Summary Statement

Repetitive loading of degenerated human intervertebral discs in combined axial compression, flexion and axial rotation, typical of manual handling lifing activities, causes: an increase in intradiscal maximum shear strains, circumferential annular tears and nuclear seperation from the endplate.

Irradiating allograft bone may compromise the mechanical stability of the prosthesis-bone construct, potentially having adverse effects on the outcome of femoral impaction grafting at revision hip replacement. This in vitro study aimed to determine the effect of irradiation of allograft bone used in femoral impaction grafting on initial prosthesis stability.

Morsellised ovine femoral head bone was irradiated at 0 kGy (control), 15 kGy and 60 kGy. For each group, six ovine femurs were implanted with a cemented polished double taper stem following femoral impaction bone grafting. Dynamic hip joint loading was applied to the femoral head using a servo-hydraulic materials testing machine. The primary outcome was stem micromotion. Tri-axial micromotion of the stem relative to the bone at two sites was measured using linear variable differential transformers and non-contact laser motion transducers. Statistical analysis was performed using SPSS.

Compared to the control and 15 kGy groups, specimens in the 60 kGy group demonstrated statistically significant greater micromotion in the axial, antero-posterior and medio-lateral axes. A multi-factorial post-hoc power analysis based on the overall effect of group size indicated a power of 0.7. There was no difference in micromotion between the control and 15 kGy groups. The average micromotion in the axial axes was 63μm in the control and 59μm in the 15 kGy group.

The results of this study suggest that a maximum irradiation dose of 15 kGy may not affect initial prosthesis stability following femoral impaction grafting in this model and provide the basis for us to now proceed to in-vivo studies examining the effect of irradiated bone on implant stability over time.

Introduction: Disc degeneration causes structural and biochemical tissue changes resulting in altered stresses that may affect vertebral bone remodelling. We hypothesized that disc degeneration alters vertebral cortical strains and disc mechanics of the motion segment, with and without the presence of zygapophyseal joints.

Methods: Twenty human lumbar functional spinal units (FSUs) were strain gauged on the lateral and anterior vertebral cortices, below the inferior endplate. Each FSU was preloaded overnight (0.2 MPa) in a bath and subjected to dynamic compression (1 MPa), flexion/extension/lateral bending (500N + 5 Nm), and axial rotation (5 Nm), before and after removal of the zygapophyseal joints. After testing, discs were macroscopically assessed and graded (1–4) for degeneration. Stiffness, phase angle (energy absorption) and principal strains were calculated. ANOVAs with the dependent variable of principal strain/stiffness/phase angle versus disc grade were performed for each testing direction.

Results: Assessment of disc degenerative condition revealed six grade 2 discs, eight grade 3, and six grade 4. Age and degeneration were highly correlated (r=0.80, P< 0.0001). The effect of disc grade on stiffness was significant overall in most loading directions, before and after removal of zygapophyseal joints (P< 0.008), apart for axial rotation (P> 0.587). Post-hoc multiple comparisons for all loading directions apart for axial rotation revealed that the stiffness of grade 4 discs was significantly larger than grades 2 and 3 discs in most loading directions.

For phase angle (approximate magnitude 5°), no significant overall effects due to degeneration were found across any loading direction (P> 0.2). ANOVA analyses on maximum/minimum principal strains found no significant effect due to disc grade (P> 0.063). However, a small number of significant effects due to disc grade were found at particular strain gauge locations for the isolated disc in flexion, the intact FSU in extension, and the intact FSU/isolated disc in right lateral bending.

Discussion: This study represents the first of its kind to investigate the effects of disc degeneration on vertebral bone cortical strain and disc mechanical properties. Significant increases in stiffness were found with increasing degeneration in all test directions apart for axial rotation. Changes in disc stiffness were consistent with other studies and may be a result of the structural and biochemical changes within the disc that accompany the degenerative process.

The non-significant small phase angles suggest that the disc behaves more like an elastic solid than a poroelastic material, and that dehydration associated with degeneration does not adversely affect damping. Principal strains were not significantly affected by disc degeneration overall, suggesting that the cortical shell adjacent to the disc-endplate boundary maintains a relatively homeostatic condition, with more dramatic architectural changes probably occurring within the trabecular bone. Applications of this research include providing important validation data for analytical/finite element models of the intact FSU and isolated disc segment, and a better understanding of the magnitudes of cortical strains that need to be maintained in order to avoid damaging vertebral bone stress-shielding effects after treatments for disc degeneration.

The energy-storing human Achilles tendon and equine superficial digital flexor tendon (SDFT) show no adaptation to exercise unlike muscle and bone, and are prone to injury. Injury involves microdamage accumulation until there is sufficient weakening for rupture to occur during normal athletic activity. Anatomically opposing positional tendons, such as the common digital extensor tendon (CDET) in the horse rarely suffer exercise–induced injury. Tenocytes maintain the extra-cellular matrix, but in energy-storing tendons they appear unable to adequately repair microdamage as it occurs. Tenocytes have been classified subjectively into 3 subtypes on the basis of histological nuclear morphology. Long, thin type 1 cells are thought to be less synthetically active than cigar-shaped type 2 cells, but their exact morphology and relative proportions in different tendon sites and ages has not been clearly defined. We hypothesised that tenocytes are separable into morphologically distinct subtypes, reflecting differences in age and functional requirements within and between specific tendons. Samples were taken from tensional and compressed regions of the SDFT and CDET of 5 neonates, 5 foals (1–6 m), 5 young adults (2–6 y) and 5 old horses (18–33 y) Cell nuclei were counted and measured in digital images from histological sections by computerised image analysis. Total tenocyte densities and proportions of the 3 subtypes were calculated for each age group, as were nuclear length:width ratios. Length:width ratio distributions for all horses were evaluated using a normality test followed by a paired t-test. There was a significantly higher total cellularity in the SDFT than the CDET, with a higher proportion of type 1 tenocytes in the CDET. With age, total cellularity decreased in all tendon sites and an increase in the proportion of type 1 tenocytes was observed in tensional regions. Foal and neonatal tendons contained significantly higher proportions of type 2 tenocytes than older tendons. The morphology of the two main subtypes in all age groups was significantly different; type 1 tenocytes had a higher nuclear length:width ratio (mean ± SD = 9.6 ± 2.5) than type 2 (mean ± SD =4.7 ±1.1) (p< 0.001). We were able to objectively separate tenocytes into 3 distinct subtypes based on nuclear length:width ratio measurements. There were significant differences in proportions of subtypes with tendon site and age. The positional tendon had significantly lower cellularity and a higher proportion of type 1 tenocytes; these cells may be less functionally active but sufficient to maintain the matrix in a tendon which is not subjected to high levels of strain. The SDFT continues to grow up to 2 years of age and is subjected to high strains, explaining the need for relatively higher proportions of type 2 cells. There is however an age-related increase in type 1 cells in both tendons which may explain an inability of the adult energy-storing tendon to adapt to exercise and to repair microdamage. Understanding the stimulus for age-related changes in tenocyte subtype proportions in tendons with different functions may help us understand the pathogenesis of exercise-induced tendon injury and to develop more appropriate training regimens.

Injury to the core region of energy-storing tendons is a frequent occurrence in both human and equine athletes, the incidence of which increases with age. Such energy-storing tendons include the human Achilles tendon (AT) and the equine superficial digital flexor tendon (SDFT). By definition, energy-storing tendons experience high strains during high-speed athletic activity. In contrast, anatomically opposing tendons (“positional” tendons), such as the common digital extensor tendon (CDET) in the horse and extensor digitorum longus tendon in man act only to transmit muscular force and rarely suffer exercise–induced injury. Functional adaptation of muscle and bone in response to exercise is well – documented, but there has been no convincing evidence to suggest that the energy-storing tendons in adults have the ability to adapt to exercise. We hypothesised that adaptive increases in tenocyte cellularity would occur in the energy-storing and positional tendons of young horses subjected to three specific exercise regimens. Samples were taken from midmeta-carpal regions of the SDFT (periphery and core) and CDET of young Thoroughbred horses from the following groups. Group 1: 6 horses exercised on a high-speed treadmill for 18 months from 21.3 months of age (SD 1.1) with 6 age-matched controls that underwent walking exercise only (long-term); Group 2: 6 horses exercised on a high-speed treadmill for 18 weeks from 19.4 months of age (SD 0.6) with 6 age-matched controls that underwent walking exercise only (short-term) and Group 3: 6 horses trained on pasture in New Zealand for 18 months beginning at 7–10 days of age, with 6 age-matched controls kept at pasture with no additional enforced exercise (Global Equine Research Alliance). Tenocyte nuclei were counted and measured in digital images from histological sections stained with haematoxylin and eosin, by computerised image analysis. Tenocyte densities (per mm2) for exercised and control groups for each study were evaluated using paired t-tests. Tenocyte density was significantly higher in the CDET of exercised horses in Group 3 (mean ± SD =260.4 ± 23.4) compared with the non – exercised controls (mean ± SD =226.9 ± 23.8) (p < 0.01). There was no such difference in the SDFT (core or periphery). There was also no significant exercise-related difference in tenocyte density in either the SDFT (core or periphery) or CDET for Groups 1 or 2. No previous data is available on the effect of exercise on tenocyte populations in equine tendons. The lack of other adaptive changes in previous studies of mature equine tendons had raised the question as to whether immature tendons would be more able to adapt to mechanical stimuli. In this study we were able to show that beginning training of horses shortly after birth (Group 3) stimulated an adaptive response by tenocytes in the positional CDET but not the SDFT. The inability of energy-storing tendons to show functional adaptation to exercise in immature or mature animals may explain the high incidence of strain-induced injury. Understanding the pathway by which exercise-related increases in tenocyte densities occur in immature positional but not energy-storing tendons may increase our understanding of the pathogenesis of strain-induced tendon injury.

Introduction The influence of annular tears on the biomechanical inter-relationship between the disc and vertebral body has a potentially important role in the mechanism of subsequent biological changes in disc and bone. The disc is a complex structure, exhibiting visco-elastic behaviour that is highly dependent on its condition and fluid content. Studies have shown that the stiffness of the disc is altered by its water content in human, ovine and bovine discs. It has also been shown that disc stiffness or modulus can be preserved if the level of water in the disc is kept constant. The importance of maintaining a reproducible state of stress in the disc during sequential testing of the same specimen is crucial to ensuring consistency of results and minimising systematic experimental errors. The aims of this study were to assess the reliability of sequential testing of the same specimen, and to determine whether stiffness, strains and pressure distribution can be restored to pre-testing levels under a uniform hydration loading environment.

Methods Six ovine FSUs with isolated discs were used in this study. Eight, 1-mm strain gauge rosettes were then bonded to the inferior VB of each FSU at lateral and anterior positions and three heights. FSUs were equilibrated for four hours in a saline bath at room temperature in a materials testing machine. A real-time pressure sensor was placed under the VB. FSUs were tested in axial compression at 0.1 Hz to 1 MPa for 5 sinusoidal cycles. Once tested, the FSU was placed under 0.25 MPa preload for one hour in the water bath for re-equilibration and tested again. Pilot studies by this group have shown that one hour is sufficient to return the disc to its original equilibrium state in a bath after testing, with no associated change in stiffness. This sequence was repeated four times to produce a total of five tests on each FSU. Outcome measures were FSU stiffness, axial strain, peak pressure, average pressure and contact area. Data was statistically analysed using intra-class correlation coefficients (ICC), and repeated measures ANOVA or paired t-tests.

Results The ICC for the five repeated stiffness measures was 0.24 (i.e 24% of the variation in the results was due to between-specimen tests with 76% of the variation due to within-specimen tests). Repeated measures ANOVA found no significant effect on stiffness due to repeating the test five times (P = 0.445). The ICC for the eight axial strains ranged from 0.8 to 0.99. There were no significant differences within any of the eight axial strains over the five repeats (P > 0.287). ICCs, and P values (in brackets) from repeated measures ANOVA, were 0.91 (0.179) for peak pressure, 0.85 (0.44) for average pressure and 0.99 (0.077) for contact area.

Discussion The largest systematic variation was seen for stiffness and this may be due to the tissue changes over the 9 hours of testing. Axial strains showed good to excellent agreement over the five repeated tests as did all pressure parameters. We conclude that the method of allowing one hour for re-equilibration in ovine discs produces a reproducible state of stress in the disc and minimises experimental errors.

Introduction The influence of annular tears on the biomechanical inter-relationship between the disc and vertebral body (VB) has a potentially important role in the mechanism of subsequent biological changes in disc and bone. It is postulated that changes in the disc may result in increased or abnormal spinal segment motion, modified load distribution across the spinal joint and altered cancellous bone architecture. There have been no studies investigating the direct effect of disc injury on functional spinal unit (FSU) stiffness and the distribution of pressure immediately adjacent to the disc inferior endplate. The aim of this study was to determine whether minor and severe radial tear injuries to the disc alters FSU stiffness and VB surface pressure distribution.

Methods Six ovine FSUs were used in this study. The posterior elements were removed leaving the isolated disc in each FSU. The inferior VB was transversely cut immediately inferior to the endplate and the neutral axis of bending (NAB) identified and marked. FSUs were equilibrated in a saline bath at room temperature for four hours under a constant preload of approximately 0.25 MPa prior to testing. After equilibration, FSUs were transferred to a saline bath in a materials testing machine (Instron 8511, Instron, High Wycombe, UK) and a real-time pressure sensor (I-Scan 5076, Tekscan Inc., MA, USA) placed under the inferior VB.

While maintaining the preload, FSUs were loaded in axial compression at 0.1 Hz through the NAB to 1 MPa in a saline bath for 5 sinusoidal cycles. Once tested, a radial tear was introduced via scalpel injury into the left postero-lateral region of the annulus and tested after one hour of re-equilibration. A final, more severe injury, in the form of removal of a 5 mm x 2 mm window of annulus at the same location was performed and tested after re-equilibration.

Outcome measures were FSU stiffness, peak pressure, average pressure, contact area, and centroid of force location. Data was statistically analysed using repeated measures ANOVA or paired t-tests.

Results No significant differences in stiffness was found as a result of disc injury (P = 0.857), nor for peak and average pressure, contact area and centroid location (P > 0.179).

Discussion These results may not be surprising given that the disc has been shown to be remarkably resilient under axial compression, even with a severe annular or nuclear injury. Further insight will be revealed when other modes of loading are performed in both ovine and human discs for the main study planned to be undertaken in the near future.

Introduction Dynamically identifying the distribution of pressure between any two given surfaces such as articulating joints is of fundamental importance in understanding their interaction. The purpose of this laboratory study was to assess the potential of a dynamic pressure measurement system, Tekscan. ( I-Scan 5076, Tekscan Inc., MA, USA) via a study which observed the changes in the load profile through the vertebral body of harvested ovine lumbar functional spinal units (FSU’s) with a created defect in the intervertebral disc.

The system was used to determine pressure distributions in isolated vertebral bodies inferior to the disc, during axial compression of normal and injured discs of an ovine functional spinal unit.

Methods Four ovine lumbar segments L1-L3 were harvested The superior vertebral body (VB) remained complete, whilst the inferior VB was sectioned 2mm from the endplate and the surface smoothed using emery paper in order to achieve maximum contact area. The neutral axis of bending for each specimen was identified and marked. In accordance with the manufacturer guidelines, the sensor was conditioned and calibrated between 20-200N of load. Testing was carried out in a materials testing machine (Instron 8511, Instron, High Wycombe, UK), where 200N of axial load was applied through the FSU and a snapshot of the instantaneous pressure distribution was taken. A 12 x 2 mm gap defect was created in the right ventro-lateral (2 specimens) and the right lateral (2 specimens) aspect of the IVD. The specimens were returned to the Instron and 200N of load was applied axially through the NAB. A recorded image of the pressure footprint was taken.

Results Comparing the recorded colour-coded images together with their centroids of force of the pre- and post-injury pressure distributions of the vertebral bodies, it was clearly evident that there was a major shift of the load through the IVD. As predicted and as seen in the pressure footprint, the pressure shifted in the opposing direction of the injury in order to maintain a balanced system. A pressure reading validation was also carried out with the use of the Instron, where the experimental pressure of the sensor was within 3% of the NATA calibrated load cell.

Discussion The system was used to sample pressure in real time and display it as a 3D colour-coded map, allowing for visualisation of normal pressure distributions. The associated software has numerous aids and functions, allowing real-time visualisation of the dynamic forces and the balance of forces across two interacting surfaces, making the system an invaluable analytical tool.

The Tekscan system will be used to observe the effect of disc injury on the pressure distribution of the adjacent vertebral body. The relationship between the pressure distribution across the vertebral body and bone architecture will also be studied

This study illustrated that this system is a valid tool for qualitatively and quantitatively assessing dynamic pressure distributions.

Introduction Vertebral deformity, disc disorganisation, and change to vertebral bone architecture are morphological features that are associated with degeneration of the spine and with back pain. Observations from our earlier studies found that the BV/TV is always a maximum in the inferior third of the vertebral body (VB), and minimum in the central third. Animal model studies have reported that the strain in loaded vertebra is a minimum in the central third of the vertebra. There have been no studies investigating the direct affect of VB removal on functional spinal unit (FSU) stiffness, strain magnitude and the distribution of pressure immediately adjacent to the sectioned VB. There were a number of aims for this study. The first aim was to determine whether the strain varies between supero-inferior locations on the VB. The second aim was to determine if strain symmetry was present across the normal VB. The third aim was to determine whether transverse sectioning of the VB alters the stiffness, strains and pressure distributions of the functional spinal unit (FSU) and VB.

Methods Six ovine FSUs with isolated discs were used in this study. Eight, 1-mm strain gauge rosettes were then bonded to the inferior VB of each FSU at lateral and anterior positions and three heights. FSUs were equilibrated in a saline bath at room temperature in a materials testing machine. A real-time pressure sensor was placed under the VB. FSUs were tested in axial compression at 0.1 Hz to 1 MPa for 5 sinusoidal cycles. The inferior VB was then sectioned transversely at 1/3 of its height and placed under preload for one hour for re-equilibration and re-tested. This procedure was repeated at 2/3 of VB height and immediately adjacent to the endplate. Outcome measures were FSU stiffness, axial strain, peak pressure and average pressure. Data was statistically analysed using repeated measures ANOVA or paired t-tests.

Results The results of the first aim found no significant difference in strains within the right lateral or left lateral (P > 0.134) columns of strain gauges. However, for the anterior column of strain gauges, the superior strain was 30% higher than the inferior strain (P = 0.047). The results of the second aim found no significant differences between laterally opposing strain gauges (P > 0.139). For the third aim, transverse sectioning of the VB over three levels produced no significant differences for FSU stiffness (P = 0.275), strains for any strain gauge (P > 0.087), or peak and average pressures (P > 0.076).

Discussion This complex pilot study has shown that overall, axial cortical strain in a normal, ovine FSU did not vary with VB supero-inferior location laterally, but did vary anteriorly. Strains were symmetrical between laterally opposing VB locations at each of three levels, and was not affected by transverse sectioning of the VB at three levels. The finding that anterior column strains differ, may relate to changes in load distribution governed by VB surface second moment of area differences (laterally compared to anteroposteriorly), and the absence of a disc inferiorly. Further insight will be revealed when other modes of loading are performed in both ovine and human discs for the main study planned to be undertaken in the near future.

The effect of screw geometry on the pullout strength of Anterior Cruciate Ligament [ACL] reconstruction is well documented. Most research has looked at the effect of screw length and diameter, however other factors such as the degree of taper may also be important. Tapered screws should in theory be associated with increased pullout strength. This has not been demonstrated either clinically or in vitro before. The aim of this study was to compare the pullout strength of ACL reconstruction with a parallel against a tapered screw.

A parallel and tapered screw were manufactured which were identical in all other respects. Sixty superficial digital flexors from the hind legs of sheep were harvested. The tendons were paired and combined to form a quadruple tendon reconstruction of approximately 7mm diameter as measured with graft sizer. An ACL reconstruction was performed on the proximal tibia of 30 bovine knees, which had been harvested in right and left knee pairs, using the quadruple tendon. Fifteen reconstructions were fixed using tapered screws and fifteen with non-tapered screws. The insertion torque of both tapered and non tapered screws were recorded using an instrumented torque screwdriver. The reconstructions were mounted in an Instron materials testing machine with an x-ray bearing system to eliminate horizontal forces, to ensure that the forces were all directed along the line of the tibial tunnel. The maximum pullout strengths were recorded in each case. Five knee pairs were subjected to bone densitometry scanning to ensure that any difference in pull out strength was not due to changes in bone density between right and left knee pairs.

Results indicated that there was no difference between right and left knee pairs [p = 0.58] and that tapered screws were associated with significantly higher pull-out strengths [p=0.007] and insertion torques [p = 0.001].

We sought to identify the tensile properties of the medial patellofemoral ligament (MPFL), and determine whether its repair was sufficient as a means of restoring stability after acute lateral patella dislocation. We also sought to establish whether there was a correlation between the tensile properties of the anterior cruciate ligament (ACL) and the MPFL.

16 hind limbs of Merino Wethers were obtained and stored fresh frozen. The specimens were thawed overnight, dissected out and then placed in a water bath at 37 degrees centigrade for 30 minutes prior to testing. All testing was carried out in the water bath to approximate a more physiological environment. For each specimen the ACL was first tested to failure on an Instron 8511. The MPFL was then tested to failure, then repaired and retested to failure. Finally a reconstruction was carried out, using a flexor tendon, which was again tested to failure.

Results:

There was no correlation between ACL and MPFL strength (p=0.677). Statistical analysis showed that the intact MPFL was significantly stronger than the repaired MPFL (P=0.001) but no different to the reconstructed MPFL (P=0.224), with no difference between repaired and reconstructed (P=0.174). A Power analysis showed that there was not adequate power to detect a significant difference between the last two pairs, and that we would have needed over 35 specimens to show a difference.

This study does not support carrying out a repair of the MPFL following an acute lateral patella dislocation, as it does not restore its tensile properties. It further suggests that a reconstruction may better restore the tensile properties of this ligament.

Aim: To determine the intra operative biomechanical properties of a semitendinosus graft used in ACL reconstruction.

Introduction: ACL reconstruction has become a commonly performed operation with 1,139 of these procedures being performed in South Australia in 1997 (SA Health Commission)

The majority of the scientific literature is based on data obtained from elderly cadaveric material. Little is known about the biomechanical properties of the soft tissue grafts currently used prior to implantation. The correct preconditioning and intraoperative tensioning of the soft tissue grafts has also not been investigated.

The initial graft biomechanical properties are important. Inadequate tension will lead to continuing instability whilst excessive tension may cause accelerated joint arthrosis. The tension in the graft may decrease by 30% if it has not been cyclically pretensioned.

Methods: A machine has been designed that will allow the intraoperative biomechanical testing of soft tissue grafts immediately prior to their implantation into the patient during ACL reconstruction. Data will be available on creep, stress relaxation, and tensile testing.

This device will also allow the accurate preconditioning of the graft, providing objective data that can then be compared to the subsequent clinical progress of the patient.

All testing will be accomplished during the time it takes to prepare the tunnels for insertion of the graft, and as such will not prolong unnecessarily the operative time.

Procedure: Once the graft has been prepared prior to fixation, it will be placed between two clamps. One is fixed to a load cell whilst the other is coupled to a linear actuator. The linear actuator will be driven by a computer controlled stepper motor under close-loop control. Custom software will cyclically load the autograft between two definable load points. A linear variable differential transformer (LVDT) will be used to monitor displacement of the autograft and load will be monitored with a load cell of capacity 125Kg.

This set-up will be immersed in a saline water bath maintained at body temperature during testing. The load cell will be hermetically sealed, with clamps and water bath being autoclavable. With the facilities for draping, the test area will remain sterile. The auto graft clamps will be designed to allow fixation of various graft materials (eg semitendinosus, gracilis, bone-patella tendon-bone) and adjustable for graft lengths. The water bath will house a thermocouple, heating mat and controller to maintain the saline temperature to within 1°C.

The testing system will be mounted on a stainless steel trolley for mobility in the operating room with an underlying shelf to house the associated electronics and a retractable side draw for storage of the laptop computer.

The autograft will be preconditioned between two known loads for 20 cycles recording load and displacement simultaneously on a laptop computer. Once preconditioned, the autograft will then be used for the ACL reconstruction in the standard way.

Summary: Objective data on preconditioning of ACL grafts, has never before been available intra-operatively. We outline the experimental set-up which has been designed and is undergoing testing prior to its use in a prospective study.