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A new approach to soft tissue elastography is presented. The work was motivated by the need to understand and mitigate the effects of anthropogenic sound on marine mammals. These efforts have been hampered by a lack of knowledge of in vivo tissue viscoelastic moduli. To address this problem, a measurement system concept was developed to non-invasively determine shear viscoelastic properties at tissue depths of over 12 cm – well beyond the capabilities of existing systems. The central design feature of the measurement system is a focused, sectored, annular ultrasonic source that generates a ring-like pressure field. This in turn produces a ring-like radiation force distribution in soft tissue, the response of which is primarily observable as a shear wave field that converges to the center of the force pattern. A second confocal transducer nested inside the shear wave generation source is used to measure the component of the shear wave motion along the beam axis. Propagation speed is estimated from displacement phase changes resulting from drive frequency induced dilation of the forcing radius. Forcing beams are modulated in order to establish shear speed frequency dependence, allowing quantification of shear speed dispersion. This concept for convergent field elastography (CFE) is intended to significantly improve the overall ability to estimate soft tissue shear speeds in thick, complex tissues while keeping within FDA-mandated ultrasound exposure limits. A prototype system was developed and tested in tissue mimicking materials for which properties were independently determined. Experiments were first carried out in a homogeneous material, and subsequently in a material containing elastic contrast inclusions. Transmission experiments with re-hydrated samples of bottlenose dolphin skull and mandibular bone samples were conducted to quantify ultrasonic beam attenuation and distortion effects, and their cumulative impact on CFE shear estimation performance. In addition to supporting marine mammal studies, the techniques developed in this thesis may enable or extend a wide range of human medical diagnostics.

Fracture non-unions and bone defects represent a recalcitrant problem in the field of orthopaedic surgery. Although the current gold-standard treatment, autologous bone grafting, has a relatively high success rate, the technique is not without serious problems. The emerging field of regenerative medicine may have the potential to provide an alternative treatment. One promising strategy involves the delivery of both cells and multiple growth factors with different release profiles. A range of scaffolds was developed from Poly( -caprolactone) (PCL), Poly(lactideco- glycolide) (PLGA), and two blends of PCL (Mn 42,500) and PLGA. The scaffolds were manufactured utilising a novel modified fused deposition modelling system, using polymer/dichloromethane solutions. The scaffolds were found to have pore sizes suitable for bone regenerative medical applications (373±9.5 μm in the Ydirection and 460±13 μm in the X-direction). However, the scaffolds were found to be only 52±3 μm in height. This means that the two-layer scaffolds were relatively flat. This was undesirable, as direct control of the complete 3D geometry was the favoured strategy, though it may not be a necessary requirement. Five scaffold coatings were also developed from alginate, chitosan (crosslinked using sodium hydroxide or tripolyphosphate), Type-I collagen and Type-A gelatin. The scaffold coatings were screened in vitro for their cell-compatibility with human marrow stromal cells (hMSCs), human osteoblasts and MG63 cells. This was assessed using an assay for cell death, and assessing total cell counts. From these studies, Type-I collagen was found to be the optimum coating. For hMSCs, their death rates were found to be 19.1±6.3% for alginate, 5.3±3.6% and 2.9±1.4% for chitosan crosslinked with tripolyphosphate and sodium hydroxide respectively, compared to 0.11±0.07% for Type-I collagen, and 0.15±0.13% and 0.16±0.12% for 0.1% and 0.2% gelatin respectively. Type-I collagen was found to be the most cellcompatible coating, as it was consistently associated with higher cell counts than Type-A gelatin. Similarly, PCL scaffolds vacuum dried for 1 hr were found to be cell-compatible. No detectable clinically significant difference was found in either total cell counts, or the proportion of cell death in; hMSCs exposed to PCL scaffolds processed with dichloromethane, hMSCs either exposed to scaffolds known to be biocompatible, or hMSCs cultured in the absence of scaffolds. When cell morphology was compared, scaffolds vacuum dried for 1 hr or more were found to have a similar morphology to the cells cultured in the absence of scaffolds. It was therefore concluded that a vacuum drying time of 1 hr was sufficient for cell-compatibility. The scaffold materials were screened both for their encapsulation efficiencies and release characteristics using the model drug, methylene blue. The encapsulation efficiency was found to be both relatively high and consistent for both Mn 42,500 and 80,000 PCL as well as PCL:PLGA 66:33, at 71±6%, 71±5%, and 78±10% respectively, relative to the low efficiencies recorded for both PCL:PLGA 66:33 and PLGA: 57±5% and 38±10% respectively. The release rate of methylene blue from PCL (Mn 42,500), was found to be relatively slow, controlled, and consistent between batches (between 21±2% and 20±3% released in the first 24 hr). Despite the release rate being consistent for PCL (Mn 80,000), the release rate was thought to be too high, since between 29±3% and 39±5% of the test compound was released in the first 24 hr period. The release rate of methylene blue from the PCL/PLGA blends (between 17±2% – 30±7% and 18±4% – 31±6% in the first 24 hr) and PLGA (between 7.1±3.4% – 9.3±2.9% in the first 24 hr) were found to be inconsistent, and low in the case of PLGA, even taking the different loading efficiencies into account. Therefore, PCL (Mn 42,500) was selected as the favoured candidate scaffold material. The loading content and release profiles from methylene blue loaded collagen scaffold coatings were also evaluated. The drug loading capacity was found to be suitable for use as a drug delivery system (65±5 μg/g of methylene blue per unit scaffold mass). The release of methylene blue was observed to be rapid (between 54±10% – 70±17% in the first 24 hr), which was thought to be desirable for the coating delivery system. Recombinant human bone morphogenetic protein-7 (rhBMP-7) was used as a representative growth factor of interest for bone regenerative medical applications. It was loaded in collagen scaffold coatings (CoatBMP 1.25) and encapsulated within PCL (Mn 42,500) scaffolds (ScaffBMP 1.25). Control coatings and scaffolds were designated CoatPBS and ScaffPBS respectively. Both delivery systems were found to release detectable quantities of rhBMP-7 (releasing 2.8±0.2 μg/g and 87±7 ng/g respectively in the first 24 hr), even after 14 days. The release rate of the growth factor from the scaffold coating was higher than that from the encapsulating scaffolds. However, the cumulative release profiles were found to deviate from the desired ideal release profiles, and burst release was observed from both delivery systems. Although differences were observed for the two delivery systems, this difference may not be of clinical significance. Nevertheless, scaffolds with less than ideal delivery properties may still be of potential clinical use. The bioactivity of the rhBMP-7 released from the test scaffolds was therefore assessed by quantifying the area of normalised ALP staining of hMSCs. The release of rhBMP-7 from the collagen coating of the PCL (Mn 42,500) scaffolds (CoatBMP 1.25ScaffPBS) was capable of statistically significantly increasing hMSC normalised ALP expression, although the actual differences were often relatively small. Therefore, at least a proportion of the growth factor released is likely to have been bioactive. The release from scaffolds encapsulating rhBMP-7 (CoatPBSScaffBMP 1.25) did not have this effect on the hMSCs, indicating that either the concentration released was too low, or the growth factor released was no longer bioactive. However, when the cells were seeded directly onto the scaffolds, the activity of ALP, normalised by a DNA assay, was statistically significantly increased for the CoatPBSScaffBMP 1.25 scaffolds, in hMSCs from all three test patient donors (by 35±10% on the control). ALP activity was also significantly increased in hMSCs from two of the three patients seeded onto CoatBMP 1.25ScaffBMP 1.25 scaffolds (by 39±10% on the control). ALP activity was only statistically significantly increased for one of the hMSC patients when seeded onto CoatBMP 1.25ScaffPBS scaffolds (by 35±14% on the control). The functional osteoinductive capacity of Type-I collagen coated PCL (Mn 42,500) scaffolds loaded with rhBMP-7 was assessed using C2C12 cells seeded onto the scaffolds, and quantified using qRT-PCR. The genes of interest were; Type-I collagen (Col1), osteopontin (OP), ALP, osteocalcin (OC) and runt related transcription factor 2 (Runx2). The CoatBMP 1.25ScaffPBS scaffolds had an early osteoinductive effect on the C2C12 cells, as ALP, OC and Runx2 were elevated during the first 2 days only, compared to the control (e.g. by 44±12%, 128±42%, 60±25% and 46±25% respectively at the 24 hr mark). The CoatPBSScaffBMP 1.25 scaffolds also had an osteoinductive effect on the cells, which was more sustained than that observed for the CoatBMP 1.25ScaffPBS group. While OP, ALP and Runx2 were up-regulated in the first 24 hr compared to the control (by 38±10%, 208±82% and 72±31% respectively), statistically significant up-regulation of the late marker OC was delayed until the 48 hr mark (by 73±49%). The effect was found to be sustained until day 7, when OC and Runx2 were both statistically significantly up-regulated compared to the control (by 151±91% and 93±27% respectively). The CoatBMP 1.25ScaffBMP 1.25 scaffolds were found to combine the early effect of the CoatBMP 1.25ScaffPBS scaffolds, with the more sustained effect of the CoatPBSScaffBMP 1.25 scaffolds. ALP, OC and Runx2 were all up-regulated at the 24 hr mark (by 312±56%, 329±39% and 96±25% respectively). This osteoinductive effect was sustained until day 7 when Col1, ALP and Runx2 were still up-regulated compared to the control (by 174±78%, 72±24% and 178±78% respectively). These data suggest that the scaffolds containing rhBMP-7 have a weak osteoinductive effect on the cells seeded onto them. The different delivery systems were found to affect the cells differently. The clinical significance of this was not assessed in these studies. 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) was used as a model drug to assess the feasibility of releasing lipid-soluble active factors from the scaffolds. This was assessed by quantifying the area of normalised ALP staining of hMSCs. The release of 1,25(OH)2D3 from the loaded collagen scaffold coatings and the encapsulating scaffolds significantly increased ALP expression compared to the control scaffold groups (by 115±28% and 69±25% respectively). Furthermore, ALP expression was significantly increased when the two delivery systems were used together, when compared to either delivery system on its own. These data suggest that the delivery of lipid-soluble active factors is feasible from collagen coated PCL scaffolds, and that the coating and encapsulating delivery systems are mutually compatible.

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published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

This thesis describes the isolation, immobilisation and permeabilisation of <i>Digitalis lanata</i> (foxglove) and <i>Nicotiana tabacum</i> (tobacco) plant cells and protoplasts. Protoplasts were isolated from leaves of <i>Digitalis lanata</i> and <i>Nicotiana tabacum</i> and cultured in conditions of varying osmotic potential, illumination and cell density in order to achieve maximum cell stability. The regeneration of cellulose cell walls in <i>Nicotiana tabacum</i> protoplasts could be inhibited by the addition of dilute concentrations of the herbicide 2,6-Dichlorobenzonitrile. At concentrations of 2-10 mg/l in culture medium cell wall regeneration could be prevented for up to 6-8 weeks. Isolated cells and protoplasts were stabilised by entrapment within a beaded agarose support matrix. <i>Nicotiana tabacum</i> protoplasts and cells, immobilised within an agarose beaded support matrix were used to develop a technique to permeabilise and destabilise the cell membranes. Cells or protoplasts pre-loaded with 6-Carboxy-Fluorescein, Neutral Red stains and <SUP>14</SUP>C-Sucrose or <SUP>86</SUP>Rb<SUP></SUP>+ radioactive tracers were employed as markers for cell permeability or leakiness. Efflux or Compartmental analysis was used to determine the influence of various selected permeabilising agents on the integrity and the leakiness of either protoplast or cell membranes. Immobilised protoplasts or cells were subjected to a three - step procedure involving initial loading with <SUP>86</SUP>Rb<SUP></SUP>+ tracer, a membrane permeabilisation step and finally a recovery step. Protoplast membrane stability could not be regained after the recovery step, following a 30 minute period of permeabilisation with 50 mM acetic acid in culture medium. Immobilised cells could, however, regain membrane integrity with good intracellular retention of <SUP>86</SUP>Rb<SUP></SUP>+ tracer ion during the efflux experiment. Thus, immobilised <i>Nicotiana tabacum</i> cells could be made reversibly permeable with the use of specific agents. It is anticipated that such techniques which encourage reversible permeabilisation of immobilised cells have potential in larger scale plant cell culture systems to effect the release of intracellularly stored, useful secondary metabolites. There are also possibilities for the inclusion of exogenous precursors, cofactors and foreign genetic material which would increase compound yield and may produce novel secondary metabolites.

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