Combined marrow stromal cell-sheet techniques and high-strength biodegradable composite scaffolds for engineered functional bone grafts

In this study, cell sheets comprising multilayered porcine bone marrow stromal cells (BMSC) were assembled with fully interconnected scaffolds made from medical-grade polycaprolactone–calcium phosphate (mPCL–CaP), for the engineering of structural and functional bone grafts. The BMSC sheets were harvested from culture flasks and wrapped around pre-seeded composite scaffolds. The layered cell sheets integrated well with the scaffold/cell construct and remained viable, with mineralized nodules visible both inside and outside the scaffold for up to 8 weeks culture. Cells within the constructs underwent classical in vitro osteogenic differentiation with the associated elevation of alkaline phosphatase activity and bone-related protein expression. In vivo, two sets of cell-sheet-scaffold/cell constructs were transplanted under the skin of nude rats. The first set of constructs (554mm3) were assembled with BMSC sheets and cultured for 8 weeks before implantation. The second set of constructs (10104mm3) was implanted immediately after assembly with BMSC sheets, with no further in vitro culture. For both groups, neo cortical and well-vascularised cancellous bone were formed within the constructs with up to 40% bone volume. Histological and immunohistochemical examination revealed that neo bone tissue formed from the pool of seeded BMSC and the bone formation followed predominantly an endochondral pathway, with woven bone matrix subsequently maturing into fully mineralized compact bone; exhibiting the histological markers of native bone. These findings demonstrate that large bone tissues similar to native bone can be regenerated utilizing BMSC sheet techniques in conjunction with composite scaffolds whose structures are optimized from a mechanical, nutrient transport and vascularization perspective.

An alginate-based hybrid system for growth factor delivery in the functional repair of large bone defects

The treatment of challenging fractures and large osseous defects presents a formidable problem for orthopaedic surgeons. Tissue engineering/regenerative medicine approaches seek to solve this problem by delivering osteogenic signals within scaffolding biomaterials. In this study, we introduce a hybrid growth factor delivery system that consists of an electrospun nanofiber mesh tube for guiding bone regeneration combined with peptide-modified alginate hydrogel injected inside the tube for sustained growth factor release. We tested the ability of this system to deliver recombinant bone morphogenetic protein-2 (rhBMP-2) for the repair of critically-sized segmental bone defects in a rat model. Longitudinal [mu]-CT analysis and torsional testing provided quantitative assessment of bone regeneration. Our results indicate that the hybrid delivery system resulted in consistent bony bridging of the challenging bone defects. However, in the absence of rhBMP-2, the use of nanofiber mesh tube and alginate did not result in substantial bone formation. Perforations in the nanofiber mesh accelerated the rhBMP-2 mediated bone repair, and resulted in functional restoration of the regenerated bone. [mu]-CT based angiography indicated that perforations did not significantly affect the revascularization of defects, suggesting that some other interaction with the tissue surrounding the defect such as improved infiltration of osteoprogenitor cells contributed to the observed differences in repair. Overall, our results indicate that the hybrid alginate/nanofiber mesh system is a promising growth factor delivery strategy for the repair of challenging bone injuries.

In vivo evaluation of an ultra-thin polycaprolactone film as a wound dressing

The use of ultra-thin films as dressings for cutaneous wounds could prove advantageous in terms of better conformity to wound topography and improved vapour transmission. For this purpose, ultra-thin poly(epsilon-caprolactone) (PCL) films of 5-15 microm thickness were fabricated via a biaxial stretching technique. To evaluate their in vivo biocompatibility and feasibility as an external wound dressing, PCL films were applied over full and partial-thickness wounds in rat and pig models. Different groups of PCL films were used: untreated, NaOH-treated, untreated with fibrin, NaOH-treated with perforations, and NaOH-treated with fibrin and S-nitrosoglutathione. Wounds with no external dressings were used as controls. Wound contraction rate, histology and biomechanical analyses were carried out. Wounds re-epithelialized completely at a comparable rate. Formation of a neo-dermal layer and re-epithelialization were observed in all the wounds. A lower level of fibrosis was observed when PCL films were used, compared to the control wounds. Ultimate tensile strength of the regenerated tissue in rats reached 50-60% of that in native rat skin. Results indicated that biaxially-stretched PCL films did not induce inflammatory reactions when used in vivo as a wound dressing and supported the normal wound healing process in full and partial-thickness wounds.

Sodium removal from Maramarua CSG waters using Ngakuru zeolites

Coal seam gas (CSG) waters are a by-product of natural gas extraction from un derground coal seams. The main issue with these waters is their elevated sodium content, which in conjunction with their low calcium and magnesium concentrations can generate soil infiltration problems in the long run , as well as short term toxicity effects in plants due to the sodium ion itself. Zeolites are minerals having a porous structure, crystalline characteristics, and an alumino-silicate configuration resulting in an overall negative charge which is balanced by loosely held cations. In New Zealand, Ngakuru zeolites have been mined for commercial use in wastewater treatment applications, cosmetics, and pet litter. This research focuses on assessing the capacity of Ngakuru zeolites to reduce sodium concentrations of CSG waters from Maramarua. Batch and column test (flow through) experiments revealed that Ngakuru zeolites are capable of sorbing sodium cations from concentrated solutions of sodium. In b atch tests, the sodium adsorption capacity ranged from 5.0 to 34.3meq/100g depending on the solution concentration and on the number of times the zeolite had been regenerated. Regeneration with CaCl2 was foun d to be effective. The calculated sodium adsorption capacity of Ngakuru zeolites under flow-through conditions ranged from 11 to 42meq/100g depending on the strength of the solution being treated and on w hether the zeolites had been previously regenerated. The slow kinetics and low cost of the zeolities, coupled with potentially remote sites for gas extraction, could make semi-batch operational processes without regeneration more favourable than in more industrial ion exchange situations.

Periodontal regeneration and mesenchymal stem cells

The ultimate goal of periodontal therapy is to regenerate periodontal supporting tissues, but this is hard to achieve as the results of periodontal techniques for regeneration are clinically unpredictable. Stem cells owing to their plasticity and proliferation potential provides a new paradigm for periodontal regeneration. Stem cells from mesenchyme can self renew and generate new dental tissues (including dentin and cementum), alveolar bone and periodontal ligament, and thus they have great potential in periodontal regeneration. This chapter presents an insight into mesenchymal stem cells and their potential use in periodontal regeneration. In this chapter the cellular and molecular biology in periodontal regeneration will be introduced, followed by a range of conventional surgical procedures for periodontal regeneration will be discussed. Mesenchymal stem cells applied in regenerated periodontal tissue and their biological characterizations in vitro will be also introduced. Lastly, the use of mesenchymal stem cell to repair periodontal tissues in large animal models will be also reviewed.

Towards the biofortification of banana fruit for enhanced micronutrient content

In Uganda, vitamin A deficiency (VAD) and iron deficiency anaemia (IDA) are major public health problems with between 15-32% of children under 5 years of age showing VAD and 73% being anaemic. This is largely due to the fact that the staple food crop of the country, banana, is low in pro-vitamin A and iron, therefore leading to dietary deficiencies. Although worldwide progress has been made to control VAD and IDA through supplementation, food fortification and diet diversification, their long term sustainability and impact in developing countries such as Uganda is limited. The approach taken by researchers at Queensland University of Technology (QUT), Australia, in collaboration with the National Agricultural Research Organization (NARO), Uganda, to address this problem, is to generate consumer acceptable banana varieties with significantly increased levels of pro-vitamin A and iron in the fruit using genetic engineering techniques. Such an approach requires the use of suitable, well characterised genes and promoters for targeted transgene expression. Recently, a new banana phytoene synthase gene (APsy2a) involved in the synthesis of pro-vitamin A (pVA) carotenoids was isolated from a high â-carotene banana (F’ei cv Asupina). In addition, sequences of banana ferritin, an iron storage protein, have been isolated from Cavendish banana. The aim of the research described in this thesis was to evaluate the function of these genes to assess their suitability for the biofortification of banana fruit. In addition, a range of banana-derived promoters were characterised to determine their suitability for controlling the expression of transgenes in banana fruit. Due to the time constraints involved with generating transgenic banana fruit, rice was used as the model crop to investigate the functionality of the banana-derived APsy2a and ferritin genes. Using Agrobacterium-mediated transformation, rice callus was transformed with APsy2a +/- the bacterial-derived carotene desaturase gene (CrtI) each under the control of the constitutive maize poly-ubiquitin promoter (ZmUbi) or seed-specific rice glutelin1 (Gt1) promoter. The maize phytoene synthase (ZmPsy1) gene was included as a control. On selective media, with the exception of ZmUbi-CrtI-transgenic callus, all antibiotic resistant callus displayed a yellow-orange colour from which the presence of â-carotene was demonstrated using Raman spectroscopy. Although the regeneration of plants from yellow-orange callus was difficult, 16 transgenic plants were obtained and characterised from callus transformed with ZmUbi-APys2a alone. At least 50% of the T1 seeds developed a yellow-orange coloured callus which was found to contain levels of â-carotene ranging from 4.6-fold to 72-fold higher than that in non-transgenic rice callus. Using the seed-specific Gt1 promoter, 38 transgenic rice plants were generated from APsy2a-CrtI-transformed callus while 32 plants were regenerated from ZmPsy1-CrtI-transformed callus. However, when analysed for presence of transgene by PCR, all transgenic plants contained the APsy2a, ZmPsy1 or CrtI transgene, with none of the plants found to be co-transformed. Using Raman spectroscopy, no â-carotene was detected in-situ in representative T1 seeds. To investigate the potential of the banana-derived ferritin gene (BanFer1) to enhance iron content, rice callus was transformed with constitutively expressed BanFer1 using the soybean ferritin gene (SoyFer) as a control. A total of 12 and 11 callus lines independently transformed with BanFer1 and SoyFer, respectively, were multiplied and transgene expression was verified by RT-PCR. Pearl’s Prussian blue staining for in-situ detection of ferric iron showed a stronger blue colour in rice callus transformed with BanFer1 compared to SoyFer. Using flame atomic absorption spectrometry, the highest mean amount of iron quantified in callus transformed with BanFer1 was 30-fold while that obtained using the SoyFer was 14-fold higher than the controls. In addition, ~78% of BanFer1-transgenic callus lines and ~27% of SoyFer-transgenic callus lines had significantly higher iron content than the non-transformed controls. Since the genes used for enhancing micronutrient content need to be expressed in banana fruit, the activity of a range of banana-derived, potentially fruit-active promoters in banana was investigated. Using uidA (GUS) as a reporter gene, the function of the Expansin1 (MaExp1), Expansin1 containing the rice actin intron (MaExp1a), Expansin4 (MaExp4), Extensin (MaExt), ACS (MaACS), ACO (MaACO), Metallothionein (MaMT2a) and phytoene synthase (APsy2a) promoters were transiently analysed in intact banana fruit using two transformation methods, particle bombardment and Agrobacterium-mediated infiltration (agro-infiltration). Although a considerable amount of variation in promoter activity was observed both within and between experiments, similar trends were obtained using both transformation methods. The MaExp1 and MaExp1a directed high levels of GUS expression in banana fruit which were comparable to those observed from the ZmUbi and Banana bunchy top virus-derived BT4 promoters that were included as positive controls. Lower levels of promoter activity were obtained in both methods using the MaACO and MaExt promoters while the MaExp4, MaACS, and APsy2a promoters directed the lowest GUS activity in banana fruit. An attempt was subsequently made to use agro-infiltration to assess the expression of pVA biosynthesis genes in banana fruit by infiltrating fruit with constructs in which the ZmUbi promoter controlled the expression of APsy2a +/- CrtI, and with the maize phytoene synthase gene (ZmPsy1) included as a control. Unfortunately, the large amount of variation and inconsistency observed within and between experiments precluded any meaningful conclusions to be drawn. The final component of this research was to assess the level of promoter activity and specificity in non-target tissue. These analyses were done on leaves obtained from glasshouse-grown banana plants stably transformed with MaExp1, MaACO, APsy2a, BT4 and ZmUbi promoters driving the expression of the GUS gene in addition to leaves from a selection of the same transgenic plants which were growing in a field trial in North Queensland. The results from both histochemical and fluorometric GUS assays showed that the MaExp1 and MaACO promoters directed very low GUS activities in leaves of stably transformed banana plants compared to the constitutive ZmUbi and BT4 promoters. In summary, the results from this research provide evidence that the banana phytoene synthase gene (APsy2a) and the banana ferritin gene (BanFer1) are functional, since the constitutive over-expression of each of these transgenes led to increased levels of pVA carotenoids (for APsy2a) and iron content (for BanFer1) in transgenic rice callus. Further work is now required to determine the functionality of these genes in stably-transformed banana fruit. This research also demonstrated that the MaExp1 and MaACO promoters are fruit-active but have low activity in non-target tissue (leaves), characteristics that make them potentially useful for the biofortification of banana fruit. Ultimately, however, analysis of fruit from field-grown transgenic plants will be required to fully evaluate the suitability of pVA biosynthesis genes and the fruit-active promoters for fruit biofortification.

Development of a high-level gene expression system for the production of bioplastics in sugarcane

Despite various approaches, the production of biodegradable plastics such as polyhydroxybutyrate (PHB) in transgenic plants has met with limited success due largely to low expression levels. Even in the few instances where high levels of protein expression have been reported, the transgenic plants have been stunted indicating PHB is phytotoxic (Poirier 2002). This PhD describes the application of a novel virus-based gene expression technology, termed InPAct („In Plant Activation.), for the production of PHB in tobacco and sugarcane. InPAct is based on the rolling circle replication mechanism by which circular ssDNA viruses replicate and provides a system for controlled, high-level gene expression. Based on these features, InPAct was thought to represent an ideal system to enable the controlled, high-level expression of the three phb genes (phbA, phbB and phbC) required for PHB production in sugarcane at a preferred stage of plant growth. A Tobacco yellow dwarf virus (TbYDV)-based InPAct-phbA vector, as well as linear vectors constitutively expressing phbB and phbC were constructed and different combinations were used to transform tobacco leaf discs. A total of four, eight, three and three phenotypically normal tobacco lines were generated from discs transformed with InPAct-phbA, InPAct-phbA + p1300-TaBV P-phbB/phbC- 35S T, p1300-35S P-phbA-NOS T + p1300-TaBV P-phbB/phbC-35S T and InPAct-GUS, respectively. To determine whether the InPAct cassette could be activated in the presence of the TbYDV Rep, leaf samples from the eight InPActphbA + p1300-TaBV P-phbB/phbC-35S T plants were agroinfiltrated with p1300- TbYDV-Rep/RepA. Three days later, successful activation was indicated by the detection of episomes using both PCR and Southern analysis. Leaf discs from the eight InPAct-phbA + p1300-TaBV P-phbB/phbC-35S T transgenic plant lines were agroinfiltrated with p1300-TbYDV-Rep/RepA and leaf tissue was collected ten days post-infiltration and examined for the presence of PHB granules. Confocal microscopy and TEM revealed the presence of typical PHB granules in five of the eight lines, thus demonstrating the functionality of InPActbased PHB production in tobacco. However, analysis of leaf extracts by HPLC failed to detect the presence of PHB suggesting only very low level expression levels. Subsequent molecular analysis of three lines revealed low levels of correctly processed mRNA from the catalase intron contained within the InPAct cassette and also the presence of cryptic splice sites within the intron. In an attempt to increase expression levels, new InPAct-phb cassettes were generated in which the castorbean catalase intron was replaced with a synthetic intron (syntron). Further, in an attempt to both increase and better control Rep/RepA-mediated activation of InPAct cassettes, Rep/RepA expression was placed under the control of a stably integrated alc switch. Leaf discs from a transgenic tobacco line (Alc ML) containing 35S P-AlcR-AlcA P-Rep/RepA were supertransformed with InPAct-phbAsyn or InPAct-GUSsyn using Agrobacterium and three plants (lines) were regenerated for each construct. Analysis of the RNA processing of the InPAct-phbAsyn cassette revealed highly efficient and correct splicing of the syntron, thus supporting its inclusion within the InPAct system. To determine the efficiency of the alc switch to activate InPAct, leaf material from the three Alc ML + InPAct-phbAsyn lines was either agroinfiltrated with 35S P-Rep/RepA or treated with ethanol. Unexpectedly, episomes were detected not only in the infiltrated and ethanol treated samples, but also in non-treated samples. Subsequent analysis of transgenic Alc ML + InPAct-GUS lines, confirmed that the alc switch was leaky in tissue culture. Although this was shown to be reversible once plants were removed from the tissue culture environment, it made the regeneration of Alc ML + InPAct-phbsyn plant lines extremely difficult, due to unintentional Rep expression and therefore high levels of phb expression and phytotoxic PHB production. Two Alc ML + InPAct-phbAsyn + p1300-TaBV P-phbB/phbC-35S T transgenic lines were able to be regenerated, and these were acclimatised, alcohol-treated and analysed. Although episome formation was detected as late as 21 days post activation, no PHB was detected in the leaves of any plants using either microscopy or HPLC, suggesting the presence of a corrupt InPAct-phbA cassette in both lines. The final component of this thesis involved the application of both the alc switch and the InPAct systems to sugarcane in an attempt to produce PHB. Initial experiments using transgenic Alc ML + InPAct-GUS lines indicated that the alc system was not functional in sugarcane under the conditions tested. The functionality of the InPAct system, independent of the alc gene switch, was subsequently examined by bombarding the 35S Rep/RepA cassette into leaf and immature leaf whorl cells derived from InPAct-GUS transgenic sugarcane plants. No GUS expression was observed in leaf tissue, whereas weak and irregular GUS expression was observed in immature leaf whorl tissue derived from two InPAct- GUS lines and two InPAct-GUS + 35S P-AlcR-AlcA P-GUS lines. The most plausible reason to explain the inconsistent and low levels of GUS expression in leaf whorls is a combination of low numbers of sugarcane cells in the DNA replication-conducive S-phase and the irregular and random nature of sugarcane cells bombarded with Rep/RepA. This study details the first report to develop a TbYDV-based InPAct system under control of the alc switch to produce PHB in tobacco and sugarcane. Despite the inability to detect quantifiable levels of PHB levels in either tobacco or sugarcane, the findings of this study should nevertheless assist in the further development of both the InPAct system and the alc system, particularly for sugarcane and ultimately lead to an ethanol-inducible InPAct gene expression system for the production of bioplastics and other proteins of commercial value in plants.

Structural and thermal performance of cold-formed steel stud wall systems under fire conditions

Cold-formed steel stud walls are a major component of Light Steel Framing (LSF) building systems used in commercial, industrial and residential buildings. In the conventional LSF stud wall systems, thin steel studs are protected from fire by placing one or two layers of plasterboard on both sides with or without cavity insulation. However, there is very limited data about the structural and thermal performance of stud wall systems while past research showed contradicting results, for example, about the benefits of cavity insulation. This research was therefore conducted to improve the knowledge and understanding of the structural and thermal performance of cold-formed steel stud wall systems (both load bearing and non-load bearing) under fire conditions and to develop new improved stud wall systems including reliable and simple methods to predict their fire resistance rating. Full scale fire tests of cold-formed steel stud wall systems formed the basis of this research. This research proposed an innovative LSF stud wall system in which a composite panel made of two plasterboards with insulation between them was used to improve the fire rating. Hence fire tests included both conventional steel stud walls with and without the use of cavity insulation and the new composite panel system. A propane fired gas furnace was specially designed and constructed first. The furnace was designed to deliver heat in accordance with the standard time temperature curve as proposed by AS 1530.4 (SA, 2005). A compression loading frame capable of loading the individual studs of a full scale steel stud wall system was also designed and built for the load-bearing tests. Fire tests included comprehensive time-temperature measurements across the thickness and along the length of all the specimens using K type thermocouples. They also included the measurements of load-deformation characteristics of stud walls until failure. The first phase of fire tests included 15 small scale fire tests of gypsum plasterboards, and composite panels using different types of insulating material of varying thickness and density. Fire performance of single and multiple layers of gypsum plasterboards was assessed including the effect of interfaces between adjacent plasterboards on the thermal performance. Effects of insulations such as glass fibre, rock fibre and cellulose fibre were also determined while the tests provided important data relating to the temperature at which the fall off of external plasterboards occurred. In the second phase, nine small scale non-load bearing wall specimens were tested to investigate the thermal performance of conventional and innovative steel stud wall systems. Effects of single and multiple layers of plasterboards with and without vertical joints were investigated. The new composite panels were seen to offer greater thermal protection to the studs in comparison to the conventional panels. In the third phase of fire tests, nine full scale load bearing wall specimens were tested to study the thermal and structural performance of the load bearing wall assemblies. A full scale test was also conducted at ambient temperature. These tests showed that the use of cavity insulation led to inferior fire performance of walls, and provided good explanations and supporting research data to overcome the incorrect industry assumptions about cavity insulation. They demonstrated that the use of insulation externally in a composite panel enhanced the thermal and structural performance of stud walls and increased their fire resistance rating significantly. Hence this research recommends the use of the new composite panel system for cold-formed LSF walls. This research also included steady state tensile tests at ambient and elevated temperatures to address the lack of reliable mechanical properties for high grade cold-formed steels at elevated temperatures. Suitable predictive equations were developed for calculating the yield strength and elastic modulus at elevated temperatures. In summary, this research has developed comprehensive experimental thermal and structural performance data for both the conventional and the proposed non-load bearing and load bearing stud wall systems under fire conditions. Idealized hot flange temperature profiles have been developed for non-insulated, cavity insulated and externally insulated load bearing wall models along with suitable equations for predicting their failure times. A graphical method has also been proposed to predict the failure times (fire rating) of non-load bearing and load bearing walls under different load ratios. The results from this research are useful to both fire researchers and engineers working in this field. Most importantly, this research has significantly improved the knowledge and understanding of cold-formed LSF walls under fire conditions, and developed an innovative LSF wall system with increased fire rating. It has clearly demonstrated the detrimental effects of using cavity insulation, and has paved the way for Australian building industries to develop new wall panels with increased fire rating for commercial applications worldwide.

Properties of lignin and poly(hydroxybutyrate) blends

The Queensland University of Technology (QUT) allows the presentation of a thesis for the Degree of Doctor of Philosophy in the format of published or submitted papers, where such papers have been published, accepted or submitted during the period of candidature. This thesis is composed of Seven published/submitted papers and one poster presentation, of which five have been published and the other two are under review. This project is financially supported by the QUTPRA Grant. The twenty-first century started with the resurrection of lignocellulosic biomass as a potential substitute for petrochemicals. Petrochemicals, which enjoyed the sustainable economic growth during the past century, have begun to reach or have reached their peak. The world energy situation is complicated by political uncertainty and by the environmental impact associated with petrochemical import and usage. In particular, greenhouse gasses and toxic emissions produced by petrochemicals have been implicated as a significant cause of climate changes. Lignocellulosic biomass (e.g. sugarcane biomass and bagasse), which potentially enjoys a more abundant, widely distributed, and cost-effective resource base, can play an indispensible role in the paradigm transition from fossil-based to carbohydrate-based economy. Poly(3-hydroxybutyrate), PHB has attracted much commercial interest as a plastic and biodegradable material because some its physical properties are similar to those of polypropylene (PP), even though the two polymers have quite different chemical structures. PHB exhibits a high degree of crystallinity, has a high melting point of approximately 180°C, and most importantly, unlike PP, PHB is rapidly biodegradable. Two major factors which currently inhibit the widespread use of PHB are its high cost and poor mechanical properties. The production costs of PHB are significantly higher than for plastics produced from petrochemical resources (e.g. PP costs $US1 kg-1, whereas PHB costs $US8 kg-1), and its stiff and brittle nature makes processing difficult and impedes its ability to handle high impact. Lignin, together with cellulose and hemicellulose, are the three main components of every lignocellulosic biomass. It is a natural polymer occurring in the plant cell wall. Lignin, after cellulose, is the most abundant polymer in nature. It is extracted mainly as a by-product in the pulp and paper industry. Although, traditionally lignin is burnt in industry for energy, it has a lot of value-add properties. Lignin, which to date has not been exploited, is an amorphous polymer with hydrophobic behaviour. These make it a good candidate for blending with PHB and technically, blending can be a viable solution for price and reduction and enhance production properties. Theoretically, lignin and PHB affect the physiochemical properties of each other when they become miscible in a composite. A comprehensive study on structural, thermal, rheological and environmental properties of lignin/PHB blends together with neat lignin and PHB is the targeted scope of this thesis. An introduction to this research, including a description of the research problem, a literature review and an account of the research progress linking the research papers is presented in Chapter 1. In this research, lignin was obtained from bagasse through extraction with sodium hydroxide. A novel two-step pH precipitation procedure was used to recover soda lignin with the purity of 96.3 wt% from the black liquor (i.e. the spent sodium hydroxide solution). The precipitation process is presented in Chapter 2. A sequential solvent extraction process was used to fractionate the soda lignin into three fractions. These fractions, together with the soda lignin, were characterised to determine elemental composition, purity, carbohydrate content, molecular weight, and functional group content. The thermal properties of the lignins were also determined. The results are presented and discussed in Chapter 2. On the basis of the type and quantity of functional groups, attempts were made to identify potential applications for each of the individual lignins. As an addendum to the general section on the development of composite materials of lignin, which includes Chapters 1 and 2, studies on the kinetics of bagasse thermal degradation are presented in Appendix 1. The work showed that distinct stages of mass losses depend on residual sucrose. As the development of value-added products from lignin will improve the economics of cellulosic ethanol, a review on lignin applications, which included lignin/PHB composites, is presented in Appendix 2. Chapters 3, 4 and 5 are dedicated to investigations of the properties of soda lignin/PHB composites. Chapter 3 reports on the thermal stability and miscibility of the blends. Although the addition of soda lignin shifts the onset of PHB decomposition to lower temperatures, the lignin/PHB blends are thermally more stable over a wider temperature range. The results from the thermal study also indicated that blends containing up to 40 wt% soda lignin were miscible. The Tg data for these blends fitted nicely to the Gordon-Taylor and Kwei models. Fourier transform infrared spectroscopy (FT-IR) evaluation showed that the miscibility of the blends was because of specific hydrogen bonding (and similar interactions) between reactive phenolic hydroxyl groups of lignin and the carbonyl group of PHB. The thermophysical and rheological properties of soda lignin/PHB blends are presented in Chapter 4. In this chapter, the kinetics of thermal degradation of the blends is studied using thermogravimetric analysis (TGA). This preliminary investigation is limited to the processing temperature of blend manufacturing. Of significance in the study, is the drop in the apparent energy of activation, Ea from 112 kJmol-1 for pure PHB to half that value for blends. This means that the addition of lignin to PHB reduces the thermal stability of PHB, and that the comparative reduced weight loss observed in the TGA data is associated with the slower rate of lignin degradation in the composite. The Tg of PHB, as well as its melting temperature, melting enthalpy, crystallinity and melting point decrease with increase in lignin content. Results from the rheological investigation showed that at low lignin content (.30 wt%), lignin acts as a plasticiser for PHB, while at high lignin content it acts as a filler. Chapter 5 is dedicated to the environmental study of soda lignin/PHB blends. The biodegradability of lignin/PHB blends is compared to that of PHB using the standard soil burial test. To obtain acceptable biodegradation data, samples were buried for 12 months under controlled conditions. Gravimetric analysis, TGA, optical microscopy, scanning electron microscopy (SEM), differential scanning calorimetry (DSC), FT-IR, and X-ray photoelectron spectroscopy (XPS) were used in the study. The results clearly demonstrated that lignin retards the biodegradation of PHB, and that the miscible blends were more resistant to degradation compared to the immiscible blends. To obtain an understanding between the structure of lignin and the properties of the blends, a methanol-soluble lignin, which contains 3× less phenolic hydroxyl group that its parent soda lignin used in preparing blends for the work reported in Chapters 3 and 4, was blended with PHB and the properties of the blends investigated. The results are reported in Chapter 6. At up to 40 wt% methanolsoluble lignin, the experimental data fitted the Gordon-Taylor and Kwei models, similar to the results obtained soda lignin-based blends. However, the values obtained for the interactive parameters for the methanol-soluble lignin blends were slightly lower than the blends obtained with soda lignin indicating weaker association between methanol-soluble lignin and PHB. FT-IR data confirmed that hydrogen bonding is the main interactive force between the reactive functional groups of lignin and the carbonyl group of PHB. In summary, the structural differences existing between the two lignins did not manifest itself in the properties of their blends.

Apoptosis-related genes confer resistance to Fusarium wilt in transgenic ‘Lady Finger’ bananas

Fusarium wilt, caused by Fusarium oxysporum f. sp. cubense (Foc), is one of the most devastating diseases of banana (Musa spp.). Apart from resistant cultivars, there are no effective control measures for the disease. We investigated whether the transgenic expression of apoptosis-inhibition related genes in banana could be used to confer disease resistance. Embryogenic cell suspensions of the banana cultivar, ‘Lady Finger’, were stably transformed with animal genes that negatively regulate apoptosis, namely Bcl-xL, Ced-9 and Bcl-2 3’ UTR, and independently transformed plant lines were regenerated for testing. Following a 12 week exposure to Foc race 1 in small-plant glasshouse bioassays, seven transgenic lines (2 x Bcl-xL, 3 x Ced-9 and 2 x Bcl-2 3’ UTR) showed significantly less internal and external disease symptoms than the wild-type susceptible ‘Lady Finger’ banana plants used as positive controls. Of these, one Bcl-2 3’ UTR line showed resistance that was equivalent to that of wild-type Cavendish bananas that were included as resistant negative controls. Further, the resistance of this line continued for 23 weeks post-inoculation at which time the experiment was terminated. Using TUNEL assays, Foc race 1 was shown to induce apoptosis-like features in the roots of wild-type ‘Lady Finger’ plants consistent with a necrotrophic phase in the lifecycle of this pathogen. This was further supported by the observed reduction of these effects in the roots of the resistant Bcl-2 3’ UTR transgenic line. This is the first report on the generation of transgenic banana plants with resistance to Fusarium wilt.

Evaluation of silk sericin as a biomaterial: In vitro growth of human corneal limbal epithelial cells on Bombyx mori sericin membranes

Sericin and fibroin are the two major proteins in the silk fibre produced by the domesticated silkworm, Bombyx mori. Fibroin has been extensively investigated as a biomaterial. We have previously shown that fibroin can function successfully as a substratum for growing cells of the eye. Sericin has been so far neglected as a biomaterial because of suspected allergenic activity. However, this misconception has now been dispelled, and sericin’s biocompatibility is currently indisputable. Aiming at promoting sericin as a possible substratum for the growth of corneal cells in order to make tissue-engineered constructs for the restoration of the ocular surface, in this study we investigated the attachment and growth in vitro of human corneal limbal epithelial cells (HLECs) on sericin-based membranes. Sericin was isolated and regenerated from the silkworm cocoons by an aqueous procedure, manufactured into membranes, and characterized (mechanical properties, structural analysis, contact angles). Primary cell cultures from two donors were established in serum-supplemented media in the presence of murine feeder cells. Membranes made of sericin and fibroin-sericin blends were assessed in vitro as substrata for HLECs in a serum-free medium, in a cell attachment assay and in a 3-day cell growth experiment. While the mechanical characteristics of sericin were found to be inferior to those of fibroin, its ability to enhance the attachment of HLECs was significantly superior to fibroin, as revealed by the PicoGreen® assay. Evidence was also obtained that cells can grow and differentiate on these substrata.

Reliability of clinical measurement for assessing spinal fusion: An experimental sheep study

Study Design. A sheep study designed to compare the accuracy of static radiographs, dynamic radiographs, and computed tomographic (CT) scans for the assessment of thoracolumbar facet joint fusion as determined by micro-CT scanning. Objective. To determine the accuracy and reliability of conventional imaging techniques in identifying the status of thoracolumbar (T13-L1) facet joint fusion in a sheep model. Summary of Background Data. Plain radiographs are commonly used to determine the integrity of surgical arthrodesis of the thoracolumbar spine. Many previous studies of fusion success have relied solely on postoperative assessment of plain radiographs, a technique lacking sensitivity for pseudarthrosis. CT may be a more reliable technique, but is less well characterized. Methods. Eleven adult sheep were randomized to either attempted arthrodesis using autogenous bone graft and internal fixation (n = 3) or intentional pseudarthrosis (IP) using oxidized cellulose and internal fixation (n = 8). After 6 months, facet joint fusion was assessed by independent observers, using (1) plain static radiography alone, (2) additional dynamic radiographs, and (3) additional reconstructed spiral CT imaging. These assessments were correlated with high-resolution micro-CT imaging to predict the utility of the conventional imaging techniques in the estimation of fusion success. Results. The capacity of plain radiography alone to correctly predict fusion or pseudarthrosis was 43% and was not improved using plain radiography and dynamic radiography with also a 43% accuracy. Adding assessment by reformatted CT imaging to the plain radiography techniques increased the capacity to predict fusion outcome to 86% correctly. The sensitivity, specificity, and accuracy of static radiography were 0.33, 0.55, and 0.43, respectively, those of dynamic radiography were 0.46, 0.40, and 0.43, respectively, and those of radiography plus CT were 0.88, 0.85, and 0.86, respectively. Conclusion. CT-based evaluation correlated most closely with high-resolution micro-CT imaging. Neither plain static nor dynamic radiographs were able to predict fusion outcome accurately. © 2012 Lippincott Williams & Wilkins.

Agrobacterium-mediated transformation of indica rice cultivars using binary and superbinary vectors

The Agrobacterium-mediated transformation system was extended to two indica cultivars: a widely cultivated breeding line IR-64 and an elite basmati cultivar Karnal Local. Root tips and shoot tips of seedlings, and scutellar-calli derived from mature seeds showed high-efficiency Agrobacterium tumefaciens infection and stable transformation. In addition to the superbinary vector pTOK233 in Agrobacterium strain LBA4404, almost equally high levels of transformation were achieved with a relatively much smaller (13.1 kb) binary vector (pCAMBIA1301) in a supervirulent host strain AGL1. In both cases, as well as in both cultivars, while 60–90% of the infected explants produced calli resistant to the selectable agent hygromycin, 59–75% of such calli tested positive for GUS. A high level (400 μM) of acetosyringone in the preinduction medium for Agrobacterium and a higher level (500 μM) in the cocultivation medium was necessary for an enhancement in transformation frequency of the binary vector to levels comparable to a superbinary. Hygromycin-resistant calli could be produced from all the explants used. Transformants could be regenerated for both cultivars using the superbinary and binary vector, but only for calli of scutellar origin. In addition to the molecular confirmation of hpt and gus gene transfer and transcription, absence of gene sequences outside the transferred DNA (T-DNA) region confirmed absence of any long T-DNA transfer.

A study of ionic liquids for dissolution of sugarcane bagasse

Over the last decade, Ionic Liquids (ILs) have been used for the dissolution and derivatization of isolated cellulose. This ability of ILs is now sought for their application in the selective dissolution of cellulose from lignocellulosic biomass, for the manufacture of cellulosic ethanol. However, there are significant knowledge gaps in the understanding of the chemistry of the interaction of biomass and ILs. While imidazolium ILs have been used successfully to dissolve both isolated crystalline cellulose and components of lignocellulosic biomass, phosphonium ILs have not been sufficiently explored for the use in dissolution of lignocellulosic biomass. This thesis reports on the study of the chemistry of sugarcane bagasse with phosphonium ILs. Qualitative and quantitative measurements of biomass components dissolved in the phosphonium ionic liquids (ILs), trihexyltetradecylphosphonium chloride ([P66614]Cl) and tributylmethylphosphonium methylsulphate ([P4441]MeSO4) are obtained using attenuated total reflectance-Fourier Transform Infra Red (FTIR). Absorption bands related to cellulose, hemicelluloses and lignin dissolution monitored in situ in biomass-IL mixtures indicate lignin dissolution in both ILs and some holocellulose dissolution in the hydrophilic [P4441]MeSO4. The kinetics of lignin dissolution reported here indicate that while dissolution in the hydrophobic IL [P66614]Cl appears to follow an accepted mechanism of acid catalysed β-aryl ether cleavage, dissolution in the hydrophilic IL [P4441]MeSO4 does not appear to follow this mechanism and may not be followed by condensation reactions (initiated by reactive ketones). The quantitative measurement of lignin dissolution in phosphonium ILs based on absorbance at 1510 cm-1 has demonstrated utility and greater precision than the conventional Klason lignin method. The cleavage of lignin β-aryl ether bonds in sugarcane bagasse by the ionic liquid [P66614]Cl, in the presence of catalytic amounts of mineral acid. (ca. 0.4 %). The delignification process of bagasse is studied over a range of temperatures (120 °C to 150 °C) by monitoring the production of β-ketones (indicative of cleavage of β-aryl ethers) using FTIR spectroscopy and by compositional analysis of the undissolved fractions. Maximum delignification is obtained at 150 °C, with 52 % of lignin removed from the original lignin content of bagasse. No delignification is observed in the absence of acid which suggests that the reaction is acid catalysed with the IL solubilising the lignin fragments. The rate of delignification was significantly higher at 150 °C, suggesting that crossing the glass transition temperature of lignin effects greater freedom of rotation about the propanoid carbon-carbon bonds and leads to increased cleavage of β-aryl ethers. An attempt has been made to propose a probable mechanism of delignifcation of bagasse with the phosphonuim IL. All polymeric components of bagasse, a lignocellulosic biomass, dissolve in the hydrophilic ionic liquid (IL) tributylmethylphosphonium methylsulfate ([P4441]MeSO4) with and without a catalytic amount of acid (H2SO4, ca. 0.4 %). The presence of acid significantly increases the extent of dissolution of bagasse in [P4441]MeSO4 (by ca. 2.5 times under conditions used here). The dissolved fractions can be partially recovered by the addition of an antisolvent (water) and are significantly enriched in lignin. Unlike acid catalysed dissolution in the hydrophobic IL tetradecyltrihexylphosphonium chloride there is little evidence of cleavage of β-aryl ether bonds of lignin dissolving in [P4441]MeSO4 (with and without acid), but this mechanism may play some role in the acid catalysed dissolution. The XRD of the undissolved fractions suggests that the IL may selectively dissolve the amorphous cellulose component, leaving behind crystalline material.