Search

Intra‐synovial tendon injuries are a common orthopedic problem with limited treatment options. The synovium is a specialized connective tissue forming the inner encapsulating lining of diarthrodial joints and intra‐synovial tendons. It contains multipotent mesenchymal stromal cells that render it a viable source of progenitors for tendon repair. This study evaluated the effects of autologous implantation of cells derived from normal synovium (synovial membrane cells [SMCs]) in augmenting repair in an ovine model of intra‐synovial tendon injury. For this purpose, synovial biopsies were taken from the right digital flexor tendon sheath following creation of a defect to the lateral deep digital flexor tendon. Mononuclear cells were isolated by partial enzymatic digestion and assessed for MSC characteristics. Cell tracking and tendon repair were assessed by implanting 5 × 106 cells into the digital flexor tendon sheath under ultrasound guidance with the effects evaluated using magnetic resonance imaging and histopathology. Synovial biopsies yielded an average 4.0 × 105 ± 2.7 × 105 SMCs that exhibited a fibroblastic morphology, variable osteogenic, and adipogenic responses but were ubiquitously strongly chondrogenic. SMCs displayed high expression of CD29 with CD271NEGATIVE and MHC‐IILOW cell‐surface marker profiles, and variable expression of CD73, CD90, CD105, CD166, and MHC‐I. Implanted SMCs demonstrated engraftment within the synovium, though a lack of repair of the tendon lesion over 24 weeks was observed. We conclude healthy synovium is a viable source of multipotent cells, but that the heterogeneity of synovium underlies the variability between different SMC populations, which while capable of engraftment and persistence within the synovium exhibit limited capacity of influencing tendon repair. © 2019 The Authors. Journal of Orthopaedic Research® published by Wiley Periodicals, Inc. on behalf of Orthopaedic Research Society J Orthop Res 38:128–138, 2020 more...

Background Intra-synovial tendon injuries display poor healing, which often results in reduced functionality and pain. A lack of effective therapeutic options has led to experimental approaches to augment natural tendon repair with autologous mesenchymal stem cells (MSCs) although the effects of the intra-synovial environment on the distribution, engraftment and functionality of implanted MSCs is not known. This study utilised a novel sheep model which, although in an anatomically different location, more accurately mimics the mechanical and synovial environment of the human rotator cuff, to determine the effects of intra-synovial implantation of MSCs. Methods A lesion was made in the lateral border of the lateral branch of the ovine deep digital flexor tendon within the digital sheath and 2 weeks later 5 million autologous bone marrow MSCs were injected under ultrasound guidance into the digital sheath. Tendons were recovered post mortem at 1 day, and 1–2, 4, 12 and 24 weeks after MSC injection. For the 1-day and 1–2-week groups, MSCs labelled with fluorescent-conjugated magnetic iron-oxide nanoparticles (MIONs) were tracked with MRI, histology and flow cytometry. The 4, 12 and 24-week groups were implanted with non-labelled cells and compared with saline-injected controls for healing. Results The MSCs displayed no reduced viability in vitro to an uptake of 20.0 ± 4.6 pg MIONs per cell, which was detectable by MRI at minimal density of ~ 3 × 104 cells. Treated limbs indicated cellular distribution throughout the tendon synovial sheath but restricted to the synovial tissues, with no MSCs detected in the tendon or surgical lesion. The lesion was associated with negligible morbidity with minimal inflammation post surgery. Evaluation of both treated and control lesions showed no evidence of healing of the lesion at 4, 12 and 24 weeks on gross and histological examination. Conclusions Unlike other laboratory animal models of tendon injury, this novel model mimics the failed tendon healing seen clinically intra-synovially. Importantly, however, implanted stem cells exhibited homing to synovium niches where they survived for at least 14 days. This phenomenon could be utilised in the development of novel physical or biological approaches to enhance localisation of cells in augmenting intra-synovial tendon repair. more...

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. more...

Fragility fractures, those fractures which result from low level trauma, have a large and growing socio-economic cost in countries with aging populations. Bone-density-based assessment techniques are vital for identifying populations that are at higher risk of fracture, but do not have high sensitivity when it comes to identifying individuals who will go on to have their first fragility fracture. We are developing Spatially Offset Raman Spectroscopy (SORS) as a tool for retrieving chemical information from bone non-invasively in vivo. Unlike X-ray-based techniques SORS can retrieve chemical information from both the mineral and protein phases of the bone. This may enable better discrimination between those who will or will not go on to have a fragility fracture because both phases contribute to bone's mechanical properties. In this study we analyse excised bone with Raman spectroscopy and multivariate analysis, and then attempt to look for similar Raman signals in vivo using SORS. We show in the excised work that on average, bone fragments from the necks of fractured femora are more mineralised (by 5-10%) than (cadaveric) non-fractured controls, but the mineralisation distributions of the two cohorts are largely overlapped. In our in vivo measurements, we observe similar, but as yet statistically underpowered, differences. After the SORS data (the first SORS measurements reported of healthy and diseased human cohorts), we identify methodological developments which will be used to improve the statistical significance of future experiments and may eventually lead to more sensitive prediction of fragility fractures. © 2015 The Authors. Journal of Raman Spectroscopy Published by John Wiley & Sons, Ltd. more...

Binary blend polymers offer the opportunity to combine different desirable properties into a single scaffold, to enhance function within the field of tissue engineering. Previous in vitro and murine in vivo analysis identified a polymer blend of poly(l-lactic acid)–poly(e-caprolactone) (PLLA:PCL 20:80) to have characteristics desirable for bone regeneration. Polymer scaffolds in combination with marrow-derived skeletal stem cells (SSCs) were implanted into mid-shaft ovine 3.5 cm tibial defects, and indices of bone regeneration were compared to groups implanted with scaffolds alone and with empty defects after 12 weeks, including micro-CT, mechanical testing and histological analysis. The critical nature of the defect was confirmed via all modalities. Both the scaffold and scaffold/SSC groups showed enhanced quantitative bone regeneration; however, this was only found to be significant in the scaffold/SSCs group (p = 0.04) and complete defect bridging was not achieved in any group. The mechanical strength was significantly less than that of contralateral control tibiae (p < 0.01) and would not be appropriate for full functional loading in a clinical setting. This study explored the hypothesis that cell therapy would enhance bone formation in a critical-sized defect compared to scaffold alone, using an external fixation construct, to bridge the scale-up gap between small animal studies and potential clinical translation. The model has proved a successful critical defect and analytical techniques have been found to be both valid and reproducible. Further work is required with both scaffold production techniques and cellular protocols in order to successfully scale-up this stem cell/binary blend polymer scaffold. © 2015 The Authors. Journal of Tissue Engineering and Regenerative Medicine published by John Wiley & Sons, Ltd. more...

The ability of Spatially Offset Raman Spectroscopy (SORS) to obtain chemically specific information from below the sample surface makes it a promising technique for non-invasive in vivo diagnosis of bone conditions by sampling bone through the skin. The depth below a surface interrogated by SORS depends on the system's optical properties and is difficult to estimate for complex bone material. This paper uses 830 nm laser excitation to investigate the influence of bone mineralization on photon migration properties in deer antler cortex, equine metacarpal cortex and whale tympanic bulla. Thin slices form each type of bone (thickness: 0.6-1.0 mm) were cut and put together on top of each other forming stacks with a total thickness of 4.1-4.7 mm. A 0.38 mm thin slice of polytetrafluoroethylene (PTFE) served as a test material for Raman signal recovery and was placed in between the individual bone slices within the stack. At SORS offsets of 8.0-9.5 mm Raman bands of materials not present in healthy bone (e.g. PTFE as an example) can be recovered through 4.4-4.7 mm of cortical bone tissue, depending on mineralization level and porosity. These findings significantly increase our understanding of SORS analysis through bones of different composition and provide information that is vital to determine the value of SORS as a medical diagnostic technique. The data serve to define which SORS offset is best deployed for the non-invasive detection of chemically specific markers associated with infection, degeneration and disease or cancer within bone. more...

The development of an osteogenic bone graft substitute has important practical and cost implications in many branches of medicine where bone regeneration is required. Previous in vitro and small animal (murine) in vivo studies highlighted a porous hydroxyapatite/poly (DL-lactic acid) composite scaffold in combination with skeletal stem cells (SSCs) as a potential bone graft substitute candidate. The aim of the current study was to scale up the bone cell-scaffold construct to large animals and examine the potential for repair of a critical-sized defect via an ovine model. SSC seeded scaffolds (and unseeded scaffold controls) were implanted bilaterally into ovine femoral condyle critical defects for 3 months. A parallel in vitro analysis of ovine SSC seeded scaffolds was also performed. Post mortem mechanical indentation testing showed the bone strengths of the defect sites were 20% (controls) and 11% (SSC seeded scaffolds) those of normal cancellous bone (p < 0.01). MicroCT analysis demonstrated new bone formation within all defects with a mean increase of 13.4% in the control scaffolds over the SSC seeded scaffolds (p = 0.14). Histological examination confirmed these findings, with enhanced quality new bone within the control defects. This study highlights important issues and steps to overcome in scale-up and translation of tissue engineered products. The scaffold demonstrated encouraging results as an osteoconductive matrix; however, further work is required with cellular protocols before any human trials. more...

The purpose of this work was to validate isolation methods for sheep mesenchymal stem cells (MSC) from different sources and to explore the hypothesis that MSC exhibit markers of the same phenotype independent from tissue source. Cells derived from ovine bone marrow, synovial membrane and adipose tissue were characterized using the following markers: CD44, CD45, CD11b and MHC-I. The isolated MSC were cultivated, went through osteogenic, chondrogenic and adipogenic differentiation, and were characterized by flow cytometry using mouse anti-ovine CD44, CD45 and MHC-I monoclonal antibody (mAb), and mouse anti-bovine CD11b mAb. Ovine MSC from all three sources differentiated under chondorgenic, osteogenic and adipogenic conditions. Also, MSC from the three tissues were found to express CD44 and MHC-I but lack of CD11b and CD45. The results obtained revealed that our isolation methods for the different tissues tested are valid and that MSC from the three sources studied have same immunophenotic characteristics. more...

Raman spectroscopy was used to show that across 10 cm of diaphyseal (mid-shaft) cortical bone the phosphate-to-amide I ratio (a measure of the mineral to collagen ratio) can vary by as much as 8%, and the phosphate-to-carbonate ratio (a measure of carbonate inclusion in mineral crystals) by as much as 5%. The data are preliminary but are important because they reveal a spatial variation at a scale that is much larger than many of the spectral maps reported in the literature to date. Thus they illustrate natural variation in chemical composition that could have been overlooked in such studies or could have appeared as an undue error where the overall composition of the bone was investigated. Quantifying the variation in mid-shaft cortical bone at the millimeter/centimeter scale reduces the possibility of natural heterogeneity obscuring the average bone composition, or being mistaken for experimental signal, and results in an improvement in the sampling accuracy analogous to that obtained by switching from micrometer-size point spectra of bones to spectral images obtained across hundreds of micrometers. Although the study was carried out using Raman spectroscopy, the underlying cause of the variation is ascribed to the variation of the chemical composition of the bone; therefore the findings have direct implications for other chemically specific analytical methods such as Fourier transform infrared spectroscopy or nuclear magnetic resonance spectroscopy. more...

The decomposition of spatially offset Raman spectra for complex multilayer systems, such as biological tissues, requires advanced techniques such as multivariate analyses. Often, in such situations, the decomposition methods can reach their limits of accuracy well before the limits imposed by signal-to-noise ratios. Consequently, more effective reconstruction methods could yield more accurate results with the same data set. In this study we process spatially offset Raman spectroscopy (SORS) data with three different multivariate techniques (band-target entropy minimization (BTEM), multivariate curve resolution and parallel factor analysis (PARAFAC)) and compare their performance when analysing a spectrally challenging plastic model system and an even more challenging problem, the analysis of human bone transcutaneously in vivo. For the in vivo measurements, PARAFAC's requirement of multidimensional orthogonal data is addressed by recording SORS spectra both at different spatial offsets and at different anatomical points, the latter providing added dimensionality through the variation of skin/soft tissue thickness. The BTEM and PARAFAC methods performed the best on the plastic system with the BTEM more faithfully reconstructing the major Raman bands and PARAFAC the smaller more heavily overlapped features. All three methods succeeded in reconstructing the bone spectrum from the transcutaneous data and gave good figures for the phosphate-to-carbonate ratio (within 2% of excised human tibia bone); the PARAFAC gave the most accurate figure for the mineral-to-collagen ratio (20% less than excised human tibia bone). Previous studies of excised bones have shown that certain bone diseases (such as osteoarthritis, osteoporosis and osteogenesis imperfecta) are accompanied by compositional abnormalities that can be detected with Raman spectroscopy, the utility of a technique which could reconstruct bone spectra accurately is manifest. The results have relevance on the use of SORS in general. © 2014 Crown copyright. Journal of Raman Spectroscopy published by John Wiley & Sons, Ltd. Spatially offset Raman spectroscopy data from a spectrally challenging plastic model system and from the transcutaneous measurement of bone in vivo are processed using three different multivariate techniques (band-target entropy minimization, multivariate curve resolution and parallel factor analysis) and the performance of each technique is compared. © 2014 Crown copyright. Journal of Raman Spectroscopy published by John Wiley & Sons, Ltd. more...

WOS:000324536200013; International audience; The present study shows no adverse effects of the anti-diabetic drug metformin on bone mass and fracture healing in rodents but demonstrates that metformin is not osteogenic in vivo, as previously proposed. Introduction In view of the increased incidence of fractures in patients with type 2 diabetes mellitus (T2DM), we investigated the effects of metformin, a widely used T2DM therapy, on bone mass and fracture healing in vivo using two different rodent models and modes of metformin administration. Methods We first subjected 12-week-old female C57BL/6 mice to ovariectomy (OVX). Four weeks after OVX, mice received either saline or metformin administered by gavage (100 mg/kg/daily). After 4 weeks of treatment, bone micro-architecture and cellular activity were determined in tibia by micro-CT and bone histomorphometry. In another experiment, female Wistar rats aged 3 months were given only water or metformin for 8 weeks via the drinking water (2 mg/ml). After 4 weeks of treatment, a mid-diaphyseal osteotomy was performed in the left femur. Rats were sacrificed 4 weeks after osteotomy and bone architecture analysed by micro-CT in the right tibia while fracture healing and callus volume were determined in the left femur by X-ray analysis and micro-CT, respectively. Results In both models, our results show no significant differences in cortical and trabecular bone architecture in metformin-treated rodents compared to saline. Metformin had no effect on bone resorption but reduced bone formation rate in trabecular bone. Mean X-ray scores assessed on control and metformin fractures showed no significant differences of healing between the groups. Fracture callus volume and mineral content after 4 weeks were similar in both groups. Conclusions Our results indicate that metformin has no effect on bone mass in vivo or fracture healing in rodents. more...

Meniscal cartilage tears are common and predispose to osteoarthritis (OA). Most occur in the avascular portion of the meniscus where current repair techniques usually fail. We described previously the use of undifferentiated autologous mesenchymal stem cells (MSCs) seeded onto a collagen scaffold (MSC/collagen-scaffold) to integrate meniscal tissues in vitro. Our objective was to translate this method into a cell therapy for patients with torn meniscus, with the long-term goal of delaying or preventing the onset of OA. After in vitro optimization, we tested an ovine-MSC/collagen-scaffold in a sheep meniscal cartilage tear model with promising results after 13 weeks, although repair was not sustained over 6 months. We then conducted a single center, prospective, open-label first-in-human safety study of patients with an avascular meniscal tear. Autologous MSCs were isolated from an iliac crest bone marrow biopsy, expanded and seeded into the collagen scaffold. The resulting human-MSC/collagen-scaffold implant was placed into the meniscal tear prior to repair with vertical mattress sutures and the patients were followed for 2 years. Five patients were treated and there was significant clinical improvement on repeated measures analysis. Three were asymptomatic at 24 months with no magnetic resonance imaging evidence of recurrent tear and clinical improvement in knee function scores. Two required subsequent meniscectomy due to retear or nonhealing of the meniscal tear at approximately 15 months after implantation. No other adverse events occurred. We conclude that undifferentiated MSCs could provide a safe way to augment avascular meniscal repair in some patients. Registration: EU Clinical Trials Register, 2010-024162-22. more...

Summary: Loading increases bone mass and strength in a site-specific manner; however, possible effects of loading on bone matrix composition have not been evaluated. Site-specific structural and material properties of mouse bone were analyzed on the macro- and micro/molecular scale in the presence and absence of axial loading. The response of bone to load is heterogeneous, adapting at molecular, micro-, and macro-levels. Introduction: Osteoporosis is a degenerative disease resulting in reduced bone mineral density, structure, and strength. The overall aim was to explore the hypothesis that changes in loading environment result in site-specific adaptations at molecular/micro- and macro-scale in mouse bone. Methods: Right tibiae of adult mice were subjected to well-defined cyclic axial loading for 2 weeks; left tibiae were used as physiologically loaded controls. The bones were analyzed with μCT (structure), reference point indentation (material properties), Raman spectroscopy (chemical), and small-angle X-ray scattering (mineral crystallization and structure). Results: The cranial and caudal sites of tibiae are structurally and biochemically different within control bones. In response to loading, cranial and caudal sites increase in cortical thickness with reduced mineralization (−14 and −3%, p < 0.01, respectively) and crystallinity (−1.4 and −0.3%, p < 0.05, respectively). Along the length of the loaded bones, collagen content becomes more heterogeneous on the caudal site and the mineral/collagen increases distally at both sites. Conclusion: Bone structure and composition are heterogeneous, finely tuned, adaptive, and site-specifically responsive at the micro-scale to maintain optimal function. Manipulation of this heterogeneity may affect bone strength, relative to specific applied loads. more...

Bone diseases and disorders are a growing challenge in aging populations; so effective diagnostic and therapeutic solutions are now essential to manage the demands of healthcare sectors effectively. Spatially offset Raman spectroscopy (SORS) allows for chemically specific sub-surface probing and has a great potential to become an in vivo tool for early non-invasive detection of bone conditions. Bone is a complex hierarchical material and the volume probed by SORS is dependent on its optical properties. Understanding and taking into account the variations in diffuse scattering properties of light in various bone types is essential for the effective development and optimization of SORS as a diagnostic in vivo tool for characterizing bone disease. This study presents SORS investigations at 830 nm excitation on two specific types of bone with differing mineralization levels. Thin slices of bone from horse metacarpal cortex (0.6 mm thick) and whale bulla (1.0 mm thick) were cut and stacked on top of each other (4-7 layers with a total thickness of 4.1 mm). To investigate the depth origin of the detected Raman signal inside the bone a 0.38 mm thin Teflon slice was used as test sample and inserted in between the layers of stacked bone slices. For both types of bone it could be demonstrated that chemically specific Raman signatures different from those of normal bone can be retrieved through 3.8-4.0 mm of overlying bone material with a spatial offset of 7-8 mm. The determined penetration depths can be correlated with the mechanical and optical properties of the specimens. The findings of this study increase our understanding of SORS analysis of bone and thus have impact for medical diagnostic applications e.g. enabling the non-invasive detection of spectral changes caused by degeneration, infection or cancer deep inside the bone matrix. more...

Made available in DSpace on 2022-04-29T07:37:27Z (GMT). No. of bitstreams: 0 Previous issue date: 2016-01-01 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-Growths® 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. Institute of Orthopaedics and Musculoskeletal Science University College London Royal National Orthopaedic Hospital Department of Veterinary Surgery and Anaesthesiology School of Veterinary Medicine and Animal Science São Paulo State University Research and Development Department Eurocoating Spa Department of Veterinary Surgery and Anaesthesiology School of Veterinary Medicine and Animal Science São Paulo State University more...

Bone is a composite material comprising a collagen fibril scaffold surrounded by crystals of carbonated-hydroxyapatite mineral. It is well established that the relative proportions of mineral and collagen in mature bone are not definite and are adapted in order to ‘tune’ its mechanical properties. It is not known, however, how the mineral to collagen ratio is controlled. This paper uses Raman spectroscopy (which permits the probing of both the mineral and the collagen phases of bone) to explore the hypothesis that the control mechanism is related to the nature of the collagen and that bones with different levels of mineralisation have qualitatively different collagen. Raman spectra of functionally adapted bones with varying levels of mineralisation are presented and features that indicate the differences in the collagen's secondary structure (amide I band profiles) and post-translational modification (hydroxyproline/proline ratios) are highlighted. The study demonstrates that Raman spectroscopy can provide a means to investigate the mechanisms that control the mineral to collagen ratio of bone. Understanding these mechanisms could pave the way towards the therapeutic alteration of the mineral to collagen ratio and, thus, the control of the mechanical properties of bone. Copyright © 2012 John Wiley & Sons, Ltd. more...

Objective To determine mechanical differences between two plates with different requirements for supplementary casting after pancarpal arthrodesis (PCA): the Veterinary Instrumentation Hybrid Dynamic Compression Plate (HDCP), and the OrthoMed CastLess Arthrodesis Plate (CLP). Study Design In vitro mechanical analysis. Sample Population HDCP(n = 10), CLP(10). Methods Single-cycle load to failure using a materials-testing machine and cyclic loading between 38 and 380 N ± 5% to simulate estimated in vivo loads until failure or 106 cycles. Results Single-cycle to failure: bending stiffness was significantly higher for the HDCP(2269 ± 175 N/mm) than CLP(1754 ± 88 N/mm; P < .001). Bending structural stiffness was higher for the HDCP(3.8 ± 0.3 Nm2) versus CLP(2.9 ± 0.2 Nm2; P= .0022). A difference between the 2 plates for bending strength was not demonstrated; HDCP= 13.9 ± 1.4 Nm, CLP13.2 ± 0.5 Nm (P= .24). Cyclic Loading: no failures occurred with either plate type when plates were cycled to 106 cycles. Conclusion There is no mechanical advantage in bending resistance afforded by the CLPover the HDCP. Fatigue failure of either plate during the convalescent period of an estimated 150,000–250,000 cycles is unlikely. Based on the bending performance, there is no evidence to support the use of the CLPover the HDCPfor castless PCA. more...

Exercise or lack of it in early life affects chondro-osseous development. Two groups of horses were used to investigate the effects of age and exercise regimen on bone parameters of diaphyseal, metaphyseal, epiphyseal and cuboidal bones of the distal limb of Thoroughbreds. One group had exercised only spontaneously from an early age at pasture (PASTEX group), while the other group of horses were exposed to a 30% greater workload through additional defined exercise (CONDEX). Longitudinal data from peripheral quantitative computed tomography (pQCT) were obtained from eight scan sites of the left forelimb (proximal phalangeal (P(p); 1 site), third metacarpal (Mc3; six sites) and third carpal (C(3); one site) bones) of 32 Thoroughbred foals scanned five times from ∼3 weeks to 17 months of age. The primary outcome measures were bone mineral content (BMC), bone area (BA), and periosteal circumference (Peri C) in diaphyseal bone, with cortical thickness (CortTh), volumetric bone mineral density (BMD(v)) and a bone strength index (SSI) also being analysed. At the P(p) site within the model there was a significant effect (P=0.00-0.025) of conditioning exercise increasing bone parameters, except endosteal circumference (Endo C) and BMD(v). The BMC, BA, and SSI of P(p) were significantly greater in the CONDEX than PASTEX groups at 12 and 17 months (P=0.015-0.042) and CortTh at 17 months (P=0.033). At the M55 site of Mc3 BMC, BA and SSI (P=0.02-0.04), and at the M33 site, SSI (P=0.05) were higher in the CONDEX than PASTEX group. The adaptive responses, consistent with diaphyseal strengthening, were more marked in the diaphysis of P(p) than Mc3. In the Mc3, metaphysis, trabecular BMD(v) was less in the CONDEX than PASTEX group, associated with greater bone mineral accretion in the outer cortical-sub-cortical bone in the CONDEX group. There were no significant between-group differences in any epiphyseal or cuboidal bone parameter. Although the early imposed exercise regimen was not intensive, it had significant effects on diaphyseal bone strength, through change in size but not bone density. more...

Objective—To determine whether histopathologic characteristics of the osteochondral units of equine distal tarsal joints were associated with exercise history in horses without lameness. Sample Population—30 cadaver tarsi from horses without lameness and with known exercise history were separated into 3 groups: nonridden, pasture exercise (group P); low-intensity, ridden exercise (group L); and high-intensity, elite competition exercise (group E). Procedures—Standardized sites from the centrodistal and tarsometatarsal joints under went histologic preparation. A grading system was adapted to describe location, depth, and shape of lesions; cellular arrangement; organization at cartilage and subchondral bone (SCB) junctions; and organization of SCB. A high score signified a more severe pathological change than a low score. Exercise groups were compared by calculation of Spearman rank correlations. Results—In the centrodistal joint, lesions were present in groups L and E but only medially. Cellular arrangement scores were higher at the dorsomedial location in group P than in groups L and E. Groups L and E had higher scores than group P for the organization of the cartilage, SCB junctions, and SCB, with higher scores at the dorsomedial location. In the tarsometatarsal joint, lesions were evident across the whole joint surface, with more severe lesions located laterally in all 3 groups. Overall, group E had higher scores for cellular arrangement and SCB organization than groups P and L. Conclusions and Clinical Relevance—Ridden exercise may increase the risk of osteochondral lesions at distal tarsal sites predisposed to osteoarthritis relative to the risk with nonridden exercise. more...

P>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. more...

P>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. more...

P>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. more...

Summary Loading increases bone mass and strength in a site-specific manner; however, possible effects of loading on bone matrix composition have not been evaluated. Site-specific structural and material properties of mouse bone were analyzed on the macro- and micro/molecular scale in the presence and absence of axial loading. The response of bone to load is heterogeneous, adapting at molecular, micro-, and macro-levels. Introduction Osteoporosis is a degenerative disease resulting in reduced bone mineral density, structure, and strength. The overall aim was to explore the hypothesis that changes in loading environment result in site-specific adaptations at molecular/micro- and macro-scale in mouse bone. Methods Right tibiae of adult mice were subjected to well-defined cyclic axial loading for 2 weeks; left tibiae were used as physiologically loaded controls. The bones were analyzed with μCT (structure), reference point indentation (material properties), Raman spectroscopy (chemical), and small-angle X-ray scattering (mineral crystallization and structure). Results The cranial and caudal sites of tibiae are structurally and biochemically different within control bones. In response to loading, cranial and caudal sites increase in cortical thickness with reduced mineralization (−14 and −3%, p more...

In human athletes, conditioning, training and competition are commenced before skeletal maturity. Yet in equine athletics, racing of young (age 2 years) horses remains contentious. Tendon injury persists as major causes of wastage in equine athletes. Minimising injury and associated welfare issues could involve a radical approach to the timing and implementation of conditioning and training. Tendons were examined from Thoroughbreds, Dutch Warmblood foals, working horses and also a group of wild horses to evaluate effects of age, function and exercise. Gross mechanical properties did not differ significantly with age or exercise, but showed a high variance within each group. Mechanical properties of tendon tissue showed significant differences as a function of age and location. The collagen fibril crimp angle and length showed a regional reduction in the central core with exercise and age, with a synergistic effect. Regional differences in collagen fibril diameter were seen in long-term exercised older horses, but not in short-term exercised, or younger, horses. The higher proportion of small fibrils in the central region of the long-term exercised horses did not correlate with new collagen formation and therefore appear to result from disassembly of the larger diameter fibrils. Fibril diameter distributions were influenced by exercise regimens in the growing foal. Changes in molecular composition occurred in longer-term exercise and older horses, in the centre of the tendon, with higher levels of type III collagen and changes in glycosaminoglycan (GAG) content. Cartilage Oligomeric Matrix Protein (COMP) levels also appear to be modulated by age, function and superimposition of exercise. These changes were all exacerbated with age and exercise, suggesting appropriate exercise in young horses may lead to a lower incidence of injury than in older horses. An hypothesis is advanced that immature tendon can respond to exercise while mature tendon has limited, if any, ability to do so. These findings support potentially controversial earlier conditioning and racing of younger, rather than older, equine athletes. more...

Most skeletal tissues are thought to adapt to the mechanical environment they experience. While this has been demonstrated for muscle and bone, previous studies in the mature horse have failed to demonstrate adaptation in the superficial digital flexor tendon (SDFT), which suffers a high frequency of injury. This study tested the hypothesis that imposed exercise during growth would result in an increase in SDFT cross-sectional area (CSA). Fourteen Thoroughbred foals were divided into 2 sex-matched groups. A control group received 4 h pasture exercise and an exercise group had the same amount of pasture exercise with an additional short period of treadmill exercise daily from age 2-15 months. Activity at pasture was assessed objectively using a visual system. There was no significant difference in pasture activity between groups, although males were more active than females. The exercise programme resulted in a significantly larger tendon CSA in the exercise group at several, but not all, timepoints, which may be attributed to levels of variance. However, there was a significantly greater rate of increase in tendon CSA as a function of time in the exercised compared to the control group. This is the first evidence to suggest that tendon development can be modulated by exercise during growth in the horse, potentially increasing the ability of tendon to withstand the rigours of later athletic activity. more...

This study evaluated the effects of metrenperone on healing of unilateral, collagenase-induced lesions in the Superficial Digital Flexor Tendons (SDFT) of rabbits. After controlled injury of the left SDFT, nine rabbits received daily treatment with metrenperone for 28 days. Another nine were untreated controls; in both groups the contra-lateral tendons served as uninjured controls. Histological and ultrastructural changes, mechanical properties, dry weight, collagen content, and amount of DNA in healing and control tendons were assessed 28 days after injury. Restoration of structural hierarchy was more organized in treated than in untreated tendons while cellularity was greater in the latter. At the ultrastructural level, collagen in treated lesions was predominantly in the form of small-diameter, new fibrils, with few large, old fibrils; in untreated lesions there was a high proportion of large, old fibrils but relatively few small, new ones. The amount of DNA in untreated injuries was much greater than in normal tendons, while in treated lesions it was not significantly different from that of uninjured controls. There were no significant differences in total collagen, stiffness and ultimate strength of injured, treated, and untreated tendons 28 days after injury. Both were significantly weaker than their corresponding contralaterals. The findings suggest that metrenperone had positive effects on collagen turnover, remodelling, and organization during acute inflammation and fibroplasia. Provided that the new fibrils subsequently matured in a normal manner, mechanical characteristics of the organized scar should be better than those of an untreated lesion. more...

Reasons for performing study: Osteoarthritis (OA) is a common cause of distal tarsal pain, but disease development is poorly understood. Awareness of normal tarsal structure and function is important in order to understand the pathogenesis of OA. Thickening of the subchondral bone (SCB) plate has been related to the development of OA, but SCB plate patterns in the equine tarsus have not been documented.Hypotheses: There is a repeatable pattern of SCB thickness across the distal tarsal joints, and specifically that thickness would be greatest dorsally and laterally.Methods: Twenty cadaver tarsi were collected from mature horses that had undertaken low-level exercise only with no history of hindlimb lameness. Magnetic resonance images were acquired using a high-resolution sagittal 3-dimensional T1-weighted spoiled gradient echo sequence. Subchondral bone thickness was measured on sagittal images at dorsal and plantar locations on the proximal and distal aspects of the central (CT) and third (T3) tarsal bones and proximal aspect of the third metatarsal bone (MO).Results: On the proximal aspect of CT, medial and lateral SCB thickness were significantly greater than midline. On the distal aspect of CT and T3 and proximal Mt3, lateral SCB thickness was significantly greater than medial and midline sites. Dorsal SCB thickness was greatest on the proximal and distal aspects of CT and proximal Mt3. Subehondral bone accounted for a greater proportion of CT and T3 on the dorsal aspect than the plantar.Conclusions: There is a repeatable pattern of SCB thickness in the distal tarsal bones of horses with no history of hindlimb lameness. This reflects the pattern of loading across the joints.Potential relevance: This study provides evidence of a consistent osteochondral pattern in the equine tarsus for reference in identification of osteoarticular pathologies. more...

Objective—To investigate effects of exercise on hyaline cartilage (HC), calcified cartilage (CC), and subchondral bone (SCB) thickness patterns of equine tarsi. Sample Population—30 tarsi from cadavers of horses with known exercise history. Procedures—Tarsi were assigned to 3 groups according to known exercise history as follows: pasture exercise only (PE tarsi), low-intensity general-purpose riding exercise (LE tarsi), and high-intensity elite competition riding exercise (EE tarsi). Osteochondral tissue from distal tarsal joints underwent histologic preparation. Hyaline cartilage, CC, and SCB thickness were measured at standard sites at medial, midline, and lateral locations across joints with a histomorphometric technique. Results—HC, CC, and SCB thickness were significantly greater at all sites in EE tarsi, compared with PE tarsi; this was also true when LE tarsi were compared with PE tarsi. At specific sites, HC, CC, and SCB were significantly thicker in EE tarsi, compared with LE tarsi. Along the articular surface of the proximal aspect of the third metatarsal bone, SCB was thickest in EE tarsi and thinnest in LE tarsi; increases were greatest at sites previously reported to undergo peak strains and osteochondral damage. Conclusions and Clinical Relevance—Increased exercise was associated with increased HC, CC, and SCB thickness in mature horses. At sites that undergo high compressive strains, with a reported predisposition to osteoarthritic change, there was increased CC and SCB thickness. These results may provide insight into the interaction between adaptive response to exercise and pathological change. more...

Bone tissue has long been recognized as sensitive to mechanical loading, in a manner which can have either beneficial (anabolic) or destructive (catabolic) consequences. Extrapolating this adaptive capacity to the role of mechanical signals on bone healing, it should not be surprising that loading also has a critical influence on the repair of skeletal fractures,1,2 either in enhancing the response,3,4 or, in cases of too much loading, derailing the healing process.5,6 While these “form follows function” rules of the skeleton, originally formulated by observing the morphologic patterns of healing bones and outlined in “Wolff’s Law of Bone Remodeling,”7 only very few guidelines exist which help define the ideal mechanical environment of healing tissue towards the restoration of biomechanical function.8 Bone healing is subject to mechanical signals, as regulated via stiffness of fixation, rigidity of cast immobilization, control of weight bearing, or even applied loading of the fracture site, can influence the quality, rate, and progression of repair.9,10 For example, too much loading can compromise the healing response, as evidenced by a delayed union as exacerbated by early weight bearing on a fracture protected only by flexible external fixation.5 While decreased frame stiffness stimulates periosteal callus, excessive motion is permissive to chondrogenesis, particularly where high levels of intermittent shear occurs,11,12 and may lead to hypertrophic nonunion. Frames with low stiffness characteristics may also result in high pin/bone interface stresses that induce local resorption and associated pin loosening.13 At the other extreme, external fixation with high frame stiffness can suppress the osteogenic response at the periosteum, and is permissive to a delayed or atrophic nonunion,4,6 while internal fracture fixation, via plates which are too stiff, can cause osteopenia below the device.14 The paradox between harnessing mechanical signals as beneficial to bone healing and avoiding those which damage the process further incites a challenge to identify a mechanical environment which is both safe and enhances the repair process. A strategy to establish the relevant window between compliant and rigid fixation has been approached by using external fixation with high rigidity except in the axial direction, and thus permitting passive, controlled loading of the fracture gap, or the external application of directed, dynamic axial inter-fragmentary motion.1 Indirect fracture repair can be enhanced by applying axial cyclical interfragmentary motions with specific characteristics for short daily periods, without potentiating risk to the fracture gap which might arise during functional loading but poor protection afforded by low rigidity of the fixator.10,15–17 While limited interfragmentary motion is accepted as anabolic to the healing response, it remains unclear if the potential benefits of loading in the laboratory setting can exceed the risks inherent in the clinic of overloading the repair site and thus promoting a pathologic response.18 The osteogenic potential of strain magnitude is well recognized,19 but does not, in isolation, govern bone’s response to mechanical loading. Other factors such as the number of loading cycles,20 strain rate,21 and spatial strain gradient22 also play a significant role in the osteogenic capacity of mechanical loading. It has been shown previously that extremely low magnitude ( more...

The beneficial effects of serotonin S(2)-receptor blockers on healing skin and muscle ulcers and refractory lesions such as leprosy and diabetic and ischemic ulcers have been reported previously, but their mechanisms of actions are not clear. The present study sought to elucidate the action of an S(2)-receptor blocker, metrenperone, on the healing of collagenase-induced injuries in superficial digital flexor tendons of two groups of rabbits. In one group, oral and topical therapy for 28 days with metrenperone, was started within 48 hr of a single acute injury. The animals were then left untreated for another 60 days, when it was found that most of the morphological, biochemical, and biomechanical characteristics of the healed tendons matched those of their normal uninjured controls. Injured, untreated controls showed poor healing. In the second group of animals, tendon injury was induced by four separate injections of collagenase at weekly intervals. The rabbits were left for another 60 days, before being treated with metrenperone for 26 days. This delayed treatment had no apparent effect on the biomechanical, biochemical, or morphological characteristics of the healing tendons. It appeared that metrenperone had a significant effect on collagen turnover and organization of scar tissue, but only while the inflammatory and fibroplastic processes were active in the early stages of healing. S(2)-receptor blockers may, therefore, be of potential value for modulating repair in acutely injured collagenous tissue. more...

This study explores the hypothesis that high-intensity exercise induces degenerative changes in the injury-prone equine superficial digital flexor tendon (SDFT), but not in the rarely injured common digital extensor tendon (CDET). The horse represents a large-animal model that is applicable to human tendon and ligament physiology and pathology. Twelve age-matched female horses undertook galloping exercise three times a week with trotting exercise on alternative days (high-intensity group, n = 6) or only walking exercise (low-intensity group, n = 6) for 18 mo. The SDFT, suspensory ligament, deep digital flexor tendon, and CDET were harvested from the forelimb. Tissue from the mid-metacarpal region of the right limb tendons was analyzed for water, DNA, sulfated glycosaminoglycan and collagen content, collagen type III-to-I ratios, collagen cross-links, and tissue fluorescence. Left limb tendons were mechanically tested to failure. The analyses showed matrix composition to have considerable diversity between the functionally different structures. In addition, the specific structures responded differently to the imposed exercise. High-intensity training resulted in a significant decrease in the GAG content in the SDFT, but no change in collagen content, despite a decrease in collagen fibril diameters. There were no signs of degeneration or change in mechanical properties of the SDFT. The CDET had a lower water content following high-intensity training and a higher elastic modulus. Long-term, high-intensity training in skeletally mature individuals results in changes that suggest accelerated aging in the injury-prone SDFT and adaptation in the CDET. more...

Conditioning by early training may influence the composition of certain musculoskeletal tissues, but very few data exist on its effect during growth on tendon structure and function.To investigate whether conditioning exercise in young foals would lead to any ultrasonographically detectable damage to the superficial digital flexor tendon or an increase in cross-sectional area (CSA).Thirty-three Thoroughbred foals reared at pasture were allocated to 2 groups: control (PASTEX) allowed exercise freely at pasture; and CONDEX, also at pasture, began conditioning exercise from mean age 21 days over 1030 m on a purpose-built oval grass track, for 5 days/week until mean age 18 months. Foals were observed daily, and underwent orthopaedic examination monthly. Ultrasonographic images of the superficial digital flexor tendon (SDFT) at the mid-metacarpal level of both forelimbs were obtained in all foals at ages 5, 8, 12, 15 and 18 months. CSA was validated (r(2) = 0.89) by determining CSA from digital photographs of the transected SDFT surface from 12 of the horses necropsied at age 17.1 months.here was no clinical or ultrasonographic evidence of tendonopathy in either group and the greatest increase in mean CSA in both groups occurred between age 5 and 8 months. Across all age categories, there was no significant difference in mean CSA between the left and right limbs, or colts and fillies; there was a trend towards a larger CSA in the CONDEX group (P = 0.058).There was no conclusive evidence for a structural adaptive hypertrophy of the SDFT, probably because the regimen was insufficiently rigorous or because spontaneous pasture exercise may induce maximal development of energy storing tendons.A moderate amount of early conditioning exercise against a background of constant exercise at pasture is not harmful to the development of the flexor tendons. more...

Some hibernating animals are known to reduce muscle and bone loss associated with mechanical unloading during prolonged immobilisation, compared to humans. However, here we show that wild pregnant polar bears (Ursus maritimus) are the first known animals to avoid significant bone loss altogether, despite six months of continuous hibernation. Using serum biochemical markers of bone turnover, we showed that concentrations for bone resorption are not significantly increased as a consequence of hibernation in wild polar bears. This is in sharp contrast to previous studies on other hibernating species, where for example, black bears (Ursus americanus), show a 3-4 fold increase in serum bone resorption concentrations post-hibernation, and must compensate for this loss through rapid bone recovery on remobilisation, to avoid the risk of fracture. In further contrast to black bears, serum concentrations of bone formation markers were highly significantly increased in pregnant female polar bears compared to non-pregnant, thus non-hibernating females both prior to and after hibernation. However, bone formation concentrations in new mothers were significantly reduced compared to pre-hibernation concentrations. The de-coupling of bone turnover in favour of bone formation prior to hibernation, suggests that wild polar bears may posses a unique physiological mechanism for building bone in protective preparation against expected osteopenia associated with disuse, starvation, and hormonal drives to mobilise calcium for reproduction, during hibernation. Understanding this physiological mechanism could have profound implications for a natural solution for the prevention of osteoporosis in animals subjected to captivity with inadequate space for exercise, humans subjected to prolonged bed rest while recovering from illness, or astronauts exposed to antigravity during spaceflight. (C) 2008 Elsevier Inc. All rights reserved. more...

The hypothesis tested in this study was that perioperative administration of the bisphosphonate zoledronate will reduce strain protection-related calcar osteopenia and maintain functional integration of the femoral component in an ovine hemiarthroplasty model. Twelve sheep received a unilateral cemented hemiarthroplasty where six animals were given nine intravenous infusions of zoledronate (10 microg/kg) pre-, peri-, and postsurgery over 8 months. Control animals received physiological saline only. Implants remained in vivo for 9 months. Ground reaction force (GRF) was used to assess functional loading of the implanted limb, bone mineral density (BMD) was quantified using dual energy X-ray absorptiometry (DEXA). Cortical bone area, thickness, and viable osteocytes were assessed histologically. No significant differences in GRF data between groups was identified. Results demonstrated a significant drop in BMD values in the control group (9.7%) when compared with the bisphosphonate-treated group (3.2%) (p = 0.0159). Histological results showed that cortical area, thickness, and the percentage of lacunae with viable osteocytes was significantly greater in the bisphosphonate-treated group when compared with control (p = 0.002, p = 0.001, p = 0.003, respectively). The administration of zoledronate reduced cortical osteopenia in the calcar region of the proximal femur and this therapy could be used as a preventive measure to combat strain protection osteopenia and its contribution to associated aseptic loosening in total hip replacement surgery. more...

The tendons in the turkey leg have specific well-defined areas which become mineralized as the animal ages and they are a thoroughly characterized model system for studying the mineralization process of bone. In this study, nondestructive Raman spectroscopic analysis was used to explore the hypothesis that regions of the turkey tendon that are associated with mineralization exhibit distinct and observable chemical modifications of the collagen prior to the onset of mineralization. The Raman spectroscopy features associated with mineralization were identified by probing (on the micrometer scale) the transition zone between mineralized and nonmineralized regions of turkey leg tendons. These features were then measured in whole tendons and identified in regions of tendon which are destined to become rapidly mineralized around 14 weeks of age. The data show there is a site-specific difference in collagen prior to the deposition of mineral, specifically the amide III band at 1270 cm(-1) increases as the collagen becomes more ordered (increased amide III:amide I ratio) in regions that become mineralized compared to collagen destined to remain nonmineralized. If this mechanism were present in materials of different mineral fraction (and thus material properties), it could provide a target for controlling mineralization in metabolic bone disease. more...

Background: Although favorable integration occurs with immediately loaded implants, the relationship between implant outcome, levels of occlusion, and diet requires optimization. Purpose: Pertubating load on single implant restorations immediately after placement by a hard food diet will increase the strains at the bone-implant interface, increasing the risk for failure. Materials and Methods: Forty-eight implants replaced the first and third mandibular premolars in 12 pigs, allocated into two groups based on soft- and hard-diet feeding. Cylindrical and tapered implants replaced the first and third premolars, respectively. Each animal received at random four different masticatory loading conditions (group 1 [control]: implant with either a cover screw or a healing abutment, and group 2 [test]: implant with a crown either with or without occlusal contacts). Results: Thirteen implants out of 44 failed in 11 animals (one with a cover screw, one with a healing abutment, three with nonocclusal, and eight with occlusal restorations). The failure rate of restored implants (either in occlusion or not) was significantly higher in the third premolar sites (p = .007), although diet had no significant effect (p = .421). Conclusions: While diet had no effect on the failure pattern of immediately loaded single implants, the position and type of load under the masticatory mode were significant. Immediately loaded implants both in and out of occlusion were less successful than the controls, and this is probably attributed to detrimental strain induced on the bone-implant interface. more...

Adaptation of osteochondral tissues is based on the strains experienced during exercise at each location within the joint. Different exercise intensities and types may induce particular site-specific strains, influencing osteochondral adaptation and potentially predisposing to injury. Our hypotheses were that patterns of equine distal tarsal subchondral bone (SCB) thickness relate to the type and intensity of exercise, and that high-intensity exercise leads to site-specific increases in thickness. SCB thickness was measured at defined dorsal and plantar locations on magnetic resonance images of cadaver tarsi collected from horses with a history of low [general purpose ( n = 20) and horse walker ( n = 6)] or high [elite competition ( n = 12), race training ( n = 15), and treadmill training ( n = 4)] exercise intensity. SCB thickness was compared between sites within each exercise group and between exercise groups. SCB thickness in elite competition and race training, but not treadmill training, was greater than low-intensity exercise. For general purpose horses, lateral SCB thickness was greater than medial throughout. Horse walker exercise led to relatively thicker lateral and medial SCB compared with the midline. Elite competition was associated with increased SCB thickness of the proximal small tarsal bones medially and the distal bones laterally. For race training and treadmill training, there were minimal differences between sites overall, although the lateral aspect was greater than medial, and medial greater than midline at a few sites for race training. In conclusion, different types of high-intensity exercise were associated with different patterns of SCB thickness across the joints from medial to lateral and proximal to distal, indicating that both exercise intensity and type of exercise affect the SCB response at any particular site within the equine distal tarsal joints. more...

Noninvasive imaging modalities for in vivo monitoring of distal tarsal osteochondral structure are needed. The objectives of this paper were to use high-field magnetic resonance (MR) imaging to describe tarsal structure, variations in association with age, exercise, and distal tarsal pain. Cadaver tarsi of specific ages and exercise histories, and from horses with a history of distal tarsal pain, were examined using MR imaging. Quantitative and subjective analysis of subchondral bone (SCB) was performed according to predefined criteria. There was a repeatable SCB thickness pattern across normal tarsi, with greatest thickness at dorsal and lateral sites. Subchondral bone thickness increased with age. High intensity exercise was associated with altered mediolateral and proximodistal thickness patterns. There was loss of the repeatable SCB thickness pattern in painful tarsi, and MR imaging detected many different types of pathology. This paper provides new information regarding structure and pathological changes in the equine tarsus, which can be used as reference material for clinical situations. more...

Raman Spectroscopy has become an important technique for assessing the composition of excised sections of bone, and is currently being developed as an in vivo tool for transcutaneous detection of bone disease using spatially offset Raman spectroscopy (SORS). The sampling volume of the Raman technique (and thus the amount of bone material interrogated by SORS) depends on the nature of the photon scattering in the probed tissue. Bone is a complex hierarchical material and to date little is known regarding its diffuse scattering properties which are important for the development and optimization of SORS as a diagnostic tool for characterizing bone disease in vivo. SORS measurements at 830 nm excitation wavelength are carried out on stratified samples to determine the depth from which the Raman signal originates within bone tissue. The measurements are made using a 0.38 mm thin Teflon slice, to give a pronounced and defined spectral signature, inserted in between layers of stacked 0.60 mm thin equine bone slices. Comparing the stack of bone slices with and without underlying bone section below the Teflon slice illustrated that thin sections of bone can lose appreciable number of photons through the unilluminated back surface. The results show that larger SORS offsets lead to progressively larger penetration depth into the sample; different Raman spectral signatures could be retrieved through up to 3.9 mm of overlying bone material with a 7 mm offset. These findings have direct impact on potential diagnostic medical applications; for instance on the detection of bone tumors or areas of infected bone. more...

Conventional amputation prosthetics are problematic because they rely on the stump-socket interface for attachment. Intraosseous transcutaneous amputation prostheses (ITAP) could solve these problems; however they rely on the integrity of the soft tissue-implant interface as a barrier to exogenous agents, and in the prevention of downgrowth and marsupilisation. We have used an in vivo animal model to study the soft tissue interfaces around bone-anchored transcutaneous implants. We hypothesise that by facilitating and increasing the area of dermal attachment to the implant epithelial down-growth will be reduced. A flange with a series of 24, 0.7 mm holes positioned immediately below the epithelium was used to increase dermal attachment. This significantly reduced downgrowth and optimised the integrity of the collagenous tissue-implant interface at the dermal level. We postulate that the flange reduces relative interfacial movement at the epithelium-implant interface by providing increased surface area for dermal tissue attachment. A tight seal at the dermal tissue level reduces the degree of downgrowth around ITAP, eliminating marsupilisation as a potential failure modality. Surface topography and coatings did not affect the degree of downgrowth or dermal attachment to straight or flanged implants. A significant negative correlation was observed between downgrowth and both epithelial and dermal attachment. This study shows that a soft tissue-implant interface capable of preventing downgrowth and marsupilisation can develop around a bone-anchored transcutaneous implant, given the incorporation of a porous flange positioned in the dermal tissues immediately below the epithelium. This will benefit applications where bone-anchored transcutaneous implants are used. more...

We report the first transcutaneous Raman spectrum of human bone in vivo obtained at skin-safe laser illumination levels. The spectrum of thumb distal phalanx was obtained using spatially offset Raman spectroscopy (SORS), which provides chemically specific information on deep layers of human tissue, well beyond the reach of existing comparative approaches. The spectroscopy is based on collecting Raman spectra away from the point of laser illumination using concentric rings of optical fibers. As a generic analytical tool this approach paves the way for a range of uses including disease diagnosis, noninvasive probing of pharmaceutical products, biofilms, catalysts, paints, and in dermatological applications. more...

To evaluate soft tissue reactions and biofilm formation on percutaneous external fixator screws coated with diamond-like carbon (DLC) and hydroxyapatite (HA) coatings on stainless-steel (SS) pins in an ovine loaded osteotomy model, an Orthofix external fixator was used to stabilize a 3-mm tibial midshaft osteotomy with six tapered pins inserted into the right tibia of 32 skeletally mature Friesland ewes. Animals were divided into four groups; SS, fully coated HA, DLC, and HA-coated threads. At 10 weeks, specimens were harvested and the pins were removed en bloc to examine the interfaces between the surface coatings and the tissues. Fully coated HA pins had a significantly higher percentage of dermal contact with the pin surface than HA-coated threads (p=0.028). The presence of a biofilm was evident on all pin surfaces except DLC-coated pins. Significantly greater numbers of bacteria were present on fully coated HA and plain stainless-steel pins compared with DLC. The surface of DLC-coated pins had a significantly lower number of bacterial colonies compared to SS (p=0.028) and fully coated HA pins (p=0.005). Fully coated HA pins have greater dermal attachment to the pin surface than the other pin coatings investigated. DLC-coated pins have the potential to prevent biofilm formation and bacterial colonization that may reduce infection and consequent pin loosening. An external fixator pin that is partially coated with HA to encourage bone and soft tissue integration and with DLC to reduce biofilm formation is advocated. more...

The main problem facing the longevity of total joint replacements is wear particle-induced osteolysis, particularly around the acetabular component. Ovine Total Hip Replacement surgery was performed with roughened femoral heads in order to enhance wear debris generation in vivo. The resistance to aseptic loosening of acetabular components with different surface coatings was investigated. Implants remained in vivo for 1 year.Sheep were randomly assigned to one of six experimental groups where the acetabular cup was fixed utilising an: (a) cemented polyethylene acetabular cup, (b) metal backed grit blasted surface, (c) metal backed plasma sprayed titanium porous coating, (d) metal backed sintered beaded coating, (e) Hydroxyapatite (HA)-coated grit blasted surface (f) and HA-coated porous components. Ground Reaction Force (GRF) was used to asses the functional performance of the implants and data was collected pre-operatively and at 12, 24, 36 and 52 weeks post op. Wear debris generated was analysed and radiographs taken prior to preparation of thin sections. Fibrous tissue (FT) thickness and bone contact at 1 mm intervals along the acetabular bone-implant interface was calculated.GRF data demonstrated significant differences between experimental groups. In all groups there was an increase in the function of the hip after surgery and up to 24 weeks but thereafter the function of the group with the grit blasted surface reduced whereas the function of the other groups did not significantly change. Average wear particles generated were < 1 mum in size. The cemented group demonstrated a significantly thicker average FT layer (2.69 mm) when compared with all other groups (p < 0.05 in all cases) except the grit blasted group (1.56 mm). HA porous coated cups demonstrated significantly least fibrous tissue adjacent to its interface when compared with all other groups (cemented p < 0.05, grit blasted p = 0.029, porous p < 0.05, sintered beads p < 0.05 and HA grit blasted p < 0.05). Significantly increased bone contact to HA-coated porous cups (73.33%) when compared with all groups was identified except HA-coated grit blasted cups where no significant difference was demonstrated. Radiographic signs of loosening were visible in all groups except the HA-coated porous group.Results demonstrated that HA porous coated acetabular components significantly enhanced bone ingrowth in the presence of wear particles, preventing their migration and reducing osteolysis. Non-HA-coated porous and sintered beaded components provided a more effective seal against the ingress of wear debris when compared with cemented cups. (C) 2004 Elsevier Ltd. All rights reserved. more...

We developed an in vivo model of the attachment of a patellar tendon to a metal implant to simulate the reconstruction of an extensor mechanism after replacement of the proximal tibia. In 24 ewes, the patellar tendon was attached to a hydroxyapatite (HA)-coated titanium prosthesis. In 12, the interface was augmented with autograft containing cancellous bone and marrow. In the remaining ewes, the interface was not grafted. Kinematic gait analysis showed nearly normal function of the joint by 12 weeks. Force-plate assessment showed a significant increase in functional weight-bearing in the grafted animals (p = 0.043). The tendon-implant interface showed that without graft, encapsulation of fibrous tissue occurred. With autograft, a developing tendon-bone-HA-implant interface was observed at six weeks and by 12 weeks a layered tendon-fibrocartilage-bone interface was seen which was similar to a direct-type enthesis. With stable mechanical fixation, an appropriate bioactive surface and biological augmentation the development of a functional tendon-implant interface can be achieved. more...

We describe a simple methodology for the effective retrieval of Raman spectra of subsurface layers in diffusely scattering media. The technique is based on the collection of Raman scattered light from surface regions that are laterally offset away from the excitation laser spot on the sample. The Raman spectra obtained in this way exhibit a variation in relative spectral intensities of the surface and subsurface layers of the sample being investigated. The data set is processed using a multivariate data analysis to yield pure Raman spectra of the individual sample layers, providing a method for the effective elimination of surface Raman scatter. The methodology is applicable to the retrieval of pure Raman spectra from depths well in excess of those accessible with conventional confocal microscopy. In this first feasibility study we have differentiated between surface and subsurface Raman signals within a diffusely scattering sample composed of two layers: trans-stilbene powder beneath a 1 mm thick over-layer of PMMA (poly(methyl methacrylate)) powder. The improvement in contrast of the subsurface trans-stilbene layer without numerical processing was 19 times. The potential applications include biomedical subsurface probing of specific tissues through different overlying tissues such as assessment of bone quality through skin, providing an effective noninvasive means of screening for bone degeneration, other skeletal disease diagnosis, and dermatology studies, as well as materials and catalyst research. more...

This study investigates the use of porous biphasic ceramics as graft extenders in impaction grafting of the femur during revision hip surgery. Impaction grafting of the femur was performed in four groups of sheep. Group one received pure allograft, group two 50% allograft and 50% BoneSave, group three 50% allograft and 50% BoneSave type 2 and group four 10% allograft and 90% BoneSave as the graft material. Function was assessed using an index of pre- and post-operative peak vertical ground reaction force ratios. Changes in bone mineral density were measured by dual energy X ray absorptiometry (DEXA) scanning. Loosening and subsidence were assessed radiographically and by histological examination of the explanted specimens. There was no statistically significant difference between the four groups after 18 months of unrestricted functional loading for all outcome measures. more...