Background: The present study aimed to explore if risk factors associated with loading at knee were associated with degenerative changes in ACL injured groups. Methods: Part 1: Biomechanics were investigated for gait, jogging and single legged squatting (SLS) in Anterior Crucitate Ligament Reconstructed (ACLR) (n=30), Anterior Cruciate Ligament Deficient (ACLD) (n=28) and controls (n=30). Analysis of biomechanics was also undertaken on a subgroup of ACLR (ACLR2) (n=10) 12.9±1.8 months after their first assessment. From the ACLR2 those with MRi (ACLM) were recruited (n=8). Part 2: Comparison between the ACLM groups NHS diagnostic scans and a follow up scan was undertaken 27±11.7 months apart. Quantitative measurement of cartilage thickness and a semi-quantitative analysis developed from the Whole-Organ Magnetic Resonance Imaging Score (WORMS) was undertaken. Part 3: Used a case series analysis incorporating individual participants’ outcomes from the first two parts of the study. Statistical analysis: Differences between ACLR, ACLD and control groups was performed using ANOVA. Longitudinal analysis was performed using a paired t-test and changes in MRi using a Wilcoxen signed-rank test. Results: Biomechanics: No significant differences between groups existed for gait. For jogging ACLD and ACLR demonstrated reductions in peak knee extensor moment. The SLS showed a reduction in sagittal plane knee range of motion in the ACLD. The ACLD group had lower self-reported measures of function compared to the ACLR group. Quantitative MRi: No significant differences in regional cartilage thickness between diagnostic and follow up scans was observed. Semi-quantitative MRi: Significant improvement in total knee score was observed in ACLM. Discussion: Despite increased loading being associated with the development of OA, the ACLD and ACLR groups maintained or decreased knee moments. Interestingly, the one ACLM participant with worsening of total semi-quantitative score had evidence of decreased extensor moment. However, reductions in net moment caused by a stiffening strategy may still lead to increased compression forces that may have implications for knee health in the full ACLR and ACLD. Conclusion: No evidence of degenerative changes was found in ACLM. However, individual’s demonstrated degenerative changes in some features; this may suggest that OA is an end point but initiated and developed through different mechanisms.

Congenital heart disease (CHD) is the most common birth defect, causing an important rate of morbidity and mortality. Treatment of CHD requires surgical correction in a significant percentage of cases which exposes patients to cardiac and end organ injury. Cardiac surgical procedures often require the utilisation of cardiopulmonary bypass (CPB), a system that replaces heart and lungs function by diverting circulation into an external circuit. The use of CPB can initiate potent inflammatory responses, in addition a proportion of procedures require a period of aortic cross clamp during which the heart is rendered ischaemic and is exposed to injury. High O2 concentrations are used during cardiac procedures and when circulation is re-established to the heart which had adjusted metabolically to ischaemia, further injury is caused in a process known as ischaemic reperfusion injury (IRI). Several strategies are in place in order to protect the heart during surgery, however injury is still caused, having detrimental effects in patients at short and long term. Remote ischaemic preconditioning (RIPC) is a technique proposed as a potential cardioprotective measure. It consists of exposing a remote tissue bed to brief episodes of ischaemia prior to surgery in order to activate protective pathways that would act during CPB, ischaemia and reperfusion. This study aimed to assess RIPC in paediatric patients requiring CHD surgical correction with a translational approach, integrating clinical outcome, marker analysis, cardiac function parameters and molecular mechanisms within the cardiac tissue. A prospective, single blinded, randomized, controlled trial was conducted applying a RIPC protocol to randomised patients through episodes of limb ischaemia on the day before surgery which was repeated right before the surgery started, after anaesthesia induction. Blood samples were obtained before surgery and at three post-operative time points from venous lines, additional pre and post-bypass blood samples were obtained from the right atrium. Myocardial tissue was resected during the ischaemic period of surgery. Echocardiographic images were obtained before the surgery started after anaesthetic induction and the day after surgery, images were stored for later off line analysis. PICU surveillance data was collected including ventilation parameters, inotrope use, standard laboratory analysis and six hourly blood gas analysis. Pre and post-operative quantitation of markers in blood specimens included cardiac troponin I (cTnI) and B-type natriuretic peptide (BNP), inflammatory mediators including interleukins IL-6, IL-8, IL-10, tumour necrosis factor (TNF-α), and the adhesion molecules ICAM-1 and VCAM-1; the renal marker Cystatin C and the cardiovascular markers asymmetric dymethylarginine (ADMA) and symmetric dymethylarginine (SDMA). Nitric oxide (NO) metabolites and cyclic guanosine monophosphate (cGMP) were measured before and after bypass. Myocardial tissue was processed at baseline and after incubation at hyperoxic concentration during four hours in order to mimic surgical conditions. Expression of genes involved in IRI and RIPC pathways was analysed including heat shock proteins (HSPs), toll like receptors (TLRs), transcription factors nuclear factor κ-B (NF- κ-B) and hypoxia inducible factor 1 (HIF-1). The participation of hydrogen sulfide enzymatic genes, apelin and its receptor were explored. There was no significant difference according to group allocation in any of the echocardiographic parameters. There was a tendency for higher cTnI values and inotropic score in control patients post-operatively, however this was not statistically significant. BNP presented no significant difference according to group allocation. Inflammatory parameters tended to be higher in the control group, however only TNF- α was significantly higher. There was no difference in levels of Cystatin C, NO metabolites, cGMP, ADMA or SDMA. RIPC patients required shorter PICU stay, all other clinical and laboratory analysis presented no difference related to the intervention. Gene expression analysis revealed interesting patterns before and after incubation. HSP-60 presented a lower expression at baseline in tissue corresponding to RIPC patients, no other differences were found. This study provided with valuable descriptive information on previously known and newly explored parameters in the study population. Demographic characteristics and the presence of cyanosis before surgery influenced patterns of activity in several parameters, numerous indicators were linked to the degree of injury suffered by the myocardium. RIPC did not reduce markers of cardiac injury or improved echocardiographic parameters and it did not have an effect on end organ function; some effects were seen in inflammatory responses and gene expression analysis. Nevertheless, an important clinical outcome indicator, PICU length of stay was reduced suggesting benefit from the intervention. Larger studies with more statistical power could determine if the tendency of lower injury and inflammatory markers linked to RIPC is real. The present results mostly support findings of larger multicentre trials which have reported no cardiac benefit from RIPC in paediatric cardiac surgery.

With an ageing population putting ever increasing demands on the musculoskeletal system there is a growing need for the development of regenerative medical strategies to provide for the healthcare needs of the future. With increasing numbers of joint arthroplasty occurring in younger and younger patients there is likely to be a growing need for therapeutic strategies to replace lost bone stock in the coming decades This thesis aims to explore strategies to enhance the biological and mechanical properties of impaction bone grafting and the effects of skeletal stem cell (SSC) concentration. In order for SSC to be effective in replacing new bone stock new strategies looking to enhance osteogenic differentiation have been examined. Section I: An in vitro Impaction Bone Graft (lBG) model of SSC seeded onto human allograft was used to study the biomechanical effects of altering SSC concentration. The use of concentrated SSC was then used in the treatment of patients with avascular necrosis (AVN) of the femoral head and fracture non union with parallel in vitro analysis of the samples. Section 11: In vitro and murine in vivo analysis of the biomechanical effects of type 1 Collagen and Hydroxyapatite nanoparticles primarily looking at differences in shear strength and osteogenic differentiation compared to plain allograft and basal cultured SSC. Section Ill: An in vitro acetabular model was used to study the effect of vibration IBG compared to standard techniques in revision hip surgery. The graft compaction, force of impaction, fracture risk and rate of subsidence post cyclical loading was assessed. This thesis has demonstrated in vitro and in vivo strategies that are clinically translatable and have demonstrated that: • Skeletal Stem Cell concentration plays a pivotal role in the biomechanical enhancement of Impacted bone graft (IBG) • Translation of these strategies into the successful treatment of fracture non union and Avascular necrosis of the hip. • Type 1 Collagen and Hydroxyapatite nanoparticles both enhance the osteogenic differentiation and shear strength of the IBG / SSC construct • Vibration impaction bone grafting is a novel technique that significantly reduces the intraoperative risk of acetabular fracture or catastrophic subsidence This thesis has demonstrated novel techniques for the biomechanical enhancement of I BG with most techniques being readily transferable to clinical practice with the potential to form part of a surgeon's armament for regenerative medical techniques of the future.