Size and habit of mineral particles in bone and mineralized callus during bone healing in sheep

Bone healing is known to occur through the successive formation and resorption of various tissues with different structural and mechanical properties. To get a better insight into this sequence of events, we used environmental scanning electron microscopy (ESEM) together with scanning small-angle X-ray scattering (sSAXS) to reveal the size and orientation of bone mineral particles within the regenerating callus tissues at different healing stages (2, 3, 6, and 9 weeks). Sections of 200 µm were cut from embedded blocks of midshaft tibial samples in a sheep osteotomy model with an external fixator. Regions of interest on the medial side of the proximal fragment were chosen to be the periosteal callus, middle callus, intercortical callus, and cortex. Mean thickness (T parameter), degree of alignment (ρ parameter), and predominant orientation (ψ parameter) of mineral particles were deduced from resulting sSAXS patterns with a spatial resolution of 200 µm. 2D maps of T and ρ overlapping with ESEM images revealed that the callus formation occurred in two waves of bone formation, whereby a highly disordered mineralized tissue was deposited first, followed by a bony tissue with more lamellar appearance in the ESEM and where the mineral particles were more aligned, as revealed by sSAXS. As a consequence, degree of alignment and mineral particle size within the callus increased with healing time, whereas at any given moment there were structural gradients, for example, from periosteal toward the middle callus.

Pressure, oxygen tension and temperature in the periosteal callus during bone healing - An in vivo study in sheep

Adequate blood supply and sufficient mechanical stability are necessary for timely fracture healing. Damage to vessels impairs blood supply; hindering the transport of oxygen which is an essential metabolite for cells involved in repair. The degree of mechanical stability determines the mechanical conditions in the healing tissues. The mechanical conditions can influence tissue differentiation and may also inhibit revascularization. Knowledge of the actual conditions in a healing fracture in vivo is extremely limited. This study aimed to quantify the pressure, oxygen tension and temperature in the external callus during the early phase of bone healing. Six Merino-mix sheep underwent a tibial osteotomy. The tibia was stabilized with a standard mono-lateral external fixator. A multi-parameter catheter was placed adjacent to the osteotomy gap on the medial aspect of the tibia. Measurements of oxygen tension and temperature were performed for ten days post-op. Measurements of pressure were performed during gait on days three and seven. The ground reaction force and the interfragmentary movements were measured simultaneously. The maximum pressure during gait increased (p=0.028) from three (41.3 [29.2-44.1] mm Hg) to seven days (71.8 [61.8-84.8] mm Hg). During the same interval, there was no change (p=0.92) in the peak ground reaction force or in the interfragmentary movement (compression: p=0.59 and axial rotation: p=0.11). Oxygen tension in the haematoma (74.1 mm Hg [68.6-78.5]) was initially high post-op and decreased steadily over the first five days. The temperature increased over the first four days before reaching a plateau at approximately 38.5 degrees C on day four. This study is the first to report pressure, oxygen tension and temperature in the early callus tissues. The magnitude of pressure increased even though weight bearing and IFM remained unchanged. Oxygen tensions were initially high in the haematoma and fell gradually with a low oxygen environment first established after four to five days. This study illustrates that in bone healing the local environment for cells may not be considered constant with regard to oxygen tension, pressure and temperature.

Ovine Enzootic Abortion (OEA): a comparison of antibody responses in vaccinated and naturally-infected swiss sheep over a two year period

Background Prevention and control of ovine enzootic abortion (OEA) can be achieved by application of a live vaccine. In this study, five sheep flocks with different vaccination and infection status were serologically tested using a competitive enzyme-linked immunosorbent assay (cELISA) specific for Chlamydophila (Cp.) abortus over a two-year time period. Results Sheep in Flock A with recent OEA history had high antibody values after vaccination similar to Flock C with natural Cp. abortus infections. In contrast, OEA serology negative sheep (Flock E) showed individual animal-specific immunoreactions after vaccination. Antibody levels of vaccinated ewes in Flock B ranged from negative to positive two and three years after vaccination, respectively. Positive antibody values in the negative control Flock D (without OEA or vaccination) are probably due to asymptomatic intestinal infections with Cp. abortus. Excretion of the attenuated strain of Cp. abortus used in the live vaccine through the eye was not observed in vaccinated animals of Flock E. Conclusion The findings of our study indicate that, using serology, no distinction can be made between vaccinated and naturally infected sheep. As a result, confirmation of a negative OEA status in vaccinated animals by serology cannot be determined.

Polycaprolactone-based scaffold plus rhBMP-2 in a sheep thoracic spine fusion model

We report the application of a novel scaffold design in a sheep thoracic spine model for spine deformity correction. The combination of the calcium-phosphate coated polycaprolactone scaffolds with recombinant human bone morphogenic protein-2 (rhBMP-2) are intended as a future bone graft substitute in ensuring the stability of bony intervertebral fusion. A solid free-form fabrication process based on melt extrusion has been utilized in the manufacturing of these scaffolds. To date there are no studies examining the use of such biodegradable implants in a sheep thoracic spine model. The success of anterior scoliosis surgery in humans depends on achieving a solid bony fusion between adjacent vertebrae after the intervertebral discs have been surgically cleared and the disc spaces filled with graft material. Due to limited availability of autograft, there is much current interest in the development of synthetic scaffolds in combination with growth factors such as recombinant human bone morphogenetic protein (rhBMP-2) to achieve a solid bony fusion following scoliosis surgery.

Reliability of clinical measurement for assessing spinal fusion: An experimental sheep study

Study Design. A sheep study designed to compare the accuracy of static radiographs, dynamic radiographs, and computed tomographic (CT) scans for the assessment of thoracolumbar facet joint fusion as determined by micro-CT scanning. Objective. To determine the accuracy and reliability of conventional imaging techniques in identifying the status of thoracolumbar (T13-L1) facet joint fusion in a sheep model. Summary of Background Data. Plain radiographs are commonly used to determine the integrity of surgical arthrodesis of the thoracolumbar spine. Many previous studies of fusion success have relied solely on postoperative assessment of plain radiographs, a technique lacking sensitivity for pseudarthrosis. CT may be a more reliable technique, but is less well characterized. Methods. Eleven adult sheep were randomized to either attempted arthrodesis using autogenous bone graft and internal fixation (n = 3) or intentional pseudarthrosis (IP) using oxidized cellulose and internal fixation (n = 8). After 6 months, facet joint fusion was assessed by independent observers, using (1) plain static radiography alone, (2) additional dynamic radiographs, and (3) additional reconstructed spiral CT imaging. These assessments were correlated with high-resolution micro-CT imaging to predict the utility of the conventional imaging techniques in the estimation of fusion success. Results. The capacity of plain radiography alone to correctly predict fusion or pseudarthrosis was 43% and was not improved using plain radiography and dynamic radiography with also a 43% accuracy. Adding assessment by reformatted CT imaging to the plain radiography techniques increased the capacity to predict fusion outcome to 86% correctly. The sensitivity, specificity, and accuracy of static radiography were 0.33, 0.55, and 0.43, respectively, those of dynamic radiography were 0.46, 0.40, and 0.43, respectively, and those of radiography plus CT were 0.88, 0.85, and 0.86, respectively. Conclusion. CT-based evaluation correlated most closely with high-resolution micro-CT imaging. Neither plain static nor dynamic radiographs were able to predict fusion outcome accurately. © 2012 Lippincott Williams & Wilkins.

Qualitative inquiry as transformation and agency : the black sheep and workplace mobbing

This chapter discusses an action research project into the lived experience of the workplace mobbing phenomenon. The action research methodology is based on the exemplarian model (Coenen & Khonraad, 2003) from the Netherlands Group. This model requires positive outcomes for those immersed in the problem to reduce the adversity of their circumstances. The findings challenge the psychological perspective of the existing bullying literature that tends to focus on individual behaviour. This research, undertaken over a three year period with 212 participants, identified the dysfunctional nature of public sector bureaucracies and the power gained through gossip and rumour as some of the key emergent themes to explain the workplace mobbing problem. In addition, resistance, conscientisation, and agency were identified as the key to transformation for those targeted. The discussion focuses on the crystallisation phase of the exemplarian model where the participants identified themselves as the Black Sheep and adopted the motto that “a black sheep is a biting beast” (Bastard, 1565 or 6-1618, p. 90), reflecting a sense of empowerment, individual agency, and a sense of humour in dealing with the serious yet seemingly absurd reality of their situations. The identity of the Black Sheep was consolidated when the group organised a 2 day conference with over 200 attendees to discuss how best to prevent workplace mobbing. This self-affirming action was a proactive step towards metaphorically “biting back” at the problem. A number of positive outcomes were achieved including the conference with over 200 attending leading to national media coverage across Australia and additional interviews with magazines, newspapers, and radio.

Investigating the potential value of individual parameters of histological grading systems in a sheep model of cartilage damage : the modified Mankin method

A total histological grade does not necessarily distinguish between different manifestations of cartilage damage or degeneration. An accurate and reliable histological assessment method is required to separate normal and pathological tissue within a joint during treatment of degenerative joint conditions and to sub-classify the latter in meaningful ways. The Modified Mankin method may be adaptable for this purpose. We investigated how much detail may be lost by assigning one composite score/grade to represent different degenerative components of the osteoarthritic condition. We used four ovine injury models (sham surgery, anterior cruciate ligament/medial collateral ligament instability, simulated anatomic anterior cruciate ligament reconstruction and meniscal removal) to induce different degrees and potentially 'types' (mechanisms) of osteoarthritis. Articular cartilage was systematically harvested, prepared for histological examination and graded in a blinded fashion using a Modified Mankin grading method. Results showed that the possible permutations of cartilage damage were significant and far more varied than the current intended use that histological grading systems allow. Of 1352 cartilage specimens graded, 234 different manifestations of potential histological damage were observed across 23 potential individual grades of the Modified Mankin grading method. The results presented here show that current composite histological grading may contain additional information that could potentially discern different stages or mechanisms of cartilage damage and degeneration in a sheep model. This approach may be applicable to other grading systems.

Polycaprolactone-based scaffold plus recombinant human bone morphogenic protein rhBMP-2) in a sheep thoracic spine fusion model

Adolescent idiopathic scoliosis is a complex three dimensional deformity affecting 2-3% of the general population. The resulting spinal deformity consists of coronal curvature, hypokyphosis of the thoracic spine and vertebral rotation in the axial plane with posterior elements turned into the curve concavity. The potential for curve progression is heightened during the adolescent growth spurt. Success of scoliosis deformity correction depends on solid bony fusion between adjacent vertebrae after the intervertebral (IV) discs have been surgically cleared and the disc spaces filled with graft material. Recently a bioactive and resorbable scaffold fabricated from medical grade polycaprolactone has been developed for bone regeneration at load bearing sites. Combined with rhBMP-2, this has been shown to be successful in acting as a bone graft substitute in a porcine lumbar interbody fusion model when compared to autologous bone graft alone. The study aimed to establish a large animal thoracic spine interbody fusion model, develop spine biodegradable scaffolds (PCL) in combination with biologics (rhBMP-2) and to establish a platform for research into spine tissue engineering constructs. Preliminary results demonstrate higher grades of radiologically evident bony fusion across all levels when comparing fusion scores between the 3 and 6 month postop groups at the PCL CaP coated scaffold level, which is observed to be a similar grade to autograft, while no fusion is seen at the scaffold only level. Results to date suggest that the combination of rhBMP-2 and scaffold engineering actively promotes bone formation, laying the basis of a viable tissue engineered constructs.