Chirgwin, D Gaddy, and L.J Suva, Tumor-derived interleukin-8 stimulates osteolysis independent of the receptor activator of nuclear factor-kappaB ligand pathway Cancer Res, 2005 65(23): p 11001-9 Cowey, S., A.A Szafran, J Kappes, K.R Zinn, G.P Siegal, R.A Desmond, H Kim, L Evans, and R.W Hardy, Breast cancer 236 113 114 115 116 117 118 119 120 121 122 123 metastasis to bone: evaluation of bioluminescent imaging and microSPECT/CT for detecting bone metastasis in immunodeficient mice Clin Exp Metastasis, 2007 24(5): p 389-401 Phadke, P.A., R.R Mercer, J.F Harms, Y Jia, A.R Frost, J.L Jewell, K.M Bussard, S Nelson, C Moore, J.C Kappes, C.V Gay, A.M Mastro, and D.R Welch, Kinetics of metastatic breast cancer cell trafficking in bone Clin Cancer Res, 2006 12(5): p 1431-40 Chanda, D., T Isayeva, S Kumar, G.P Siegal, A.A Szafran, K.R Zinn, V.V Reddy, and S Ponnazhagan, Systemic osteoprotegerin gene therapy restores tumor-induced bone loss in a therapeutic model of breast cancer bone metastasis Mol Ther, 2008 16(5): p 871-8 Bandyopadhyay, A., J.K Agyin, L Wang, Y Tang, X Lei, B.M Story, J.E Cornell, B.H Pollock, G.R Mundy, and L.Z Sun, Inhibition of pulmonary and skeletal metastasis by a transforming growth factorbeta type I receptor kinase inhibitor Cancer Res, 2006 66(13): p 6714-21 Richert, M.M., K.S Vaidya, C.N Mills, D Wong, W Korz, D.R Hurst, and D.R Welch, Inhibition of CXCR4 by CTCE-9908 inhibits breast cancer metastasis to lung and bone Oncol Rep, 2009 21(3): p 7617 Price, J.T., J.M Quinn, N.A Sims, J Vieusseux, K Waldeck, S.E Docherty, D Myers, A Nakamura, M.C Waltham, M.T Gillespie, and E.W Thompson, The heat shock protein 90 inhibitor, 17-allylamino17-demethoxygeldanamycin, enhances osteoclast formation and potentiates bone metastasis of a human breast cancer cell line Cancer Res, 2005 65(11): p 4929-38 Kim, M.Y., T Oskarsson, S Acharyya, D.X Nguyen, X.H Zhang, L Norton, and J Massague, Tumor self-seeding by circulating cancer cells Cell, 2009 139(7): p 1315-26 Dunn, L.K., K.S Mohammad, P.G Fournier, C.R McKenna, H.W Davis, M Niewolna, X.H Peng, J.M Chirgwin, and T.A Guise, Hypoxia and TGF-beta drive breast cancer bone metastases through parallel signaling pathways in tumor cells and the bone microenvironment PLoS One, 2009 4(9): p e6896 Huck, L., S.M Pontier, D.M Zuo, and W.J Muller, beta1-integrin is dispensable for the induction of ErbB2 mammary tumors but plays a critical role in the metastatic phase of tumor progression Proc Natl Acad Sci U S A, 2010 107(35): p 15559-64 Han, H.J., J Russo, Y Kohwi, and T Kohwi-Shigematsu, SATB1 reprogrammes gene expression to promote breast tumour growth and metastasis Nature, 2008 452(7184): p 187-93 Lam, P., W Yang, Y Amemiya, H Kahn, A Yee, C Holloway, and A Seth, A human bone NOD/SCID mouse model to distinguish metastatic potential in primary breast cancers Cancer Biol Ther, 2009 8(11): p 1010-7 Sangai, T., H Fujimoto, S Miyamoto, H Maeda, M Nakamura, G Ishii, K Nagai, T Nagashima, M Miyazaki, and A Ochiai, Roles of osteoclasts and bone-derived IGFs in the survival and growth of human breast cancer cells in human adult bone implanted into 237 124 125 126 127 128 129 130 131 132 133 134 135 nonobese diabetic/severe combined immunodeficient mice Clin Exp Metastasis, 2008 25(4): p 401-10 Shtivelman, E and R Namikawa, Species-specific metastasis of human tumor cells in the severe combined immunodeficiency mouse engrafted with human tissue Proc Natl Acad Sci U S A, 1995 92(10): p 4661-5 Nemeth, J.A., J.F Harb, U Barroso, Jr., Z He, D.J Grignon, and M.L Cher, Severe combined immunodeficient-hu model of human prostate cancer metastasis to human bone Cancer Res, 1999 59(8): p 198793 Yonou, H., T Yokose, T Kamijo, N Kanomata, T Hasebe, K Nagai, T Hatano, Y Ogawa, and A Ochiai, Establishment of a novel species- and tissue-specific metastasis model of human prostate cancer in humanized non-obese diabetic/severe combined immunodeficient mice engrafted with human adult lung and bone Cancer Res, 2001 61(5): p 2177-82 Kuperwasser, C., S Dessain, B.E Bierbaum, D Garnet, K Sperandio, G.P Gauvin, S.P Naber, R.A Weinberg, and M Rosenblatt, A mouse model of human breast cancer metastasis to human bone Cancer Res, 2005 65(14): p 6130-8 Yang, W., P Lam, R Kitching, H.J Kahn, A Yee, J.E Aubin, and A Seth, Breast cancer metastasis in a human bone NOD/SCID mouse model Cancer Biol Ther, 2007 6(8): p 1289-94 Moreau, J.E., K Anderson, J.R Mauney, T Nguyen, D.L Kaplan, and M Rosenblatt, Tissue-engineered bone serves as a target for metastasis of human breast cancer in a mouse model Cancer Res, 2007 67(21): p 10304-8 Balbin, M., A Fueyo, A.M Tester, A.M Pendas, A.S Pitiot, A Astudillo, C.M Overall, S.D Shapiro, and C Lopez-Otin, Loss of collagenase-2 confers increased skin tumor susceptibility to male mice Nat Genet, 2003 35(3): p 252-7 Husemann, Y and C.A Klein, The analysis of metastasis in transgenic mouse models Transgenic Res, 2009 18(1): p 1-5 Backlund, M.G., S.L Trasti, D.C Backlund, V.L Cressman, V Godfrey, and B.H Koller, Impact of ionizing radiation and genetic background on mammary tumorigenesis in p53-deficient mice Cancer Res, 2001 61(17): p 6577-82 Miller, F.R., B.E Miller, and G.H Heppner, Characterization of metastatic heterogeneity among subpopulations of a single mouse mammary tumor: heterogeneity in phenotypic stability Invasion Metastasis, 1983 3(1): p 22-31 Eckhardt, B.L., B.S Parker, R.K van Laar, C.M Restall, A.L Natoli, M.D Tavaria, K.L Stanley, E.K Sloan, J.M Moseley, and R.L Anderson, Genomic analysis of a spontaneous model of breast cancer metastasis to bone reveals a role for the extracellular matrix Mol Cancer Res, 2005 3(1): p 1-13 Yang, J., S.A Mani, J.L Donaher, S Ramaswamy, R.A Itzykson, C Come, P Savagner, I Gitelman, A Richardson, and R.A Weinberg, Twist, a master regulator of morphogenesis, plays an essential role in tumor metastasis Cell, 2004 117(7): p 927-39 238 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 Siveen, K.S and G Kuttan, Role of macrophages in tumour progression Immunol Lett, 2009 123(2): p 97-102 Gabrilovich, D., Mechanisms and functional significance of tumourinduced dendritic-cell defects Nat Rev Immunol, 2004 4(12): p 94152 Gallimore, A and A Godkin, Regulatory T cells and tumour immunity - observations in mice and men Immunology, 2008 123(2): p 15763 Wang, C.Y and Y.W Chang, A model for osseous metastasis of human breast cancer established by intrafemur injection of the MDAMB-435 cells in nude mice Anticancer Res, 1997 17(4A): p 2471-4 Buijs, J.T., I Que, C.W Lowik, S.E Papapoulos, and G van der Pluijm, Inhibition of bone resorption and growth of breast cancer in the bone microenvironment Bone, 2009 44(2): p 380-6 DeSantis, C., A Jemal, E Ward, and M.J Thun, Temporal trends in breast cancer mortality by state and race Cancer Causes Control, 2008 19(5): p 537-45 Yoneda, T., A Sasaki, C Dunstan, P.J Williams, F Bauss, Y.A De Clerck, and G.R Mundy, Inhibition of osteolytic bone metastasis of breast cancer by combined treatment with the bisphosphonate ibandronate and tissue inhibitor of the matrix metalloproteinase-2 J Clin Invest, 1997 99(10): p 2509-17 Peyruchaud, O., B Winding, I Pecheur, C.M Serre, P Delmas, and P Clezardin, Early detection of bone metastases in a murine model using fluorescent human breast cancer cells: application to the use of the bisphosphonate zoledronic acid in the treatment of osteolytic lesions J Bone Miner Res, 2001 16(11): p 2027-34 Canon, J.R., M Roudier, R Bryant, S Morony, M Stolina, P.J Kostenuik, and W.C Dougall, Inhibition of RANKL blocks skeletal tumor progression and improves survival in a mouse model of breast cancer bone metastasis Clin Exp Metastasis, 2008 25(2): p 119-29 Price, J.E., A Polyzos, R.D Zhang, and L.M Daniels, Tumorigenicity and metastasis of human breast carcinoma cell lines in nude mice Cancer Res, 1990 50(3): p 717-21 Shultz, L.D., P.A Schweitzer, S.W Christianson, B Gott, I.B Schweitzer, B Tennent, S McKenna, L Mobraaten, T.V Rajan, D.L Greiner, and et al., Multiple defects in innate and adaptive immunologic function in NOD/LtSz-scid mice J Immunol, 1995 154(1): p 180-91 Mercadante, S., Malignant bone pain: pathophysiology and treatment Pain, 1997 69(1-2): p 1-18 Mundy, G.R., Metastasis to bone: causes, consequences and therapeutic opportunities Nat Rev Cancer, 2002 2(8): p 584-93 Solomayer, E.F., I.J Diel, G.C Meyberg, C Gollan, and G Bastert, Metastatic breast cancer: clinical course, prognosis and therapy related to the first site of metastasis Breast Cancer Res Treat, 2000 59(3): p 271-8 Bellahcene, A., M Kroll, F Liebens, and V Castronovo, Bone sialoprotein expression in primary human breast cancer is associated 239 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 with bone metastases development J Bone Miner Res, 1996 11(5): p 665-70 Naujoks C, B.K., Meyer U, Laboratory Procedures - Culture of Cells and Tissues, in Fundamentals of Tissue Engineering and Regenerative Medicine, Springer: New York p 565-565 Reichert, J.C., V.M Quent, L.J Burke, S.H Stansfield, J.A Clements, and D.W Hutmacher, Mineralized human primary osteoblast matrices as a model system to analyse interactions of prostate cancer cells with the bone microenvironment Biomaterials, 2010 31(31): p 792836 Gentleman, E., R.J Swain, N.D Evans, S Boonrungsiman, G Jell, M.D Ball, T.A Shean, M.L Oyen, A Porter, and M.M Stevens, Comparative materials differences revealed in engineered bone as a function of cell-specific differentiation Nat Mater, 2009 8(9): p 76370 Zaragosi, L.E., G Ailhaud, and C Dani, Autocrine fibroblast growth factor signaling is critical for self-renewal of human multipotent adipose-derived stem cells Stem Cells, 2006 24(11): p 2412-9 Shevchenko, A., H Tomas, J Havlis, J.V Olsen, and M Mann, In-gel digestion for mass spectrometric characterization of proteins and proteomes Nat Protoc, 2006 1(6): p 2856-60 Olszta, M.J., X Cheng, S.S Jee, R Kumar, Y.-Y Kim, M.J Kaufman, E.P Douglas, and L.B Gower, Bone structure and formation: A new perspective Materials Science and Engineering: R: Reports, 2007 58(3-5): p 77-116 Deshpande, A.S and E Beniash, Bioinspired synthesis of mineralized collagen fibrils Crystal Growth & Design, 2008 8(8): p 3084-3090 Mastro, A.M and E.A Vogler, A three-dimensional osteogenic tissue model for the study of metastatic tumor cell interactions with bone Cancer Res, 2009 69(10): p 4097-100 Rubin, M.A., I Jasiuk, J Taylor, J Rubin, T Ganey, and R.P Apkarian, TEM analysis of the nanostructure of normal and osteoporotic human trabecular bone Bone, 2003 33(3): p 270-82 Wagner, H.D and S Weiner, On the relationship between the microstructure of bone and its mechanical stiffness J Biomech, 1992 25(11): p 1311-20 Yin, J.J., C.B Pollock, and K Kelly, Mechanisms of cancer metastasis to the bone Cell Res, 2005 15(1): p 57-62 Price, P.A., A.A Otsuka, J.W Poser, J Kristaponis, and N Raman, Characterization of a gamma-carboxyglutamic acid-containing protein from bone Proc Natl Acad Sci U S A, 1976 73(5): p 1447-51 Boskey, A.L., S Gadaleta, C Gundberg, S.B Doty, P Ducy, and G Karsenty, Fourier transform infrared microspectroscopic analysis of bones of osteocalcin-deficient mice provides insight into the function of osteocalcin Bone, 1998 23(3): p 187-96 Kelm, R.J., Jr., N.A Swords, T Orfeo, and K.G Mann, Osteonectin in matrix remodeling A plasminogen-osteonectin-collagen complex J Biol Chem, 1994 269(48): p 30147-53 Ross, F.P., J Chappel, and J.I Alvarez, Interactions between the bone matrix proteins osteopontin and bone sialoprotein and the 240 166 167 168 169 170 171 172 173 174 175 176 177 osteoclast integrin {alpha}vß3 potentiate bone resorption J Biol Chem, 1993 268: p 9901-7 Zhang, J.H., J Tang, J Wang, W Ma, W Zheng, T Yoneda, and J Chen, Over-expression of bone sialoprotein enhances bone metastasis of human breast cancer cells in a mouse model Int J Oncol, 2003 23(4): p 1043-8 Fedarko, N.S., B Fohr, P.G Robey, M.F Young, and L.W Fisher, Factor H binding to bone sialoprotein and osteopontin enables tumor cell evasion of complement-mediated attack J Biol Chem, 2000 275(22): p 16666-72 Chiquet-Ehrismann, R and R.P Tucker, Connective tissues: signalling by tenascins Int J Biochem Cell Biol, 2004 36(6): p 10859 Mackie, E.J and S Ramsey, Modulation of osteoblast behaviour by tenascin J Cell Sci, 1996 109 ( Pt 6): p 1597-604 Jones, F.S and P.L Jones, The tenascin family of ECM glycoproteins: structure, function, and regulation during embryonic development and tissue remodeling Dev Dyn, 2000 218(2): p 23559 Tang, J., Y.M Wu, P Zhao, J.L Jiang, and Z.N Chen, Betaig-h3 interacts with alpha3beta1 integrin to promote adhesion and migration of human hepatoma cells Exp Biol Med (Maywood), 2009 234(1): p 35-9 Genge, B.R., L.N Wu, and R.E Wuthier, Differential fractionation of matrix vesicle proteins Further characterization of the acidic phospholipid-dependent Ca2(+)-binding proteins J Biol Chem, 1990 265(8): p 4703-10 Selicharova, I., M Sanda, J Mladkova, S.S Ohri, A Vashishta, M Fusek, J Jiracek, and V Vetvicka, 2-DE analysis of breast cancer cell lines 1833 and 4175 with distinct metastatic organ-specific potentials: comparison with parental cell line MDA-MB-231 Oncol Rep, 2008 19(5): p 1237-44 Shimizu-Sasaki, E., M Yamazaki, S Furuyama, H Sugiya, J Sodek, and Y Ogata, Identification of FGF2-response element in the rat bone sialoprotein gene promoter Connect Tissue Res, 2003 44 Suppl 1: p 103-8 Ribatti, D., A Vacca, M Rusnati, and M Presta, The discovery of basic fibroblast growth factor/fibroblast growth factor-2 and its role in haematological malignancies Cytokine Growth Factor Rev, 2007 18(3-4): p 327-34 Levental, K.R., H Yu, L Kass, J.N Lakins, M Egeblad, J.T Erler, S.F Fong, K Csiszar, A Giaccia, W Weninger, M Yamauchi, D.L Gasser, and V.M Weaver, Matrix crosslinking forces tumor progression by enhancing integrin signaling Cell, 2009 139(5): p 891-906 Goetz, J.G., S Minguet, I Navarro-Lerida, J.J Lazcano, R Samaniego, E Calvo, M Tello, T Osteso-Ibanez, T Pellinen, A Echarri, A Cerezo, A.J Klein-Szanto, R Garcia, P.J Keely, P Sanchez-Mateos, E Cukierman, and M.A Del Pozo, Biomechanical 241 178 179 180 181 182 183 184 185 186 187 188 189 190 remodeling of the microenvironment by stromal caveolin-1 favors tumor invasion and metastasis Cell, 2011 146(1): p 148-63 Soucy, P.A., J Werbin, W Heinz, J.H Hoh, and L.H Romer, Microelastic properties of lung cell-derived extracellular matrix Acta biomaterialia, 2011 7(1): p 96-105 Guise, T.A., Antitumor effects of bisphosphonates: promising preclinical evidence Cancer Treat Rev, 2008 34 Suppl 1: p S19-24 Maity, G., P.R Choudhury, T Sen, K.K Ganguly, H Sil, and A Chatterjee, Culture of human breast cancer cell line (MDA-MB-231) on fibronectin-coated surface induces pro-matrix metalloproteinase-9 expression and activity Tumour Biol, 2011 32(1): p 129-38 Kostenuik, P.J., O Sanchez-Sweatman, F.W Orr, and G Singh, Bone cell matrix promotes the adhesion of human prostatic carcinoma cells via the alpha beta integrin Clin Exp Metastasis, 1996 14(1): p 19-26 http://www.atcc.org 2008; Available from: ATCC http://www.atcc.org/ATCCAdvancedCatalogSearch/ProductDetails/tab id/452/Default.aspx?ATCCNum=HTB-22&Template=cellBiology Soule, H.D., T.M Maloney, S.R Wolman, W.D Peterson, Jr., R Brenz, C.M McGrath, J Russo, R.J Pauley, R.F Jones, and S.C Brooks, Isolation and characterization of a spontaneously immortalized human breast epithelial cell line, MCF-10 Cancer Res, 1990 50(18): p 6075-86 Chang, S.E., J Keen, E.B Lane, and J Taylor-Papadimitriou, Establishment and characterization of SV40-transformed human breast epithelial cell lines Cancer Res, 1982 42(5): p 2040-53 Neve, R.M., K Chin, J Fridlyand, J Yeh, F.L Baehner, T Fevr, L Clark, N Bayani, J.P Coppe, F Tong, T Speed, P.T Spellman, S DeVries, A Lapuk, N.J Wang, W.L Kuo, J.L Stilwell, D Pinkel, D.G Albertson, F.M Waldman, F McCormick, R.B Dickson, M.D Johnson, M Lippman, S Ethier, A Gazdar, and J.W Gray, A collection of breast cancer cell lines for the study of functionally distinct cancer subtypes Cancer Cell, 2006 10(6): p 515-27 Cailleau, R., M Olive, and Q.V Cruciger, Long-term human breast carcinoma cell lines of metastatic origin: preliminary characterization In Vitro, 1978 14(11): p 911-5 Yoneda, T., P.J Williams, T Hiraga, M Niewolna, and R Nishimura, A bone-seeking clone exhibits different biological properties from the MDA-MB-231 parental human breast cancer cells and a brain-seeking clone in vivo and in vitro J Bone Miner Res, 2001 16(8): p 1486-95 Ross, D.T and C.M Perou, A comparison of gene expression signatures from breast tumors and breast tissue derived cell lines Dis Markers, 2001 17(2): p 99-109 Lacroix, M and G Leclercq, Relevance of breast cancer cell lines as models for breast tumours: an update Breast Cancer Res Treat, 2004 83(3): p 249-89 Taubenberger, A.V., M.A Woodruff, H Bai, D.J Muller, and D.W Hutmacher, The effect of unlocking RGD-motifs in collagen I on preosteoblast adhesion and differentiation Biomaterials, 2010 31(10): p 2827-35 242 191 192 193 194 195 196 197 198 199 200 201 202 203 204 205 Ulrich, F., M Krieg, E.M Schotz, V Link, I Castanon, V Schnabel, A Taubenberger, D Mueller, P.H Puech, and C.P Heisenberg, Wnt11 functions in gastrulation by controlling cell cohesion through Rab5c and E-cadherin Developmental cell, 2005 9(4): p 555-64 Muller, D.J., J Helenius, D Alsteens, and Y.F Dufrene, Force probing surfaces of living cells to molecular resolution Nature chemical biology, 2009 5(6): p 383-90 Gail, M.H and C.W Boone, The locomotion of mouse fibroblasts in tissue culture Biophys J, 1970 10(10): p 980-93 Guo, W and F.G Giancotti, Integrin signalling during tumour progression Nat Rev Mol Cell Biol, 2004 5(10): p 816-26 Rudland, P.S., A Platt-Higgins, M El-Tanani, S De Silva Rudland, R Barraclough, J.H Winstanley, R Howitt, and C.R West, Prognostic significance of the metastasis-associated protein osteopontin in human breast cancer Cancer Res, 2002 62(12): p 3417-27 Ronnov-Jessen, L., O.W Petersen, and M.J Bissell, Cellular changes involved in conversion of normal to malignant breast: importance of the stromal reaction Physiol Rev, 1996 76(1): p 69-125 Ross, D.T., U Scherf, M.B Eisen, C.M Perou, C Rees, P Spellman, V Iyer, S.S Jeffrey, M Van de Rijn, M Waltham, A Pergamenschikov, J.C Lee, D Lashkari, D Shalon, T.G Myers, J.N Weinstein, D Botstein, and P.O Brown, Systematic variation in gene expression patterns in human cancer cell lines Nat Genet, 2000 24(3): p 227-35 Castello-Cros, R., D.R Khan, J Simons, M Valianou, and E Cukierman, Staged stromal extracellular 3D matrices differentially regulate breast cancer cell responses through PI3K and beta1integrins BMC Cancer, 2009 9: p 94 Castello-Cros, R and E Cukierman, Stromagenesis during tumorigenesis: characterization of tumor-associated fibroblasts and stroma-derived 3D matrices Methods Mol Biol, 2009 522: p 275-305 Hakkinen, K.M., J.S Harunaga, A.D Doyle, and K.M Yamada, Direct comparisons of the morphology, migration, cell adhesions, and actin cytoskeleton of fibroblasts in four different three-dimensional extracellular matrices Tissue Eng Part A, 2011 17(5-6): p 713-24 Doyle, A.D., F.W Wang, K Matsumoto, and K.M Yamada, Onedimensional topography underlies three-dimensional fibrillar cell migration J Cell Biol, 2009 184(4): p 481-90 Larsen, M., V.V Artym, J.A Green, and K.M Yamada, The matrix reorganized: extracellular matrix remodeling and integrin signaling Curr Opin Cell Biol, 2006 18(5): p 463-71 Berrier, A.L and K.M Yamada, Cell-matrix adhesion J Cell Physiol, 2007 213(3): p 565-73 Felding-Habermann, B., Integrin adhesion receptors in tumor metastasis Clin Exp Metastasis, 2003 20(3): p 203-13 Zhao, Y., R Bachelier, I Treilleux, P Pujuguet, O Peyruchaud, R Baron, P Clement-Lacroix, and P Clezardin, Tumor alphavbeta3 integrin is a therapeutic target for breast cancer bone metastases Cancer Res, 2007 67(12): p 5821-30 243 206 207 208 209 210 211 212 213 214 215 216 217 218 219 220 221 Wong, N.C., B.M Mueller, C.F Barbas, P Ruminski, V Quaranta, E.C Lin, and J.W Smith, Alphav integrins mediate adhesion and migration of breast carcinoma cell lines Clin Exp Metastasis, 1998 16(1): p 50-61 Rolli, M., E Fransvea, J Pilch, A Saven, and B Felding-Habermann, Activated integrin alphavbeta3 cooperates with metalloproteinase MMP-9 in regulating migration of metastatic breast cancer cells Proc Natl Acad Sci U S A, 2003 100(16): p 9482-7 Rae, J.M., C.J Creighton, J.M Meck, B.R Haddad, and M.D Johnson, MDA-MB-435 cells are derived from M14 melanoma cells a loss for breast cancer, but a boon for melanoma research Breast Cancer Res Treat, 2007 104(1): p 13-9 Lundstrom, A., J Holmbom, C Lindqvist, and T Nordstrom, The role of alpha2 beta1 and alpha3 beta1 integrin receptors in the initial anchoring of MDA-MB-231 human breast cancer cells to cortical bone matrix Biochem Biophys Res Commun, 1998 250(3): p 735-40 Liapis, H., A Flath, and S Kitazawa, Integrin alpha V beta expression by bone-residing breast cancer metastases Diagn Mol Pathol, 1996 5(2): p 127-35 Ridley, A.J., M.A Schwartz, K Burridge, R.A Firtel, M.H Ginsberg, G Borisy, J.T Parsons, and A.R Horwitz, Cell migration: integrating signals from front to back Science, 2003 302(5651): p 1704-9 Friedl, P and K Wolf, Tumour-cell invasion and migration: diversity and escape mechanisms Nat Rev Cancer, 2003 3(5): p 362-74 Hynes, R.O., Integrins: bidirectional, allosteric signaling machines Cell, 2002 110(6): p 673-87 Palmer, T.D., Ashby, W.J., Lewis, J.D., Zijlstra, A., Targeting tumor cell motility to prevent matastasis Advanced Drug Delivery Reviews, 2011 Provenzano, P.P., D.R Inman, K.W Eliceiri, J.G Knittel, L Yan, C.T Rueden, J.G White, and P.J Keely, Collagen density promotes mammary tumor initiation and progression BMC Med, 2008 6: p 11 Petrie, R.J., A.D Doyle, and K.M Yamada, Random versus directionally persistent cell migration Nat Rev Mol Cell Biol, 2009 10(8): p 538-49 Yamaguchi, H., J Wyckoff, and J Condeelis, Cell migration in tumors Curr Opin Cell Biol, 2005 17(5): p 559-64 Condeelis, J and J.E Segall, Intravital imaging of cell movement in tumours Nat Rev Cancer, 2003 3(12): p 921-30 Bailly, M., L Yan, G.M Whitesides, J.S Condeelis, and J.E Segall, Regulation of protrusion shape and adhesion to the substratum during chemotactic responses of mammalian carcinoma cells Exp Cell Res, 1998 241(2): p 285-99 Morini, M., M Mottolese, N Ferrari, F Ghiorzo, S Buglioni, R Mortarini, D.M Noonan, P.G Natali, and A Albini, The alpha beta integrin is associated with mammary carcinoma cell metastasis, invasion, and gelatinase B (MMP-9) activity Int J Cancer, 2000 87(3): p 336-42 Baum, O., R Hlushchuk, A Forster, R Greiner, P Clezardin, Y Zhao, V Djonov, and G Gruber, Increased invasive potential and up244 222 223 224 225 226 227 228 229 230 231 232 233 234 regulation of MMP-2 in MDA-MB-231 breast cancer cells expressing the beta3 integrin subunit Int J Oncol, 2007 30(2): p 325-32 McLean, G.W., N.O Carragher, E Avizienyte, J Evans, V.G Brunton, and M.C Frame, The role of focal-adhesion kinase in cancer - a new therapeutic opportunity Nat Rev Cancer, 2005 5(7): p 505-15 Carragher, N.O., M.A Westhoff, V.J Fincham, M.D Schaller, and M.C Frame, A novel role for FAK as a protease-targeting adaptor protein: regulation by p42 ERK and Src Curr Biol, 2003 13(16): p 1442-50 Ilic, D., Y Furuta, S Kanazawa, N Takeda, K Sobue, N Nakatsuji, S Nomura, J Fujimoto, M Okada, and T Yamamoto, Reduced cell motility and enhanced focal adhesion contact formation in cells from FAK-deficient mice Nature, 1995 377(6549): p 539-44 Ren, X.D., W.B Kiosses, D.J Sieg, C.A Otey, D.D Schlaepfer, and M.A Schwartz, Focal adhesion kinase suppresses Rho activity to promote focal adhesion turnover J Cell Sci, 2000 113 ( Pt 20): p 3673-8 Montagut, C and J Settleman, Targeting the RAF-MEK-ERK pathway in cancer therapy Cancer Lett, 2009 283(2): p 125-34 Tuck, A.B., F.P O'Malley, H Singhal, K.S Tonkin, J.F Harris, D Bautista, and A.F Chambers, Osteopontin and p53 expression are associated with tumor progression in a case of synchronous, bilateral, invasive mammary carcinomas Arch Pathol Lab Med, 1997 121(6): p 578-84 Tuck, A.B., F.P O'Malley, H Singhal, J.F Harris, K.S Tonkin, N Kerkvliet, Z Saad, G.S Doig, and A.F Chambers, Osteopontin expression in a group of lymph node negative breast cancer patients Int J Cancer, 1998 79(5): p 502-8 Fox, C.F., Skalak, R , Tissue engineering: proceedings of a workshop held at Granlibakken, Lake Thoe, California, February 26-291988, Liss, New York Hutmacher, D.W., J.T Schantz, C.X Lam, K.C Tan, and T.C Lim, State of the art and future directions of scaffold-based bone engineering from a biomaterials perspective J Tissue Eng Regen Med, 2007 1(4): p 245-60 Hutmacher, D.W., R.E Horch, D Loessner, S Rizzi, S Sieh, J.C Reichert, J.A Clements, J.P Beier, A Arkudas, O Bleiziffer, and U Kneser, Translating tissue engineering technology platforms into cancer research J Cell Mol Med, 2009 13(8A): p 1417-27 Hutmacher, D.W., Scaffolds in tissue engineering bone and cartilage Biomaterials, 2000 21(24): p 2529-43 Zhou, Y., F Chen, S.T Ho, M.A Woodruff, T.M Lim, and D.W Hutmacher, Combined marrow stromal cell-sheet techniques and high-strength biodegradable composite scaffolds for engineered functional bone grafts Biomaterials, 2007 28(5): p 814-24 Hutmacher, D.W and S Cool, Concepts of scaffold-based tissue engineering the rationale to use solid free-form fabrication techniques J Cell Mol Med, 2007 11(4): p 654-69 245 235 236 237 238 239 240 241 242 243 244 245 246 Arthur, A., A Zannettino, and S Gronthos, The therapeutic applications of multipotential mesenchymal/stromal stem cells in skeletal tissue repair J Cell Physiol, 2009 218(2): p 237-45 Biggs, M.J., R.G Richards, N Gadegaard, C.D Wilkinson, R.O Oreffo, and M.J Dalby, The use of nanoscale topography to modulate the dynamics of adhesion formation in primary osteoblasts and ERK/MAPK signalling in STRO-1+ enriched skeletal stem cells Biomaterials, 2009 30(28): p 5094-103 Santos, M.I., R.E Unger, R.A Sousa, R.L Reis, and C.J Kirkpatrick, Crosstalk between osteoblasts and endothelial cells co-cultured on a polycaprolactone-starch scaffold and the in vitro development of vascularization Biomaterials, 2009 30(26): p 4407-15 Ripamonti, U., B Van Den Heever, T.K Sampath, M.M Tucker, D.C Rueger, and A.H Reddi, Complete regeneration of bone in the baboon by recombinant human osteogenic protein-1 (hOP-1, bone morphogenetic protein-7) Growth Factors, 1996 13(3-4): p 27389,color plates III-VIII,pre bk Ramoshebi, L.N and U Ripamonti, Osteogenic protein-1, a bone morphogenetic protein, induces angiogenesis in the chick chorioallantoic membrane and synergizes with basic fibroblast growth factor and transforming growth factor-beta1 Anat Rec, 2000 259(1): p 97-107 Dominici, M., K Le Blanc, I Mueller, I Slaper-Cortenbach, F Marini, D Krause, R Deans, A Keating, D Prockop, and E Horwitz, Minimal criteria for defining multipotent mesenchymal stromal cells The International Society for Cellular Therapy position statement Cytotherapy, 2006 8(4): p 315-7 Larson, B.L., J Ylostalo, and D.J Prockop, Human multipotent stromal cells undergo sharp transition from division to development in culture Stem cells, 2008 26(1): p 193-201 Colter, D.C., I Sekiya, and D.J Prockop, Identification of a subpopulation of rapidly self-renewing and multipotential adult stem cells in colonies of human marrow stromal cells Proceedings of the National Academy of Sciences of the United States of America, 2001 98(14): p 7841-5 Sekiya, I., B.L Larson, J.R Smith, R Pochampally, J.G Cui, and D.J Prockop, Expansion of human adult stem cells from bone marrow stroma: conditions that maximize the yields of early progenitors and evaluate their quality Stem cells, 2002 20(6): p 530-41 Reichert, J.C., V.M Quent, U Noth, and D.W Hutmacher, Ovine cortical osteoblasts outperform bone marrow cells in an ectopic bone assay J Tissue Eng Regen Med, 2011 Lam, C.X., D.W Hutmacher, J.T Schantz, M.A Woodruff, and S.H Teoh, Evaluation of polycaprolactone scaffold degradation for months in vitro and in vivo J Biomed Mater Res A, 2009 90(3): p 906-19 Erlebacher, A and R Derynck, Increased expression of TGF-beta in osteoblasts results in an osteoporosis-like phenotype J Cell Biol, 1996 132(1-2): p 195-210 246 247 248 249 250 251 252 253 254 255 256 257 258 259 260 Sheehan, D.C.H., B.B., Theory and practice of histotechnology 2nd ed1980, St Louis: Mosby, C.V Reichert, J.C., M.A Woodruff, T Friis, V.M Quent, S Gronthos, G.N Duda, M.A Schutz, and D.W Hutmacher, Ovine bone- and marrowderived progenitor cells and their potential for scaffold-based bone tissue engineering applications in vitro and in vivo J Tissue Eng Regen Med, 2010 4(7): p 565-76 Harada, H., S Tagashira, M Fujiwara, S Ogawa, T Katsumata, A Yamaguchi, T Komori, and M Nakatsuka, Cbfa1 isoforms exert functional differences in osteoblast differentiation The Journal of biological chemistry, 1999 274(11): p 6972-8 Kern, B., J Shen, M Starbuck, and G Karsenty, Cbfa1 contributes to the osteoblast-specific expression of type I collagen genes The Journal of biological chemistry, 2001 276(10): p 7101-7 Ducy, P., R Zhang, V Geoffroy, A.L Ridall, and G Karsenty, Osf2/Cbfa1: a transcriptional activator of osteoblast differentiation Cell, 1997 89(5): p 747-54 Bonewald, L.F and S.L Dallas, Role of active and latent transforming growth factor beta in bone formation Journal of cellular biochemistry, 1994 55(3): p 350-7 Centrella, M., M.C Horowitz, J.M Wozney, and T.L McCarthy, Transforming growth factor-beta gene family members and bone Endocrine reviews, 1994 15(1): p 27-39 Noda, M., Transcriptional regulation of osteocalcin production by transforming growth factor-beta in rat osteoblast-like cells Endocrinology, 1989 124(2): p 612-7 Frank, O., M Heim, M Jakob, A Barbero, D Schafer, I Bendik, W Dick, M Heberer, and I Martin, Real-time quantitative RT-PCR analysis of human bone marrow stromal cells during osteogenic differentiation in vitro Journal of cellular biochemistry, 2002 85(4): p 737-46 Niyibizi, C and D.R Eyre, Structural characteristics of cross-linking sites in type V collagen of bone Chain specificities and heterotypic links to type I collagen European journal of biochemistry / FEBS, 1994 224(3): p 943-50 Viguet-Carrin, S., P Garnero, and P.D Delmas, The role of collagen in bone strength Osteoporosis international : a journal established as result of cooperation between the European Foundation for Osteoporosis and the National Osteoporosis Foundation of the USA, 2006 17(3): p 319-36 Kamiya, N and M Takagi, Differential expression of dentin matrix protein 1, type I collagen and osteocalcin genes in rat developing mandibular bone The Histochemical journal, 2001 33(9-10): p 54552 Suda, N., M.T Gillespie, K Traianedes, H Zhou, P.W Ho, D.K Hards, E.H Allan, T.J Martin, and J.M Moseley, Expression of parathyroid hormone-related protein in cells of osteoblast lineage Journal of cellular physiology, 1996 166(1): p 94-104 Albo, D., V.L Rothman, D.D Roberts, and G.P Tuszynski, Tumour cell thrombospondin-1 regulates tumour cell adhesion and invasion 247 261 262 263 264 265 266 267 268 269 270 271 through the urokinase plasminogen activator receptor Br J Cancer, 2000 83(3): p 298-306 Miao, D., X.K Tong, G.K Chan, D Panda, P.S McPherson, and D Goltzman, Parathyroid hormone-related peptide stimulates osteogenic cell proliferation through protein kinase C activation of the Ras/mitogen-activated protein kinase signaling pathway The Journal of biological chemistry, 2001 276(34): p 32204-13 Cornish, J., K.E Callon, C Lin, C Xiao, J.M Moseley, and I.R Reid, Stimulation of osteoblast proliferation by C-terminal fragments of parathyroid hormone-related protein Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research, 1999 14(6): p 915-22 Martinez, M.E., A Garcia-Ocana, M Sanchez, S Medina, T del Campo, A Valin, M.J Sanchez-Cabezudo, and P Esbrit, C-terminal parathyroid hormone-related protein inhibits proliferation and differentiation of human osteoblast-like cells Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research, 1997 12(5): p 778-85 Zerega, B., S Cermelli, P Bianco, R Cancedda, and F.D Cancedda, Parathyroid hormone [PTH(1-34)] and parathyroid hormone-related protein [PTHrP(1-34)] promote reversion of hypertrophic chondrocytes to a prehypertrophic proliferating phenotype and prevent terminal differentiation of osteoblast-like cells Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research, 1999 14(8): p 1281-9 Thomas, G.P., S.U Baker, J.A Eisman, and E.M Gardiner, Changing RANKL/OPG mRNA expression in differentiating murine primary osteoblasts The Journal of endocrinology, 2001 170(2): p 451-60 Paul, S., J.C Lee, and L.C Yeh, A comparative study on BMPinduced osteoclastogenesis and osteoblastogenesis in primary cultures of adult rat bone marrow cells Growth Factors, 2009 27(2): p 121-31 Ristiniemi, J., T Flinkkila, P Hyvonen, M Lakovaara, H Pakarinen, and P Jalovaara, RhBMP-7 accelerates the healing in distal tibial fractures treated by external fixation J Bone Joint Surg Br, 2007 89(2): p 265-72 Yeh, L.C., A.D Tsai, and J.C Lee, Osteogenic protein-1 (OP-1, BMP7) induces osteoblastic cell differentiation of the pluripotent mesenchymal cell line C2C12 J Cell Biochem, 2002 87(3): p 292304 Hauschka, P.V., J.B Lian, D.E Cole, and C.M Gundberg, Osteocalcin and matrix Gla protein: vitamin K-dependent proteins in bone Physiol Rev, 1989 69(3): p 990-1047 Hauschka, P.V and F.H Wians, Jr., Osteocalcin-hydroxyapatite interaction in the extracellular organic matrix of bone Anat Rec, 1989 224(2): p 180-8 Ljusberg, J., B Ek-Rylander, and G Andersson, Tartrate-resistant purple acid phosphatase is synthesized as a latent proenzyme and activated by cysteine proteinases Biochem J, 1999 343 Pt 1: p 63-9 248 272 273 274 275 276 277 278 279 280 281 282 283 Ljusberg, J., Y Wang, P Lang, M Norgard, R Dodds, K Hultenby, B Ek-Rylander, and G Andersson, Proteolytic excision of a repressive loop domain in tartrate-resistant acid phosphatase by cathepsin K in osteoclasts J Biol Chem, 2005 280(31): p 28370-81 Minkin, C., Bone acid phosphatase: tartrate-resistant acid phosphatase as a marker of osteoclast function Calcif Tissue Int, 1982 34(3): p 285-90 Angel, N.Z., N Walsh, M.R Forwood, M.C Ostrowski, A.I Cassady, and D.A Hume, Transgenic mice overexpressing tartrate-resistant acid phosphatase exhibit an increased rate of bone turnover J Bone Miner Res, 2000 15(1): p 103-10 Hayman, A.R., S.J Jones, A Boyde, D Foster, W.H Colledge, M.B Carlton, M.J Evans, and T.M Cox, Mice lacking tartrate-resistant acid phosphatase (Acp 5) have disrupted endochondral ossification and mild osteopetrosis Development, 1996 122(10): p 3151-62 Kaneko, H., T Arakawa, H Mano, T Kaneda, A Ogasawara, M Nakagawa, Y Toyama, Y Yabe, M Kumegawa, and Y Hakeda, Direct stimulation of osteoclastic bone resorption by bone morphogenetic protein (BMP)-2 and expression of BMP receptors in mature osteoclasts Bone, 2000 27(4): p 479-86 Hentunen, T.A., P.T Lakkakorpi, J Tuukkanen, P.P Lehenkari, T.K Sampath, and H.K Vaananen, Effects of recombinant human osteogenic protein-1 on the differentiation of osteoclast-like cells and bone resorption Biochem Biophys Res Commun, 1995 209(2): p 433-43 Schantz, J.T., D.W Hutmacher, C.X Lam, M Brinkmann, K.M Wong, T.C Lim, N Chou, R.E Guldberg, and S.H Teoh, Repair of calvarial defects with customised tissue-engineered bone grafts II Evaluation of cellular efficiency and efficacy in vivo Tissue Eng, 2003 Suppl 1: p S127-39 Schantz, J.T., T.C Lim, C Ning, S.H Teoh, K.C Tan, S.C Wang, and D.W Hutmacher, Cranioplasty after trephination using a novel biodegradable burr hole cover: technical case report Neurosurgery, 2006 58(1 Suppl): p ONS-E176; discussion ONS-E176 Rai, B., S.H Teoh, K.H Ho, D.W Hutmacher, T Cao, F Chen, and K Yacob, The effect of rhBMP-2 on canine osteoblasts seeded onto 3D bioactive polycaprolactone scaffolds Biomaterials, 2004 25(24): p 5499-506 Rai, B., S.H Teoh, D.W Hutmacher, T Cao, and K.H Ho, Novel PCL-based honeycomb scaffolds as drug delivery systems for rhBMP2 Biomaterials, 2005 26(17): p 3739-48 Sawyer, A.A., S.J Song, E Susanto, P Chuan, C.X Lam, M.A Woodruff, D.W Hutmacher, and S.M Cool, The stimulation of healing within a rat calvarial defect by mPCL-TCP/collagen scaffolds loaded with rhBMP-2 Biomaterials, 2009 30(13): p 2479-88 Kolambkar, Y.M., K.M Dupont, J.D Boerckel, N Huebsch, D.J Mooney, D.W Hutmacher, and R.E Guldberg, An alginate-based hybrid system for growth factor delivery in the functional repair of large bone defects Biomaterials, 2011 32(1): p 65-74 249 284 285 286 287 288 Kolambkar, Y.M., A Peister, A.K Ekaputra, D.W Hutmacher, and R.E Guldberg, Colonization and osteogenic differentiation of different stem cell sources on electrospun nanofiber meshes Tissue Eng Part A, 2010 16(10): p 3219-30 Yang, F., J.G.C Wolke, and J.A Jansen, Biomimetic calcium phosphate coating on electrospun poly (epsilon-caprolactone) scaffolds for bone tissue engineering Chemical Engineering Journal, 2008 137(1): p 154-161 Ehrbar, M., S.C Rizzi, R.G Schoenmakers, B.S Miguel, J.A Hubbell, F.E Weber, and M.P Lutolf, Biomolecular hydrogels formed and degraded via site-specific enzymatic reactions Biomacromolecules, 2007 8(10): p 3000-7 Roldan, J.C., R Detsch, S Schaefer, E Chang, M Kelantan, W Waiss, T.E Reichert, G.C Gurtner, and U Deisinger, Bone formation and degradation of a highly porous biphasic calcium phosphate ceramic in presence of BMP-7, VEGF and mesenchymal stem cells in an ectopic mouse model Journal of cranio-maxillo-facial surgery : official publication of the European Association for Cranio-MaxilloFacial Surgery, 2010 38(6): p 423-30 Wang, X.J., H Huang, F Yang, L.G Xia, W.J Zhang, X.Q Jiang, and F.Q Zhang, Ectopic study of tissue-engineered bone complex with enamel matrix proteins, bone marrow stromal cells in porous calcium phosphate cement scaffolds, in nude mice Cell proliferation, 2011 44(3): p 274-82 250 ... List of abbreviations 2D Two dimensional 3D Three dimensional AFM Atomic force microscopy AIWH Australian Institute of Health and Welfare ALP Alkaline phosphatase AM Adrenomedullin ANOVA Analysis... engineered bone construct 214 List of Tables Chapter Table 1: Major components of human bone and their overall function 15 Table 2: Main integrins and their ECM ligands that participate in bone metastasis. .. of breast cancer- related bone metastasis – overview of currently used in vitro and in vivo models 2.1 Epidemiology – facts about breast cancer 2.2 Clinical presentation of bone metastases