Type-2 Diabetic (T2D) patients experience up to a 3-fold increase in bone fracture risk[1]. Paradoxically, T2D-patients have a normal or increased bone mineral density when compared to non-diabetic patients. This implies that T2D has a deleterious effect on bone quality, whereby the intrinsic material properties of the bone matrix are altered. Creating clinical challenges as current diagnostic techniques are unable to accurately predict the fracture probability in T2D-patients. To date, the relationship between cyclic fatigue loading, mechanical properties and microdamage accumulation of T2D-bone tissue has not yet been examined and thus our objective is to investigate this relationship. Ethically approved femoral heads were obtained from patients, with (n=8) and without (n=8) T2D. To obtain the mechanical properties of the sample, one core underwent a monotonic compression test to 10% strain, the other core underwent a cyclic compression test at a normalized stress ratio between 0.0035mm/mm and 0.016mm/mm to a maximum strain of 3%. Microdamage was evaluated by staining the tissue with barium sulfate precipitate [2] and conducting microcomputed tomography scanning with a voxel size of 10μm. The monotonically tested T2D-group showed no statistical difference in mechanical properties to the non-T2D-group, even when normalised against BV/TV. There was also no difference in BV/TV. For the cyclic test, the T2D-group had a significantly higher initial modulus (p<0.01) and final modulus (p<0.05). There was no difference in microdamage accumulation. Previous population-level studies have found that T2D-patients have been shown to have an increased fracture risk when compared to non-T2D-patients. This research indicates that T2D does not impair the mechanical properties of trabecular bone from the femoral heads of T2D-patients, suggesting that other mechanisms may be responsible for the increased fracture risk seen in T2D-patients.
This study aims to assess the fracture mechanics of type-2 diabetic (T2D) femoral bone using innovative site-specific tests, whilst also examining the cortical and trabecular bone microarchitecture from various regions using micro-computed tomography (CT) of the femur as the disease progresses. Male [Zucker Diabetic Fatty (ZDF: fa/fa) (T2D) and Zucker Lean (ZL: fa/+) (Control)] rats were euthanized at 12-weeks of age, thereafter, right and left femora were dissected (Right femora: n = 6, per age, per condition; Left femora: n=8-9, per age, per condition). Right femurs were notched in the posterior of the midshaft. Micro-CT was used to scan the proximal femur, notched and unnotched femoral midshaft (cortical) of the right femur and the distal metaphysis (trabecular) of the left femur to investigate microarchitecture and composition. Right femurs were fracture toughness tested to measure the stress intensity factor (Kic) followed by a sideways fall test using a custom-made rig to investigate femoral neck mechanical properties. There was no difference in trabecular and cortical tissue material density (TMD) between T2D and control rats. Cortical thickness was unchanged, but trabeculae were thinner (p<0.01) in T2D rats versus controls. However, T2D rats had a greater number of trabeculae (p<0.05) although trabecular spacing was not different to controls. T2D rats had a higher connectivity distribution (p<0.05) and degree of anisotropy (p<0.05) in comparison to controls. There was no difference in the mechanical properties between strains. At 12-weeks of age, rats are experiencing early-stage T2Ds and the disease impact is currently not very clear. Structural and material properties are unchanged between strains, but the trabecular morphology shows that T2D rats have more trabecular struts present in order to account for the thinner trabeculae.
Individuals with type 2 diabetes (T2D) have a 3-fold increased risk of bone fracture compared to non-diabetics, with the majority of fractures occurring in the hip, vertebrae and wrists. However, unlike osteoporosis, in T2D, increased bone fragility is generally not accompanied by a reduction in bone mineral density (BMD). This implies that T2D is explained by poorer bone quality, whereby the intrinsic properties of the bone tissue itself are impaired, rather than bone mass. Yet, the mechanics remain unclear. The objective of this study is to (1) assess the fracture mechanics of bone at the structural and tissue level; and (2) investigate for changes in the composition of bone tissue along with measuring total fluorescent advanced glycation end products (fAGEs) from the skin, as T2D progresses with age in Zucker diabetic fatty (ZDF (fa/fa)) and lean Zucker (ZL (fa/+)) rats. Right ulnae and skin sections were harvested from ZDF (fa/fa) (T2D) and ZL (fa/+) (Control) rats at 12 and 46 weeks (wks) of age (n = 8, per strain and age) and frozen. Right ulnae were thawed for 12 hrs before micro-CT (μCT) scanning to assess the microstructure and measure BMD. After scanning, ulnae were loaded until failure via three-point bending. Fourier transform-infrared microspectroscopy (FTIR) was used to measure various bone mineral- and collagen-related parameters such as, mineral-to-matrix ratio and nonenzymatic cross-link ratio. Finally, fAGEs were measured from skin sections using fluorescence spectrometry and an absorbance assay, reported in units of ng quinine/ mg collagen.Introduction and Objective
Materials and Methods
Bone remodelling is a continuous process whereby osteocytes regulate the activity of osteoblasts and osteoclasts to repair loading-induced microdamage. While many in vitro studies have established the role of paracrine factors (e.g., RANKL/OPG) and cellular pathways involved in bone homeostasis, these techniques are generally limited to two-dimensional cell culture, which neglects the role of the native extracellular matrix in maintaining the phenotype of osteocyte. Recently, ex vivo models have been used to understand cell physiology and mechanobiology in the presence of the native matrix. Such approaches could be applicable to study the mechanisms of bone repair, whilst also enabling exploration of biomechanical cues. However, to date an ex vivo model of bone remodelling in cortical bone has not been developed. In this study, the objective was to develop an ex vivo model where cortical bone was subjected to cyclic strains to study the remodelling of bone. Ex vivo model of bone remodelling induced by cyclic loading: At the day of culling, beam-shape bovine bone samples were cut and preserved in PBS + 5% Pen/Strep + 2 mM L-Glut overnight at 37°C. Cyclic strains were applied with a three-point bend system to induce damage with a regime at 16.66 mm/min for 5,000 cycles in sterile PBS in Evolve® bags (maximum strain 6%). A control group was cultured under static conditions. Metabolic activity: Alamar Blue assays were performed after 1 and 7 days of ex vivo culture for each group (Static, Loaded) and normalized to weight. Bone remodelling: ALP activity was assessed in the media at day 1 and 7. After 24 hours cell culture conditioned media (CM) was collected from each group and stored at −80°C. RAW264.7 cells were cultured with CM for 6 days, after which the samples were stained for TRAP, to determine osteoclastogenesis, and imaged. Histomorphometry: Samples were cultured with calcein for 3 days to label bone formation between day 4 and 7. Fluorescent images were captured at day 7. μCT scanning was performed at 3 μm resolution after labelling samples with BaSO4 precipitate to quantify bone damage.Introduction and Objective
Materials and Methods
Although osteoporosis reduces overall bone mass causing bone fragility, our recent studies have shown that bone tissue composition is altered at the microscopic level, which is undetectable by conventional diagnostic techniques (DEXA) but may contribute to bone fracture. However, the time sequence of changes in bone microarchitecture, mechanical environment and mineral distribution are not yet fully understood. This study quantified the longitudinal effects of estrogen deficiency on the trabecular microarchitecture and mineral distribution in the tibia of Female Wistar rats (6 months) that underwent ovariectomy (OVX, n=10) or sham surgery (SHAM, n=10). Weekly micro-CT scans of the proximal tibia were conducted at 15µm resolution for the first month of estrogen deficiency. Morphometric analysis was conducted to characterise the trabecular bone microarchitecture. The bone mineral composition was characterised with analysis of bone mineral density distributions (BMDD). There was significantly reduced trabecular bone volume fraction at 2 weeks in OVX rats compared to controls (p<0.01). There was no difference in mineral distribution between the OVX and control animals. This study provides the first evidence in uncovering the temporal nature of changes in bone microarchitecture and mineral distribution, showing that structure changes before composition.
Osteoporosis has long been associated with weak bones but recent studies have shown that bone tissue mineral becomes more heterogeneous and the expression of mechanosensors are altered during estrogen deficiency in an animal model of osteoporosis. However, whether these changes occur as a primary response to estrogen deficiency is unknown. In this study we investigate whether matrix production and mineralisation by mechanically-stimulated osteoblasts are impaired as a direct consequence of estrogen depletion. Osteoblast-like MC3T3-E1 cells were cultured for 14 days with 10−8M of 17β-estradiol and subsequently cultured with osteogenic media only, or supplemented with estrogen or an estrogen antagonist (Fulvestrant, 10−7M). Physiological shear stress (1Pa) was applied using an orbital shaker (290rpm, 40min/day), which allows long-term culture and induces oscillatory flow on cells. Osteoblasts phenotype, extracellular matrix (ECM), mineralisation and mechanosensors were tracked by qRT-PCR (Runx2, Col1a1, Col1a2, Cox2, Bglap2, FN1), by biochemical assays (ALP activity, DNA and calcium content), by immunostaining (integrin αv, BSP2, fibronectin) and by labelling with calcein the calcium. The results of this study demonstrate that after 7 days, estrogen depleted cells had less integrin αv mechanosensors compared to those that received continuous estrogen treatment. By 14 days the ECM formation (calcium, fibronectin) by osteoblasts was altered under estrogen depletion, when compared to cells that were cultured continuously with estrogen. This study provides evidence of changes in osteoblast behaviour under estrogen depletion, which might explain the alteration in tissue mineral content and the decrease of integrins observed previously in ovariectomized rats
