Danijela Djonic

Decrease in the osteocyte lacunar density accompanied by hypermineralized lacunar occlusion reveals failure and delay of remodeling in aged human bone

Aging decreases the human femur’s fatigue resistance, impact energy absorption, and the ability to withstand load. Changes in the osteocyte distribution and in their elemental composition might be involved in age‐related bone impairment. To address this question, we carried out a histomorphometric assessment of the osteocyte lacunar distribution in the periosteal and endosteal human femoral cortexes of 16 female and 16 male donors with regard to age‐ and sex‐related bone remodeling. Measurements of the bone mineral density distribution by quantitative backscattered electron imaging and energy dispersive X‐ray analysis were taken to evaluate the osteocyte lacunar mineral composition and characteristics. Age‐dependent decreases in the total osteocyte lacunar number were measured in all of the cases. This change signifies a risk for the bone’s safety. Cortical subdivision into periosteal and endosteal regions of interest emphasized that, in both sexes, primarily the endosteal cortex is affected by age‐dependent reduction in number of osteocyte lacunae, whereas the periosteal compartment showed a less pronounced osteocyte lacunar deficiency. In aged bone, osteocyte lacunae showed an increased amount of hypermineralized calcium phosphate occlusions in comparison with younger cases. With respect to Frost’s early delineation of micropetrosis, our microanalyses revealed that the osteocyte lacunae are subject to hypermineralization. Intralacunar hypermineralization accompanied by a decrease in total osteocyte lacunar density may contribute to failure or delayed bone repair in aging bone. A decreased osteocyte lacunar density may cause deteriorations in the canalicular fluid flow and reduce the detection of microdamage, which counteracts the bone’s structural integrity, while hypermineralized osteocyte lacunae may increase bone brittleness and render the bone fragile.

Analysis of neck injuries in hanging

Retrospective study of suicidal hangings was made on 175 cases (133 male victims, 42 female victims) for a 5-year period. The mean age was 47.33 ± 17.51 years. The sample was divided in 4 groups according to the position of the ligature knot (anterior, right, left, and posterior hanging). The authors analyzed all visible injuries of soft tissues and bones and cartilage of the neck, and in 150 cases (85.7%), we established that there was at least one injury of these structures (hematoma or fracture for example). The most frequent injury was to sternocleidomastoid muscles. Fracture of throat skeleton was detected in 119 cases (68%). The proportion of fractures increases with age of the deceased. There was no clear correlation between frequency of neck injuries and type of hanging.

Micro-structural basis for particular vulnerability of the superolateral neck trabecular bone in the postmenopausal women with hip fractures

In this study we analyzed the trabecular bone micro-architecture in the inferomedial and superolateral subregions of the femoral neck in a group with hip fractures and a control group of elderly women, with aim to clarify the micro-structural basis of bone fragility. Proximal femora from 29 Caucasian female cadavers were collected at Institute of Forensic Medicine in Belgrade (15 women with hip fracture: age 79.5±8.5 yrs.; and 14 women without hip fractures: age 74.1±9.3 yrs.). The femoral neck section was scanned in dry conditions using a micro-computed tomography (Scanco μCT 40), at 70 kV, 114 μA, 300 ms integration time, 36 μm resolution, isotropic, 1024×1024 pixels per slice, automatically evaluating trabecular micro-architecture using the built-in program of the micro-CT with direct 3D morphometry. The samples were foam padded to avoid any movement artifacts during scanning. Analysis of the neck section in the fracture group compared to the control cases demonstrated significantly lower bone volume fraction (mean: 6.3% vs. 11.2%, p=0.002), lower connectivity density (0.33/mm(3) vs. 0.74/mm(3), p=0.019) and higher trabecular separation (0.87 mm vs. 0.83 mm, p=0.030). Division into the superolateral and inferomedial regions of interest revealed that the superolateral neck displayed even more differences in micro-architectural properties between the fracture and non-fracture groups. Namely, while in the inferomedial neck only bone volume fraction and degree of anisotropy displayed significant inter-group variability (lower BV/TV with higher degree of anisotropy in the fracture group), in the superolateral neck almost all parameters were different between the fracture cases and the controls, where the fracture group showed a lower trabecular bone volume fraction (3.6% vs. 8.2%, p=0.001), lower connectivity (0.21 vs. 0.63/mm(3), p=0.008), more rod like trabecular structure (SMI: 2.94 vs. 2.62, p=0.049), higher separation and the thinned trabeculae (Tb.Sp: 0.89 vs. 0.85 mm, p=0.013; Tb.Th: 0.17 vs. 0.20 mm, p=0.05). In addition, after adjusting for the effects of BV/TV, the majority of differences disappeared, demonstrating that the bone loss manifests itself via the changes in micro-architectural parameters: trabecular thinning, rising the spacing between individual trabeculae, reducing trabecular connectivity and accentuating trabecular perforations leading to predominance of rod-like trabecular elements. Preferential impairment of the superolateral neck trabecular structure and organization in women with hip fracture reveals the region-dependent micro-structural basis of bone fragility in elderly women.

Evaluation of the Suchey–Brooks Method for Aging Skeletons in the Balkans

ABSTRACT: This study has been carried out to examine whether the Suchey–Brooks (S&B) methods could be successfully applied in age assessment of populations from the Balkans. The known‐age sample consists of 33 females and 52 males pairs of pubic bones collected from the autopsy cases. Age estimation by S&B method showed an accuracy of 89.74% in males and 72.0% in females. Statistical analysis showed a positive correlation between the actual age of the investigated individuals and age phases obtained by the S&B method, although the mean values of the sixth age category differed significantly compared with the original model. The most reliable indicators in both sexes were the relief of the symphyseal surface, lipping, symphyseal rim, and dorsal margin. The discriminating power of these indicators was the least reliable in distinguishing S&B phases 2 and 3. Based on these results, the appropriate recommendations for aging Serbian populations are made. There was a good agreement between two observers (κ=0.726).

Multi-level characterization of human femoral cortices and their underlying osteocyte network reveal trends in quality of young, aged, osteoporotic and antiresorptive-treated bone

Characterization of bones hierarchical structure in aging, disease and treatment conditions is imperative to understand the architectural and compositional modifications to the material and its mechanical integrity. Here, cortical bone sections from 30 female proximal femurs - a frequent fracture site - were rigorously assessed to characterize the osteocyte lacunar network, osteon density and patterns of bone matrix mineralization by backscatter-electron imaging and Fourier-transform infrared spectroscopy in relation to mechanical properties obtained by reference-point indentation. We show that young, healthy bone revealed the highest resistance to mechanical loading (indentation) along with higher mineralization and preserved osteocyte-lacunar characteristics. In contrast, aging and osteoporosis significantly alter bone material properties, where impairment of the osteocyte-lacunar network was evident through accumulation of hypermineralized osteocyte lacunae with aging and even more in osteoporosis, highlighting increased osteocyte apoptosis and reduced mechanical competence. But antiresorptive treatment led to fewer mineralized lacunae and fewer but larger osteons signifying rejuvenated bone. In summary, multiple structural and compositional changes to the bone material were identified leading to decay or maintenance of bone quality in disease, health and treatment conditions. Clearly, antiresorptive treatment reflected favorable effects on the multifunctional osteocytic cells that are a prerequisite for bones structural, metabolic and mechanosensory integrity.

Age-related deterioration in trabecular bone mechanical properties at material level: Nanoindentation study of the femoral neck in women by using AFM

Despite general belief that the mechanical properties of bone material contribute to whole bone strength, it is still obscure what the age effects are on mechanical behavior of the bone material, particularly in the case of the femoral neck trabeculae. In this study, atomic force microscopy was used for imaging and measuring the size of mineral grains, as well as nano-scale mechanical characterization (nanoindentation) of the bone mineralized matrix of trabeculae, with the aim to explore the age effects on bone elasticity and give new insight into age-related bone fragility. The bone samples in this study comprised trabecular bone specimens of the femoral neck region, collected from eight skeletal healthy women (five young adults: 27-38yrs., three elderly: 83-94yrs.) at autopsy. Bone trabeculae in the elderly displayed a higher modulus and nanohardness, signifying a decreased amount of energy that can be accommodated by the bone tissue during loading. Regression analysis revealed that nearly 65% of variability in the bone matrix elastic modulus can be statistically explained by the changes in size of the matrix mineral grains. This study revealed that the bone trabeculae of elderly women express less elastic behavior at the material level, which makes them more vulnerable to unusual impact loads originating from a fall. The observed age-related structural and mechanical alteration at the bone material level adds new evidence for understanding why hip fractures are more frequent in elderly women.

Nano-structural, compositional and micro-architectural signs of cortical bone fragility at the superolateral femoral neck in elderly hip fracture patients vs. healthy aged controls.

To unravel the origins of decreased bone strength in the superolateral femoral neck, we assessed bone structural features across multiple length scales at this cortical fracture initiating region in postmenopausal women with hip fracture and in aged-matched controls. Our combined methodological approach encompassed atomic force microscopy (AFM) characterization of cortical bone nano-structure, assessment of mineral content/distribution via quantitative backscattered electron imaging (qBEI), measurement of bone material properties by reference point indentation, as well as evaluation of cortical micro-architecture and osteocyte lacunar density. Our findings revealed a wide range of differences between the fracture group and the controls, suggesting a number of detrimental changes at various levels of cortical bone hierarchical organization that may render bone fragile. Namely, mineral crystals at external cortical bone surfaces of the fracture group were larger (65.22nm±41.21nm vs. 36.75nm±18.49nm, p<0.001), and a shift to a higher mineral content and more homogenous mineralization profile as revealed via qBEI were found in the bone matrix of the fracture group. Fracture cases showed nearly 35% higher cortical porosity and showed significantly reduced osteocyte lacunar density compared to controls (226±27 vs. 247±32#/mm(2), p=0.05). Along with increased crystal size, a shift towards higher mineralization and a tendency to increased cortical porosity and reduced osteocyte lacunar number delineate that cortical bone of the superolateral femoral neck bears distinct signs of fragility at various levels of its structural organization. These results contribute to the understanding of hierarchical bone structure changes in age-related fragility.

Nanostructure and mineral composition of trabecular bone in the lateral femoral neck: implications for bone fragility in elderly women.

Despite interest in investigating age-related hip fractures, the determinants of decreased bone strength in advanced age are not clear enough. Hitherto it has been obscure how the aging process affects the femoral neck nanostructure and composition, particularly in the lateral subregion of the femoral neck, which is considered as a fracture-initiating site. The femoral bone samples used in this study were obtained at autopsy in 10 women without skeletal disease (five younger: aged 20-40 years, and five elderly: aged 73-94 years). Atomic force microscopy (AFM) was applied to explore the mineral grain size in situ in young vs. old trabecular bone samples from the lateral femoral neck. The chemical compositions of the samples were determined using inductively coupled plasma optical emission spectroscopy and direct current argon arc plasma optical emission spectrometry. Our AFM study revealed differences in trabecular bone nanostructure between young and elderly women. The mineral grain size in the trabeculae of the old women was larger than that in the young (median: 95 vs. 59nm), with a particular bimodal distribution: 45% were small grains (similar to the young) and the rest were larger. Since chemical analyses showed that levels of calcium and phosphorus were unchanged with age, our study suggests that during aging the existing bone mineral is reorganized and forms larger aggregates. Given the mechanical disadvantage of large-grained structures (decreased material strength), the observed nanostructural differences contribute to our understanding of the increased fragility of the lateral femoral neck in aged females. Moreover, increasing data on mineral grains in natural bone is essential for advancing calcium-phosphate ceramics for bone tissue replacement.

Enhanced trabecular micro-architecture of the femoral neck in hip osteoarthritis vs. healthy controls: a micro-computer tomography study in postmenopausal women

PurposeA controversial relationship between osteoarthritis (OA) and bone fragility has been attracting considerable attention. However, despite interest in the effects of OA on femoral neck fracture risk and numerous studies analysing the changes in the arthritic femoral head, there is insufficient data about femoral neck 3D bone micro-architecture in individuals with hip osteoarthritis. We compared trabecular micro-architecture of the femoral neck between postmenopausal women with coxarthrosis and controls to explore whether coxarthrosis may indicate reduced bone fragility from the trabecular micro-architectural perspective.MethodsThe study sample included nine women with hip osteoarthritis and 13 age-matched controls. The femoral neck sections were scanned using micro-computed tomography, evaluating the cancellous bone from the superolateral and inferomedial neck subregions.ResultsOsteoarthritic subjects demonstrated a general trend of improved trabecular micro-architecture in both analysed subregions when compared with age-matched controls. In particular, several architectural properties that are important predictors of cancellous bone strength showed significantly better values in the OA group, even after adjusting for bone volume fraction. Namely, the OA group expressed higher trabecular connectivity (p = 0.008), lower SMI indicating more plate-like structure (p = 0.005), and reduced anisotropy (p = 0.006) particularly in the inferomedial neck. Osteoarthritic cases also trended towards higher BV/TV, particularly in the superolateral neck. All micro-architectural parameters displayed significant regional heterogeneity (p ≤ 0.01), with the inferomedial neck region showing more favourable values than the superolateral region.ConclusionsEnhanced trabecular micro-architecture of the femoral neck in postmenopausal osteoarthritic subjects suggests reduced cancellous bone fragility in comparison with their age-matched healthy controls.

Systemic fat embolism and the patent foramen ovale—A prospective autopsy study

A fat embolism is a known and common complication of blunt force injuries, especially pelvic and long bones fractures. The aim of this study was to determine the importance of a patent foramen ovale (PFO) in developing systemic fat embolism (SFE) and eventually fat embolism syndrome (FES) in patients suffering from orthopaedic blunt injuries and consequent lung fat embolism. The sample was divided: 32 subjects with a sealed foramen ovale (SFO), and 20 subjects with a PFO. In our sample, there was no difference in either the incidence of renal fat embolism in subjects with PFO compared to those with SFO (Fishers exact test 0.228, p=0.154) or in the grade of renal fat embolism (Pearson Chi-square 2.728, p=0.435). However, there was a statistically significant correlation between the grade of lung fat embolism and the number of fractured bones for the whole sample (Spearmans rho 0.271, p=0.052), but no correlation between the grade of lung fat embolism and the ISS or NISS (Pearson correlation 0.048, p=0.736, and 0.108, p=0.445, respectively). In our study, the presence of fat emboli in the kidney, i.e. SFE, could effectively be predicted by the grade of lung fat embolism (the moderate and slight grades of lung fat embolism were better predictors than the massive one: logistic regression - Wald. Coeff.=11.446, p=0.003, Wald. Coeff.=10.553, p=0.001, and Wald. Coeff.=4.128, p=0.042), and less effectively by presence of PFO (Wald. Coeff.=2.850, p=0.091). This study pointed out that lung and SFE are not pure biomechanical events, so the role of a PFO is not crucial in developing a lung fat embolism into a systemic embolism: the fat embolism is more of a biochemical and pathophsyiological event, than a biomechanical one. The appearance of a patent foramen ovale associated with a systemic fat embolism should be less emphasised: maybe arteriovenous shunts and anastomosis between the functional and nutritive, i.e. systemic circulation of lungs play a more important role in developing a SFE than a PFO.