Young children as regenerative tissue donors : considering the need for legal reform in light of divergent ethical approaches

In Australia, young children who lack decision-making capacity can have regenerative tissue removed to treat another person suffering from a severe or life-threatening disease. While great good can potentially result from this as the recipient’s life may be saved, ethical unease remains over the ‘use’ of young children in this way. This paper examines the ethical approaches that have featured in the debate over the acceptability and limits of this practice, and how these are reflected in Australia’s legal regime governing removal of tissue from young children. This analysis demonstrates a troubling dichotomy within the Australia’s laws that requires decision-makers to adopt inconsistent ethical approaches depending on where a donor child is situated. It is argued that this inconsistency in approach warrants legal reform of this ethically sensitive issue.

The genetics of cutaneous melanoma

Although germline mutations in CDKN2A are present in approximately 25% of large multicase melanoma families, germline mutations are much rarer in the smaller melanoma families that make up most individuals reporting a family history of this disease. In addition, only three families worldwide have been reported with germline mutations in a gene other than CDKN2A (i.e., CDK4). Accordingly, current genomewide scans underway at the National Human Genome Research Institute hope to reveal linkage to one or more chromosomal regions, and ultimately lead to the identification of novel genes involved in melanoma predisposition. Both CDKN2A and PTEN have been identified as genes involved in sporadic melanoma development; however, mutations are more common in cell lines than uncultured tumors. A combination of cytogenetic, molecular, and functional studies suggests that additional genes involved in melanoma development are located to chromosomal regions 1p, 6q, 7p, 11q, and possibly also 9p and 10q. With the near completion of the human genome sequencing effort, combined with the advent of high throughput mutation analyses and new techniques including cDNA and tissue microarrays, the identification and characterization of additional genes involved in melanoma pathogenesis seem likely in the near future.

Microarray expression profiling in melanoma reveals a BRAF mutation signature

We have used microarray gene expression profiling and machine learning to predict the presence of BRAF mutations in a panel of 61 melanoma cell lines. The BRAF gene was found to be mutated in 42 samples (69%) and intragenic mutations of the NRAS gene were detected in seven samples (11%). No cell line carried mutations of both genes. Using support vector machines, we have built a classifier that differentiates between melanoma cell lines based on BRAF mutation status. As few as 83 genes are able to discriminate between BRAF mutant and BRAF wild-type samples with clear separation observed using hierarchical clustering. Multidimensional scaling was used to visualize the relationship between a BRAF mutation signature and that of a generalized mitogen-activated protein kinase (MAPK) activation (either BRAF or NRAS mutation) in the context of the discriminating gene list. We observed that samples carrying NRAS mutations lie somewhere between those with or without BRAF mutations. These observations suggest that there are gene-specific mutation signals in addition to a common MAPK activation that result from the pleiotropic effects of either BRAF or NRAS on other signaling pathways, leading to measurably different transcriptional changes.

Mussel-inspired porous SiO2 scaffolds with improved mineralization and cytocompatibility for drug delivery and bone tissue engineering

Porous SiO2 scaffolds with mesopore structure (named as MS scaffolds) have been proposed as suitable for bone tissue engineering due to their excellent drug-delivery ability; however, the mineralization and cytocompatibility of MS scaffolds are far from optimal for bone tissue engineering, and it is also unclear how the delivery of drugs from MS scaffolds affects osteoblastic cells. The aims of the present study were to improve the mineralization and cytocompatibility of MS scaffolds by coating mussel-inspired polydopamine on the pore walls of scaffolds. The effects of polydopamine modification on MS scaffolds was investigated with respect to apatite mineralization and the attachment, proliferation and differentiation of bone marrow stromal cells (BMSCs), as was the release profile of the drug dexamethasone (DEX). Our results show that polydopamine can readily coat the pore walls of MS scaffolds and that polydopamine-modified MS scaffolds have a significantly improved apatite-mineralization ability as well as better attachment and proliferation of BMSCs in the scaffolds, compared to controls. Polydopamine modification did not alter the release profile of DEX from MS scaffolds but the sustained delivery of DEX significantly improved alkaline phosphatase (ALP) activity of BMSCs in the scaffolds. These results suggest that polydopamine modification is a viable option to enhance the bioactivity of bone tissue engineering scaffolds and, further, that DEX-loaded polydopamine MS scaffolds have potential uses as a release system to enhance the osteogenic properties of bone tissue engineering applications.

Nanostructured mesoporous bioglass coated zirconia scaffolds for bone tissue engineering

For the filling and reconstruction of non-healing bone defects, the application of porous ceramic scaffold as bone substitutes is considered to be a reasonable choice. In bone tissue engineering, an ideal scaffold must satisfy several criterias such as open porosity, having high compressive strength (it depends where in body, and if external fixatures are used) and the practicability for cell migration. Many researchers have focused on enhancing the mechanical properties of hydroxyapatite scaffolds by combining it with other biomaterials, such as bioglass and polymers. Nevertheless, there is still a lack of suitable scaffolds based on porous biomaterials. In this study, zirconia scaffolds from two different templates (polyurethane (PU) and Acrylonitrile Butadiene Styrene (ABS) templates) were successfully fabricated with dissimilar fabrication techniques. The scaffold surfaces were further modified with mesoporous bioglass for the purpose of bone tissue engineering. In the study of PU template scaffold, high porosity (~88%) sol-gel derived yttria-stabilized zirconia (YSZ) scaffold was prepared by a polyurethane (PU) foam replica method using sol-gel derived zirconia for the first time, and double coated with Mesoporous Bioglass (MBGs) coating. For the ABS template scaffold, two types of templates (cube and cylinder) with different strut spacings were used and fabricated by a 3D Rapid Prototyper. Subsequently, zirconia scaffolds with low porosity (63±2.8% to 68±2.5%) were fabricated by embedding the zirconia powder slurry into the ABS templates and burning out the ABS to produce a uniform porous structure. The zirconia scaffolds were double coated with mesoporous bioglass by dip coating for the first time. The porosities of the scaffolds were calculated before and after coating. The microstructures were then examined using scanning electron microscopy and the mechanical properties were evaluated using compressive test. Accordingly, relationships between microstructure, processing and mechanical behaviour of the porous zirconia was discussed. Scaffold biocompatibility and bioactivity was also evaluated using a bone marrow stromal cell (BMSC) proliferation test and a simulated body fluid test.

Preparation and characterisation of tri-calcium phosphate scaffolds with tunnel-like macro-pores for bone tissue engineering

Calcium Phosphate ceramics have been widely used in tissue engineering due to their excellent biocompatibility and biodegradability. In the physiological environment, they are able to gradually degrade, absorbed and promote bone growth. Ultimately, they are capable of replacing damaged bone with new tissue. However, their low mechanical properties limit calcium phosphate ceramics in load-bearing applications. To obtain sufficient mechanical properties as well as high biocompatibility is one of the main focuses in biomaterials research. Therefore, the current project focuses on the preparation and characterization of porous tri-calcium phosphate (TCP) ceramic scaffolds. Hydroxapatite (HA) was used as the raw material, and normal calcium phosphate bioglass was added to adjust the ratio between calcium and phosphate. It was found that when 20% bioglass was added to HA and sintered at 1400oC for 3 hours, the TCP scaffold was obtained and this was confirmed by X-ray diffraction (XRD) analysis. Test results have shown that by applying this method, TCP scaffolds have significantly higher compressive strength (9.98MPa) than those made via TCP powder (<3MPa). Moreover, in order to further increase the compressive strength of TCP scaffolds, the samples were then coated with bioglass. For normal bioglass coated TCP scaffold, compressive strength was 16.69±0.5MPa; the compressive strength for single layer mesoporous bioglass coated scaffolds was 15.03±0.63MPa. In addition, this project has also concentrated on sizes and shapes effects; it was found that the cylinder scaffolds have more mechanical property than the club ones. In addition, this project performed cell culture within scaffold to assess biocompatibility. The cells were well distributed in the scaffold, and the cytotoxicity test was performed by 3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-di- phenytetrazoliumromide (MTT) assay. The Alkaline Phosphatase (Alp) activity of human bone marrow mesenchymal stem cell system (hBMSCs) seeded on scaffold expressed higher in vitro than that in the positive control groups in osteogenic medium, which indicated that the scaffolds were both osteoconductive and osteoinductive, showing potential value in bone tissue engineering.

Temporal tracking of mineralization and transcriptional developments of shell formation during the early life history of pearl oyster Pinctada maxima

Molluscan larval ontogeny is a highly conserved process comprising three principal developmental stages. A characteristic unique to each of these stages is shell design, termed prodissoconch I, prodissoconch II and dissoconch. These shells vary in morphology, mineralogy and microstructure. The discrete temporal transitions in shell biomineralization between these larval stages are utilized in this study to investigate transcriptional involvement in several distinct biomineralization events. Scanning electron microscopy and X-ray diffraction analysis of P. maxima larvae and juveniles collected throughout post-embryonic ontogenesis, document the mineralogy and microstructure of each shelled stage as well as establishing a timeline for transitions in biomineralization. P. maxima larval samples most representative of these biomineralization distinctions and transitions were analyzed for differential gene expression on the microarray platform PmaxArray 1.0. A number of transcripts are reported as differentially expressed in correlation to the mineralization events of P. maxima larval ontogeny. Some of those isolated are known shell matrix genes while others are novel; these are discussed in relation to potential shell formation roles. This interdisciplinary investigation has linked the shell developments of P. maxima larval ontogeny with corresponding gene expression profiles, furthering the elucidation of shell biomineralization.

Hypoxia-mimicking mesoporous bioactive glass scaffolds with controllable cobalt ion release for bone tissue engineering

Low oxygen pressure (hypoxia) plays an important role in stimulating angiogenesis; there are, however, few studies to prepare hypoxia-mimicking tissue engineering scaffolds. Mesoporous bioactive glass (MBG) has been developed as scaffolds with excellent osteogenic properties for bone regeneration. Ionic cobalt (Co) is established as a chemical inducer of hypoxia-inducible factor (HIF)-1α, which induces hypoxia-like response. The aim of this study was to develop hypoxia-mimicking MBG scaffolds by incorporating ionic Co2+ into MBG scaffolds and investigate if the addition of Co2+ ions would induce a cellular hypoxic response in such a tissue engineering scaffold system. The composition, microstructure and mesopore properties (specific surface area, nano-pore volume and nano-pore distribution) of Co-containing MBG (Co-MBG) scaffolds were characterized and the cellular effects of Co on the proliferation, differentiation, vascular endothelial growth factor (VEGF) secretion, HIF-1α expression and bone-related gene expression of human bone marrow stromal cells (BMSCs) in MBG scaffolds were systematically investigated. The results showed that low amounts of Co (< 5%) incorporated into MBG scaffolds had no significant cytotoxicity and that their incorporation significantly enhanced VEGF protein secretion, HIF-1α expression, and bone-related gene expression in BMSCs, and also that the Co-MBG scaffolds support BMSC attachment and proliferation. The scaffolds maintain a well-ordered mesopore channel structure and high specific surface area and have the capacity to efficiently deliver antibiotics drugs; in fact, the sustained released of ampicillin by Co-MBG scaffolds gives them excellent anti-bacterial properties. Our results indicate that incorporating cobalt ions into MBG scaffolds is a viable option for preparing hypoxia-mimicking tissue engineering scaffolds and significantly enhanced hypoxia function. The hypoxia-mimicking MBG scaffolds have great potential for bone tissue engineering applications by combining enhanced angiogenesis with already existing osteogenic properties.

Characterisation of the TaNF-Y family of transcription factors in wheat

Light plays a unique role for plants as it is both a source of energy for growth and a signal for development. Light captured by the pigments in the light harvesting complexes is used to drive the synthesis of the chemical energy required for carbon assimilation. The light perceived by photoreceptors activates effectors, such as transcription factors (TFs), which modulate the expression of light-responsive genes. Recently, it has been speculated that increasing the photosynthetic rate could further improve the yield potential of three carbon (C3) crops such as wheat. However, little is currently known about the transcriptional regulation of photosynthesis genes, particularly in crop species. Nuclear factor Y (NF-Y) TF is a functionally diverse regulator of growth and development in the model plant species, with demonstrated roles in embryo development, stress response, flowering time and chloroplast biogenesis. Furthermore, a light-responsive NF-Y binding site (CCAAT-box) is present in the promoter of a spinach photosynthesis gene. As photosynthesis genes are co-regulated by light and co-regulated genes typically have similar regulatory elements in their promoters, it seems likely that other photosynthesis genes would also have light-responsive CCAAT-boxes. This provided the impetus to investigate the NF-Y TF in bread wheat. This thesis is focussed on wheat NF-Y members that have roles in light-mediated gene regulation with an emphasis on their involvement in the regulation of photosynthesis genes. NF-Y is a heterotrimeric complex, comprised of the three subunits NF-YA, NF-YB and NF-YC. Unlike the mammalian and yeast counterparts, each of the three subunits is encoded by multiple genes in Arabidopsis. The initial step taken in this study was the identification of the wheat NF-Y family (Chapter 3). A search of the current wheat nucleotide sequence databases identified 37 NF-Y genes (10 NF-YA, 11 NF-YB, 14 NF-YC & 2 Dr1). Phylogenetic analysis revealed that each of the three wheat NF-Y (TaNF-Y) subunit families could be divided into 4-5 clades based on their conserved core regions. Outside of the core regions, eleven motifs were identified to be conserved between Arabidopsis, rice and wheat NF-Y subunit members. The expression profiles of TaNF-Y genes were constructed using quantitative real-time polymerase chain reaction (RT-PCR). Some TaNF-Y subunit members had little variation in their transcript levels among the organs, while others displayed organ-predominant expression profiles, including those expressed mainly in the photosynthetic organs. To investigate their potential role in light-mediated gene regulation, the light responsiveness of the TaNF-Y genes were examined (Chapters 4 and 5). Two TaNF-YB and five TaNF-YC members were markedly upregulated by light in both the wheat leaves and seedling shoots. To identify the potential target genes of the light-upregulated NF-Y subunit members, a gene expression correlation analysis was conducted using publically available Affymetrix Wheat Genome Array datasets. This analysis revealed that the transcript expression levels of TaNF-YB3 and TaNF-YC11 were significantly correlated with those of photosynthesis genes. These correlated express profiles were also observed in the quantitative RT-PCR dataset from wheat plants grown under light and dark conditions. Sequence analysis of the promoters of these wheat photosynthesis genes revealed that they were enriched with potential NF-Y binding sites (CCAAT-box). The potential role of TaNF-YB3 in the regulation of photosynthetic genes was further investigated using a transgenic approach (Chapter 5). Transgenic wheat lines constitutively expressing TaNF-YB3 were found to have significantly increased expression levels of photosynthesis genes, including those encoding light harvesting chlorophyll a/b-binding proteins, photosystem I reaction centre subunits, a chloroplast ATP synthase subunit and glutamyl-tRNA reductase (GluTR). GluTR is a rate-limiting enzyme in the chlorophyll biosynthesis pathway. In association with the increased expression of the photosynthesis genes, the transgenic lines had a higher leaf chlorophyll content, increased photosynthetic rate and had a more rapid early growth rate compared to the wild-type wheat. In addition to its role in the regulation of photosynthesis genes, TaNF-YB3 overexpression lines flower on average 2-days earlier than the wild-type (Chapter 6). Quantitative RT-PCR analysis showed that there was a 13-fold increase in the expression level of the floral integrator, TaFT. The transcript levels of other downstream genes (TaFT2 and TaVRN1) were also increased in the transgenic lines. Furthermore, the transcript levels of TaNF-YB3 were significantly correlated with those of constans (CO), constans-like (COL) and timing of chlorophyll a/b-binding (CAB) expression 1 [TOC1; (CCT)] domain-containing proteins known to be involved in the regulation of flowering time. To summarise the key findings of this study, 37 NF-Y genes were identified in the crop species wheat. An in depth analysis of TaNF-Y gene expression profiles revealed that the potential role of some light-upregulated members was in the regulation of photosynthetic genes. The involvement of TaNF-YB3 in the regulation of photosynthesis genes was supported by data obtained from transgenic wheat lines with increased constitutive expression of TaNF-YB3. The overexpression of TaNF-YB3 in the transgenic lines revealed this NF-YB member is also involved in the fine-tuning of flowering time. These data suggest that the NF-Y TF plays an important role in light-mediated gene regulation in wheat.

Study of tissue damage during mechanical peeling of tough skinned vegetables

Peeling is an essential phase of post harvesting and processing industry; however the undesirable losses and waste rate that occur during peeling stage are always the main concern of food processing sector. There are three methods of peeling fruits and vegetables including mechanical, chemical and thermal, depending on the class and type of fruit. By comparison, the mechanical method is the most preferred; this method keeps edible portions of produce fresh and creates less damage. Obviously reducing material losses and increasing the quality of the process has a direct effect on the whole efficiency of food processing industry which needs more study on technological aspects of this industrial segment. In order to enhance the effectiveness of food industrial practices it is essential to have a clear understanding of material properties and behaviour of tissues under industrial processes. This paper presents the scheme of research that seeks to examine tissue damage of tough skinned vegetables under mechanical peeling process by developing a novel FE model of the process using explicit dynamic finite element analysis approach. In the proposed study a nonlinear model which will be capable of simulating the peeling process specifically, will be developed. It is expected that unavailable information such as cutting force, maximum shearing force, shear strength, tensile strength and rupture stress will be quantified using the new FEA model. The outcomes will be used to optimize and improve the current mechanical peeling methods of this class of vegetables and thereby enhance the overall effectiveness of processing operations. Presented paper aims to review available literature and previous works have been done in this area of research and identify current gap in modelling and simulation of food processes.

Tissue engineering bone - reconstruction of critical sized segmental bone defects in a large animal model

Currently, well established clinical therapeutic approaches for bone reconstruction are restricted to the transplantation of autografts and allografts, and the implantation of metal devices or ceramic-based implants to assist bone regeneration. Bone grafts possess osteoconductive and osteoinductive properties, their application, however, is associated with disadvantages. These include limited access and availability, donor site morbidity and haemorrhage, increased risk of infection, and insufficient transplant integration. As a result, recent research focuses on the development of complementary therapeutic concepts. The field of tissue engineering has emerged as an important alternative approach to bone regeneration. Tissue engineering unites aspects of cellular biology, biomechanical engineering, biomaterial sciences and trauma and orthopaedic surgery. To obtain approval by regulatory bodies for these novel therapeutic concepts the level of therapeutic benefit must be demonstrated rigorously in well characterized, clinically relevant animal models. Therefore, in this PhD project, a reproducible and clinically relevant, ovine, critically sized, high load bearing, tibial defect model was established and characterized as a prerequisite to assess the regenerative potential of a novel treatment concept in vivo involving a medical grade polycaprolactone and tricalciumphosphate based composite scaffold and recombinant human bone morphogenetic proteins.

Endogenous musculoskeletal tissue engineering : a focussed perspective

Two major difficulties facing widespread clinical implementation of existing Tissue Engineering (TE) strategies for the treatment of musculoskeletal disorders are (1) the cost, space and time required for ex vivo culture of a patient’s autologous cells prior to re-implantation as part of a TE construct, and (2) the potential risks and availability constraints associated with transplanting exogenous (foreign) cells. These hurdles have led to recent interest in endogenous TE strategies, in which the regenerative potential of a patient’s own cells is harnessed to promote tissue regrowth without ex vivo cell culture. This article provides a focused perspective on key issues in the development of endogenous TE strategies, progress to date, and suggested future research directions toward endogenous repair and regeneration of musculoskeletal tissues and organs.

Investigation of the expression of the EphB4 receptor tyrosine kinase in prostate carcinoma

Background: The EphB4 receptor tyrosine kinase has been reported as increased in tumours originating from several different tissues and its expression in a prostate cancer xenograft model has been reported. Methods: RT-PCR, western blotting and immunohistochemical techniques were used to examine EphB4 expression and protein levels in human prostate cancer cell lines LNCaP, DU145 and PC3. Immunohistochemistry was also used to examine localisation of EphB4 in tissue samples from 15 patients with prostate carcinomas. Results: All three prostate cancer cell lines expressed the EphB4 gene and protein. EphB4 immunoreactivity in vivo was significantly greater in human prostate cancers as compared with matched normal prostate epithelium and there appeared to be a trend towards increased expression with higher grade disease. Conclusions: EphB4 is expressed in prostate cancer cell lines with increased expression in human prostate cancers when compared with matched normal tissue. EphB4 may therefore be a useful anti-prostate cancer target. © 2005 Lee et al., licensee BioMed Central Ltd.