Julia C. Shelton

An investigation into the effects of the hierarchical structure of tendon fascicles on micromechanical properties

Abstract During physiological loading, a tendon is subjected to tensile strains in the region of up to 6 per cent. These strains are reportedly transmitted to cells, potentially initiating specific mechano-transduction pathways. The present study examines the local strain fields within tendon fascicles subjected to tensile strain in order to determine the mechanisms responsible for fascicle extension. A hierarchical approach to the analysis was adopted, involving micro and macro examination. Micro examination was carried out using a custom-designed rig, to enable the analysis of local tissue strains in isolated fascicles, using the cell nuclei as strain markers. In macro examination, a video camera was used to record images of the fascicles during mechanical testing, highlighting the point of crimp straightening and macro failure. Results revealed that local tensile strains within a collagen fibre were consistently smaller than the applied strain and showed no further increase once fibres were aligned. By contrast, between-group displacements, a measure of fibre sliding, continued to increase beyond crimp straightening, reaching a mean value of 3.9 per cent of the applied displacement at 8 per cent strain. Macro analysis displayed crimp straightening at a mean load of 1 N and sample failure occurred through the slow unravelling of the collagen fibres. Fibre sliding appears to provide the major mechanism enabling tendon fascicle extension within the rat-tail tendon. This process will necessarily affect local and cellular strains and consequently mechanotransduction pathways.

Temporal regulation of chondrocyte metabolism in agarose constructs subjected to dynamic compression

The temporal response of chondrocyte metabolism in agarose constructs subjected to different dynamic compression regimes was investigated. The current study explored the effects of continuous or intermittent compression using various duty cycles of dynamic compressive loading, over a 48 h culture period. For the continuous compression experiments, duty cycles ranged from 5400 to 172,800 and intermittent compression delivered a total of 86,400 cycles. Large numbers of duty cycles significantly stimulated proteoglycan synthesis with maximal levels obtained for constructs subjected to 12h of intermittent compression. The shortest duration of intermittent compression suggested that further cycles are inhibitory for cell proliferation. Nitrite release was independent of the length or type of compressive regime applied. The uncoupled nature of the metabolic response determined in this study suggests that mechanical conditioning regimes may be fine tuned to selectively stimulate key metabolic parameters of relevance to cartilage tissue engineering.

The influence of noncollagenous matrix components on the micromechanical environment of tendon fascicles

Tendon is composed of type I collagen fibers, interspersed with proteoglycan matrix and cells. Glycosaminoglycans may play a role in maintaining the structural integrity of tendon, preventing excessive shearing between collagen components. This study tests the hypothesis that tendon extension mechanisms can be altered by modifying the composition of noncollagenous matrix. Tendon explants were treated with phosphate buffered saline (PBS) or PBS + 0.5 U ml−1 chondroitinase ABC. Structural changes were examined using TEM and biochemical analysis, while strain response was examined using confocal microscopy and gross mechanical characterization. Chondroitinase ABC removed 90% of glycosaminoglycans from the matrix. Results demonstrated significant swelling of fibrils and surrounding matrix when incubated in either solution. In response to applied strain, PBS incubated samples demonstrated significantly less sliding between adjacent fibers than nonincubated, and a 33% reduction in maximum force. By contrast, fascicles incubated in chondroitinase ABC demonstrated a similar strain response to nonincubated. Data indicate that collagen-proteoglycan binding characteristics can be influenced by incubation and this, in turn, can influence the preferred extension mechanisms adopted by fascicles. This highlights the importance of maintaining fascicles within their natural environment to prevent structural or mechanical changes prior to subsequent analysis.

Influence of cup orientation on the wear performance of metal-on-metal hip replacements.

Abstract There are a number of factors that determine the overall outcome of total hip replacement (THR) surgery, some of which appear to be related to the surgical procedure. In particular, the inclination angle at which the acetabular component is placed has been reported to influence the long-term successful performance of THR. The present study assessed the influence of cup orientation on the wear of 40 mm diameter metal-on-metal (MoM) hip bearings tested in a hip simulator. The bearings had a mean radial clearance of 150 μm; the cups oriented at 35°, 50°, and 60° to the horizontal were loaded for up to 6×106 cycles. In each test the wear rates during the run-in phase were higher than in the steady state phase; the wear rates during the run-in phase were not significantly different for each cup orientation. However, at cup angles of 50° and 60°, the steady state wear rates were 0.69 mm3/106 cycles and 1.7 mm3/106 cycles respectively, significantly higher than at 35° (0.24 mm3/106 cycles). The results indicated that larger cup inclination angles not only move the position of the wear scar but also, more significantly in MoM bearings, increase the wear rates and total wear volume generated.

Influence of external uniaxial cyclic strain on oriented fibroblast-seeded collagen gels.

This study investigates the influence of cyclic tensile strain, applied to fully contracted fibroblast-seeded collagen constructs. The constructs were preloaded to either 2 or 10 mN. The preloaded constructs were subsequently subjected to a further 10% cyclic strain (0-10%) at 1 Hz, using a triangular waveform, or were cultured in the preloaded state. In all cases cellular viability was maintained during the conditioning period. Cell proliferation was enhanced by the application of cyclic strain within constructs preloaded to both 2 and 10 mN. Collagen synthesis was enhanced by cyclic strain within constructs preloaded at 2 mN only. The profile of matrix metalloproteinase (MMP) expression, determined by zymography, was broadly similar in constructs preloaded at 2 mN with or without the application of cyclic strain. By contrast, constructs preloaded at 10 mN and subjected to cyclic strain expressed enhanced levels of staining for latent MMP-1, latent MMP-9, and both latent and active MMP-2, when compared with the other conditioning regimens. The structural stiffness of constructs preloaded at 2 mN and subjected to cyclic strain was enhanced compared with control specimens, reflecting the increase in collagen synthesis. By contrast, the initial failure loads for cyclically strained constructs preloaded at 10 mN were reduced, potentially because of enhanced catabolic activity.

'Severe' wear challenge to 'as-cast' and 'double heat-treated' large-diameter metal-on-metal hip bearings.

Abstract The wear generation of double-heat-treated and as-cast large-diameter metal-on-metal (MOM) hip bearings was investigated using standard- and ‘severe’-gait simulations. The test hypothesis was that double heat treatment would change MOM hip wear compared with the as-cast condition. Two groups of high-carbon MOM bearings of 40 mm diameter were manufactured and subjected to either hot isostatic pressing (HIP) and solution annealing (SA) or no heat treatment (as cast). The results showed no statistical difference between the two groups under both running-in and steady state conditions. Even under the most ‘severe’-gait simulation published to date, the mean volumetric wear rates were 2.9 and 3.9 mm3 per 106 cycles for the HIP-SA and as-cast bearings respectively, showing a ten-fold increase in wear compared with walking. These differences were not statistically different; therefore our hypothesis was negated. Changes in alloy microstructure do not appear to influence the wear behaviour of high-carbon cast MOM articulations with similar chemical compositions. This is in sharp contrast with the published significance of bearing diameter and radial clearance on the wear of MOM hip bearings.

Metal-on-metal hip simulator study of increased wear particle surface area due to 'severe' patient activity.

Abstract This study investigated changes in metal-on-metal (MOM) hip wear and wear particle characteristics arising from a more aggressive patient activity level compared with normal walking. The test hypothesis was that ‘severe’-gait conditions will change wear, wear particle sizes, and morphology owing to a decline in joint lubrication. Four carbon MOM hip bearings 40 mm high were subjected to normal-walking and fast-jogging simulations in an orbital hip joint simulator with 25 per cent α-calf serum as a lubricant. Co-Cr-Mo wear particles were extracted using an enzymatic method, and prolate ellipsoid equations were used to estimate particle volume and surface area. Fast-jogging simulations generated a sevenfold increase in volumetric wear, a 33 per cent increase in mean wear particle size, and a threefold increase in the number of larger (needle) particles compared with walking. This resulted in a twentyfold increase in total wear particle surface area per 106 cycles compared with walking, thereby confirming our hypothesis. The clinical significance of this result suggests that highly active MOM patients may exhibit greater ion release than less active patients.

Investigation and quantification of the blood trauma caused by the combined dynamic forces experienced during cardiopulmonary bypass.

Blood is exposed to various dynamic forces during cardiopulmonary bypass (CPB). Understanding the damaging nature of these forces is paramount for research and development of the CPB circuit. The object of this study was to identify the most damaging dynamic non-physiological forces and then quantify this damage. A series of in vitro experiments simulated the different combinations of dynamic forces experienced during CPB while damage to the blood was closely monitored. A combination of air interface (a) and negative pressure (P) caused the greatest rate of change in plasma Hb (Δp Hb) (4.94 10-3 mg/dl/s) followed by negative pressure and then an air interface. Shear stresses, positive pressures, wall impact forces and a blood-nonendothelial surface caused the least damage (0.26 10-3 mg/dl/s). An air interface showed no threshold value for blood damage, with the relationship between the size of the interface and the blood damage modelled by a second-order polynomial. However, negative pressure did exhibit a threshold value at -120 mmHg, beyond which point there was a linear relationship. Investigating the reasons for the increased blood trauma caused by the low-pressure suction (LPS) system makes it clear how research into minimizing or completely avoiding certain forces must be the next step to advancing extracorporeal technology.

A system for monitoring the response of uniaxial strain on cell seeded collagen gels.

The success of cell seeded constructs for the repair of collagenous tissues may be improved by the use of mechanical stimulation in vitro. A mechanical loading apparatus, termed the cell straining system, was developed according to a set of design criteria, to enable cell seeded constructs to be cyclically loaded in tension. A suitable cell seeded collagen gel model system was used to characterise the apparatus. These gels were subjected to a cyclic strain of 10% superimposed on two separate tare loads of 2 and 10 mN, while being maintained in cell culture conditions. The computer controlled apparatus was shown to be capable of monitoring the individual loads on six specimens simultaneously, to an accuracy of 0.02 mN. Results indicated a wide variability between individual specimens. Following cyclic loading, the cell seeded collagen gels exhibited an increase in structural stiffness compared with the unloaded controls. This novel and versatile apparatus will provide a means of enhancing structural and mechanical integrity of tissue engineered repair systems.

Hydrogels as an interface between bone and an implant

The use of fully hydrated hydrogels in the body has been well established. The forces a hydrogel generates on swelling when it is placed in a constrained space were investigated with a view to providing a mechanism for fixing a prosthesis in the intramedullary cavity. A cross-linked poly(2-hydroxyethyl methacrylate) [p(HEMA)] hydrogel was investigated as a potential material. In vitro mechanical tests were carried out to determine the stresses generated in the p(HEMA) when it was placed in water and not allowed to swell. Pull out loads of up to 375 N indicated that the system could be used successfully in vivo. Consequently, the material was placed intraosseously at two sites in a rabbit animal model, in the mid-shaft (diaphysis) and the lower end (metaphysis) of the femur. Histological examination showed there was no adverse bone response; bone was growing from the endosteal surface up to and into the hydrogel in the diaphyseal implants and surrounded the hydrogel in the metaphysis. As a result of the shape and size variations in the rabbit femur, in vivo mechanical tests were found to give lower values than those obtained in vitro.