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The Biological and Physical Performance of High Strength Dicalcium Phosphate Cement in Physiologically Relevant Models

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The Biological and Physical Performance of High Strength Dicalcium Phosphate Cement in Physiologically Relevant Models Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology 1502 The biological and physical performance of high strength dicalcium phosphate cement in physiologically relevant models MICHAEL PUJARI-PALMER ACTA UNIVERSITATIS UPSALIENSIS ISSN 1651-6214 ISBN 978-91-554-9885-6 UPPSALA urn:nbn:se:uu:diva-319495 2017 Dissertation presented at Uppsala University to be publicly examined in Å2001, Ångströmlaboratoriet, Lägerhyddsvägen 1, Uppsala, Friday, 2 June 2017 at 09:15 for the degree of Doctor of Philosophy. The examination will be conducted in English. Faculty examiner: Professor Christele Combes ( Inter-University Center for Research and Materials Engineering, University of Toulouse). Abstract Pujari-Palmer, M. 2017. The biological and physical performance of high strength dicalcium phosphate cement in physiologically relevant models. Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology 1502. 78 pp. Uppsala: Acta Universitatis Upsaliensis. ISBN 978-91-554-9885-6. The chemical properties of calcium phosphate cements (CPCs) are very similar to the mineral phase of bone. CPCs are, consequently, very effective substrates (scaffolds) for tissue engineering; bone and stem cells attach readily, and can proliferate and differentiate to form new bone tissue. Unlike other CPCs that may remain largely unchanged in the body for years, such as hydroxyapatite, dicalcium phosphates are remodelled by the body and rapidly converted to new bone. Unfortunately, the dicalcium phosphates are also typically too weak to support load bearing in the human body. Our laboratory has recently developed a novel, high strength brushite CPC, (hsCPC), which can reach 10-50 fold higher failure strength than many commercially available CPCs. The aim of this thesis was to investigate the physical, chemical and biological performance of hsCPCs in physiologically relevant model of drug release, load bearing, osteoconductivity, and as a scaffold for bone tissue engineering. Multiple CPCs were compared in a model of screw augmentation to determine whether the physical properties of the cement, such as bulk strength and porosity, affected orthopedic screw holding strength. In an in vitro model of bone regeneration stem cells were grown on macroporous scaffolds that were fabricated from hsCPC. Drug releasing scaffolds were fabricated to examine whether the low porosity of hsCPC impeded drug release during a 4 week incubation period. The biological activity of an incorporated drug, Rebamipide, was examined after acute and chronic incubation periods. In the drug release study it was noted that the biological response to hsCPC was significantly better than tissue culture grade polystyrene, even in groups without drug. The mechanism underlying this biological response was further investigated by testing the effect of pyrophosphate, a common cement additive, on bone cell proliferation and differentiation. This thesis concludes that a high strength cement can produce significant improvement in screw augmentation strength, if there is sufficient cortical bone near the augmentation site. The hsCPC is also cytocompatible, and can support bone and stem cell proliferation and differentiation. hsCPC scaffolds stimulated osteogenic gene expression comparable to native bone scaffolds. hsCPC scaffolds are also capable of delivering drug for up to 4 weeks, in vitro. Finally, a cement additive, pyrophosphate, stimulated differentiation, but not proliferation of bone cells. Keywords: biomaterial, bioceramic, osteoinduction, calcium phosphate, cement, osteoblast, pyrophosphate, Rebamipide, drug delivery, screw augmentation Michael Pujari-Palmer, Department of Engineering Sciences, Applied Materials Sciences, Box 534, Uppsala University, SE-75121 Uppsala, Sweden. © Michael Pujari-Palmer 2017 ISSN 1651-6214 ISBN 978-91-554-9885-6 urn:nbn:se:uu:diva-319495 (http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-319495) For my family List of Papers This thesis is based on the following papers, which are referred to in the text by their roman numerals. I Pujari-Palmer, M., Pujari-Palmer, S., Engqvist, H., Karlsson Ott, M. (2015) Rebamipide delivered by brushite cement enhances osteoblast and macro- phage proliferation. PLOS one, 10(5):e0128324. II Sladkova, M., Palmer, M., Öhman, C., Alhaddad, RJ., Esmael, A., Engqvist, H., dePeppo, GM. (2016) Fabrication of macroporous cement scaffolds using PEG particles: In vitro evaluation with induced pluripotent stem cell-derived mesenchymal progenitors. Mater Sci. Eng. C Mater Biol Appl., 69:640-52. III Pujari-Palmer, M., Pujari-Palmer, S., Liu, X., Lind, T., Melhus, H., Eng- strand T., Karlsson Ott, M., Engqvist, H.(2016) Pyrophosphate stimulates differentiation, matrix gene expression and alkaline phosphatase activity in osteoblasts. PLOS one, 11(10):e0163530. IV Pujari-Palmer, M., Robo, C., Procter, P., Persson, C., Engqvist, H. (2017) Influence of cement compressive strength and porosity on augmentation performance in a model of orthopedic screw pull-out. Submitted to the jour- nal the mechanical behavior of biomedical materials. Author’s Contributions Paper I Major part of the planning, experimental work and writing. Paper II Major part of the planning, experimental work and writing. Paper III Major part of the planning, experimental work and writing. Paper IV Major part of the planning, experimental work and writing. Reprints were made with permission from the respective publishers. Papers not included in the thesis V Sladkova, M., Palmer, M., Öhman, C., Cheng, J., Al-Ansari, S., Saad, M., Engqvist, H., dePeppo, G. (2017) Engineering Human Bone Grafts with New Macroporous Calcium Phosphate Cement Scaffolds. (in prepration) VI Pujari-Palmer, S., Lu, X., Singh, V.P., Engmanb, L., Pujari-Palmer, M., Karlsson Ott, M. (2017) Incorporation and delivery of an organoselenium antioxidant from a brushite cement. Materials letters. 197(15):115-119. VII Pujari-Palmer, S., Pujari-Palmer, M., Karlsson Ott, M. (2016) Reduced oxidative stress in primary human cells by antioxidant released from na- noporous alumina. J Biomed Mater Res B Appl Biomater. 104(3):568-75. VIII Tran, RT., Palmer, M., Tang, SJ., Abell TL., Yang, J. (2012) Injectable drug-eluting elastomeric polymer: a novel submucosal injection material. Gastrointest Endosc. 75(5):1092-7. Contents Aim of the thesis ........................................................................................... 11 Introduction ................................................................................................... 12 Bone Architecture ......................................................................................... 15 Macroscale architecture ............................................................................ 15 Microscale architecture ............................................................................. 15 Osteoblasts ........................................................................................... 15 Osteoclasts ........................................................................................... 16 Osteocytes ............................................................................................ 16 Nanoscale architecture .............................................................................. 16 Atomic scale architecture ......................................................................... 17 Summary .............................................................................................. 17 Bone cellular biology .................................................................................... 19 Role of matrix proteins and enzymes ....................................................... 19 Bone Matrix proteins ........................................................................... 20 Calcium and phosphate and the effect of ions .......................................... 23 Mineralization process ......................................................................... 25 Wound healing in bone ........................................................................ 28 Summary .............................................................................................. 28 Bone Tissue Engineering and Calcium Phosphate Biomaterials .................. 30 Biomaterials .............................................................................................. 30 Bone tissue engineering parameters ......................................................... 31 Bone mechanics ................................................................................... 31 Porosity ................................................................................................ 32 Calcium phosphate biomaterials .......................................................... 35 Physical properties of hsCPC cements ..................................................... 37 Drug delivery ....................................................................................... 40 Osteoinduction ..................................................................................... 41 Summary .............................................................................................. 42 Results and Discussion .................................................................................. 44 Mechanical strength of hsCPC ................................................................. 44 Porosity of hsCPC ...................................................................................
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