Evaluating transglutaminase crosslinked collagen gel systems for hard and soft tissue repair

Tissue Transglutaminase (TG2) and FXIIIa, members of the transglutaminase (TG) family, catalyses a transamidating reaction and form covalent bond between or within proteins. In bone development, both enzymes expressions correlate with the initial of the mineralisation process by osteoblasts and chondrocytes. Exogenous TG2 also promotes maturation of chondrocytes and mineralisation in pre-osteoblasts. To understand the role of endogenous TG2 in osteoblast mineralisation, the TG2 expression was examined during the human osteoblast (HOB) mineralisation. The expression of the endogenous TG2 increased during the mineralisation, yet, its expression was not essential for mineral deposition due to the compensation effect by other members in the TG family. The extracellular transamidating activity of HOBs was found increased during mineralisation and a shift from FXIIIa dominant- to TG2-dominant crosslinking activity was suggested after differentiation. However, the transamidating activity of both TG2 and FXIIIa were not critical for cell mineralisation. On the other hand, Exogenous TG2 was found to enhance wild type HOB and TG2 knockdown HOB mineral deposition. The transamidating activity of TG2 was not required but most likely a close conformation was essential for this enhancement. Results also demonstrated that exogenous TG2 may activate the ß-catenin pathway through LRP5 receptor thus contribute in cell mineralisation. This enhancement could be abolished by addition of ß-catenin inhibitors. Finally, using of TG2 crosslinked collagen gel for bone and cornea repair was evaluated. Crosslinked collagen gel showed promising results in improving HOB mineralisation, human corneal fibroblast (hCF) proliferation and migration. These effects might be resulted from the trapped TG2 within the collagen matrix and the alteration of matrix topography by TG2.

Transducer diffraction theory and development of an ultrasonic tomogram

The work described in this thesis is the development of an ultrasonic tomogram to provide outlines of cross-sections of the ulna in vivo. This instrument, used in conjunction with X-ray densitometry previously developed in this department, would provide actual bone mineral density to a high resolution. It was hoped that the accuracy of the plot obtained from the tomogram would exceed that of existing ultrasonic techniques by about five times. Repeat measurements with these instruments to follow bone mineral changes would involve very low X-ray doses. A theoretical study has been made of acoustic diffraction, using a geometrical transform applicable to the integration of three different Green's functions, for axisymmetric systems. This has involved the derivation of one of these in a form amenable to computation. It is considered that this function fits the boundary conditions occurring in medical ultrasonography more closely than those used previously. A three dimensional plot of the pressure field using this function has been made for a ring transducer, in addition to that for disc transducers using all three functions. It has been shown how the theory may be extended to investigate the nature and magnitude of the particle velocity, at any point in the field, for the three functions mentioned. From this study. a concept of diffraction fronts has been developed, which has made it possible to determine energy flow also in a diffracting system. Intensity has been displayed in a manner similar to that used for pressure. Plots have been made of diffraction fronts and energy flow direction lines.

Development and deployment of an autonomous micro-drilling system for cochleostomy

This thesis describes the design and development of an autonomous micro-drilling system capable of accurately controlling the penetration of complaint tissues and its application to the drilling of the cochleostomy; a key stage in the cochlea implant procedure. The drilling of the cochleostomy is a precision micro-surgical task in which the control of the burr penetration through the outer bone tissue of the cochlea is vital to prevent damage to the structures within and requires a high degree of skill to perform successfully. The micro-drilling system demonstrates that the penetration of the cochlea can be achieved consistently and accurately. Breakthrough can be detected and controlled to within 20µm of the distal surface and the hole completed without perforation of the underlying endosteal membrane, leaving the membranous cochlea intact. This device is the first autonomous surgical tool successfully deployed in the operating theatre. The system is unique due to the way in which it uses real-time data from the cutting tool to derive the state of the tool-tissue interaction. Being a smart tool it uses this state information to actively control the way in which the drilling process progresses. This sensor guided strategy enables the tool to self-reference to the deforming tissue and navigate without the need for pre-operative scan data. It is this capability that enables the system to operate in circumstances where the tissue properties and boundary conditions are unknown, without the need to restrain the patient.

Characterization of human mesenchymal stem cells from multiple donors and the implications for large scale bioprocess development

Cell-based therapies have the potential to contribute to global healthcare, whereby the use of living cells and tissues can be used as medicinal therapies. Despite this potential, many challenges remain before the full value of this emerging field can be realized. The characterization of input material for cell-based therapy bioprocesses from multiple donors is necessary to identify and understand the potential implications of input variation on process development. In this work, we have characterized bone marrow derived human mesenchymal stem cells (BM-hMSCs) from multiple donors and discussed the implications of the measurable input variation on the development of autologous and allogeneic cell-based therapy manufacturing processes. The range of cumulative population doublings across the five BM-hMSC lines over 30 days of culture was 5.93, with an 18.2% range in colony forming efficiency at the end of the culture process and a 55.1% difference in the production of interleukin-6 between these cell lines. It has been demonstrated that this variation results in a range in the process time between these donor hMSC lines for a hypothetical product of over 13 days, creating potential batch timing issues when manufacturing products from multiple patients. All BM-hMSC donor lines demonstrated conformity to the ISCT criteria but showed a difference in cell morphology. Metabolite analysis showed that hMSCs from the different donors have a range in glucose consumption of 26.98 pmol cell−1 day−1, Lactate production of 29.45 pmol cell−1 day−1 and ammonium production of 1.35 pmol cell−1 day−1, demonstrating the extent of donor variability throughout the expansion process. Measuring informative product attributes during process development will facilitate progress towards consistent manufacturing processes, a critical step in the translation cell-based therapies.

Serum-free process development:improving the yield and consistency of human mesenchymal stromal cell production

Background aims: The cost-effective production of human mesenchymal stromal cells (hMSCs) for off-the-shelf and patient specific therapies will require an increasing focus on improving product yield and driving manufacturing consistency. Methods: Bone marrow-derived hMSCs (BM-hMSCs) from two donors were expanded for 36 days in monolayer with medium supplemented with either fetal bovine serum (FBS) or PRIME-XV serum-free medium (SFM). Cells were assessed throughout culture for proliferation, mean cell diameter, colony-forming potential, osteogenic potential, gene expression and metabolites. Results: Expansion of BM-hMSCs in PRIME-XV SFM resulted in a significantly higher growth rate (P < 0.001) and increased consistency between donors compared with FBS-based culture. FBS-based culture showed an inter-batch production range of 0.9 and 5 days per dose compared with 0.5 and 0.6 days in SFM for each BM-hMSC donor line. The consistency between donors was also improved by the use of PRIME-XV SFM, with a production range of 0.9 days compared with 19.4 days in FBS-based culture. Mean cell diameter has also been demonstrated as a process metric for BM-hMSC growth rate and senescence through a correlation (R² = 0.8705) across all conditions. PRIME-XV SFM has also shown increased consistency in BM-hMSC characteristics such as per cell metabolite utilization, in vitro colony-forming potential and osteogenic potential despite the higher number of population doublings. Conclusions: We have increased the yield and consistency of BM-hMSC expansion between donors, demonstrating a level of control over the product, which has the potential to increase the cost-effectiveness and reduce the risk in these manufacturing processes.