Barbieri Thesis - BioMedical Materials program (BMM)

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Chapter 7 – Polymer molecular weight and instructive composites recommended that such tests, to be valid, should be performed at temperatures lower than the glass transition temperature (i.e. Tg) of the considered polymer to avoid unwanted effects due to polymer state transitions. [354–357, 368] A phosphate buffered saline (PBS; Invitrogen, Darmstadt, Germany) solution having initial pH of 7.5±0.05 was used. Sterile granules of each composite (75±0.5 mg) were carefully weighed before use (m0) and soaked in 30 mL PBS at 55±0.5°C for five weeks. The temperature was set referring to the polymers Tg of the copolymers used here (i.e. 55–60°C). For each of the three time points considered, i.e. 1, 3 and 5 weeks, three samples of each material were used. Every day the pH of the degrading solution was recorded with a pH–meter (Orion 4 Star, Thermo Scientific, USA) and the medium was refreshed in case the pH fell below 7.0. With the solution removed at every refreshment, the amounts (in M) of calcium and phosphate ion released from the samples were measured using appropriate biochemical kits (QuantiChrom TM Calcium assay kit, BioAssay Systems, USA; PhosphoWorks TM Colorimetric Phosphate Assay kit Blue Color, Bioquest Inc, USA) with the help of a spectrophotometer (AnthosZenyth 3100, Anthos Labtec Instruments GmbH, Salzburg, Austria) and absorbance filter of 620 nm for both assays. At each of the considered time points, i.e. 1, 3 and 5 weeks, the granules were removed from the degrading media, the excess PBS was wiped away and their wet weight was carefully weighed (mwet). Afterwards they have been vacuum–dried at room temperature until their weight was stable and then were weighed again (mdry). The mass loss and fluid uptake of the composites was determined as: mass loss = 100 · (m0 – mdry) / m0 fluid uptake = 100 · (mwet – mdry) / mdry Part of the degraded samples were then was heated at 900°C to burn the polymer phase out and determine the final effective apatite and polymer percentage contents (in %wt., pap and ppol respectively). The remaining part of the samples was dissolved in chloroform (Sigma–Aldrich) and, after separating apatite from the polymer, we determined the intrinsic viscosity () using an Ubbelohde viscometer as explained in §7.2.2. As already mentioned in Chapter 6 (i.e. §6.2.5. for theory background), to evaluate the hydrolysis rate of the polymer phase in the two composites we estimated the autocatalysed hydrolytic degradation rate constant k as: k · t = –ln( / ) where is the initial intrinsic viscosity and is the intrinsic viscosity at the considered time point t (t=0, 7, 21 and 35 days) for the considered polymer phase. For convenience, the results were plotted on a graph ln(/) against time t (in days). 7a.2.4. Dynamic mechanical analysis It has been done following the protocol described in §7.2.7. 174
Chapter 7 – Polymer mole ecular weight annd instructive co omposites 7a.2.5. Aniimal study To evaluatte the in vivvo biological response, grranules of the e two composites were intramuscuularly implanteed in five shee ep for six monnths (with the e permission of o the local animal carre committee: Animal Cent ter, Sichuan UUniversity, Ch hengdu, China; protocol #P11030). The surgery procedure (b but no fluoresscent injections were done e) and the histological analysis were similar to those t describbed in Chapter 2 (§2.2.4 an nd §2.2.5). Unfortunately, no samplee was kept for degradation analyses (e.g g. burn test an nd viscosity measurements). 7a.3. Resuults 7a.3.1. Preeparation and characterizati ion of compossites Extrusion pprocess, as expected, significantly decreeased the intr rinsic viscos sity of both polymer phhases, and thee addition of apatite a furtherr decreased it (Table 5). Th he two final materials sstill had significant differen nt intrinsic visscosity. The composites had h similar surface chhemistry (in tterms of apat tite and monoomer chemistry) as demon nstrated by XRD patterns (Figure 100) and, from past p experiencce (described in other Chap pters of this thesis), it wwas expected that they also had the simmilar bulk chemistry. As res sulted from the burn teest, they hadd similar starting phase coomposition (Table ( 6). The eir surface topographhy was similaar, with random mly disordereed texture due e to the alkali i treatment (Figure 10) ). Figure 10. ( (a) and (b) are SSEM images of the surface of MM38 and M60 re espectively. Similar randomly disordered ssurface topograpphies between the t two compossites are visible e. (c) XRD spe ectra for the copolymers, aapatite and compposites. It may be e observed that thhe composites ha ave almost identi ical patterns. 175
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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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Chapter 6 - Fluid uptake as instruc
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Chapter 6 - Fluid uptake as instruc
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Page 221 and 222: References [102] McBeath R, Pirone
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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
Page 243: 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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