Abstract The goal of this study is to investigate the causes of skeletal fragility in Type 1 Diabetic (T1D) women. We hypothesize that bone fragility in diabetic individuals is partly due to changes in mineral and/or intrinsic material properties in the osteocyte lacunar/peri-lacunar regions of bone tissue. Studies of bone material properties in T1D are limited, and bone mineral density (BMD) alone does not explain the elevated fracture risk in T1D women (Cases). Innovative instruments with nanoscale resolution, including a laser scanning microscope (LSM), an atomic force microscope that is integrated with infrared spectroscopy (AFM-IR), and a nanoindenter were used for the characterization of the material properties surrounding osteocyte lacunae. In trabecular bone tissue, the compositional mineral matrix and Mineral Matrix Peak ratios (MMA, MMP for both near- & far-lacunae) along with material strength variables (Modulus, Hardness for both near- & far-lacunae) were lower in T1D compared to Controls. While material compositional ratio of mineral maturity crystallinity area (MMCA) was higher, the mineral maturity crystallinity peak (MMCP) and hardness tended to be higher in “lacunae-near” than “lacunae-far” for all the biopsies. Furthermore, the higher mineral matrix (MMA) and material strength (modulus, hardness) in peri-lacunar regions of Controls may suggest its increased brittleness or differences in properties as compared to T1D. We postulate that difference between Cases (T1D) and Controls in modulus and hardness could be due to the variation in mineral exchange (diffusion) rate within the peri lacunar space.

Bone's resistance to fracture depends on its amount and quality, the latter including its structural and material/ compositional properties. Bone material properties are dependent on bone turnover rates, which are significantly elevated immediately following menopause. Previously published data reported that following menopause, the amount of organic matrix synthesized at actively forming surfaces is significantly decreased, while glycosaminoglycan content was also modulated at resorbing surfaces, in the cancellous compartment. In the present study, we used Raman microspectroscopic analysis of paired iliac crest biopsies obtained before and shortly after menopause (1 year after cessation of menses) in healthy females to investigate changes in material/compositional properties due to menopause, in the cortical compartment. Specifically, the mineral/ matrix ratio, the relative proteoglycan content, the mineral maturity/crystallinity, and the relative pyridinoline collagen cross-link content were determined at actively forming intracortical surfaces (osteons) as a function of tissue age, as well as in interstitial bone. Results indicated that it is the freshly synthesized organic matrix content that significantly declines following menopause, in agreement with what was previously reported for the cancellous compartment. This decline was not evident in the freshly deposited mineral content. None of the compositional/quality properties were altered following menopause either. Finally, no differences in any of the monitored parameters were evident in cortical interstitial bone.

Increased fracture risk in type 1 diabetes (T1D) patients is not fully captured by bone mineral density (BMD) by DXA. Advanced glycation end-products (AGEs) have been implicated in the increased fracture risk in T1D, yet recent publications question this. To test the hypothesis that enzymatic collagen cross-links rather than AGEs correlate with fracture incidence in T1D, we analyzed iliac crest biopsies from sex-matched, fracturing T1D patients (N = 5; T1DFx), 6 non-fracturing T1D patients (T1DNoFx), and 6 healthy subjects, by Raman microspectroscopy as a function of tissue age (based on double fluorescent labels), in intracortical and trabecular bone, to determine pyridinoline (Pyd), ε-N-Carboxymethyl-L-lysine, and pentosidine (PEN)). There were no differences in the clinical characteristics between the T1DFx and T1DNoFx groups. At trabecular forming surfaces, T1DFx patients had higher PEN and Pyd content compared to T1DNoFx ones. Previous studies have shown that elevated PEN does not necessarily correlate with fracture incidence in postmenopausal, long-term T1D patients. On the other hand, the elevated Pyd content in the T1DFx patients would be consistent with published studies showing a significant correlation between elevated trivalent enzymatic collagen cross-links and fracture occurrence independent of BMD. Collagen fibers with high Pyd content are more brittle. Thus, a plausible suggestion is that it is the enzymatic collagen cross-links that either by themselves or in combination with the adverse effects of increased AGE accumulation that result in fragility fracture in T1D.

Pregnancy and lactation-associated osteoporosis (PLO) is a rare presentation of early-onset osteoporosis characterized by low trauma and spontaneous fractures during late pregnancy/lactation. Herein, we report areal BMD (aBMD) by DXA and volumetric BMD (vBMD), microarchitecture, and strength at the distal radius and tibia by HR-pQCT in 59 women with PLO-in comparison to both healthy premenopausal controls (n = 28) and premenopausal women with idiopathic osteoporotic fractures not associated with pregnancy/lactation (non-PLO IOP; n = 50). Women with PLO (aged 34 ± 6 yr) had a more severe clinical presentation than non-PLO IOP: 80% had vertebral and 92% had multiple fractures (p<.001). They had lower DXA aBMD at all sites vs Controls (all p<.001) and non-PLO IOP (all p<.05). By HR-pQCT, PLO had deficits in all radial/tibial density and most microarchitecture parameters and lower bone strength than controls (all p<.001). Compared to non-PLO IOP, PLO had lower total and trabecular density at radius and tibia (all p ≤ .01) and significant deficits in trabecular microstructure and cortical thickness at the radius only. We studied PLO subgroups with clinical factors potentially related to bone physiology: Within PLO, women with vertebral fractures had lower spine aBMD and higher tibial cortical porosity but were otherwise structurally similar to the nonvertebral group. Those with prior heparin exposure had larger bone size and trabecular area, and those with renal stones had smaller bone size and lower 1/3 radius aBMD. We also compared groups based on postpartum timing: Recent PLO (n = 25) evaluated ≤12 M postpartum, before expected recovery of pregnancy/lactation bone loss, had significantly lower aBMD than distant PLO (n = 34) evaluated >12 M postpartum. However, radial/tibial HR-pQCT measures did not differ, suggesting pre-existing and/or persistent structural deficits. This structural study increases our mechanistic understanding of the severe bone fragility presentation that characterizes PLO and also highlights areas of potential mechanistic heterogeneity that require additional investigation.

Context: We previously reported that sequential teriparatide followed by denosumab substantially increases bone mineral density (BMD) in premenopausal idiopathic osteoporosis (PremenIOP). Objective: To determine whether administration of bisphosphonates after denosumab cessation is associated with stable BMD in PremenIOP Design: Open-label extension study. Participants: Twenty-four PremenIOP Teriparatide-Denosumab Study participants. Interventions: Oral alendronate (ALN), 70 mg weekly, or intravenous zoledronic acid (ZOL), 5 mg once (patient choice), was administered 7 months (M) after final denosumab dose. Outcomes: BMD by dual-energy x-ray absorptiometry and serum C-telopeptide (CTX) q6M; Vertebral Fracture Assessment (VFA), and high-resolution peripheral quantitative computed tomography (HR-pQCT) q12 M. Results: Twenty-four women with PremenIOP (aged 43 +/- 8 years), severely affected with low trauma adult fractures (range 0-12; 9 with vertebral fractures) and/or very low BMD, had large BMD increases on sequential teriparatide-denosumab (spine: 25 +/- 9%; total hip: 11 +/- 6%). During the Bisphosphonate Extension, mean BMD and CTX changes in the entire group were small and not statistically significant at 6 or 12 M. Women choosing ZOL (n = 6) vs ALN (n = 18) did not differ by baseline age, body mass index, fractures, BMD, or CTX. On ZOL, there were small lumbar spine BMD declines and CTX increases, particularly between 6 M and 12 M, while greater stability was observed on ALN. Changes in BMD and CTX did not differ by duration of denosumab (36 M vs <36 M) or between 20 women who remained premenopausal and 4 who transitioned into menopause. Higher pre-teriparatide CTX, likely reflecting baseline remodeling status, predicted more spine and hip bone loss. No new vertebral (clinical or vertebral fraction assessment screening) or nonvertebral fractures occurred. Conclusion: BMD remained stable in women with PremenIOP who received bisphosphonates after sequential teriparatide-denosumab therapy.

Premenopausal women with idiopathic osteoporosis (PreMenIOP) have marked deficits in skeletal microstructure. We have reported that sequential treatment with teriparatide and denosumab improves central skeletal bone mineral density (BMD) by dual‐energy X‐ray absorptiometry and central QCT in PreMenIOP. We conducted preplanned analyses of high‐resolution peripheral quantitative computed tomography (HR‐pQCT) scans from teriparatide and denosumab extension studies to measure effects on volumetric BMD (vBMD), microarchitecture, and estimated strength at the distal radius and tibia. Of 41 women enrolled in the parent teriparatide study (20 mcg daily), 34 enrolled in the HR‐pQCT study. HR‐pQCT participants initially received teriparatide (N = 24) or placebo (N = 10) for 6 months; all then received teriparatide for 24 months. After teriparatide, 26 enrolled in the phase 2B denosumab extension (60 mg q6M) for 24 months. Primary outcomes were percentage change in vBMD, microstructure, and stiffness after teriparatide and after denosumab. Changes after sequential teriparatide and denosumab were secondary outcomes. After teriparatide, significant improvements were seen in tibial trabecular number (3.3%, p = 0.01), cortical area and thickness (both 2.7%, p < 0.001), and radial trabecular microarchitecture (number: 6.8%, thickness: 2.2%, separation: −5.1%, all p < 0.02). Despite increases in cortical porosity and decreases in cortical density, whole‐bone stiffness and failure load increased at both sites. After denosumab, increases in total (3.5%, p < 0.001 and 3.3%, p = 0.02) and cortical vBMD (1.7% and 3.2%; both p < 0.01), and failure load (1.1% and 3.6%; both p < 0.05) were seen at tibia and radius, respectively. Trabecular density (3.5%, p < 0.001) and number (2.4%, p = 0.03) increased at the tibia, while thickness (3.0%, p = 0.02) increased at the radius. After 48 months of sequential treatment, significant increases in total vBMD (tibia: p < 0.001; radius: p = 0.01), trabecular microstructure (p < 0.05), cortical thickness (tibia: p < 0.001; radius: p = 0.02), and whole bone strength (p < 0.02) were seen at both sites. Significant increases in total vBMD and bone strength parameters after sequential treatment with teriparatide followed by denosumab support the use of this regimen in PreMenIOP. © 2022 American Society for Bone and Mineral Research (ASBMR).

The incidence of diabetes mellitus and the associated complications are growing worldwide, affecting the patients' quality of life and exerting a considerable burden on health systems. Yet, the increase in fracture risk in type 1 diabetes (T1D) patients is not fully captured by bone mineral density (BMD), leading to the hypothesis that alterations in bone quality are responsible for the increased risk. Material/compositional properties are important aspects of bone quality, yet information on human bone material/compositional properties in T1D is rather sparse. The purpose of the present study is to measure both the intrinsic material behaviour by nanoindentation, and material compositional properties by Raman spectroscopy as a function of tissue age and microanatomical location (cement lines) in bone tissue from iliac crest biopsies from postmenopausal women diagnosed with long-term T1D (N = 8), and appropriate sex-, age-, BMD- and clinically-matched controls (postmenopausal women; N = 5). The results suggest elevation of advanced glycation endproducts (AGE) content in the T1D and show significant differences in mineral maturity / crystallinity (MMC) and glycosaminoglycan (GAG) content between the T1D and control groups. Furthermore, both hardness and modulus by nanoindentation are greater in T1D. These data suggest a significant deterioration of material strength properties (toughness) and compositional properties in T1D compared with controls.

Osteocytes act as bone mechanosensors, regulators of osteoblast/osteoclast activity and mineral homeostasis, however, knowledge about their functional/morphological changes throughout life is limited.We used quantitative backscattered electron imaging (qBEI) to investigate osteocyte lacunae sections (OLS) as a 2D-surrogate characterizing the osteocytes. OLS characteristics, the density of mineralized osteocyte lacunae (i.e., micropetrotic osteocytes, md.OLS-Density in nb/mm 2 ) and the average degree of mineralization (Ca Mean in weight% calcium) of cortex and spongiosa were analyzed in transiliac biopsy samples from healthy individuals under 30 (n = 59) and over 30 years (n = 50) (i.e., before and after the age of peak bone mass, respectively).We found several differences in OLS-characteristics: 1). Inter-individually between the age groups: OLS-Density and OLS-Porosity were reduced by about 20% in older individuals in spongiosa and in cortex versus younger probands (both, p < 0.001).2). Intra-individually between bone compartments: OLS-Density was higher in the cortex, + 18.4%, p < 0.001 for younger and + 7.6%, p < 0.05 for older individuals.Strikingly, the most frequent OLS nearest-neighbor distance was about 30 mu m in both age groups and at both bone sites revealing a preferential organization of osteocytes in clusters. OLS-Density was negatively correlated with Ca Mean in both spongiosa and cortex (both, p < 0.001). Few mineralized OLS were found in young individuals along with an increase of md.OLS-Density with age.In summary, this transiliac bone sample analysis of 20 0 0 0 0 OLS from 109 healthy individuals throughout lifespan reveals several age-related differences in OLS characteristics. Moreover, our study provides reference data from healthy individuals for different ages to be used for diagnosis of bone abnormalities in diseases.

The goal of this study is to investigate the causes of osteoporosis-related skeletal fragility in postmenopausal women. We hypothesize that bone fragility in these individuals is largely due to mineral, and/or intrinsic material properties in the osteocyte lacunar/peri-lacunar regions of bone tissue. Innovative measurements with nanoscale resolution, including scanning electron microscope (SEM), an atomic force microscope that is integrated with infrared spectroscopy (AFM-IR), and nanoindentation, were used to characterize osteocyte lacunar and peri-lacunar properties in bone biopsies from fracturing (Cases) and matched (Age, BMD), non-fracturing (Controls) postmenopausal healthy women. In the peri-lacunar space, the nanoindentation results show that the modulus and hardness of the Controls are lower than the Cases. The AFM-IR results conclusively show that the mineral matrix, maturity (peak) (except in outer/far regions in Controls) were greater in Controls than in Cases. Furthermore, these results indicated that while mineral-to-matrix area ratio tended to be greater, the mineral maturity and crystallinity peak ratio 'near' lacunae is greater than at regions 'far' or more distance from lacunae in the Controls only. Due to the heterogeneity of bone structure, additional measurements are needed to provide more convincing evidence of altered lacunar characteristics and changes in the peri-lacunar bone as mechanisms related to postmenopausal women and fragility. Such findings would motivate new osteocyte-targeted treatments to reduce fragility fracture risks in these groups.

Macro- and microarchitectural, bone material property, dynamic histomorphometric, and bone turnover marker data were studied in normal bone mineral density (BMD) post-menopausal women with fragility fracture. Women with fracture had thinner iliac cortices and more homogeneous bone material properties in cortical bone than age/BMD-matched non-fracture women. Low cortical thickness and bone tissue heterogeneity in normal BMD women are associated with prevalent fragility fracture. Bone mass (bone mineral density, (BMD)) of the spine and hip is today’s best single measurement for evaluating future fragility fracture risk. However, the majority of fragility fractures occur in women with BMD T-score above the WHO osteoporotic BMD threshold of − 2.5, indicating that non-BMD endpoints may play a role in their fragility fractures. We hypothesize that in non-osteoporotic women, bone micoarchitecture, bone material properties, dynamic histomorphometric endpoints, and bone turnover markers are related to fragility fracture. Two groups (N = 60 each) of post-menopausal women with total hip BMD T-score ranging from + 0.3 to –2.49 were recruited: fragility fracture and age/BMD-matched, non-fragility fracture women. Normal (T-score >  − 0.99) and osteopenic (T-score ≤  − 1.0) BMD cohorts were designated within both the fracture and non-fracture groups. Transiliac biopsy specimens were obtained to evaluate dynamic histomorphometric and microarchitectural endpoints and bone material properties by static and dynamic nanoindentation testing. All variables for fracture and non-fracture women within each BMD cohort were compared by the Wilcoxon signed-rank test (P < 0.01). Compared to non-fracture/normal BMD women, fracture/normal BMD women display lower iliac cortical thickness (− 12%, P = 0.0041) and lower heterogeneity of hardness (− 27%, P = 0.0068), elastic modulus (− 35%, P = 0.0009), and storage modulus (− 23%, P = 0.0054) in the cortical bone tissue, and lower heterogeneity of hardness (− 13%, P = 0.0088) in the trabecular bone tissue. Osteopenic women had no abnormalities related to fracture status. Post-menopausal women with normal BMD and fragility fracture have low cortical thickness and heterogeneity of several bone material properties in cortical and trabecular mineralized bone tissue. These differences may explain a portion of the excess bone fragility in women with normal BMD and fragility fracture.