Comprehensive Associations between Acidosis and the Skeleton in Patients with Kidney Disease.

SIGNIFICANCE STATEMENT: Renal osteodystrophy (ROD) contributes substantially to morbidity in CKD, including increased fracture risk. Metabolic acidosis (MA) contributes to the development of ROD, but an up-to-date skeletal phenotype in CKD-associated acidosis has not been described. We comprehensively studied associations between acidosis and bone in patients with CKD using advanced methods to image the skeleton and analyze bone-tissue, along with biochemical testing. Cross-sectionally, acidosis was associated with higher markers of bone remodeling and female-specific impairments in cortical and trabecular bone quality. Prospectively, acidosis was associated with cortical expansion and trabecular microarchitectural deterioration. At the bone-tissue level, acidosis was associated with deficits in bone mineral content. Future work investigating acidosis correction on bone quality is warranted. BACKGROUND: Renal osteodystrophy is a state of impaired bone quality and strength. Metabolic acidosis (MA) is associated with alterations in bone quality including remodeling, microarchitecture, and mineralization. No studies in patients with CKD have provided a comprehensive multimodal skeletal phenotype of MA. We aim to describe the structure and makeup of bone in patients with MA in the setting of CKD using biochemistry, noninvasive imaging, and histomorphometry. METHODS: The retrospective cross-sectional analyses included 180 patients with CKD. MA was defined as bicarbonate ≤22 mEq/L. We evaluated circulating bone turnover markers and skeletal imaging with dual energy x-ray absorptiometry and high-resolution peripheral computed tomography. A subset of 54 participants had follow-up. We assessed associations between baseline and change in bicarbonate with change in bone outcomes. Histomorphometry, microCT, and quantitative backscatter electron microscopy assessed bone biopsy outcomes in 22 participants. RESULTS: The mean age was 68±10 years, 54% of participants were male, and 55% were White. At baseline, acidotic subjects had higher markers of bone turnover, lower areal bone mineral density at the radius by dual energy x-ray absorptiometry, and lower cortical and trabecular volumetric bone mineral density and impaired trabecular microarchitecture. Over time, acidosis was associated with opposing cortical and trabecular effects: cortical expansion but trabecular deterioration. Bone-tissue analyses showed reduced tissue mineral density with increased heterogeneity of calcium distribution in acidotic participants. CONCLUSIONS: MA is associated with multiple impairments in bone quality. Future work should examine whether correction of acidosis improves bone quality and strength in patients with CKD.

Bone Material Properties in Bone Diseases Affecting Children.

PURPOSE OF REVIEW: Metabolic and genetic bone disorders affect not only bone mass but often also the bone material, including degree of mineralization, matrix organization, and lacunar porosity. The quality of juvenile bone is moreover highly influenced by skeletal growth. This review aims to provide a compact summary of the present knowledge on the complex interplay between bone modeling and remodeling during skeletal growth and to alert the reader to the complexity of bone tissue characteristics in children with bone disorders. RECENT FINDINGS: We describe cellular events together with the characteristics of the different tissues and organic matrix organization (cartilage, woven and lamellar bone) occurring during linear growth. Subsequently, we present typical alterations thereof in disorders leading to over-mineralized bone matrix compared to those associated with low or normal mineral content based on bone biopsy studies. Growth spurts or growth retardation might amplify or mask disease-related alterations in bone material, which makes the interpretation of bone tissue findings in children complex and challenging.

A Mild Case of Autosomal Recessive Osteopetrosis Masquerading as the Dominant Form Involving Homozygous Deep Intronic Variations in the CLCN7 Gene.

Osteopetrosis is a heterogeneous group of rare hereditary diseases characterized by increased bone mass of poor quality. Autosomal-dominant osteopetrosis type II (ADOII) is most often caused by mutation of the CLCN7 gene leading to impaired bone resorption. Autosomal recessive osteopetrosis (ARO) is a more severe form and is frequently accompanied by additional morbidities. We report an adult male presenting with classical clinical and radiological features of ADOII. Genetic analyses showed no amino-acid-converting mutation in CLCN7 but an apparent haploinsufficiency and suppression of CLCN7 mRNA levels in peripheral blood mononuclear cells. Next generation sequencing revealed low-frequency intronic homozygous variations in CLCN7, suggesting recessive inheritance. In silico analysis of an intronic duplication c.595-120_595-86dup revealed additional binding sites for Serine- and Arginine-rich Splicing Factors (SRSF), which is predicted to impair CLCN7 expression. Quantitative backscattered electron imaging and histomorphometric analyses revealed bone tissue and material abnormalities. Giant osteoclasts were present and additionally to lamellar bone, and abundant woven bone and mineralized cartilage were observed, together with increased frequency and thickness of cement lines. Bone mineralization density distribution (BMDD) analysis revealed markedly increased average mineral content of the dense bone (CaMean T-score + 10.1) and frequency of bone with highest mineral content (CaHigh T-score + 19.6), suggesting continued mineral accumulation and lack of bone remodelling. Osteocyte lacunae sections (OLS) characteristics were unremarkable except for an unusually circular shape. Together, our findings suggest that the reduced expression of CLCN7 mRNA in osteoclasts, and possibly also osteocytes, causes poorly remodelled bone with abnormal bone matrix with high mineral content. This together with the lack of adequate bone repair mechanisms makes the material brittle and prone to fracture. While the skeletal phenotype and medical history were suggestive of ADOII, genetic analysis revealed that this is a possible mild case of ARO due to deep intronic mutation.

No evidence of mineralization abnormalities in iliac bone of premenopausal women with type 2 diabetes mellitus.

OBJECTIVES: Patients with type-2 diabetes mellitus (T2DM) have increased risk for bone fractures which points towards impaired bone quality. METHODS: We measured bone mineralization density distribution (BMDD) and osteocyte lacunae section (OLS) characteristics based on quantitative backscattered electron images of transiliac biopsy samples from n=26 premenopausal women with T2DM. Outcomes were compared to those from reference cohorts as well as between T2DM subgroups defined by clinical characteristics. RESULTS: Comparison to references did not reveal any differences in BMDD (all p>0.05) but a lowered OLS-density in cancellous bone in T2DM (-14.9%, p<0.001). Neither BMDD nor OLS-characteristics differed in T2DM subgroups defined by HbA1c (<7% versus >7%). The average degree of bone mineralization (CaMean) was higher (0.44 wt%Ca in T2DM, 0.30 wt%Ca in reference) and consistently the calcium concentration between the tetracycline double labels (CaYoung) was higher (0.76 wt%Ca, all p<0.001) in cancellous versus cortical bone. CONCLUSIONS: Our findings suggest that bone matrix mineralization was neither affected by the presence nor by the glycemic control of T2DM in our study cohort. The intra-individual differences between cancellous and cortical bone mineralization gave evidence for differences in the time course of the early mineralization process in these compartments in general.

Quantitative Backscattered Electron Imaging of Bone Using a Thermionic or a Field Emission Electron Source.

Quantitative backscattered electron imaging is an established method to map mineral content distributions in bone and to determine the bone mineralization density distribution (BMDD). The method we applied was initially validated for a scanning electron microscope (SEM) equipped with a tungsten hairpin cathode (thermionic electron emission) under strongly defined settings of SEM parameters. For several reasons, it would be interesting to migrate the technique to a SEM with a field emission electron source (FE-SEM), which, however, would require to work with different SEM parameter settings as have been validated for DSM 962. The FE-SEM has a much better spatial resolution based on an electron source size in the order of several 100 nanometers, corresponding to an about [Formula: see text] to [Formula: see text] times smaller source area compared to thermionic sources. In the present work, we compare BMDD between these two types of instruments in order to further validate the methodology. We show that a transition to higher pixel resolution (1.76, 0.88, and 0.57 µm) results in shifts of the BMDD peak and BMDD width to higher values. Further the inter-device reproducibility of the mean calcium content shows a difference of up to 1 wt% Ca, while the technical variance of each device can be reduced to [Formula: see text] wt% Ca. Bearing in mind that shifts in calcium levels due to diseases, e.g., high turnover osteoporosis, are often in the range of 1 wt% Ca, both the bone samples of the patients as well as the control samples have to be measured on the same SEM device. Therefore, we also constructed new reference BMDD curves for adults to be used for FE-SEM data comparison.

Clinical Phenotype and Bone Biopsy Characteristics in a Child with Proteus Syndrome.

Proteus syndrome is a rare genetic disorder, which is characterized by progressive, segmental, or patchy overgrowth of diverse tissues of all germ layers, including the skeleton. Here, we present a 9-year-old girl with a somatic-activating mutation (c.49G > A; p.Glu17Lys) in AKT1 gene in a mosaic status typical for Proteus syndrome. She presented with hemihypertrophy of the right lower limb and a "moccasin" lesion among others. A transiliac bone biopsy was analyzed for bone histology/histomorphometry as well as bone mineralization density distribution (BMDD) and osteocyte lacunae sections (OLS) characteristics based on quantitative backscattered electron imaging. Bone histomorphometry revealed highly increased mineralizing surface (Z-score + 2.3) and mineral apposition rate (Z-score + 19.3), no osteoclasts (Z-score - 2.1), and an increased amount of primary bone in the external cortex. BMDD abnormalities included a decreased mode calcium concentration in cancellous bone (Z-score - 1.7) and an increased percentage of highly mineralized cortical bone area (Z-score + 2.4) compared to reference. OLS characteristics showed several differences compared to reference data; among them, there were the highly increased OLS-porosity, OLS-area, and OLS-perimeter on the external cortex (Z-scores + 6.8, + 4.4 and 5.4, respectively). Our findings suggest that increased bone formation reduced matrix mineralization in cancellous bone while the enhanced amount of primary bone in the external cortex increased the portion of highly mineralized cortical bone and caused OLS-characteristics abnormalities. Our results indicate further that remodeling of primary bone might be disturbed or delayed in agreement with the decreased number of osteoclasts observed in this child with Proteus syndrome.

Increased Osteocyte Lacunae Density in the Hypermineralized Bone Matrix of Children with Osteogenesis Imperfecta Type I.

Osteocytes are terminally differentiated osteoblasts embedded within the bone matrix and key orchestrators of bone metabolism. However, they are generally not characterized by conventional bone histomorphometry because of their location and the limited resolution of light microscopy. OI is characterized by disturbed bone homeostasis, matrix abnormalities and elevated bone matrix mineralization density. To gain further insights into osteocyte characteristics and bone metabolism in OI, we evaluated 2D osteocyte lacunae sections (OLS) based on quantitative backscattered electron imaging in transiliac bone biopsy samples from children with OI type I (n = 19) and age-matched controls (n = 24). The OLS characteristics were related to previously obtained, re-visited histomorphometric parameters. Moreover, we present pediatric bone mineralization density distribution reference data in OI type I (n = 19) and controls (n = 50) obtained with a field emission scanning electron microscope. Compared to controls, OI has highly increased OLS density in cortical and trabecular bone (+50.66%, +61.73%; both p < 0.001), whereas OLS area is slightly decreased in trabecular bone (-10.28%; p = 0.015). Correlation analyses show a low to moderate, positive association of OLS density with surface-based bone formation parameters and negative association with indices of osteoblast function. In conclusion, hyperosteocytosis of the hypermineralized OI bone matrix associates with abnormal bone cell metabolism and might further impact the mechanical competence of the bone tissue.

Bone properties in osteogenesis imperfecta: what can we learn from a bone biopsy beyond histology?

Transiliac bone biopsy samples are used to evaluate histology and bone cell activity in unclear pathological conditions. However, much additional information can be obtained from such bone samples. Using the example of osteogenesis imperfecta (OI), the current article describes how biopsy samples can be further used to study bone material characteristics including the degree of matrix mineralization, organic matrix properties, mineral particle size and bone nanoporosity. OI is a heritable collagen-related disorder that is phenotypically and genetically extremely heterogeneous. One essential finding was that OI bone is hypermineralized independently of clinical severity. Moreover, mineral particles in OI bone are of normal size or even smaller, but more densely packed than normally. Another recent finding was that in some forms of OI, collagen orientation is highly disorganized, indicating that the collagen-mineral particle network is profoundly altered in OI. These findings have contributed to the understanding of impaired bone strength in OI.

Osteoporosis Therapeutics 2020.

Numerous safe and efficient drug therapies are currently available to decrease risk of low trauma fractures in patients with osteoporosis including postmenopausal, male, and secondary osteoporosis. In this chapter, we give first an overview of the most important outcomes regarding fracture risk reduction, change in bone mineral density (BMD by DXA) and/or bone markers of the phase III clinical studies of well-established therapies (such as Bisphosphonates, Denosumab or Teriparatide) and also novel therapies (such as Romosozumab or Abaloparatide) and highlight their mechanisms of action at bone tissue/material level. The latter understanding is not only essential for the choice of drug, duration and discontinuation of treatment but also for the interpretation of the clinical outcomes (in particular of eventual changes in BMD) after drug administration. In the second part of this chapter, we focus on the management of different forms of osteoporosis and give a review of the respective current guidelines for treatment. Adverse effects of treatment such as atypical femoral fractures, osteonecrosis of the jaw or influence of fracture healing are considered also in this context.

No Role of Osteocytic Osteolysis in the Development and Recovery of the Bone Phenotype Induced by Severe Secondary Hyperparathyroidism in Vitamin D Receptor Deficient Mice.

Osteocytic osteolysis/perilacunar remodeling is thought to contribute to the maintenance of mineral homeostasis. Here, we utilized a reversible, adult-onset model of secondary hyperparathyroidism to study femoral bone mineralization density distribution (BMDD) and osteocyte lacunae sections (OLS) based on quantitative backscattered electron imaging. Male mice with a non-functioning vitamin D receptor (VDRΔ/Δ) or wild-type mice were exposed to a rescue diet (RD) (baseline) and subsequently to a low calcium challenge diet (CD). Thereafter, VDRΔ/Δ mice received either the CD, a normal diet (ND), or the RD. At baseline, BMDD and OLS characteristics were similar in VDRΔ/Δ and wild-type mice. The CD induced large cortical pores, osteomalacia, and a reduced epiphyseal average degree of mineralization in the VDRΔ/Δ mice relative to the baseline (-9.5%, p < 0.05 after two months and -10.3%, p < 0.01 after five months of the CD). Switching VDRΔ/Δ mice on the CD back to the RD fully restored BMDD to baseline values. However, OLS remained unchanged in all groups of mice, independent of diet. We conclude that adult VDRΔ/Δ animals on an RD lack any skeletal abnormalities, suggesting that VDR signaling is dispensable for normal bone mineralization as long as mineral homeostasis is normal. Our findings also indicate that VDRΔ/Δ mice attempt to correct a calcium challenge by enhanced osteoclastic resorption rather than by osteocytic osteolysis.

Bone matrix mineralization and osteocyte lacunae characteristics in patients with chronic kidney disease - mineral bone disorder (CKD-MBD).

OBJECTIVES: Little is known about bone mineralization and osteocyte lacunae properties in chronic kidney disease mineral bone disorder (CKD-MBD). METHODS: In this retrospective study, we measured the bone mineralization density distribution (BMDD) and osteocyte lacunar section (OLS) 2D-characteristics by quantitative backscatter electron imaging in Straumann drill biopsy samples from n=58 patients with CKD-MBD. Outcomes were studied in relation to serum parathyroid hormone (PTH), alkaline phosphatase (APH), histomorphometric bone turnover and treatment with cinacalcet or phosphate binders. RESULTS: Lower calcium concentrations in bone from high turnover (average degree of bone mineralization -6.2%, p<0.001) versus low turnover patients were observed. OLS-characteristics were distinctly different (p<0.01 to p<0.05) in patients with highest compared to those with lowest turnover. Patients with cinacalcet had different OLS-characteristics (p<0.05) compared to those without cinacalcet. Furthermore, patients with phosphate binders had differences in BMDD and OLS-characteristics (p<0.05) compared to patients without phosphate binders. CONCLUSIONS: Our findings suggest that in patients with CKD-MBD secondary hyperparathyroidism and increased bone turnover decrease the average degree of bone matrix mineralization. Conversely, density and lacunar size of the osteocytes are increased compared to adynamic bone disease pointing at distinct patterns of bone mineralization and osteocyte lacunar properties in these two disease entities.

Confocal laser scanning microscopy-a powerful tool in bone research.

The confocal laser scanning microscope (CLSM) enables the collection of images picturing selected planes in depth of thick samples, thus giving 3D information while keeping the sample intact. In this article we give an overview of our CLSM applications in bone research: (i) the characterization of osteoblasts and osteoclasts properties in cell biology, (ii) the visualization of the three dimensional (3D) osteocyte lacunar canalicular network in undemineralized plastic-embedded bone samples, (iii) the observation of tetracycline labels in bone biopsy samples from patients in combination with information on the mineralization density from quantitative backscatter electron imaging, which enables the time course of mineral accumulation in newly formed bone to be followed, (iv) the precise measurement of the thickness of thin ground bone sections, a prerequisite for the mapping of local mechanical properties by scanning acoustic microscopy.

Skeletal Implications of Chronic Obstructive Pulmonary Disease.

Chronic obstructive pulmonary disease (COPD) is associated with numerous comorbidities, among which osteoporosis is of high significance. Low bone mass and the occurrence of fragility fractures is a common finding in patients with COPD. Typical risk factors related directly or indirectly to these skeletal complications include systemic inflammation, tobacco smoking, vitamin D deficiency, and treatment with oral or inhaled corticosteroids. In particular, treatment with glucocorticoids appears to be a strong contributor to bone changes in COPD, but does not fully account for all skeletal complications. Additional to the effects of COPD on bone mass, there is evidence for COPD-related changes in bone microstructure and material properties. This review summarizes the clinical outcomes of low bone mass and increased fracture risk, and reports on recent observations in bone tissue and material in COPD patients.

Ibandronate increases sclerostin levels and bone strength in male patients with idiopathic osteoporosis.

The pathomechanism of male idiopathic osteoporosis (MIO) differs from postmenopausal osteoporosis with regard to alterations in osteoblast activity. We evaluated intravenous ibandronate (IBN) in 25 MIO patients with fragility fractures in a prospective, monocentric, single-arm, and open-label study for 24 months. The impact and changes of sclerostin (Scl), Dickkopf-1 (DKK-1), CTX, and PINP were examined. Additionally, volumetric cortical, trabecular and areal bone mineral density (BMD), trabecular bone score (TBS), and finite element analyses (FEA) were evaluated. Compared to baseline, median Scl levels were increased after 1 month (Δ 121%, p < 0.0001) and remained elevated for 12 months. DKK-1 decreased (p < 0.001) to a lesser extent until month 9 with values comparable to baseline at study endpoint. Early changes (baseline-month 1) of Scl negatively correlated with early changes of DKK-1 (-0.72), CTX (-0.82), and PINP (-0.55; p < 0.005 for all). The overall changes over the 24 months study period of Scl negatively correlated with decreased CTX (-0.32) and DKK-1 levels (-0.57, p < 0.0001 for both); CTX and PINP changes positively correlated at each time point (p < 0.001). Volumetric hip BMD increased by 12 and 18%, respectively (p < 0.0001 for both). Cross-sectional moment of inertia and section modulus for total hip significantly improved (p < 0.05 for all). Areal BMD at total hip, spine, and TBS increased. FEA displayed an increase in bone strength both in the hip (17%) and vertebrae (13%, all p < 0.0001) at anatomical sites susceptible for fragility fracture. IBN increases Scl and improves cortical and trabecular bone strength with early and ongoing vigorous suppression of bone resorption.

Bone mass and mineralization in osteogenesis imperfecta.

The main clinical features of osteogenesis imperfecta (OI) are low bone mass and high bone fragility. While the decrease in bone mass is generally regarded as an indicator of disease severity, bone fragility appears as the hallmark of the disorder. Bone has a multiscale hierarchical structural organization and is optimized to resist to fractures. In OI, modifications at the molecular level affect the total mechanical integrity of the bone. A specific characteristic in OI is that the bone matrix is abnormally high mineralized independently of the underlying mutation or clinical severity. The increased matrix mineralization affects bone material quality, leading to increased stiffness and brittleness and making bone prone to fractures. The purpose of this review is to give further insights on bone matrix mineralization in OI and to discuss advantages and pitfalls of invasive and noninvasive imaging techniques.

Classification of osteogenesis imperfecta.

Osteogenesis imperfecta (OI) is an extremely heterogeneous group of heritable connective tissue disorders. Most of the affected patients carry autosomal dominant mutations in the genes encoding for collagen type I, the most abundant protein of the bone extracellular matrix. The resulting phenotypes are extremely broad and have been classified by Sillence and colleagues into four groups according to clinical, radiological and genetic criteria.More recently, proteins have been described that interact directly or indirectly with collagen biosynthesis and their deficiency result in rare forms of mostly autosomal recessive OI sharing phenotypic features of 'classical' types but lacking primary defects in type I collagen. Consequently the Sillence classification has been gradually expanded to include novel forms based on the underlying mutations. The goal of this article is to revisit the actual OI classification and to outline current approaches in categorizing the disorder.

Changes in the degree of mineralization with osteoporosis and its treatment.

The diagnosis of osteoporosis is based on low bone mineral density (BMD) and/or the occurrence of fragility fractures. The majority of patients, however, have also abnormally low bone matrix mineralization. The latter is indicative of alterations in bone turnover rates and/or in kinetics of mineral accumulation within the newly formed bone matrix. Osteoporosis therapies can alter the bone matrix mineralization according to their action on bone turnover and/or mineralization kinetics. Antiresorptives, including the most widely used bisphosphonates, reduce the bone turnover rate resulting in a decrease in heterogeneity and an increase in the degree of mineralization toward to or even beyond normal values. Anabolic agents increase the bone volume and the amount of newly formed bone resulting in a likely transient decrease in mean degree and homogeneity of mineralization. Hence, the measurement of bone matrix mineralization is a sensitive tool to evaluate the response to therapy.

Changes in bone matrix mineralization after growth hormone treatment in children and adolescents with chronic kidney failure treated by dialysis: a paired biopsy study.

BACKGROUND: Patients with chronic kidney disease (CKD) develop renal osteodystrophy with alterations in bone turnover, mineralization, and volume (TMV). A specific skeletal complication in children is growth impairment, which currently is treated by recombinant human growth hormone (rhGH). The effects on bone material properties are poorly understood. This study assesses the effects of rhGH treatment on bone matrix mineralization. STUDY DESIGN: Observational study. SETTING & PARTICIPANTS: 18 short children and adolescents (aged 3.6-16 years) with CKD on dialysis therapy. PREDICTOR: rhGH treatment for 1 year. OUTCOMES: Tetracycline-labeled bone biopsy classified according to the TMV system. MEASUREMENTS: Bone mineralization density distribution (BMDD) was evaluated by quantitative backscattered electron imaging in trabecular and cortical compartments. Additional data for patients' height and biochemical bone serum parameters were obtained. RESULTS: Prior to rhGH treatment, our cohort showed low bone turnover and high mineralization densities versus reference data: Ca(mean) (weighted mean calcium content) in cancellous bone, +3.3% (P = 0.04); Ca(mean) in cortical bone, +6.7% (P < 0.001); Ca(peak) (mode of the BMDD) in cancellous bone, +5.0% (P < 0.001); Ca(peak) in cortical bone, +8.2% (P < 0.001); Ca(width) (heterogeneity in mineralization), no significant difference for cancellous (P = 0.2) and cortical (P = 0.1) bone; Ca(high) (portion of fully mineralized bone) in cancellous bone, 5-fold greater (P < 0.001); Ca(high) in cortical bone, 14-fold greater (P < 0.001); Ca(low) (portion of low mineralized bone) in cancellous bone, +23.9% (P = 0.02); Ca(low) in cortical bone, -22.2% (P = 0.05). After rhGH treatment, height increased by 9.1 cm (P < 0.001) and bone turnover indices to normal values or beyond. Matrix mineralization was lesser and more heterogeneous compared to baseline: Ca(width) for cancellous bone, +15.3% (P < 0.001); Ca(width) for cortical bone, +34.1% (P < 0.001). Ca(mean), Ca(peak), and Ca(high) for cancellous bone and Ca(mean) and Ca(peak) for cortical bone were no longer significantly different from reference data. Ca(high) for cortical bone dramatically decreased after treatment but was still substantially greater than reference data. LIMITATIONS: Low case number per TMV subgroup, no measurements of fibroblast growth factor 23. CONCLUSIONS: Children and adolescents with CKD and growth deficiency are at risk of having low bone turnover. rhGH treatment improves height and concomitantly bone modeling/remodeling, which appears beneficial for bone matrix mineralization.

Vitamin C Deficiency Deteriorates Bone Microarchitecture and Mineralization in a Sex-Specific Manner in Adult Mice.

Vitamin C (VitC) is essential for bone health, and low VitC serum levels increase the risk for skeletal fractures. If and how VitC affects bone mineralization is unclear. Using micro-computed tomography (µCT), histologic staining, as well as quantitative backscattered electron imaging (qBEI), we assessed the effects of VitC on femoral structure and microarchitecture, bone formation, and bone mineralization density distribution (BMDD) in the VitC incompetent Gulo-/- mouse model and wild-type mice. In particular, VitC-supplemented, 20-week-old mice were compared with age-matched counterparts where dietary VitC intake was excluded from week 15. VitC depletion in Gulo-/- mice severely reduced cortical thickness of the diaphyseal shaft and bone volume around the growth plate (eg, bone volume of the primary spongiosa -43%, p < 0.001). Loss of VitC also diminished the amount of newly formed bone tissue as visualized by histology and calcein labeling of the active mineralization front. BMDD analysis revealed a shift to higher calcium concentrations upon VitC supplementation, including higher average (~10% increase in female VitC deficient mice, p < 0.001) and peak calcium concentrations in the epiphyseal and metaphyseal spongiosa. These findings suggest higher bone tissue age. Importantly, loss of VitC had significantly more pronounced effects in female mice, indicating a higher sensitivity of their skeleton to VitC deficiency. Our results reveal that VitC plays a key role in bone formation rate, which directly affects mineralization. We propose that low VitC levels may contribute to the higher prevalence of bone-degenerative diseases in females and suggest leveraging this vitamin against these conditions. © 2023 American Society for Bone and Mineral Research (ASBMR).

Osteocyte lacunae in transiliac bone biopsy samples across life span.

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/mm2) and the average degree of mineralization (CaMean 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 µm in both age groups and at both bone sites revealing a preferential organization of osteocytes in clusters. OLS-Density was negatively correlated with CaMean 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 200000 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. STATEMENT OF SIGNIFICANCE: Osteocytes are bone cells embedded in lacunae within the mineralized bone matrix and have a key role in the bone metabolism and the mineral homeostasis. Not easily accessible, we used quantitative backscattered electron imaging to determine precisely number and shape descriptors of the osteocyte lacunae in 2D. We analyzed transiliac biopsy samples from 109 individuals with age distributed from 2 to 95 years. Compact cortical bone showed constantly higher lacunar density than cancellous bone but the lacunar density in both bone tissue decreased with age before the peak bone mass age at 30 years and stabilized or even increased after this age. This extensive study provides osteocyte lacunae reference data from healthy individuals usable for bone pathology diagnosis.