Orlaith Brennan

Biomechanical properties across trabeculae from the proximal femur of normal and ovariectomised sheep.

The elastic behaviour of trabecular bone is a function not only of bone volume and architecture, but also of tissue material properties. Variation in tissue modulus can have a substantial effect on the biomechanical properties of trabecular bone. However, the nature of tissue property variation within a single trabecula is poorly understood. This study uses nanoindentation to determine the mechanical properties of bone tissue in individual trabeculae. Using an ovariectomised ovine model, the modulus and hardness distribution across trabeculae were measured. In both normal and ovariectomised bone, the modulus and hardness were found to increase towards the core of the trabeculae. Across the width of the trabeculae, the modulus was significantly less in the ovariectomised bone than in the control bone. However, in contrast to this hardness was found not to differ significantly between the two groups. This study provides valuable information on the variation of mechanical material properties in healthy and diseased trabecular bone tissue. The results of the current study will be useful in finite element modelling where more accurate values of trabecular bone modulus will enable the prediction of the macroscale behaviour of trabecular bone.

Effects of ovariectomy on bone turnover, porosity, and biomechanical properties in ovine compact bone 12 months postsurgery

Compact bone makes up approximately 80% of the human skeleton by mass; but there are little data available on the effects of increased bone turnover on compact bone mechanical and material properties. This study addresses this question by measuring intracortical remodeling, resorption cavity number, and porosity in an ovariectomized (OVX) sheep model, and measures changes in biomechanical properties. Thirty‐eight sheep were divided into two groups. Group 1 were controls (n = 19), and Group 2 were ovariectomized (OVX; n = 19). Fluorochrome dyes were administered intravenously to both groups at five time points over 12 months post‐OVX to label sites of bone turnover. At 12 months post‐OVX all animals were euthanized. Samples were harvested from the left metatarsal and were analyzed for intracortical bone turnover at five time points, the number of resorption cavities, and the level of intracortical porosity. The effects of these parameters on bone biomechanical properties were then measured. Bone turnover was increased in the OVX group at 6, 9, and 12 months (p < 0.05). Resorption was also higher in the OVX group at 12 months (p < 0.05). Furthermore, porosity was significantly increased in the OVX group at 12 months (p < 0.05). Stiffness and yield strength were reduced in the OVX group compared to controls (p = 0.05). Ultimate compressive strength and work to fracture did not differ between groups. These findings provide new insights into the mechanisms and effects of increased bone turnover on bone material and microstructural properties.

Effects of estrogen deficiency and bisphosphonate therapy on osteocyte viability and microdamage accumulation in an ovine model of osteoporosis.

It has been proposed that osteocyte viability plays an important role in bone integrity, and that bone loss in osteoporosis may be partially due to osteocyte cell death following estrogen depletion. Osteoporosis treatments such as bisphosphonates can inhibit osteocyte apoptosis which in turn may also reduce remodeling. Consequently, microcracks in bone which are normally repaired by bone remodeling may accumulate. This study used an ovine model of osteoporosis to examine the effects of estrogen depletion and bisphosphonates on osteocyte apoptosis and microdamage accumulation. Skeletally mature ewes were randomly assigned into two equal groups; ovariectomy (OVX) and a non‐treatment group (control). Half of these animals were sacrificed 12 months post‐OVX. Twenty months post‐OVX, a number of OVX animals were randomly selected and each received a supra‐pharmacological dose of the bisphosphonate, zoledronic acid (Zol). This group and all the remaining animals were sacrificed 31 months post‐OVX. A compact bone specimen was removed from the left metacarpal of each animal; half was used for osteocyte apoptosis detection and the remainder for microdamage analysis. Estrogen deficiency resulted in significant increases in the levels of osteocyte apoptosis while zoledronic acid significantly reduced the level of apoptosis in osteocytes. Zoledronic acid treatment resulted in the formation of more microcracks. However, these cracks were shorter than in control or OVX groups which may provide one explanation as to why increased damage levels following bisphosphonate treatment have not lead to increased fractures. This study also provides additional evidence of the importance of estrogen in preserving the osteocyte network.

The effects of increased intracortical remodeling on microcrack behaviour in compact bone.

The behaviour of microdamage in bone is related to its microstructural features and thus has an important role in tissue structural properties. However, it is not known how cracks behave in areas of increased intracortical remodeling. More remodeling creates wider variation in the properties of the primary microstructural features of cortical bone, namely osteons. This situation may occur after treatment involving parathyroid hormone or events such as menopause/ovariectomy. High turnover was modeled in this study by using ovariectomy (OVX) to induce surgical menopause in sheep. We hypothesized that osteon age would influence microcrack behaviour during propagation. Five fluorochrome dyes were administered intravenously at different time-points over 12 months post-OVX to label remodeling sites and all animals were then euthanized. Compact bone specimens (2x2x36 mm) were harvested from the right metatarsal. Samples were cyclically loaded to failure and then histological analyses were carried out. Cracks were categorized by length into three groups; short (<100 mum), intermediate (100-300 mum) and long (>300 mum). Numerical crack density (Cr.Dn) of long cracks was greater in controls compared with OVX. Controls also displayed a higher crack surface density (Cr.S.Dn) compared with OVX (p<0.05). The behaviour of short cracks did not differ between old and new osteons, but intermediate and long cracks preferentially stopped at newer osteons compared with older ones (p<0.05). This mechanism may have an important role in terms of prolonging fatigue life. We conclude that recently formed secondary osteons have a unique influence on propagating microcracks compared with older osteons. Therefore localized remodeling levels should be considered when studying microcrack behaviour in bone.

Effects of High Bone Turnover on the Biomechanical Properties of the L3 Vertebra in an Ovine Model of Early Stage Osteoporosis

Study Design. Investigations of the effects of high bone turnover on the L3 vertebra were carried out, using an ovariectomized (OVX) ovine model of early stage osteoporosis. Objective. To assess the contribution of bone turnover to the biomechanics of L3. Summary of Background Data. Clinically, dual energy x-ray absorptiometry (DEXA) is used to measure bone mineral density (BMD). However, this can only predict 60% to 70% of bone strength; the remainder is due to bone quality. There is currently little information available on how strength is affected by changes in bone quality parameters, particularly bone turnover. Turnover can be assessed clinically using biochemical markers; however, this provides systemic values, whereas localized values are required to predict site-specific fracture risk. Methods. Thirty-eight sheep were assigned to 2 groups (control, n = 19; OVX, n = 19). Both groups were intravenously administered a fluorochrome dye on the day of surgery and 3, 6, 9, and 12 months thereafter, to label sites of bone turnover. After 12 months, animals were killed and the spinal columns harvested. L3 vertebrae were scanned using DEXA. Bone turnover was quantified using epifluorescence microscopy, and microarchitecture was assessed by microCT scanning. Compressive testing was used to characterize the mechanical properties of the vertebrae. Results. BMD and microarchitecture were unchanged in OVX compared with controls. However, bone turnover, as measured by fluorochrome labeled sites, was significantly increased in the OVX group in cortical and trabecular compartments. As a consequence, the biomechanical properties were significantly reduced in that group. Conclusion. These findings show that OVX resulted in changes in bone turnover, which reduced biomechanical properties in a model of early stage osteoporosis. These differences were present despite microarchitecture or BMD remaining unchanged. In the future, the ability to assess site-specific bone turnover would greatly enhance the accuracy with which fracture risk could be predicted.

Encapsulation of cardiac stem cells in superoxide dismutase-loaded alginate prevents doxorubicin-mediated toxicity

Anthracyclines are powerful drugs available for the treatment of neoplastic diseases. Unfortunately, these chemotherapy agents cause cardiomyopathy and congestive heart failure. Doxorubicin (DOX) is a widely used anthracycline and evidence indicates that DOX‐induced cardiotoxicity can be viewed as a stem cell disease, whereby the formation of reactive oxygen species (ROS) by DOX is seen to predominantly hinder cardiac stem cell (CSC) regenerative capability. Acute, early‐onset and late‐onset cardiotoxicity have been described and this may be reversible by the local administration of CSCs, which regenerate myocardial tissue and rescue the failing heart. CSCs are, however, particularly sensitive to oxidative stress and die rapidly by apoptosis in such adverse conditions. Therefore, this study aims to enhance CSC survival by encapsulation in an alginate hydrogel formulation containing superoxide dismutase (SOD), a reactive oxygen species scavenger. Cell survival was qualitatively and quantitatively assessed by fluorescent microscopy and assays measuring metabolic activity, cell viability, cytotoxicity and apoptosis. CSCs were cultured in DOX‐conditioned cell culture medium and displayed reduced live cell numbers as well as high levels of apoptosis. Encapsulation of CSCs in alginate alone failed to prevent apoptosis. Encapsulation in SOD‐loaded alginate reduced apoptosis to near‐normal levels, whilst metabolic activity was returned to baseline. In conclusion, this study demonstrates that encapsulation of CSCs in SOD‐loaded alginate hydrogel enhances CSC survival in the presence of DOX, raising the possibility of its application as a novel therapy for the treatment of acute and early onset DOX‐induced cardiotoxicity. Copyright

The effects of estrogen deficiency and bisphosphonate treatment on tissue mineralisation and stiffness in an ovine model of osteoporosis

While much research has been dedicated to understanding osteoporosis, the nature of mineral distribution and the mechanical property variation in diseased bone is poorly understood. The current study aimed to determine the effect of estrogen deficiency and bisphosphonate therapy on bone tissue properties using an ovine model of osteoporosis. Skeletally mature animals (4+ years) were divided into an ovariectomy group (ovx, n=20) and a non treatment control group (control, n=20). A zoledronic acid treated group was also included in which animals were estrogen deficient for 20 months prior to receiving treatment (Zol, n=4). Half of the control and ovx groups were euthanized 12 or 31 months post-operatively and all Zol animals were euthanised at 31 months. Individual trabeculae were removed from the proximal femur and were analysed at specific locations across the width of the trabeculae. The mineral content was measured using quantitative backscatter electron imaging and the modulus was measured using nanoindentation. The spatial distribution of tissue modulus and mineral content in bone from ovariectomised animals was similar to control. However, ovariectomy significantly reduced the overall mineral content and tissue modulus relative to the control group after 12 months. Interestingly, significant differences were not maintained 31 months post-OVX. Treatment with zoledronic acid increased the mineral content and tissue modulus relative to both the ovariectomised and control groups. Zoledronic acid was also found to alter the mineral and modulus gradients normally associated with healthy bone tissue. The current study provides evidence that both estrogen deficiency and zoledronic acid therapy significantly alter mineral content and the mechanical properties of trabecular tissue.

The Marine-derived, Multi-mineral formula, Aquamin, Enhances Mineralisation of Osteoblast Cells In Vitro.

Osteoporosis is a global health problem characterized by low bone mass and an increase in bone fragility. It is now well accepted that dietary factors play a central role in bone development and health. Diet that lacks adequate minerals is considered to be a risk factor for osteoporosis. The food supplement, Aquamin, is a natural, multi‐mineral derived from the red algae Lithothamnion corallioides, rich in calcium, magnesium and 72 other trace minerals. The aim of this study was to evaluate the effect of Aquamin on osteoblastic behaviour and mineralisation in a pre‐osteoblastic cell line. Cell number and metabolic activity were assessed using Hoescht DNA and AlamarBlue assays respectively. Osteogenic differentiation was measured using an alkaline phosphatase assay while mineralisation was determined using von Kossa and alizarin red staining. It is reported here that Aquamin promotes increased mineralisation in osteoblast cell culture. These data suggest that the nutritional supplement Aquamin plays an important role in promoting bone formation and may be useful in treating bone diseases such as osteoporosis. Copyright

Structural adaptation and intracortical bone turnover in an ovine model of osteoporosis

Compact bone makes up approximately 80% of the human skeletal mass. This study examines the effect of estrogen deficiency on compact bone turnover and associated geometrical structural adaptation over a 31‐month period in a large animal model. Twenty‐seven skeletally mature sheep were divided into control (n = 16) and ovariectomy group (OVX, n = 11). Animals were administered five different fluorochrome dyes to label intracortical bone turnover, and sacrificed at 31 months. Compact bone samples were analyzed for cortical geometry, intracortical turnover at five time points, resorption cavities, porosity, and compressive strength. Intracortical bone turnover was significantly increased in OVX, which demonstrated seasonal variation. Cross‐sectional area in OVX was significantly greater than control and was associated with an increased section modulus. Intracortical porosity was significantly increased in OVX, however, there was no significant difference in ultimate compressive strength between the groups. Our results demonstrate increased intracortical bone turnover, resportion spaces, and porosity in OVX, without adversely affecting compressive strength. Our results also support the hypothesis of geometrical adaptation of compact bone in response to estrogen deficiency. These results suggest an early structural compensatory response in compact bone, despite increased intracortical turnover.

Estrogen plus estrogen receptor antagonists alter mineral production by osteoblasts in vitro.

In early postmenopausal women, estrogen withdrawal is associated with increased bone turnover leading to bone loss and increased risk of fracture. Recent studies have suggested that the remaining bone tissue is significantly stronger, stiffer and has an increased tissue-level mineral content. Such changes may occur to compensate for bone loss or as a direct result of estrogen deficiency. To date many details of the physiology of osteoblastic cells during estrogen deficiency are vague. In this study we test the hypothesis that osteoblastic matrix mineralisation is altered at the onset of estrogen deficiency. In vitro cell culture experiments were carried out up to 28 days to compare the mineral production of MC3T3-E1 osteoblastic cells subject to estrogen deficiency (fulvestrant), enhanced estrogen supplementation (17-β-estradiol) or a combination of both. Mineralisation was detected using von Kossa staining and was quantified with alizarin red absorbance readings. The expression of osteocalcin and osteopontin proteins, markers of osteoblast differentiation and mineralisation, was monitored using immunohistochemistry. Our results demonstrate that estrogen enhancement improves matrix mineralisation by MC3T3 cells in vitro. Furthermore this study found a significant reduction in the level of mineralisation when cells were treated with a combination of estrogen and fulvestrant. In an estrogen deficient environment mineralisation by osteoblastic cells was not altered. These findings suggest that altered tissue mineralisation following estrogen deficiency is not a direct result of estrogen deficiency on osteoblasts. Rather, we propose that altered tissue mineralisation may be a compensatory mechanism by bone to counter bone loss and reduced strength.