We explored the osseointegration potential of two macroporous titanium surfaces obtained using fast plasma sintering (FPS): Ti macroporous structures with 400-600 µmØ pores (TiMac400) and 850-1000 µmØ pores (TiMac850). They were compared against two surfaces currently in clinical use: Ti-Growth® and air plasma spray (Ti-Y367). Each surface was tested, once placed over a Ti-alloy and once onto a CoCr bulk substrate. Implants were placed in medial femoral condyles in 24 sheep. Samples were explanted at four and eight weeks after surgery. Push-out loads were measured using a material-testing system. Bone contact and ingrowth were assessed by histomorphometry and SEM and EDX analyses. Histology showed early osseointegration for all the surfaces tested. At 8 weeks, TiMac400, TiMac850 and Ti-Growth® showed deep bone ingrowth and extended colonisation with newly formed bone. The mechanical push-out force was equal in all tested surfaces. Plasma spray surfaces showed greater bone-implant contact and higher level of pores colonisation with new bone than FPS produced surfaces. However, the void pore area in FPS specimens was significantly higher, yet the FPS porous surfaces allowed a deeper osseointegration of bone to implant. FPS manufactured specimens showed similar osseointegration potential to the plasma spray surfaces for orthopaedic implants. FPS is a useful technology for manufacturing macroporous titanium surfaces. Furthermore, its capability to combine two implantable materials, using bulk CoCr with macroporous titanium surfaces, could be of interest as it enables designers to conceive and manufacture innovative components. FPS delivers functional graded materials components with macroporous structures optimised for osseointegration.

Advancing age is a well‐known risk factor for tendon disease. Energy‐storing tendons [e.g., human Achilles, equine superficial digital flexor tendon (SDFT)] are particularly vulnerable and it is thought that injury occurs following an accumulation of micro‐damage in the extracellular matrix (ECM). Several authors suggest that age‐related micro‐damage accumulates due to a failure of the aging cell population to maintain the ECM or an imbalance between anabolic and catabolic pathways. We hypothesized that ageing results in a decreased ability of tendon cells to synthesize matrix components and matrix‐degrading enzymes, resulting in a reduced turnover of the ECM and a decreased ability to repair micro‐damage. The SDFT was collected from horses aged 3–30 years with no signs of tendon injury. Cell synthetic and degradative ability was assessed at the mRNA and protein levels. Telomere length was measured as an additional marker of cell ageing. There was no decrease in cellularity or relative telomere length with increasing age, and no decline in mRNA or protein levels for matrix proteins or degradative enzymes. The results suggest that the mechanism for age‐related tendon deterioration is not due to reduced cellularity or a loss of synthetic functionality and that alternative mechanisms should be considered.

Open fractures are at risk of serious infection and, if infected, require several surgical interventions and courses of systemic antibiotics. We investigated a new injectable formulation that simultaneously hardens in vivo to form a porous scaffold for bone repair and delivers antibiotics at high concentrations to the local site of infection. Duration of antimicrobial activity against Staphylococcus aureus was determined using the serial plate transfer test. Ultimate compressive strength and porosity of the material was measured with and without antibiotics. The material was evaluated in vivo in an ovine medial femoral condyle defect model contaminated with S. aureus. Sheep were sacrificed at either 2 or 13 weeks and the defect and surrounding bone assessed using micro-computed tomography and histology. Antimicrobial activity in vitro persisted for 19-21 days. Sheep with antibiotic-free material and bacteria became infected, while those with antibiotic-containing material and bacteria did not. Similarly, new bone growth was seen in uninoculated animals with plain polymer, and in those with antibiotic polymer with bacteria, but not in sheep with plain polymer and bacteria. The antibiotic-impregnated scaffolds were effective in preventing S. aureus infections whilst supporting bone growth and repair. If translated into clinical practice, this approach might reduce the need for systemic antibiotics.

METHODS: Stromal cells were isolated from sheep BM aspirates taken from the iliac crest and SM biopsies from the DFTS. Cells were selected for adherence to tissue culture plastic and characterised by trilineage differentiation and CFU-F assays. Cells were labeled with MIONs (BioPal, USA) according to supplier’s conditions; label uptake was confirmed by confocal microscopy. The sensitivity of MRI to detect different densities of cells, either clustered or evenly dispersed, was conducted. 5x106 labeled cells were injected into the DFTS of the right forelimb in which the lateral deep digital flexor tendon bore a longitudinal defect created tenoscopically 7 days earlier (n= 4). Cells were tracked in the forelimbs at 7 days post-injection with a 1.5 Tesla MRI scanner (Philips). Control sheep had PBS injected (n= 2).RESULTS: A 10 ml aspirate of ovine BM would yield~ 1x106 cells after 7 days in primary culture. By comparison, 2x105 SM-MSCs were obtainable by mild overnight digestion of~ 50 mg SM biopsy. Both populations exhibited a mean CFU count of 25% as well as tri-lineage capacity. Uniform MION labeling of cells, assessed by confocal microscopy, occurred in all instances. Addition of protamine sulfate did not enhance MION uptake, which could be detected by MRI in~ 3x104 cells suspended in low-melting point agarose. Label was detectable in cells for up to five population doublings. MION labeling did not significantly affect clonogenic replication, population doubling time or tri-lineage capacity of cells compared to control medium. MRI showed both MSC types were distributed unevenly throughout the tendon sheath, mostly to the synovial …

Unique progenitor cells have been identified recently and successfully cultured in vitro from human articular cartilage. These cells are able to maintain chondrogenic potential upon extensive expansion. In this study, we have developed a sheep, ex-vivo model of cartilage damage and repair, using these progenitor cells. This study addresses the question can such a model be used to determine factors required for progenitor cell proliferation, differentiation and integration of matrix onto bone. The hypothesis was that sheep allogenic cartilage derived progenitor cells could regenerate artificially damaged sheep articular cartilage in an osteochondral culture model. Progenitor cells were derived from ovine articular cartilage using a differential adhesion assay to fibronectin and expanded clonally. These clonal cells were marked with lentiviral vectors derived from the Human Immunodeficiency Virus-1. When a self-inactivating lentiviral vector encoding a ubiquitous phosphoglycerate kinase promoter, driving a Green Fluorescent Protein (GFP) reporter gene, was used to transduce these cells, up to 80% of these progenitor cells expressed GFP. Normal sheep medial femoral condyles containing about 2mm thick sub-condral bone were obtained and 4mm circular defects created on the cartilage surface using a biopsy punch. Condyles were cultured for two weeks in vitro with GFP labelled progenitor cells within a fibrin glue scaffold (Tisseel Lyo) and matrix production (collagen) as determined by spatially offset Raman spectroscopy and immunohistochemistry was demonstrated. Progenitor cells were able to proliferate and differentiate into collagen producing cells. Such an ex-vivo model system is an effective tool for the analysis of cartilage repair from various sources of stem cells. These ex-vivo experiments and variations on defect type, size, titration of scaffold and progenitor cell numbers requirements can further be used as a basis for screening prior to in vivo experiments.

REASONS FOR PERFORMING STUDY:Mesenchymal stem cell (MSC) therapy for orthopaedic disease is being used with increasing frequency; there is a need to define a safe, reliable and effective technique for the recovery of MSCs from the sternum of the horse.OBJECTIVES:To describe an optimised safe technique for obtaining bone marrow-derived MSCs from the sternum of the Thoroughbred horse.METHODS:The anatomical relationship of the sternum with the heart and internal anatomy was demonstrated in cadavers. Sternal anatomy was evaluated ultrasonographically and after midline sectioning. Sternebrae were examined histologically after aspiration to determine the effect of needle insertion. The quality of the aspirate was evaluated as the number of colony-forming units from sequential and separately aspirated 5 ml aliquots and assessed for their multipotency using trilineage differentiation.RESULTS:The optimal safe location for the needle was the 5th sternebra because it had a safe dorsoventral thickness and was cranial to the apex of the heart. This sternebra could be reliably identified ultrasonographically. Aspirates could also be obtained from the 4th and 6th sternebrae, although the former is between the front limbs and the latter closer to the heart. Minimal disruption of the internal bony architecture was seen after needle insertion through the thin outer cortex and the first 5 ml aliquot contained the greatest number of colony-forming units of mesenchymal stem cells with trilineage capabilities.CONCLUSIONS:Accurate placement of a Jamshidi needle into the medullary cavity of the 4th-6th individual sternebrae is facilitated by the use of ultrasonography and enables aspiration of bone marrow reliably with minimal damage to the sternum and risk to the horse.POTENTIAL CLINICAL RELEVANCE:Sternal marrow aspiration as described is a safe and reliable technique to obtain MSCs for orthopaedic cell-based therapies.

Tendon and ligament injuries have proved difficult to treat effectively. Cell-based therapies offer the potential to harness the complex protein synthetic machinery of the cell to induce a regenerative response rather than fibrous scarring. This article reviews the current state of play with respect to the clinically used cell preparations for the treatment of tendon and ligaments overstrain injuries.

The degree of osseomechanical integration of dental implants is acutely sensitive to their mechanical environment. Bone, both as a tissue and structure, adapts its mass and architecture in response to loading conditions. Therefore, application of predefined controlled loads may be considered as a treatment option to promote early maturation of bone/implant interface prior to or in conjunction with crown/prosthesis attachment. Although many studies have established that the magnitude, rate of the applied strain, and frequency have significant effects on the osteogenic response, the actual specific relationships between strain parameters and frequency have not yet been fully defined. The purpose of this study was to develop a stimulator to apply defined mechanical stimuli to individual dental implants in vivo immediately after implantation, exploring the hypothesis that immediate controlled loading could enhance implant integration. An electromechanical device was developed, based on load values obtained using a two-dimensional finite element analysis of the bone/implant interface generating 1000 to 4000 pe and operated at 30 and 3 Hz respectively. The device was then tested in a cadaveric pig mandible, and periosteal bone surface strains were recorded for potential future comparison with a three-dimensional finite element model to determine loading regimens to optimize interface strains and iterate the device for clinical use.

1. The relationship between the physical activity and leg health of broiler chickens was assessed on a semi-commercial scale. 2. Three batches of birds (2128 per batch) were raised under two lighting regimes during the photoperiod; either a step-wise change of light intensity alternating between an illuminance of 200 and 10 lx or a constant illuminance of 10 lx. The activity of focal individuals (24 per batch) was observed at 2, 4 and 6 weeks of age, and leg health assessed weekly, based on gait score, the prevalence of burns on the hock and foot pad, and angulation and rotation of the leg at the intertarsal joint. Cortical bone density and thickness and area moments of inertia of the mid-physis tibiotarsus were measured post mortem at 6 weeks of age. 3. The step-wise change in light intensity did not affect overall performance, activity or leg health. 4. An individual bird's activity did not affect its gait score, the prevalence of hock burn or foot pad burn, cortical density or thickness or shape of the tibiotarsus. Sex of the bird was the only factor to affect significantly the area moment of inertia in the horizontal and vertical planes of the tibiotarsus, with females showing a lower moment of inertia for both. No variable had a significant effect on cortical density or thickness. Mean cortical density was low across all birds and may indicate that, when allowed to move freely as much or as little as they choose, broiler chickens do not exercise enough or do not perform the higher impact activities required to affect bone quality. 5. These findings imply that the activity of broiler chickens raised on a semi-commercial scale is unaffected by step-wise changes in light intensity and that other husbandry measures are needed to raise activity and hence improve leg health.

REASONS FOR PERFORMING STUDY:Damage to the flexor tendons, particularly the superficial digital flexor tendon (SDFT), is one of the most common musculoskeletal injuries sustained by horses competing in all disciplines. Our previous work has shown that SDFTs from different individuals show a wide variation in mechanical strengths; this is important clinically as it may relate to predisposition to injury. The high mechanical strength of tendon relies on the correct orientation of collagen molecules within fibrils and stabilisation by the formation of chemical cross-links between collagen molecules. It is not known whether the variation in SDFT mechanical properties between individuals relates to differences in collagen cross-link levels.HYPOTHESIS:Enzyme-derived, intermolecular cross-linking of tendon collagen correlates with mechanical properties of the SDFT.METHODS:SDFTs were collected from 38 horses and mechanically tested to failure. Structural and material properties were calculated from the load/deformation plot and cross-sectional area for each tendon. Following mechanical testing, pyrrolic cross-link levels were measured using a spectrophotometric assay for Ehrlich's reactivity and pyridinoline levels were quantified by HPLC. Cross-link levels were correlated with mechanical properties and statistical significance tested using a Pearson's correlation test.RESULTS:Pyrrole cross-link levels showed a significant positive correlation with ultimate stress (P = 0.004), yield stress (P = 0.003) and elastic modulus (P = 0.018) of the tendons, despite being a minor cross-link in these tendons. There was no significant correlation of mechanical properties with either hydroxylysyl- or lysyl-pyridinoline levels.CONCLUSIONS:Given the low absolute levels of pyrrole, we suggest that the correlation with high mechanical strength is through an indirect mechanism. Understanding the nature of the relationships between pyrrole cross-links, other matrix characteristics and tendon material properties may allow development of strategies to identify horses at risk from tendon injury and be of value in informing training practices.

REASONS FOR PERFORMING STUDY:Injury to the superficial digital flexor tendon (SDFT) is common in competition horses. The SDFT contributes to locomotory efficiency by storing energy; such tendons have low safety margins. Tendons which merely position the limb, including the opposing common digital extensor tendon (CDET), are rarely injured. The current failure of strategies to prevent or effectively treat injury to the SDFT indicates the importance of understanding how it differs from tendons which are not injury-prone.HYPOTHESIS:That the structural and material properties and matrix composition of the SDFT and CDET differ, reflecting their specific functional requirements in vivo.METHODS:Forelimb tendons were harvested from 26 mature horses and loaded to failure prior to matrix composition analysis of specimens.RESULTS:The SDFT had a significantly higher cross-sectional area, structural stiffness, failure load and failure strain and a lower elastic modulus than the CDET (P < 0.0001).CONCLUSIONS:The SDFT has conflicting requirements for strength and elasticity; although as a whole it is a stiffer structure than the CDET, differences in the matrix molecular composition including water and total sulphated glycosaminoglycan contents allow it to remain more elastic as a material.POTENTIAL RELEVANCE:Further information on how the two tendons attain these different properties may be of use in the development of prevention and treatment strategies for SDFT rupture.

REASONS FOR PERFORMING THE STUDY:Highly prevalent superficial digital flexor tendon (SDFT) injury results in compromised tendon function through fibrosis and high frequency of re-injury due to altered biomechanical function. This study investigated the consequences of SDF tendinopathy on limb mechanics in relation to the mechanical properties of injured tendon.OBJECTIVES:To develop and validate a noninvasive in vivo assessment of tendon mechanics to investigate the effect of recent SDFT injury on limb stiffness index, providing an objective method to assess quality of healing.HYPOTHESES:Limb stiffness index would reduce as a consequence of SDFT injury and progressively increase during tendon healing and correlate with in vitro mechanical properties of the respective SDFTs.METHODS:Kinematic analysis was performed at walk in 10 horses that had sustained career-ending SDFT injury. Stiffness index was derived from limb force recorded via a series of force plates and measurement of change in metacarpophalangeal joint angle using 3D motion analysis software. Horses were subjected to euthanasia 7 months after injury, the SDFTs removed and subjected to nondestructive in vitro mechanical testing.RESULTS:Limb stiffness index was reduced following SDFT injury in comparison with the contralateral limb and increased during the convalescent period, approximating that of the contralateral limb by 7 months post injury. There was a significant positive correlation between in vivo limb stiffness index and in vitro SDFT stiffness.CLINICAL RELEVANCE:The ability to assess and monitor SDFT mechanical competence through limb stiffness measurement techniques in horses recovering from SDFT injury and the possibility of corroborating this with functional tendon healing may permit a more objective and accurate assessment of optimal tendon repair in the horse. This technique may be a useful method for assessing the efficacy of treatment regimens for tendinopathy and could be utilised to predict time to safe return to performance or re-injury.

REASON FOR PERFORMING STUDY:No data exist on the effects of conditioning exercise at foal age on workload and subsequent clinical injury rate during their 2- and 3-year-old racing careers.OBJECTIVES:To investigate the effects of subjecting TB foals to conditioning exercise prior to the start of race training on: the workload required to reach a level of fitness sufficient to compete; and the prevalence of orthopaedic injury during the first 2 seasons of their racing careers.METHODS:Twenty 18-month-old TBs, 12 subjected to conditioning exercise at foal age (CONDEX) and 8 exercised spontaneously at pasture only (PASTEX) were trained and entered in competitive events. Workload was quantified using the cumulative workload index (the product of average velocity and distance at a specific gait) and the animals were monitored clinically and radiographically for signs of musculoskeletal disorders.RESULTS:Workload to reach the desired fitness level was similar for CONDEX and PASTEX. CONDEX performed more prerace training sessions as 2-year-olds (P<0.05). The incidence of orthopaedic injuries was low in both groups and there were no differences in the occurrence of orthopaedic ailments. PASTEX animals tended to show signs of musculoskeletal disorders earlier than CONDEX animals. This time difference was significant for metacarpophalangeal joint pain on flexion, reduced carpal flexion and hindlimb lameness (P<0.05).CONCLUSIONS:Subjecting TB foals to conditioning exercise early in life does not have adverse effects on racing careers at ages 2 and 3 years, and does not influence the workload needed to reach a fitness level that is sufficient for racing.POTENTIAL RELEVANCE:The lack of negative effects and the indications of some positive effects of early conditioning exercise in the Thoroughbred encourage further large-scale comparative studies.

REASON FOR PERFORMING STUDY The injury-prone, energy-storing equine superficial digital flexor tendon (SDFT) of the mature performance horse has a limited ability to respond to exercise in contrast with the noninjury-prone, anatomically opposing common digital extensor tendon (CDET). Previous studies have indicated low levels of cellular activity in the mature SDFT, but in foal tendons the tenocytes may still have the ability to adapt positively to increased exercise. OBJECTIVES To measure tenocyte densities and types in histological sections from the SDFT and CDET of horses from controlled long-term, short-term and foal exercise studies. METHODS Specimens were collected from mid-metacarpal segments of the CDET and SDFT for each horse and processed for histology; central and peripheral regions of the SDFT cross-section were analysed separately (SDFTc, SDFTp). Tenocyte nuclei were counted in a total area of 1.59 mm(2) for each tendon region in each horse. Each nucleus was classified as type 1 (elongate and thin), type 2 (ovoid and plump) or type 3 (chondrocyte-like); type 1 cells are proposed to be less synthetically active than type 2 cells. RESULTS No significant differences were noted between exercise and control groups in any of the studies, with the exception of an exercise-related reduction in the proportion of type 1 tenocytes for all tendons combined in the long-term study. There were tendon- and site-specific differences in tenocyte densities and proportions of type 1 and 2 cells in all 3 studies. CONCLUSIONS AND POTENTIAL RELEVANCE There was no indication that exercise increased tenocyte density or proportions of the (theoretically) more active type 2 cells in immature horses (short-term and foal studies), perhaps because the training regimens did not achieve certain threshold strain levels. In the foal study these findings can still be interpreted positively as evidence that the training regimen did not induce subclinical damage.

REASON FOR PERFORMING STUDY:No data exist on the intensity of exercise required or on possible harmful effects of increasing exercise in foals over the natural level when free at pasture. OBJECTIVES:To investigate whether an increase in workload over free pasture exercise in the period from directly after birth to the start of training is tolerated by Thoroughbred (TB) foals without increasing injury rate or producing other undesired side effects. METHODS:Thirty-three TB foals were allocated to one of 2 exercise groups directly after birth. One group (PASTEX) was raised on pasture and the other (CONDEX) kept under identical circumstances, but was additionally subjected to an exercise protocol of gradually increasing intensity. Foals were monitored periodically and scored for the presence of clinical signs related to the musculoskeletal system (joint effusion, pain at flexion, occurrence of physeal swelling), and radiographs taken at the end of the conditioning phase. Also, behavioural studies were performed to detect any changes in behaviour related to the exercise programme. Cortisol levels were measured in both groups, to assess the level of stress. RESULTS:Workload in the CONDEX group was significantly higher than in the PASTEX group (approximately 30%). Conditioning increased the likelihood for joint effusion in the antebrachiocarpal joint, but reduced tarsocrural effusion and physeal swelling at the lateral distal radius, the third metacarpal bone (medial aspect) and lateral and medial aspects of the third metatarsal bone. CONCLUSIONS:The 30% increase in workload did not affect the animals' welfare, effects of conditioning exercise on clinical musculoskeletal health were few and there were no adverse effects. POTENTIAL RELEVANCE:This study supports the feasibility of imposing early conditioning exercise in horses and is a benchmark for its effects on the development of equine musculoskeletal tissues.