... Roger KW Smith, MA, VetMB, PhD, CertEO, DipECVS, MRCVS; Helen L. Birch, BSc, PhD; Emma L. Bat... more ... Roger KW Smith, MA, VetMB, PhD, CertEO, DipECVS, MRCVS; Helen L. Birch, BSc, PhD; Emma L. Batson, BSc; Janet Patterson-Kane, BVSc, PhD, DipACVP, MRCVS; Shelley Goodman, BSc, PhD; Eddy R. Cauvin, DVM, MVM, CertVR, CertES(Orth), DipECVS, MRCVS; Allen E ...
... (1970) investigated matrix synthesis in chicken digital tendons and ... trauma induced new co... more ... (1970) investigated matrix synthesis in chicken digital tendons and ... trauma induced new collagensynthesis and this was linked to an increase in mucopolysaccharide synthesis. ... may be responsible for this weakening, either through a disruption of subfascicular structure as in ...
BACKGROUND: In an attempt to establish some consensus on the proper use and design of experimenta... more BACKGROUND: In an attempt to establish some consensus on the proper use and design of experimental animal models in musculoskeletal research, AOVET (the veterinary specialty group of the AO Foundation) in concert with the AO Research Institute (ARI), and the European Academy for the Study of Scientific and Technological Advance, convened a group of musculoskeletal researchers, veterinarians, legal experts, and ethicists to discuss, in a frank and open forum, the use of animals in musculoskeletal research. METHODS: The group narrowed the field to fracture research. The consensus opinion resulting from this workshop can be summarized as follows: Results & Conclusion: Anaesthesia and pain management protocols for research animals should follow standard protocols applied in clinical work for the species involved. This will improve morbidity and mortality outcomes. A database should be established to facilitate selection of anaesthesia and pain management protocols for specific experimen...
Intra-synovial tendon injuries display poor healing, which often results in reduced functionality... more 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. 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 ...
Multipotent mesenchymal stem cells (MSCs) have enormous potential in tissue engineering and regen... more Multipotent mesenchymal stem cells (MSCs) have enormous potential in tissue engineering and regenerative medicine. However, until now, their development for clinical use has been severely limited as they are a mixed population of cells with varying capacities for lineage differentiation and tissue formation. Here, we identify receptor tyrosine kinase-like orphan receptor 2 (ROR2) as a cell surface marker expressed by those MSCs with an enhanced capacity for cartilage formation. We generated clonal human MSC populations with varying capacities for chondrogenesis. ROR2 was identified through screening for upregulated genes in the most chondrogenic clones. When isolated from uncloned populations, ROR2+ve MSCs were significantly more chondrogenic than either ROR2–ve or unfractionated MSCs. In a sheep cartilage-repair model, they produced significantly more defect filling with no loss of cartilage quality compared with controls. ROR2+ve MSCs/perivascular cells were present in developing ...
Meniscal cartilage tears are common and predispose to osteoarthritis (OA). Most occur in the avas... 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...
Raman Spectroscopy has become an important technique for assessing the composition of excised sec... 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.
Bone mass and architecture are the result of a genetically determined baseline structure, modifie... more Bone mass and architecture are the result of a genetically determined baseline structure, modified by the effect of internal hormonal/biochemical regulators and the effect of mechanical loading. Bone strain is thought to drive a feedback mechanism to regulate bone formation and resorption to maintain an optimal, but not excessive mass and organisation of material at each skeletal location. Because every site in the skeleton has different functions, we have measured bone strains induced by physiological and more unusual activities, at two different sites, the tibia and cranium of a young human male in vivo. During the most vigorous activities, tibial strains were shown to exceed 0.2%, when ground reaction exceeded 5 times body weight. However in the skull the highest strains recorded were during heading a heavy medicine/exercise ball where parietal strains were up to 0.0192%. Interestingly parietal strains during more physiological activities were much lower, often below 0.01%. Strains during biting were not dependent upon bite force, but could be induced by facial contortions of similar appearance without contact between the teeth. Rates of strain change in the two sites were also very different, where peak tibial strain rate exceeded rate in the parietal bone by more than 5 fold. These findings suggest that the skull and tibia are subject to quite different regulatory influences, as strains that would be normal in the human skull would be likely to lead to profound bone loss by disuse in the long bones.
Raman spectroscopy was used to show that across 10 cm of diaphyseal (mid-shaft) cortical bone the... 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 obta...
X-ray-based diagnostic techniques, which are by far the most widely used for diagnosing bone diso... more X-ray-based diagnostic techniques, which are by far the most widely used for diagnosing bone disorders and diseases, are largely blind to the protein component of bone. Bone proteins are important because they determine certain mechanical properties of bone and changes in the proteins have been associated with a number of bone diseases. Spatially Offset Raman Spectroscopy (SORS) is a chemically specific analytical technique that can be used to retrieve information noninvasively from both the mineral and protein phases of the bone material in vivo. Here we demonstrate that SORS can be used to detect a known compositional abnormality in the bones of a patient suffering from the genetic bone disorder, osteogenesis imperfecta, a condition which affects collagen. The confirmation of the principle that bone diseases in living patients can be detected noninvasively using SORS points the way to larger studies that focus on osteoporosis and other chronic debilitating bone diseases with large socioeconomic burdens.
Veterinary Clinics of North America: Equine Practice, 2011
Overstrain and traumatic tendon and ligament injuries are common in the horse and, for the most p... more Overstrain and traumatic tendon and ligament injuries are common in the horse and, for the most part, heal (repair) naturally by the formation of scar tissue. However, the scar tissue formed in this repair is functionally deficient compared with normal tendon, which has important consequences for the animal in terms of reduced performance and a substantial risk of reinjury, despite the multitude of treatments that have been proposed. Regenerative medicine offers the prospect of restoring normal, or close to normal, structure and function to an injured organ, thereby resulting in a successful restoration of activity without the risk of reinjury. Regenerative medicine aims to harness the combined effects of a cell source, scaffold support, and anabolic stimulus to facilitate the healing of the injury. 1 There are multiple choices for the selection of a cell source for regenerative medicine, and at this time it is not clear which source will prove to be therapeutically optimal. A logical source of cells for making new tendon tissue would be the tenocytes Disclosures: R.K.W.S. is a technical adviser of VetCell. Experimental work has been funded by the Horserace Betting Levy Board, VetCell, and the Japan Racing Association and has been performed in collaboration with
The present study shows no adverse effects of the anti-diabetic drug metformin on bone mass and f... more 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 microarchitecture 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.
Objective: To determine whether the focal susceptibility to cartilage degeneration in joints is r... more Objective: To determine whether the focal susceptibility to cartilage degeneration in joints is related to a differential response to cytokine stimulation. Methods: Compare aggrecan and collagen catabolism in in-vitro models of cartilage degradation induced by retinoic acid (RA), interleukin-1 (IL-1), tumor necrosis factor alpha (TNF) and IL-1 plus oncostatin M (OSM). Glycosaminoglycan (GAG) and hydroxyproline (HyPro) quantification and Western immunoblot analyses of aggrecan and collagen degradation products were undertaken in explant cultures of normal cartilage from regions of equine joints with a known high and low susceptibility to degeneration in disease. RNA isolation and semi quantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis were performed to determine the expression of aggrecanases, matrix metalloproteinases (MMPs) and their inhibitors. Results: Although the rate of basal cartilage aggrecan turnover was dependent on joint region there was no difference in the response of different cartilages to cytokines. Individual animals did show a significant difference in the response of certain cartilages to cytokines, with both decreased and increased aggrecan loss in cartilage with a low susceptibility to degeneration. Aggrecan release in both short-and long-term cultures from all cartilages was associated with increased cleavage by aggrecanases rather than MMPs. There was a poor correlation between expression of aggrecanases, MMPs or their inhibitors and cytokine induced aggrecan catabolism. IL-1 alone was able to stimulate collagen breakdown in equine articular cartilage and surprisingly, significantly more collagen loss was induced in cartilage from regions less susceptible to degeneration. Conclusions: Collectively, these studies suggest that a regional difference in response to catabolic cytokines is unlikely to be a factor in the initiation of focal cartilage degeneration in osteoarthritis (OA).
Metabolic bone disease has been reported in free-living red squirrels (Sciurus vulgaris) in the U... more Metabolic bone disease has been reported in free-living red squirrels (Sciurus vulgaris) in the United Kingdom but the prevalence of this disease is unknown. In this study the bone quality of free-living red squirrels in the UK was assessed by radiology and bone densitometry. The study comprised 20 red squirrels found dead and submitted to the Zoological Society of London (UK) between 1997 and 1998, 10 were from the Isle of Wight (IoW), where gray squirrels (Sciurus carolinensis) are absent, and 10 were from Cumbria (Cu), where gray squirrels are present. Gray squirrels are considered potential competitors for red squirrels. Radiologic evaluation of humerus, femur, tibia, radius, and ilium revealed a slightly lower bone density and thinner cortices in red squirrels from the IoW when compared with those from Cu. Dual-energy X-ray absorptiometry was used to measure bone mineral content and density of the isolated right humerus and femur of 19 of the 20 red squirrels. The bone densitometry study reinforced the radiographic findings. The IoW specimens had lower bone mineral density values, although statistical significance (PϽ0.05) between animals from the IoW and Cu was only reached for the proximal epiphysis of the femur and between males from the IoW and males from Cu for the proximal epiphysis of the humerus. A highly positive correlation (rϾ0.94) was found when the bone mineral content and density between the femur and the humerus among groups and within each group were compared, showing a uniform level of mineralization between upper and lower limbs. These findings suggested generalized bone loss for the IoW red squirrels that may be compatible with some degree of osteopenia. Within the wide range of causes that lead to osteopenia, malnutrition (especially protein deficiency), calcium and copper deficiencies, and genetic factors remain as possible etiologies.
The decomposition of spatially offset Raman spectra for complex multilayer systems, such as biolo... 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.
Fracture healing can be enhanced by load bearing, but the specific components of the mechanical e... more Fracture healing can be enhanced by load bearing, but the specific components of the mechanical environment which can augment or accelerate the process remain unknown. The ability of low-magnitude, high-frequency mechanical signals, anabolic in bone tissue, are evaluated here for their ability to influence fracture healing. The potential for short duration (17 min), extremely low-magnitude (25 mm), high-frequency (30 Hz) interfragmentary displacements to enhance fracture healing was evaluated in a mid-diaphyseal, 3-mm osteotomy of the sheep tibia. In a pilot study of proof of concept and clinical relevance, healing in osteotomies stabilized with rigid external fixation (Control: n ¼ 4), were compared to the healing status of osteotomies with the same stiffness of fixation, but supplemented with daily mechanical loading (Experimental: n ¼ 4). These 25-mm displacements, induced by a ferroactive shape-memory alloy (''smart'' material) incorporated into the body of the external fixator, were less than 1% of the 3-mm fracture gap, and less than 6% of the 0.45-mm displacement measured at the site during ambulation (p < 0.001). At 10-weeks post-op, the callus in the Experimental group was 3.6-fold stiffer (p < 0.03), 2.5-fold stronger (p ¼ 0.05), and 29% larger (p < 0.01) than Controls. Bone mineral content was 52% greater in the Experimental group (p < 0.02), with a 2.6-fold increase in bone mineral content (BMC) in the region of the periosteum (p < 0.001). These data reinforce the critical role of mechanical factors in the enhancement of fracture healing, and emphasize that the signals need not be large to be influential and potentially clinically advantageous to the restoration of function.
The Journal of Bone and Joint Surgery. British volume, 2009
An understanding of the remodelling of tendon is crucial for the development of scientific method... more An understanding of the remodelling of tendon is crucial for the development of scientific methods of treatment and rehabilitation. This study tested the hypothesis that tendon adapts structurally in response to changes in functional loading. A novel model allowed manipulation of the mechanical environment of the patellar tendon in the presence of normal joint movement via the application of an adjustable external fixator mechanism between the patella and the tibia in sheep, while avoiding exposure of the patellar tendon itself. Stress shielding caused a significant reduction in the structural and material properties of stiffness (79%), ultimate load (69%), energy absorbed (61%), elastic modulus (76%) and ultimate stress (72%) of the tendon compared with controls. Compared with the material properties the structural properties exhibited better recovery after re-stressing with stiffness 97%, ultimate load 92%, energy absorbed 96%, elastic modulus 79% and ultimate stress 80%. The cros...
... Roger KW Smith, MA, VetMB, PhD, CertEO, DipECVS, MRCVS; Helen L. Birch, BSc, PhD; Emma L. Bat... more ... Roger KW Smith, MA, VetMB, PhD, CertEO, DipECVS, MRCVS; Helen L. Birch, BSc, PhD; Emma L. Batson, BSc; Janet Patterson-Kane, BVSc, PhD, DipACVP, MRCVS; Shelley Goodman, BSc, PhD; Eddy R. Cauvin, DVM, MVM, CertVR, CertES(Orth), DipECVS, MRCVS; Allen E ...
... (1970) investigated matrix synthesis in chicken digital tendons and ... trauma induced new co... more ... (1970) investigated matrix synthesis in chicken digital tendons and ... trauma induced new collagensynthesis and this was linked to an increase in mucopolysaccharide synthesis. ... may be responsible for this weakening, either through a disruption of subfascicular structure as in ...
BACKGROUND: In an attempt to establish some consensus on the proper use and design of experimenta... more BACKGROUND: In an attempt to establish some consensus on the proper use and design of experimental animal models in musculoskeletal research, AOVET (the veterinary specialty group of the AO Foundation) in concert with the AO Research Institute (ARI), and the European Academy for the Study of Scientific and Technological Advance, convened a group of musculoskeletal researchers, veterinarians, legal experts, and ethicists to discuss, in a frank and open forum, the use of animals in musculoskeletal research. METHODS: The group narrowed the field to fracture research. The consensus opinion resulting from this workshop can be summarized as follows: Results & Conclusion: Anaesthesia and pain management protocols for research animals should follow standard protocols applied in clinical work for the species involved. This will improve morbidity and mortality outcomes. A database should be established to facilitate selection of anaesthesia and pain management protocols for specific experimen...
Intra-synovial tendon injuries display poor healing, which often results in reduced functionality... more 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. 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 ...
Multipotent mesenchymal stem cells (MSCs) have enormous potential in tissue engineering and regen... more Multipotent mesenchymal stem cells (MSCs) have enormous potential in tissue engineering and regenerative medicine. However, until now, their development for clinical use has been severely limited as they are a mixed population of cells with varying capacities for lineage differentiation and tissue formation. Here, we identify receptor tyrosine kinase-like orphan receptor 2 (ROR2) as a cell surface marker expressed by those MSCs with an enhanced capacity for cartilage formation. We generated clonal human MSC populations with varying capacities for chondrogenesis. ROR2 was identified through screening for upregulated genes in the most chondrogenic clones. When isolated from uncloned populations, ROR2+ve MSCs were significantly more chondrogenic than either ROR2–ve or unfractionated MSCs. In a sheep cartilage-repair model, they produced significantly more defect filling with no loss of cartilage quality compared with controls. ROR2+ve MSCs/perivascular cells were present in developing ...
Meniscal cartilage tears are common and predispose to osteoarthritis (OA). Most occur in the avas... 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...
Raman Spectroscopy has become an important technique for assessing the composition of excised sec... 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.
Bone mass and architecture are the result of a genetically determined baseline structure, modifie... more Bone mass and architecture are the result of a genetically determined baseline structure, modified by the effect of internal hormonal/biochemical regulators and the effect of mechanical loading. Bone strain is thought to drive a feedback mechanism to regulate bone formation and resorption to maintain an optimal, but not excessive mass and organisation of material at each skeletal location. Because every site in the skeleton has different functions, we have measured bone strains induced by physiological and more unusual activities, at two different sites, the tibia and cranium of a young human male in vivo. During the most vigorous activities, tibial strains were shown to exceed 0.2%, when ground reaction exceeded 5 times body weight. However in the skull the highest strains recorded were during heading a heavy medicine/exercise ball where parietal strains were up to 0.0192%. Interestingly parietal strains during more physiological activities were much lower, often below 0.01%. Strains during biting were not dependent upon bite force, but could be induced by facial contortions of similar appearance without contact between the teeth. Rates of strain change in the two sites were also very different, where peak tibial strain rate exceeded rate in the parietal bone by more than 5 fold. These findings suggest that the skull and tibia are subject to quite different regulatory influences, as strains that would be normal in the human skull would be likely to lead to profound bone loss by disuse in the long bones.
Raman spectroscopy was used to show that across 10 cm of diaphyseal (mid-shaft) cortical bone the... 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 obta...
X-ray-based diagnostic techniques, which are by far the most widely used for diagnosing bone diso... more X-ray-based diagnostic techniques, which are by far the most widely used for diagnosing bone disorders and diseases, are largely blind to the protein component of bone. Bone proteins are important because they determine certain mechanical properties of bone and changes in the proteins have been associated with a number of bone diseases. Spatially Offset Raman Spectroscopy (SORS) is a chemically specific analytical technique that can be used to retrieve information noninvasively from both the mineral and protein phases of the bone material in vivo. Here we demonstrate that SORS can be used to detect a known compositional abnormality in the bones of a patient suffering from the genetic bone disorder, osteogenesis imperfecta, a condition which affects collagen. The confirmation of the principle that bone diseases in living patients can be detected noninvasively using SORS points the way to larger studies that focus on osteoporosis and other chronic debilitating bone diseases with large socioeconomic burdens.
Veterinary Clinics of North America: Equine Practice, 2011
Overstrain and traumatic tendon and ligament injuries are common in the horse and, for the most p... more Overstrain and traumatic tendon and ligament injuries are common in the horse and, for the most part, heal (repair) naturally by the formation of scar tissue. However, the scar tissue formed in this repair is functionally deficient compared with normal tendon, which has important consequences for the animal in terms of reduced performance and a substantial risk of reinjury, despite the multitude of treatments that have been proposed. Regenerative medicine offers the prospect of restoring normal, or close to normal, structure and function to an injured organ, thereby resulting in a successful restoration of activity without the risk of reinjury. Regenerative medicine aims to harness the combined effects of a cell source, scaffold support, and anabolic stimulus to facilitate the healing of the injury. 1 There are multiple choices for the selection of a cell source for regenerative medicine, and at this time it is not clear which source will prove to be therapeutically optimal. A logical source of cells for making new tendon tissue would be the tenocytes Disclosures: R.K.W.S. is a technical adviser of VetCell. Experimental work has been funded by the Horserace Betting Levy Board, VetCell, and the Japan Racing Association and has been performed in collaboration with
The present study shows no adverse effects of the anti-diabetic drug metformin on bone mass and f... more 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 microarchitecture 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.
Objective: To determine whether the focal susceptibility to cartilage degeneration in joints is r... more Objective: To determine whether the focal susceptibility to cartilage degeneration in joints is related to a differential response to cytokine stimulation. Methods: Compare aggrecan and collagen catabolism in in-vitro models of cartilage degradation induced by retinoic acid (RA), interleukin-1 (IL-1), tumor necrosis factor alpha (TNF) and IL-1 plus oncostatin M (OSM). Glycosaminoglycan (GAG) and hydroxyproline (HyPro) quantification and Western immunoblot analyses of aggrecan and collagen degradation products were undertaken in explant cultures of normal cartilage from regions of equine joints with a known high and low susceptibility to degeneration in disease. RNA isolation and semi quantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis were performed to determine the expression of aggrecanases, matrix metalloproteinases (MMPs) and their inhibitors. Results: Although the rate of basal cartilage aggrecan turnover was dependent on joint region there was no difference in the response of different cartilages to cytokines. Individual animals did show a significant difference in the response of certain cartilages to cytokines, with both decreased and increased aggrecan loss in cartilage with a low susceptibility to degeneration. Aggrecan release in both short-and long-term cultures from all cartilages was associated with increased cleavage by aggrecanases rather than MMPs. There was a poor correlation between expression of aggrecanases, MMPs or their inhibitors and cytokine induced aggrecan catabolism. IL-1 alone was able to stimulate collagen breakdown in equine articular cartilage and surprisingly, significantly more collagen loss was induced in cartilage from regions less susceptible to degeneration. Conclusions: Collectively, these studies suggest that a regional difference in response to catabolic cytokines is unlikely to be a factor in the initiation of focal cartilage degeneration in osteoarthritis (OA).
Metabolic bone disease has been reported in free-living red squirrels (Sciurus vulgaris) in the U... more Metabolic bone disease has been reported in free-living red squirrels (Sciurus vulgaris) in the United Kingdom but the prevalence of this disease is unknown. In this study the bone quality of free-living red squirrels in the UK was assessed by radiology and bone densitometry. The study comprised 20 red squirrels found dead and submitted to the Zoological Society of London (UK) between 1997 and 1998, 10 were from the Isle of Wight (IoW), where gray squirrels (Sciurus carolinensis) are absent, and 10 were from Cumbria (Cu), where gray squirrels are present. Gray squirrels are considered potential competitors for red squirrels. Radiologic evaluation of humerus, femur, tibia, radius, and ilium revealed a slightly lower bone density and thinner cortices in red squirrels from the IoW when compared with those from Cu. Dual-energy X-ray absorptiometry was used to measure bone mineral content and density of the isolated right humerus and femur of 19 of the 20 red squirrels. The bone densitometry study reinforced the radiographic findings. The IoW specimens had lower bone mineral density values, although statistical significance (PϽ0.05) between animals from the IoW and Cu was only reached for the proximal epiphysis of the femur and between males from the IoW and males from Cu for the proximal epiphysis of the humerus. A highly positive correlation (rϾ0.94) was found when the bone mineral content and density between the femur and the humerus among groups and within each group were compared, showing a uniform level of mineralization between upper and lower limbs. These findings suggested generalized bone loss for the IoW red squirrels that may be compatible with some degree of osteopenia. Within the wide range of causes that lead to osteopenia, malnutrition (especially protein deficiency), calcium and copper deficiencies, and genetic factors remain as possible etiologies.
The decomposition of spatially offset Raman spectra for complex multilayer systems, such as biolo... 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.
Fracture healing can be enhanced by load bearing, but the specific components of the mechanical e... more Fracture healing can be enhanced by load bearing, but the specific components of the mechanical environment which can augment or accelerate the process remain unknown. The ability of low-magnitude, high-frequency mechanical signals, anabolic in bone tissue, are evaluated here for their ability to influence fracture healing. The potential for short duration (17 min), extremely low-magnitude (25 mm), high-frequency (30 Hz) interfragmentary displacements to enhance fracture healing was evaluated in a mid-diaphyseal, 3-mm osteotomy of the sheep tibia. In a pilot study of proof of concept and clinical relevance, healing in osteotomies stabilized with rigid external fixation (Control: n ¼ 4), were compared to the healing status of osteotomies with the same stiffness of fixation, but supplemented with daily mechanical loading (Experimental: n ¼ 4). These 25-mm displacements, induced by a ferroactive shape-memory alloy (''smart'' material) incorporated into the body of the external fixator, were less than 1% of the 3-mm fracture gap, and less than 6% of the 0.45-mm displacement measured at the site during ambulation (p < 0.001). At 10-weeks post-op, the callus in the Experimental group was 3.6-fold stiffer (p < 0.03), 2.5-fold stronger (p ¼ 0.05), and 29% larger (p < 0.01) than Controls. Bone mineral content was 52% greater in the Experimental group (p < 0.02), with a 2.6-fold increase in bone mineral content (BMC) in the region of the periosteum (p < 0.001). These data reinforce the critical role of mechanical factors in the enhancement of fracture healing, and emphasize that the signals need not be large to be influential and potentially clinically advantageous to the restoration of function.
The Journal of Bone and Joint Surgery. British volume, 2009
An understanding of the remodelling of tendon is crucial for the development of scientific method... more An understanding of the remodelling of tendon is crucial for the development of scientific methods of treatment and rehabilitation. This study tested the hypothesis that tendon adapts structurally in response to changes in functional loading. A novel model allowed manipulation of the mechanical environment of the patellar tendon in the presence of normal joint movement via the application of an adjustable external fixator mechanism between the patella and the tibia in sheep, while avoiding exposure of the patellar tendon itself. Stress shielding caused a significant reduction in the structural and material properties of stiffness (79%), ultimate load (69%), energy absorbed (61%), elastic modulus (76%) and ultimate stress (72%) of the tendon compared with controls. Compared with the material properties the structural properties exhibited better recovery after re-stressing with stiffness 97%, ultimate load 92%, energy absorbed 96%, elastic modulus 79% and ultimate stress 80%. The cros...
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Papers by Allen Goodship