Tendon and ligament damage causes extreme pain and decreased joint functionality. Current repair methods cannot restore original joint biomechanics nor promote regeneration of native tissue. Recent advances in tendon and ligament repair have involved engineering tissue using cell-seeded scaffolds. Self-aligned cellular structures, similar to those in ligaments and tendons, have been successfully formed, albeit with weak attachment between construct and bone. Calcium phosphates form an intimate bond with both soft and hard tissues and have successfully been used in tissue engineering bone, whilst hydrogels have often been used as cellular scaffolds. This thesis explores agarose, gelatin, carrageenan and fibrin hydrogels as potential soft tissue scaffolds. Fibrin gel exhibited high cellular compatibility with highest metabolic activity on day 14. Although the cellular gel contracted significantly, it was found that the dry weight remained stable in both the acellular and cellular forms. 3D powder printed calcium phosphate scaffolds remained structurally stable after immersion in cell culture media with immersion in protein-rich sera promoting tenocyte attachment. Bracket designs were developed to enhance grip of the cell-seeded fibrin. Ligament constructs were selfsupporting and exhibited structural characteristics similar to native connective tissue. Tenocyte density peaked on day 14, with added L-proline and ascorbic acid inducing a constant level of glycosaminoglycans and 7.4 ± 1.5 % w/w collagen. This research may significantly enhance the clinical application of tissue engineered ligaments and tendons.

Rugby is a complex, high-intensity, intermittent, collision sport with emphasis placed on players possessing high lean body-mass and low body-fat. After an 8-12 week pre-season focused on physiological adaptations, emphasis shifts towards optimizing competitive performance and recovery through periodising player’s diets and training. In Chapter 4 the physiological demands and nutritional intakes of 45 elite rugby players were assessed during a pre-season through a battery of strength and conditioning tests, quantification of training demands using global positioning system (GPS), and two 24-hour diet recalls. Mean weekly distance covered during training was 9774 ± 1404 and 11585 ± 1810 m with a total mean weekly session RPE (sRPE) of 3398 ± 335 and 2944 ± 410 arbitrary units (AU) for forwards and backs respectively. Mean daily energy intake was 14.8 ± 1.9 and 13.3 ± 1.9 MJ, carbohydrate (CHO) intake was 3.3 ± 0.7 and 4.14 ± 0.4 g·kg-1 body mass, protein intake was 2.52 ± 0.3 and 2.59 ± 0.6 g·kg-1 body mass, and fat intake was 1.0 ± 0.3 and 0.95 ± 0.3 g·kg-1 body mass for forwards and backs respectively. Markers of physical performance (1-RM strength, speed, and repeated sprint tests) and anthropometry (body fat, and estimated lean mass) significantly improved in all players, despite players’ self-selecting a ‘low’ CHO ‘high’ protein diet. It may be speculated therefore that ‘low’ CHO ‘high’ protein intakes are appropriate to fuel the pre-season, although whether these intakes are sufficient to fuel the in-season is unknown. Once the demands of the pre-season were established, the next aim of the thesis was to examine if requirements changed during the playing season, as well as quantifying energy expenditure. In Chapter 5 in-season training load using GPS and sRPE, alongside six-day assessments of energy intake (EI) and energy expenditure (EE) was measured in 44 elite Rugby Union players. Mean weekly distance covered was 7827 ± 954 m and 9572 ± 1233 m with a total mean weekly sRPE of 1776 ± 355 and 1523 ± 434 AU for forwards and backs, respectively. Mean daily EI was 16.6 ± 1.5 and 14.2 ± 1.2 MJ, and EE was 15.9 ± 0.5 and 14 ± 0.5 MJ for forwards and backs respectively. Mean CHO intake was 3.5 ± 0.8 and 3.4 ± 0.7 g·kg-1 body mass, protein intake was 2.7 ± 0.3 and 2.7 ± 0.5 g·kg-1 body mass, and fat intake was 1.4 ± 0.2 and 1.4 ± 0.3 g·kg-1 body mass for forwards and backs respectively. All players who completed the food diary self-selected a ‘low’ CHO ‘high’ protein diet during the early part of the week which increased in the days leading up to a match. EI and EE followed an inverse trend, with expenditure exceeding intake during the first four-days of the training week and then reversed in the day leading up to competition with intake exceeding expenditure. Despite this, mean EI exceeded EE which alongside no micronutrient deficiencies, suggest that the current dietary practices of these elite rugby players seem sufficient to fuel training during the in-season, providing energy intake and CHO are increased leading up to a match. Given that intakes reported in this study are still below recommended CHO intake for elite athletes (Burke et al 2011), however, it is still possible that such intakes are not optimal for match day performance. Given that in Chapters 4 and 5 it was found that elite Rugby players appear to deliberately select a low carbohydrate intake, it was deemed important to assess match-play glycogen demands following a low (the amount self selected in chapter 4) and higher (the amount self selected leading in to competition in chapter 5) carbohydrate diet. Therefore, in Chapter 6 the metabolic and physiological demands of rugby competition was assessed in 16 professional Rugby League players following either a 6g·kg (HCHO) or 3g·kg (LCHO) CHO diet for 36-hours. Muscle biopsy and blood was collected, alongside monitoring internal and external load through GPS and heart rate. Mean distance covered was 93.7 ± 12.4 and 89.4 ± 9.8 m·min-1 in the first, and 85.3 ± 13.1 and 86.9 ± 9.7 m·min-1 in the second half for HCHO and LCHO conditions respectively. Mean %HRpeak was 82.9 ± 6.1 and 81.9 ± 7.2 % in the first and 82.5 ± 7.5 and 78.4 ± 10.5 % in the second half for HCHO and LCHO conditions respectively. Mean muscle glycogen was 448.6 ± 50.8 and 444.2 ± 81.1 mmol·kg d·w-1 pre-game, and 243.4 ± 42.5 and 297.7 ± 130.5 mmol·kg d·w-1 post-game for HCHO and LCHO conditions respectively. Results demonstrate that a competitive RL match can result in ~40% muscle glycogen depletion and that match-day performance variables did not differ between conditions. It was postulated that an absolute amount of ~600 g CHO consumed 36-hours pre-match is a recommended strategy for rugby league players, although optimal dietary strategies to refuel after rugby competition are unknown. The final aim of the thesis was to examine if the current post exercise CHO guidelines are appropriate for rugby players. In Chapter 7 the magnitude of muscle glycogen repletion after consuming an immediate, or delayed re-feed post Rugby League Match Simulation Protocol (RLMSP) was assessed in 16 university rugby league players using muscle biopsy and blood letting techniques. Muscle glycogen very likely increased 48-h post-simulation (272 ± 97 cf. 416 ± 162 mmol·kg-1d.w.) after an immediate re-feed, but changes were unclear (283 ± 68 cf. 361 ± 144 mmol·kg-1d.w.) after a delayed re-feed. Creatine Kinase (CK) almost certainly increased by 77.9 ± 25.4 % (0.75 ± 0.19) post-simulation for all players. Player Load (8 ± 0.7 AU) and %HRpeak (83 ± 4.9 %) were consistent with professional RL match-play. Time to exhaustion performance test revealed no difference between conditions. This study found that simulated RL match-play elicits lower muscle glycogen utilisation (21 cf. 40 %) despite similar player load and metabolic demands to a professional RL match. This may be attributed to the difficulties of replicating extensive structural damage and physical exertion from collisions during a simulation. It was also found that substantial muscle glycogen resynthesis was possible in the immediate dietary re-feed group despite evidence of muscle damage via increased blood proteins, indicating that with appropriate feeding strategies it is possible to replenish a damaged muscle. Taken together, this thesis has characterized the training demands and energy balance of elite rugby players during the pre-season and in-season, alongside quantifying the metabolic demands of elite rugby match-play, and the most appropriate strategies to load and replenish muscle glycogen around such exercise. Future studies must now further titrate these studies and assess muscle glycogen utilisation over a number of games whilst assessing the glycogen content of individual muscle fibre types.