Petar Milovanovic

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.

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.

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.

Age-dependence of power spectral density and fractal dimension of bone mineralized matrix in atomic force microscope topography images: potential correlates of bone tissue age and bone fragility in female femoral neck trabeculae

There is an increasing interest in bone nano‐structure, the ultimate goal being to reveal the basis of age‐related bone fragility. In this study, power spectral density (PSD) data and fractal dimensions of the mineralized bone matrix were extracted from atomic force microscope topography images of the femoral neck trabeculae. The aim was to evaluate age‐dependent differences in the mineralized matrix of human bone and to consider whether these advanced nano‐descriptors might be linked to decreased bone remodeling observed by some authors and age‐related decline in bone mechanical competence. The investigated bone specimens belonged to a group of young adult women (n = 5, age: 20–40 years) and a group of elderly women (n = 5, age: 70–95 years) without bone diseases. PSD graphs showed the roughness density distribution in relation to spatial frequency. In all cases, there was a fairly linear decrease in magnitude of the power spectra with increasing spatial frequencies. The PSD slope was steeper in elderly individuals (−2.374 vs. −2.066), suggesting the dominance of larger surface morphological features. Fractal dimension of the mineralized bone matrix showed a significant negative trend with advanced age, declining from 2.467 in young individuals to 2.313 in the elderly (r = 0.65, P = 0.04). Higher fractal dimension in young women reflects domination of smaller mineral grains, which is compatible with the more freshly remodeled structure. In contrast, the surface patterns in elderly individuals were indicative of older tissue age. Lower roughness and reduced structural complexity (decreased fractal dimension) of the interfibrillar bone matrix in the elderly suggest a decline in bone toughness, which explains why aged bone is more brittle and prone to fractures.

Bone microarchitecture at muscle attachment sites: The relationship between macroscopic scores of entheses and their cortical and trabecular microstructural design

The studies of entheses in bioarchaeology attempted to reconstruct the habitual physical activities of past populations. However, the studies of microarchitecture of the underlying bone are still lacking despite well-known potential of bone internal microarchitecture to reflect mechanical loading. It is unknown whether different morphological expressions of entheseal changes (ECs) correlate with the microstructural characteristics of the underlining bone. This study analyzed bone microstructural characteristics at the entheses. Our focus was on examining the possible successive nature of the three-stage scale of entheseal macroscopic changes by comparing EC scores with the microarchitectural features at the attachment sites. The study was based on the hypothesis that mechanical loading influences the microarchitecture of the bone at the attachment site. The bone samples were taken from 24 adult male skeletons from medieval cemeteries in Serbia, with different macroscopic expression score of EC. We evaluated the macroscopic and microscopic appearance of four entheses of the lower limbs (origin of the soleus muscle and the insertions of the adductor magnus, gluteus maximus, and iliopsoas muscles). The specimens were scanned using microcomputed tomography (Scanco µCT 40). Our data showed a lack of consistent correlation between stages of the macroscopic scoring systems with microarchitecture at the entheses, only cortical thickness was significantly different between EC stages. Analyzing relationship between trabecular and cortical bone microstructure we found correlations between cortical and trabecular variables only in Stage C. Results of our study suggest that macroscopic EC might not represent distinct successive phases in bone adaptation to mechanical loading.

Representing children in excavated cemeteries: the intrinsic preservation factors

Children are often under-represented in excavated populations due to the poor survival of their bones. Using a group of medieval burials from Serbia, our researchers examine the differential survival of children and of different parts of the body within the same terrain, and rightly urge us to take these factors into consideration before attempting demographic, ritual or social interpretations.