Barbieri Thesis - BioMedical Materials program (BMM)

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Chapter 7 – Polymer molecular weight and instructive composites which would dissolve them facilitating the release of calcium and phosphate ions. However, during a 12–week period our results showed similar mass loss and molecular weight changes (in percentage), and comparable ions release profiles in vitro for the two materials as well as equivalent post–degradation phase contents (i.e. similar phase loss trends, Table 2). Further, after 12 weeks intramuscular implantation in dogs, no significant differences were found in the molecular weight of the polymers in ML and MH, indicating similar degradation behaves in vivo. This may be explained by the time course of the study (i.e. 12 weeks), which may have been too short to allow the penetrated fluids to fully hydrolyse the polymer and degrade the bulk of the composites leading to insignificant differences in the degradation patterns of the two materials. Hydrolysis may have been restrained by the buffering effect of calcium and phosphate ions on the acidity provoked by the formation of polymer residuals. From these results, we can conclude that, under the conditions of this 12–week period, the degradation of the two composites was not dependent on their different fluid uptakes. Further to this, the difference in osteoinductive potential of the two composites is not likely due to ion release. In fact, our results showed that the in vitro degradation model may be used as indication of the possible in vivo degradation trends. So, the two composites may have degraded similarly releasing comparable amounts of ions. We observed that ML had higher fluid uptake after seven days soaking in foetal bovine serum (FBS) solution and could adsorb significantly more serum proteins than MH (Figure 4b, 3c). In our situation, the higher fluid uptake in ML within short time could enhance the contact between its surfaces and FBS, increasing the probability of serum protein adsorption. Our observations support the conclusion that, in vivo, these biological molecules could have more pronouncedly been adsorbed on the surfaces of ML. As already suggested in literature, [154, 228, 251, 346] such adsorbed bio–molecules provide better conditions for (stem) cell colonisation and osteogenic differentiation that finally leads to bone matrix synthesis. The increased fluid uptake in ML, which may have facilitated the contact between the surrounding fluids and the composite surfaces, also enhanced apatite nucleation from the fluids. Besides this, the high apatite content in composites could favour the nucleation of mineralised layers onto its surfaces and thereby generating conditions that allow cells to synthesize bone. [234, 248, 249] We observed in vitro surface mineralisation on both ML and MH composites within two days, where similar three–dimensional nano–structured layers were generated (Figure 6). However, the formation of mineralized layers was faster on ML than MH as shown by the larger increase in thickness. Similarly, mineralisation occurred in vivo on both materials, where ML mineralised quicker than MH, generating thicker nucleated apatite layers (Table 3). Nano–scaled structures containing calcium can expose more binding sites for biomolecules adsorption [153, 154, 250] Thus, the formation of thicker three–dimensional nano–structured mineralised layers in ML, together with the higher fluids absorption 170
Chapter 7 – Polymer molecular weight and instructive composites ability of ML, could have allowed larger protein adsorption onto ML. Even if no differences were observed in the mineralization potential of ML and MH, the similar nano–texture on their mineralized surfaces would improve the initial anchorage of cellular filopodia [152, 195] as compared to non–surface mineralizing materials. The initial inflammatory response to the implantation of biomaterials has been proposed as a trigger for osteoinduction as well. Osteoclastogenesis and osteoblastogenesis at the biomaterial surface may be mediated by macrophage adhesion and subsequent secretion of cytokines. Histological observations over time demonstrated that, upon implantation of ceramics, fibrous tissue containing monocytes and macrophages invaded the implants at early time points, while later bone formed tight to the material and active osteoblasts were aligned with the newly synthesised bone. [218–220] Interestingly, the secretion of cytokines such as interleukin–6 (IL–6) and prostaglandin E2, [217, 386] were enhanced when macrophages were cultured on materials having micro–textures compared to smooth surfaces, [255] and the presence of IL–6 enhanced the expression of osteogenic markers (e.g. alkaline phosphatase and osteocalcin) from osteoblasts. [256] Unpublished preliminary in vitro results of members from our group showed that the culture of murine monocyte/macrophage cell line RAW 264.7 on osteoinductive ceramic resulted in significantly higher levels of secreted IL–6 and IL–4, cytokines responsible for osteoclastogenesis and foreign body giant cell fusion respectively, when compared to a non–inductive ceramic. Other data showed that osteoclastic markers in murine macrophage cell line J774.2 were significantly up–regulated during culture on osteoinductive ceramics, and stimulation of RAW 264.7 with osteoclast differentiation factor RANKL (receptor activator of NF–κB ligand) during culture on osteoinductive ceramics resulted in larger, more surface integrated osteoclast–like cells. So, by uptaking more fluids, ML could generate thicker nano–rough mineralised surfaces within few days upon implantation which further enhanced bio–molecule adsorption. Thus, more attractive conditions for macrophages colonisation may have been created on ML, and it could be that such cells may have induced to secrete various cytokines and angiogenic molecules [257] later triggering the differentiation of stem cells into an osteogenic line and leading later to bone matrix synthesis in ML. [217] However, further evaluation of these macrophage/osteoclast culture systems may shed light on the potential osteogenic effects that such cell–material interactions may have on precursor cells. In summary, the molecular weight of poly(D,L–lactide) composites can indirectly control surface phenomena occurring at the interface that will ultimately determine whether osteoinduction takes place or not. Composites with low molecular weight polymer (i.e. ML) absorbed more body fluids and activated a cascade of surface events. Serum proteins were taken up and the formation of thicker nano–structured mineralised surfaces may have allowed for even higher amounts of protein adsorption. As a 171
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INSTRUCTIVE COMPOSITES FOR BONE REG
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INSTRUCTIVE COMPOSITES FOR BONE REG
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献给潘臻 A mamma e papà A Stef
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Summary Summary Developing new biom
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Summary containing 96%mol. L-lactid
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Samenvatting De behoefte aan een de
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Samenvatting structuur, bespoedigt
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Riassunto velocità di dissoluzione
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Riassunto generale, ha migliorato i
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Chapter 1 - Introduction 1.1. Eukar
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Chap pter 1 - Introducction Interst
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Chapter 1 - Introduction bone marro
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Chap pter 1 - Introducction Figure
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Chapter 1 - Introduction regenerati
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Chapter 1 - Introduction Key concep
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Chapter 1 - Introduction grown in c
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Chapter 1 - Introduction Mainly, th
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Chapter 1 - Introduction All these
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Chap pter 1 - Introducction materia
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Chapter 1 - Introduction protein, m
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CHAPTER 2 The role of gels in bone
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Chapter 2 - The role of gels in ins
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CChapter 2 - The role r of gels in
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CHAPTER 3 Instructive composites: e
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Chapter 3 - Instructive composites:
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Chapter 3 - Instructive compos site
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CChapter 3 - Instructive composit t
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CHAPTER 4 Controlling dynamic mecha
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Chapter 4 - Conntrol of mechanical
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Chapter 4 - Control of mechanical a
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CHAPTER 5 Effects of alkali surface
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Chapter 5 - Alkali surface treatmen
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Chapter 5 - Al lkali surface treaat
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Page 215 and 216: References [1] Waggoner B. Eukaryot
Page 217 and 218: References induction of rank in ost
Page 219 and 220: References [72] Kokovay E, Wang Y,
Page 221 and 222: References [102] McBeath R, Pirone
Page 223 and 224: References molecule-mediated TGF- t
Page 225 and 226: References [159] Autumn K, Sitti M,
Page 227 and 228: References [189] Teixeira AI, McKie
Page 229 and 230: References [215] Tang L, Jennings T
Page 231 and 232: References [242] Wei G, Ma PX. Stru
Page 233 and 234: References [270] Atkinson BL, Hanks
Page 235 and 236: References [297] Sato I, Akizuki T,
Page 237 and 238: References hematopoietic stem cell
Page 239 and 240: References [352] Cullinane DM, Sali
Page 241 and 242: References [382] Norde W, Giacomell
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References differentiation of rat B
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Acknowledgments thank you for all t
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Acknowledgments desidero tutto il m
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Barbieri Thesis - BioMedical Materials program (BMM)

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