High performance additive manufactured scaffolds for bone tissue engineering application

This study demonstrates the feasibility of additive manufactured poly(3-caprolactone)/silanized tricalcium phosphate (PCL/TCP(Si)) scaffolds coated with carbonated hydroxyapatite (CHA)-gelatin composite for bone tissue engineering. In order to reinforce PCL/TCP scaffolds to match the mechanical properties of cancellous bone, TCP has been modified with 3-glycidoxypropyl trimethoxysilane (GPTMS) and incorporated into PCL to synthesize a PCL/TCP(Si) composite. The successful modification is confirmed by X-ray photoelectron spectroscopy (XPS) and Fourier transform infrared spectroscopy (FTIR) analysis. Additive manufactured PCL/TCP(Si) scaffolds have been fabricated using a screw extrusion system (SES). Compression testing demonstrates that both the compressive modulus and compressive yield strength of the developed PCL/TCP(Si) scaffolds fall within the lower ranges of mechanical properties for cancellous bone, with a compressive modulus and compressive yield strength of 6.0 times and 2.3 times of those of PCL/TCP scaffolds, respectively. To enhance the osteoconductive property of the developed PCL/TCP(Si) scaffolds, a CHA-gelatin composite has been coated onto the scaffolds via a biomimetic co-precipitation process, which is verified by using scanning electron microscopy (SEM) and XPS. Confocal laser microscopy and SEM images reveal a most uniform distribution of porcine bone marrow stromal cells (BMSCs) and cellsheet accumulation on the CHA-gelatin composite coated PCL/TCP(Si) scaffolds. The proliferation rate of BMSCs on the CHA-gelatin composite coated PCL/TCP(Si) scaffolds is 2.0 and 1.4 times higher compared to PCL/TCP(Si) and CHA coated PCL/TCP(Si) scaffolds, respectively, by day 10. Furthermore, the reverse transcription polymerase chain reaction (RT-PCR) and western blot analyses reveal that CHA-gelatin composite coated PCL/TCP(Si) scaffolds stimulate osteogenic differentiation of BMSCs the most compared to the other scaffolds. In vitro results of SEM, confocal microscopy and proliferation rate also show that there is no detrimental effect of GPTMS modification on biocompatibility of the scaffolds.

Use of first nucleotide change technology to determine the frequency of factor V Leiden in a population of Australian blood donors

Activated protein C resistance (APCR), the most common risk factor for venous thrombosis, is the result of a G to A base substitution at nucleotide 1691 (R506Q) in the factor V gene. Current techniques to detect the factor V Leiden mutation, such as determination of restriction length polymorphisms, do not have the capacity to screen large numbers of samples in a rapid, cost- effective test. The aim of this study was to apply the first nucleotide change (FNC) technology, to the detection of the factor V Leiden mutation. After preliminary amplification of genomic DNA by polymerase chain reaction (PCR), an allele-specific primer was hybridised to the PCR product and extended using fluorescent terminating dideoxynucleotides which were detected by colorimetric assay. Using this ELISA-based assay, the prevalence of the factor V Leiden mutation was determined in an Australian blood donor population (n = 500). A total of 18 heterozygotes were identified (3.6%) and all of these were confirmed with conventional MnlI restriction digest. No homozygotes for the variant allele were detected. We conclude from this study that the frequency of 3.6% is compatible with others published for Caucasian populations. In addition, the FNC technology shows promise as the basis for a rapid, automated DNA based test for factor V Leiden.

Monitoring mesenchymal stem cell cultures using image processing and pattern recognition techniques

Stem cells have attracted tremendous interest in recent times due to their promise in providing innovative new treatments for a great range of currently debilitating diseases. This is due to their potential ability to regenerate and repair damaged tissue, and hence restore lost body function, in a manner beyond the body's usual healing process. Bone marrow-derived mesenchymal stem cells or bone marrow stromal cells are one type of adult stem cells that are of particular interest. Since they are derived from a living human adult donor, they do not have the ethical issues associated with the use of human embryonic stem cells. They are also able to be taken from a patient or other donors with relative ease and then grown readily in the laboratory for clinical application. Despite the attractive properties of bone marrow stromal cells, there is presently no quick and easy way to determine the quality of a sample of such cells. Presently, a sample must be grown for weeks and subject to various time-consuming assays, under the direction of an expert cell biologist, to determine whether it will be useful. Hence there is a great need for innovative new ways to assess the quality of cell cultures for research and potential clinical application. The research presented in this thesis investigates the use of computerised image processing and pattern recognition techniques to provide a quicker and simpler method for the quality assessment of bone marrow stromal cell cultures. In particular, aim of this work is to find out whether it is possible, through the use of image processing and pattern recognition techniques, to predict the growth potential of a culture of human bone marrow stromal cells at early stages, before it is readily apparent to a human observer. With the above aim in mind, a computerised system was developed to classify the quality of bone marrow stromal cell cultures based on phase contrast microscopy images. Our system was trained and tested on mixed images of both healthy and unhealthy bone marrow stromal cell samples taken from three different patients. This system, when presented with 44 previously unseen bone marrow stromal cell culture images, outperformed human experts in the ability to correctly classify healthy and unhealthy cultures. The system correctly classified the health status of an image 88% of the time compared to an average of 72% of the time for human experts. Extensive training and testing of the system on a set of 139 normal sized images and 567 smaller image tiles showed an average performance of 86% and 85% correct classifications, respectively. The contributions of this thesis include demonstrating the applicability and potential of computerised image processing and pattern recognition techniques to the task of quality assessment of bone marrow stromal cell cultures. As part of this system, an image normalisation method has been suggested and a new segmentation algorithm has been developed for locating cell regions of irregularly shaped cells in phase contrast images. Importantly, we have validated the efficacy of both the normalisation and segmentation method, by demonstrating that both methods quantitatively improve the classification performance of subsequent pattern recognition algorithms, in discriminating between cell cultures of differing health status. We have shown that the quality of a cell culture of bone marrow stromal cells may be assessed without the need to either segment individual cells or to use time-lapse imaging. Finally, we have proposed a set of features, that when extracted from the cell regions of segmented input images, can be used to train current state of the art pattern recognition systems to predict the quality of bone marrow stromal cell cultures earlier and more consistently than human experts.