Transiliac Bone Biopsy Research Articles

Network Analysis of Brain and Bone Tissue Transcripts Reveals Shared Molecular Mechanisms Underlying Alzheimer's Disease and Related Dementias and Osteoporosis.

Alzheimer's disease and related dementias (ADRD) and osteoporosis (OP) are 2 prevalent diseases of aging with demonstrated epidemiological association, but the underlying molecular mechanisms contributing to this association are unknown. We used network analysis of bone and brain transcriptomes to discover common molecular mechanisms underlying these 2 diseases. Our study included RNA-sequencing data from the dorsolateral prefrontal cortex tissue of autopsied brains in 629 participants from ROSMAP (Religious Orders Study and the Rush Memory and Aging Project), with a subgroup of 298 meeting criteria for inclusion in 5 ADRD categories, and RNA array data from transiliac bone biopsies in 84 participants from the Oslo study of postmenopausal women. After developing each network within each tissue, we analyzed associations between modules (groups of coexpressed genes) with multiple bone and neurological traits, examined overlap in modules between networks, and performed pathway enrichment analysis to discover conserved mechanisms. We discovered 3 modules in ROSMAP that showed significant associations with ADRD and bone-related traits and 4 modules in Oslo that showed significant associations with multiple bone outcomes. We found significant module overlap between the 2 networks in modules linked to signaling, tissue homeostasis, and development, and Wingless-related integration site (Wnt) signaling was found to be highly enriched in OP and ADRD modules of interest. These results provide translational opportunities in the development of treatments and biomarkers for ADRD and OP.

Differences in tissue-level properties as assessed by nano-scratching in patients with and without atypical femur fractures on long-term bisphosphonate therapy: a proof-of-concept pilot study.

Atypical femur fractures (AFFs) are a well-established complication of long-term bisphosphonate (BP) therapy, but their pathogenesis is not fully understood. Although many patients on long-term BP therapy have severe suppression of bone turnover (SSBT), not all such patients experience AFF, even though SSBT is a major contributor to AFF. Accordingly, we evaluated tissue level properties using nano-scratch testing of trans-iliac bone biopsy specimens in 12 women (6 with and 6 without AFF matched for age and race). Nano-scratch data were analyzed using a mixed-model ANOVA with volume-normalized scratch energy as a function of AFF (Yes or No), region (periosteal or endosteal), and a first-order interaction between region and AFF. Tukey post hoc analyses of the differences of least squared means of scratch energy were performed and reported as significant if p<.05. The volume-normalized scratch energy was 10.6% higher in AFF than in non-AFF patients (p=.003) and 17.9 % higher in the periosteal than in the endosteal region (p=.004). The differences in normalized scratch energy are suggestive of a higher hardness of the bone tissue after long-term BP therapy. The results of this study are consistent with other studies in the literature and demonstrate the efficacy of using Nano-Scratch technique to evaluate bone tissue that exhibits SSBT and AFF. Further studies using nano-scratch may help quantify and elucidate underlying mechanisms for the pathogenesis of AFF.

Evaluation of romosozumab's effects on bone marrow adiposity in postmenopausal osteoporotic women: results from the FRAME bone biopsy sub-study.

Romosozumab, a humanized monoclonal antibody that binds and inhibits sclerostin, produces a marked increase in bone formation with a concomitant decreased bone resorption. This transient rise in bone formation in the first 2 months of treatment is mainly due to an increased modeling-based bone formation. This requires the recruitment and differentiation of osteoblasts, one possibility being a preferential switch in commitment of precursors to osteoblasts over adipocytes. The purpose of this study was to analyze the marrow adiposity in transiliac bone biopsies at months 2 or 12 from the FRAME biopsy sub-study in patients receiving romosozumab or placebo. The total adipocyte area, number, and density were measured on the total cancellous bone area. The size and shape at the individual adipocyte level were assessed including the mean adipocyte area, perimeter, min and max diameters, and aspect ratio. No significant difference in total adipocyte area, number, or density between placebo and romosozumab groups was observed at months 2 and 12, and no difference was observed between 2 and 12months. After 2 or 12months, romosozumab did not modify the size or shape of the adipocytes. No relationship between the adipocyte parameters and the dynamic parameters of bone formation could be evidenced. In conclusion, based on the analysis of a small number of biopsies, no effect of romosozumab on bone marrow adiposity of iliac crest was identified after 2 and 12months suggesting that the modeling-based formation observed at month 2 was not due to a preferential commitment of the precursor to osteoblast over adipocyte cell lines but may result from a reactivation of bone lining cells and from a progenitor pool independent of the marrow adipocyte population.

Identification of Transcripts with Shared Roles in the Pathogenesis of Postmenopausal Osteoporosis and Cardiovascular Disease.

Epidemiological evidence suggests existing comorbidity between postmenopausal osteoporosis (OP) and cardiovascular disease (CVD), but identification of possible shared genes is lacking. The skeletal global transcriptomes were analyzed in trans-iliac bone biopsies (n = 84) from clinically well-characterized postmenopausal women (50 to 86 years) without clinical CVD using microchips and RNA sequencing. One thousand transcripts highly correlated with areal bone mineral density (aBMD) were further analyzed using bioinformatics, and common genes overlapping with CVD and associated biological mechanisms, pathways and functions were identified. Fifty genes (45 mRNAs, 5 miRNAs) were discovered with established roles in oxidative stress, inflammatory response, endothelial function, fibrosis, dyslipidemia and osteoblastogenesis/calcification. These pleiotropic genes with possible CVD comorbidity functions were also present in transcriptomes of microvascular endothelial cells and cardiomyocytes and were differentially expressed between healthy and osteoporotic women with fragility fractures. The results were supported by a genetic pleiotropy-informed conditional False Discovery Rate approach identifying any overlap in single nucleotide polymorphisms (SNPs) within several genes encoding aBMD- and CVD-associated transcripts. The study provides transcriptional and genomic evidence for genes of importance for both BMD regulation and CVD risk in a large collection of postmenopausal bone biopsies. Most of the transcripts identified in the CVD risk categories have no previously recognized roles in OP pathogenesis and provide novel avenues for exploring the mechanistic basis for the biological association between CVD and OP.

Differences in bone histomorphometry between White postmenopausal women with and without atypical femoral fracture after long-term bisphosphonate therapy.

Bone histomorphometric endpoints in transilial biopsies may be associated with an increased risk of atypical femoral fracture (AFF) in patients with osteoporosis who take antiresorptives, including bisphosphonates (BPs). One way to test this hypothesis is to evaluate bone histomorphometric endpoints in age-, gender-, and treatment time-matched patients who either had AFF or did not have AFF. In this study, we performed transiliac bone biopsies in 52 White postmenopausal women with (n= 20) and without (n= 32) AFFs, all of whom had been treated for osteoporosis continuously with alendronate for 4-17yr. Despite the matched range of treatment duration (4-17yr), AFF patients received alendronate for significantly longer time (10.7yr) than non-AFF patients (8.0yr) (P= .014). Bone histomorphometric endpoints reflecting microstructure and turnover were assessed in cancellous, intracortical, and endocortical envelopes from transilial biopsy specimens obtained from BP-treated patients 3-6mo after AFF and from non-AFF patients with similar age-, gender-, and range of BP treatment duration. However, in both cancellous and intracortical envelopes, AFF patients had significantly lower wall thickness (W.Th) and higher osteoclast surface (Oc.S/BS) than non-AFF patients. In addition, AFF patients had significantly higher eroded surface (ES/BS) only in the intracortical envelope. None of the dynamic variables related to bone formation and turnover differed significantly between the groups. In conclusion, in the ilium of BP-treated patients with osteoporosis, AFF patients have lower thickness of superficial bone (lower W.Th) of the cancellous and cortical envelopes than non-AFF patients. AFF and non-AFF patients have a similar bone turnover rate in the ilium. Furthermore, in this population, as in previous work, AFF is more likely to occur in BP-treated patients with longer treatment duration.

Pre-treatment bone turnover does not influence the level of the response to alendronate in postmenopausal osteoporosis at the bone tissue level.

The main effect of anti-resorptive agents such as bisphosphonates is a reduction of bone resorption, with a consequent marked decrease of bone turnover. This post-hoc analysis investigated the changes of histomorphometric parameters of bone turnover after alendronate (ALN), according to the baseline turnover. Ninety postmenopausal women underwent a transiliac bone biopsy before and after 6 (n = 44) or 12 (n = 46) months of treatment with ALN (70 mg/week). The dynamic parameters reflecting the bone formation and bone turnover were mineralizing surface (MS/BS; %), bone formation rate (BFR/BS; μm3/μm2/d), and activation frequency (Ac.f; /yr). Biochemical markers sPINP and the sCTX were assessed before treatment and after 3, 6, and 12 months. Subjects were divided into quartiles based on the baseline values of BFR/BS. At baseline, MS/BS and Ac.f were significantly different (p < 0.0001) among the BFR quartiles. sCTX and sP1NP were not significantly different among quartiles. After ALN treatment, MS/BS was not significantly different among quartiles but Ac.f remained significantly lower in the first quartile compared to the third and fourth ones (p < 0.03). The absolute value of the difference between pre- and post-treatment significantly correlated with the baseline BFR/BS but when expressed in percent of the baseline value, the magnitude of the diminutions of MS/BS, Ac.f, sCTX, and sP1NP was similar in the four baseline BFR quartiles. The percentage response to ALN appeared independent of the baseline level of bone turnover. After treatment, the bone turnover tended to be similar in all BFR quartiles. This analysis investigated the influence of baseline turnover measured by bone histomorphometry on the effect of alendronate. When expressed in percent of pre-treatment values, the decreases of histomorphometric parameters and biochemical markers of bone turnover were independent of the baseline turnover.

Associations of Changes in Bone Turnover Markers with Change in Bone Mineral Density in Kidney Transplant Patients.

Bone loss after kidney transplantation is highly variable. We investigated whether changes in bone turnover markers associate with bone loss during the first post-transplant year. Bone mineral density (BMD) was measured at 0 and 12 months, with biointact parathyroid hormone, bone-specific alkaline phosphatase (BALP), intact procollagen type I N -terminal propeptide (PINP), and tartrate-resistant acid phosphatase isoform 5b (TRAP5b) measured at 0, 3, and 12 months post-transplant ( N =209). Paired transiliac bone biopsies were available in a subset ( n =49). Between-group differences were evaluated by Student's t test, Wilcoxon signed-rank test, or Pearson's chi-squared test. Changes in BMD varied from -22% to +17%/yr. Compared with patients with no change (±2.5%/yr), patients who gained BMD had higher levels of parathyroid hormone (236 versus 136 pg/ml), BALP (31.7 versus 18.8 μ g/L), and Intact PINP (121.9 versus 70.4 μ g/L) at time of transplantation; a greater decrease in BALP (-40% versus -21%) and Intact PINP (-43% versus -13%) by 3 months; and lower levels of Intact PINP (36.3 versus 60.0 μ g/L) at 12 months post-transplant. Patients who lost BMD had a less marked decrease, or even increase, in Intact PINP (+22% versus -13%) and TRAP5b (-27% versus -43%) at 3 months and higher Intact PINP (83.7 versus 60.0 μ g/L) and TRAP5b (3.89 versus 3.16 U/L) at 12 months compared with patients with no change. If none of the biomarkers decreased by the least significant change at 3 months, an almost two-fold (69% versus 36%) higher occurrence of bone loss was seen at 12 months post-transplant. Bone loss after kidney transplantation was highly variable. Resolution of high bone turnover, as reflected by decreasing bone turnover markers, associated with BMD gain, while increasing bone turnover markers associated with bone loss.

Histomorphometric reference data of transiliac bone biopsy in children from 8 to 17 years old.

Histomorphometric analysis of bone samples is a key tool for studying bone metabolism; however, only a few pediatric reference data exist. The aim of the present study is to report more reference data and to investigate if histomorphometric differences exist between age and gender. We obtained 19 transiliac bone samples previously marked with tetracycline, from children between 8 and 17 years (13 were male), with normal blood test results and urine biochemical bone markers. We evaluated bone histomorphometric parameters using a digitalizing table with osteomeasure to obtain normative data of means and standard deviations, as well as median and range. Due to the small sample, a Monte Carlo simulation was applied. Structural, static, dynamic, and resorptic histomorphometric parameters were evaluated by age and gender following the American Society for Bone and Mineral Research recommendations. Bone volume (in the older children) and mineral apposition rate (in the younger children), the eroded surface (in boys), and the new bone wall thickness (in girls) were significantly increased. On the trabecular area of mineralization front, the modeling and the remodeling bone formation were similar (16 and 18%). The rest of the histomorphometric bone parameters by age and gender showed no significant difference. In healthy children, these bone histomorphometric findings, with these techniques and for this ages could be used as reference values.

Bone Structure and Turnover in Postmenopausal Women With Long-Standing Type 1 Diabetes.

Compromised bone structural and mechanical properties are implicated in the increased fracture risk in type 1 diabetes (T1D). We investigated bone structure and turnover by histomorphometry in postmenopausal women with T1D and controls without diabetes using tetracycline double-labeled transiliac bone biopsy. After in vivo tetracycline double labeling, postmenopausal women with T1D of at least 10 years and without diabetes underwent transiliac bone biopsy. An expert blinded to the study group performed histomorphometry. Static and dynamic histomorphometry measurements were performed and compared between the two groups. The analysis included 9 postmenopausal women with T1D (mean age 58.4 ± 7.1 years with 37.9 ± 10.9 years of diabetes and HbA1c 7.1% ± 0.4%) and 7 postmenopausal women without diabetes (mean age 60.9 ± 3.3 years and HbA1c 5.4% ± 0.2%). There were no significant differences in serum PTH (38.6 ± 8.1 versus 51.9 ± 23.9 pg/mL), CTX (0.4 ± 0.2 versus 0.51 ± 0.34 ng/mL), or P1NP (64.5 ± 26.2 versus 87.3 ± 45.3 ng/mL). Serum 25-hydroxyvitamin D levels were higher in T1D than in controls (53.1 ± 20.8 versus 30.9 ± 8.2 ng/mL, p < 0.05). Bone structure metrics (bone volume, trabecular thickness, trabecular number, and cortical thickness) were similar between the groups. Indices of bone formation (osteoid volume, osteoid surface, and bone formation rate) were 40% lower in T1D and associated with lower activation frequency. However, the differences in bone formation were not statistically significant. Long-standing T1D may affect bone turnover, mainly bone formation, without significantly affecting bone structure. Further research is needed to understand bone turnover and factors affecting bone turnover in people with T1D. © 2023 The Authors. JBMR Plus published by Wiley Periodicals LLC. on behalf of American Society for Bone and Mineral Research.

Disturbed bone marrow adiposity in patients with Cushing's syndrome and glucocorticoid- and postmenopausal- induced osteoporosis.

Skeletal stem/progenitor cells (SSPCs) in the bone marrow can differentiate into osteoblasts or adipocytes in response to microenvironmental signalling input, including hormonal signalling. Glucocorticoids (GC) are corticosteroid hormones that promote adipogenic differentiation and are endogenously increased in patients with Cushing´s syndrome (CS). Here, we investigate bone marrow adiposity changes in response to endogenous or exogenous GC increases. For that, we characterize bone biopsies from patients with CS and post-menopausal women with glucocorticoid-induced osteoporosis (GC-O), compared to age-matched controls, including postmenopausal osteoporotic patients (PM-O). Transiliac crest bone biopsies from CS patients and healthy controls, and from postmenopausal women with GC-O and matched controls were analysed; an additional cohort included biopsies from women with PM-O. Plastic-embedded biopsies were sectioned for histomorphometric characterization and quantification of adipocytes. The fraction of adipocyte area per tissue (Ad.Ar/T.Ar) and marrow area (Ad.Ar/Ma.Ar), mean adipocyte profile area (Ad.Pf.Ar) and adipocyte profile density (N.Ad.Pf/Ma.Ar) were determined and correlated to steroid levels. Furthermore, the spatial distribution of adipocytes in relation to trabecular bone was characterized and correlations between bone marrow adiposity and bone remodeling parameters investigated. Biopsies from patients with CS and GC-O presented increased Ad.Ar/Ma.Ar, along with adipocyte hypertrophy and hyperplasia. In patients with CS, both Ad.Ar/Ma.Ar and Ad.Pf.Ar significantly correlated with serum cortisol levels. Spatial distribution analyses revealed that, in CS, the increase in Ad.Ar/Ma.Ar near to trabecular bone (<100 µm) was mediated by both adipocyte hypertrophy and hyperplasia, while N.Ad.Pf/Ma.Ar further into the marrow (>100 µm) remained unchanged. In contrast, patients with GC-O only presented increased Ad.Ar/Ma.Ar and mean Ad.Pf.Ar>100 µm from trabecular bone surface, highlighting the differential effect of increased endogenous steroid accumulation. Finally, the Ad.Ar/Ma.Ar and Ad.Ar/T.Ar correlated with the canopy coverage above remodeling events. Increased cortisol production in patients with CS induces increased bone marrow adiposity, primarily mediated by adipocyte hypertrophy. This adiposity is particularly evident near trabecular bone surfaces, where hyperplasia also occurs. The differential pattern of adiposity in patients with CS and GC-O highlights that bone marrow adipocytes and their progenitors may respond differently in these two GC-mediated bone diseases.

Combination of osteogenesis imperfecta and hypophosphatasia in three children with multiple fractures, low bone mass and severe osteomalacia, a challenge for therapeutic management

Osteogenesis imperfecta (OI) and hypophosphatasia (HPP) are rare skeletal disorders caused by mutations in the genes encoding collagen type I (COL1A, COL1A2) and tissue-non-specific isoenzyme of alkaline phosphatase (ALPL), respectively. Both conditions result in skeletal deformities and bone fragility although bone tissue abnormalities differ considerably. Children with OI have low bone mass and hypermineralized matrix, whereas HPP children develop rickets and osteomalacia.We report a family, father and three children, affected with growth retardation, low bone mass and recurrent fractures. None of them had rickets, blue sclera or dentinogenesis imperfecta. ALP serum levels were low and genetics revealed in the four probands heterozygous pathogenic mutations in COL1A2 c.838G > A (p.Gly280Ser) and in ALPL c.1333T > C (p.Ser445Pro). After multidisciplinary meeting, a diagnostic transiliac bone biopsy was indicated for each sibling for therapeutic decision.Bone histology and histomorphometry, as compared to reference values of children with OI type I as well as, to a control pediatric patient harboring the same COL1A2 mutation, revealed similarly decreased trabecular bone volume, increased osteocyte lacunae, but additionally severe osteomalacia. Quantitative backscattered electron imaging demonstrated that bone matrix mineralization was not as decreased as expected for osteomalacia.In summary, we observed within each biopsy samples classical features of OI and classical features of HPP. The apparent nearly normal bone mineralization density distribution results presumably from divergent effects of OI and HPP on matrix mineralization. A combination therapy was initiated with ALP enzyme-replacement and one month later with bisphosphonates. The ongoing treatment led to improved skeletal growth, increased BMD and markedly reduced fracture incidence.

#2700 RENAL OSTEODYSTROPHY AND CLINICAL OUTCOMES: RESULTS FROM THE BRAZILIAN REGISTRY OF BONE BIOPSY

Abstract Background and Aims Mineral and bone disorders caused by chronic kidney disease (CKD-MBD) encompass biochemical/hormonal abnormalities, soft tissue calcifications and bone morphological changes known as renal osteodystrophy (ROD). Data from prior studies have consistently shown that patients with CKD-MBD are susceptible to an increased risk of mortality, bone fractures and major cardiovascular outcomes. This study is a sub analysis of the Brazilian Registry of Bone Biopsy (REBRABO), a prospective, national multicenter cohort of patients with CKD who underwent bone biopsy, aimed at addressing the relationship between ROD type and the occurrence of bone fractures, hospitalizations, cardiovascular events, and a composite of all-cause mortality. Method During the period from August 2015 to December 2021, 511 patients with CKD who underwent bone biopsy and with diagnosis of ROD were included in REBRABO. Bone biopsy was indicated by medical reasons or research protocol. Were excluded from this analysis patients who lost their follow-up (N = 111), had no bone biopsy report (N = 40), had an estimated glomerular filtration rate &amp;gt; 90 mL/min (N = 28), not signed their consent (N = 24), had bone fragments inadequate for diagnosis (N = 23), had a bone biopsy indicated by a specialty other than nephrology (N = 6), or were &amp;lt; 18 years old (N = 4). Baseline was defined as the time when the patient underwent bone biopsy. The prospective analysis included data from patients who completed at least 12 months of follow-up. The mean follow- up was 1242 (693-1508) days. The following events were adjudicated: bone fractures, hospitalization, major cardiovascular events - MACEs (unstable angina, nonfatal acute myocardial infarction, elective or emergency coronary revascularization, transient ischemic attack, stroke, and cardiovascular death), and death. Bone fragments were obtained via transiliac bone biopsies using an electrical trephine after prelabeling with tetracycline (3 days) administered over two separate periods. The samples were classified as osteitis fibrosa (OF), mixed uremic osteodystrophy (MUO), adynamic bone disease (ABD), osteomalacia (OM) and normal/minor alterations, according to TMV system. Cox regression analysis was performed to detect independent determinants of clinical outcomes. Results A total of 275 patients were included. This was a population with a mean age of 52 (42– 60) years, 143 (52%) were men, 39 (14%) had diabetes, and 248 (90%) were on dialysis. During follow-up, a total of 28 bone fractures, 97 hospitalization events, 44 MACEs, and 70 deaths were reported, with incidence of 15.1% (4.4%/year), 49.5% (14.6%/year), 23.3% (6.85%/year) and 25.5% (7.5%/year), respectively. Patients who presented MACEs had lower serum hemoglobin levels [11.1 (9.6-12.6) vs. 12 (10.8-13.5; p = 0.026], higher prevalence of diabetes mellitus [11 (25%) vs. 15 (10%); p = 0.013] and previous cardiovascular disease [8 (18%) vs. 8 (5%); p = 0.008]. Age, previous cardiovascular disease, and proportion of serum phosphate levels out of the normal range were independent predictors for death [OR 1.046 (CI: 1.024-1.069), p = 0.0001; OR 1.856 (CI: 1.009-3.413), p = 0.04; OR: 1.942 (CI: 1.116-3.379), p = 0.019; respectively].Participants were grouped according to the ROD subtype as: OF (n = 113; 41%), ABD (n = 79; 29%), MUO (n = 59; 21%), OM (n = 12; 4%), and normal/minor alterations (n = 12; 4%). ROD subtypes were not related to incident outcomes. Conclusion The incidence of bone fractures, hospitalizations, cardiovascular events, and of a composite of all-cause mortality did not differ between the types of ROD.

Bone Tissue Evaluation Indicates Abnormal Mineralization in Patients with Autoimmune Polyendocrine Syndrome Type I: Report on Three Cases

Autoimmune polyendocrine syndrome type-1 (APS1) is characterized by autoimmune manifestations affecting different organs from early childhood on. Immunological abnormalities, the resulting endocrinopathies, and their treatments may compromise bone health. For the first time in APS1, we analyzed transiliac bone biopsy samples by bone histomorphometry and quantitative backscattered electron imaging in three adult patients (female P1, 38 years; male P2, 47 years; male P3, 25 years). All had biallelic mutations in the autoimmune regulator gene and in addition to endocrinopathies, also significant bone fragility. Histomorphometry showed bone volume in the lower normal range for P1 (BV/TV, − 0.98 SD) and P3 (− 1.34 SD), mainly due to reduced trabecular thickness (TbTh, − 3.63 and − 2.87 SD). In P1, osteoid surface was low (OS/BS, − 0.96 SD); active osteoblasts and double labeling were seen only on cortical bone. P3 showed a largely increased bone turnover rate (BFR/BV, + 4.53 SD) and increased mineralization lag time (Mlt, + 3.40 SD). Increased osteoid surface (OS/BS, + 2.03 and + 4.71 SD for P2 and P3) together with a large proportion of lowly mineralized bone area (Trab CaLow, + 2.22 and + 9.81 SD for P2 and P3) and focal mineralization defects were consistent with abnormal mineralization. In all patients, the density and area of osteocyte lacunae in cortical and trabecular bone were similar to healthy adults. The bone tissue characteristics were variable and included decreased trabecular thickness, increased amount of osteoid, and abnormal mineralization which are likely to contribute to bone fragility in patients with APS1.

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.

OR13-1 Long-Term Burosumab Therapy Provides Sustained Benefit in Patients with Tumor-Induced Osteomalacia: End of Study Findings From the Pivotal Phase 2 Study

Abstract Tumor-induced osteomalacia (TIO) is a rare paraneoplastic syndrome wherein small tumors secrete excess fibroblast growth factor 23 (FGF23) leading to hypophosphatemia, osteomalacia, bone pain, fractures, and muscle weakness. Burosumab is an anti-FGF23 monoclonal antibody approved for treatment of TIO. We report end of study (EOS) data from the open-label phase 2 UX023T-CL201 trial (NCT02304367) of burosumab in adults with TIO. In UX023T-CL201, participants received burosumab (0.3–2.0 mg/kg) by subcutaneous injection every 4 weeks for 48 weeks with an extension up to 300 weeks (∼6 years). Co-primary endpoints were change in serum phosphorus at Week 24 and change in excess osteoid by trans-iliac crest bone biopsy at Week 48. Additional endpoints included changes in bone biomarkers, activity on radionucleotide bone scan representing fractures/pseudofractures, patient-reported outcomes, and safety measures. This analysis included 14/17 UX023T-CL201 participants; excluding those with other diagnoses (n=3). Mean (SD) serum phosphorus improved from 1.6 (0.5) mg/dL at baseline to 2.3 (0.5) mg/dL when averaged across the mid-point of the dose interval through Week 24 (co-primary endpoint). Levels further improved (2.6 [0.4] mg/dL; n=7) to within the normal range (2.5–3.6 mg/dL) at Week 240. Most measures of osteomalacia improved at Week 48 (co-primary endpoint), including osteoid/bone volume, osteoid thickness, and mineralization lag time. However, osteoid surface/bone surface showed no change. Bone biomarkers (P1NP, CTx, BALP) followed similar trends. Upon initiation of burosumab, markers briefly increased by Week 24 as bone healing began, then gradually decreased with further treatment, reaching normal (P1NP, CTx) or near-normal (BALP) ranges by Week 240. At baseline, 249 areas of increased radionucleotide uptake indicating fracture activity were detected across 14 participants. By Week 144, 36% (72/201) and 13% (26/201) regions of abnormal uptake were fully or partially restored, respectively. By EOS, further reduction of active fractures and fewer new areas of uptake were observed. New fractures at Week 144 were observed in a single participant (considered a burosumab partial responder) and between Week 144 and EOS in another participant unrelated to burosumab (car accident). Participants reported sustained mean reduction from baseline pain at Week 240 per Brief Pain Inventory and significantly per SF-36 Bodily Pain score. Significant reductions in baseline fatigue at Week 240 were reported per Brief Fatigue Inventory and SF-36 Vitality scores. SF-36 physical health (Role Physical, Bodily Pain, Component Score) and mental health (Social Function, Vitality) significantly improved from baseline at Week 240. The observed safety profile of long-term burosumab in participants with TIO was consistent with the known safety profile of burosumab and similar to patients with XLH. Long-term burosumab therapy was associated with improved serum phosphorus, osteomalacia, bone turnover, fracture incidence and healing, health-related quality of life, and reductions in pain and fatigue in adults with TIO. Presentation: Sunday, June 12, 2022 11:00 a.m. - 11:15 a.m.

Material and nanomechanical properties of bone structural units of cortical and trabecular iliac bone tissues from untreated postmenopausal osteoporotic women

The differences in bone nanomechanical properties between cortical (Ct) and trabecular (Tb) bone remain uncertain, whereas knowing the respective contribution of each compartment is critical to understand the origin of bone strength. Our purpose was to compare bone mechanical and intrinsic properties of Ct and Tb compartments, at the bone structural unit (BSU) level, in iliac bone taken from a homogeneous untreated human population.Among 60 PMMA-embedded transiliac bone biopsies from untreated postmenopausal osteoporotic women (64 ± 7 year-old), >2000 BSUs were analysed by nanoindentation in physiological wet conditions [indentation modulus (elasticity), hardness, dissipated energy], by Fourier transform infrared (FTIRM) and Raman microspectroscopy (mineral and organic characteristics), and by X-ray microradiography (degree of mineralization of bone, DMB). BSUs were categorized based on tissue age, osteonal (Ost) and interstitial (Int) tissues location and bone compartments (Ct and Tb).Indentation modulus was higher in Ct than in Tb BSUs, both in Ost and Int. dissipated energy was higher in Ct than Tb, in Int BSUs. Hardness was not different between Ct and Tb BSUs. In Ost or Int BSUs, mineral maturity (conversion of non-apatitic into apatitic phosphates) was higher in Ct than in Tb, as well as for collagen maturity (Ost). Mineral content assessed as mineral/matrix (FTIRM and Raman) or as DMB, was lower in Ct than in Tb. Crystallinity (FTIRM) was similar in BSUs from Ct and Tb, and slightly lower in Ct than in Tb when measured by Raman, indicating that the crystal size/perfection was quite similar between Ct and Tb BSUs. The differences found between Ost and Int tissues were much higher than the difference found between Ct and Tb for all those bone material properties. Multiple regression analysis showed that Indentation modulus and dissipated energy were mainly explained by mineral maturity in Ct and by collagen maturity in Tb, and hardness by mineral content in both Ct and Tb.In conclusion, in untreated human iliac bone, Ct and Tb BSUs exhibit different characteristics. Ct BSUs have higher indentation modulus, dissipated energy (Int), mineral and organic maturities than Tb BSUs, without difference in hardness. Although those differences are relatively small compared to those found between Ost and Int BSUs, they may influence bone strength at macroscale.

Isolating mineralized bone and bone marrow mRNA from transiliac bone biopsies stored in a stabilizing solution: A comparative study

The molecular mechanisms underlying metabolic bone diseases, including renal osteodystrophy, are poorly understood. Transcriptomics are increasingly used to characterize biological molecular networks and prove promising in identifying therapeutic targets and biomarkers. A reliable method for obtaining sufficient amounts of high quality RNA from human bone biopsies is a prerequisite for the implementation of molecular diagnostics in clinical research and practice.The present study aimed to develop a simple and adequate method for isolating bone and bone marrow mRNA from transiliac bone biopsies. Several storage, separation, and extraction procedures were compared. The procedure was optimized in pig samples and subsequently validated in human samples. Appropriate amounts of mineralized bone and bone marrow mRNA of moderate to high quality were obtained from transiliac bone biopsies that were immersed in the stabilizing solution Allprotect Tissue Reagent at room temperature for up to 3 days prior to freezing. After thawing, bone marrow and mineralized bone were separated by a multistep centrifugation procedure and subsequently disrupted and homogenized by a bead crusher. Appropriate separation of mineralized bone and bone marrow was confirmed by discriminatory gene expression profiles.

Large-scale osteocyte lacunar morphological analysis of transiliac bone in normal and osteoporotic premenopausal women.

Bone's ability to adapt is governed by the network of embedded osteocytes, which inhabit individual pores called lacunae. The morphology of these lacunae and their resident osteocytes are known to change with age and diseases such as postmenopausal osteoporosis. However, it is unclear whether alterations in lacunar morphology are present in younger populations with osteoporosis. To investigate this, we implemented a previously validated methodology to image and quantify the three-dimensional morphometries of lacunae on a large scale with ultra-high-resolution micro-computed tomography (microCT) in transiliac bone biopsies from three groups of premenopausal women: control n=39; idiopathic osteoporosis (IOP) n=45; idiopathic low BMD (ILBMD) n=19. Lacunar morphometric parameters were measured in both trabecular and cortical bone such as lacunar density (Lc.N/BV), lacunar volume (Lc.V), and lacunar sphericity (Lc.Sr). These were then compared against each other and also with previously measured tissue morphometries such as bone volume density (BV/TV), trabecular separation (Tb.Sp), trabecular number (Tb.N), and others. We detected no differences in lacunar morphology between the IOP, ILBMD and healthy premenopausal women. In contrast, we did find significant differences between lacunar morphologies including Lc.N/BV, Lc. V, and Lc. Sr in cortical and trabecular regions within all three groups (p<0.001), which was consistent with our previous findings on a subgroup of the healthy group. Furthermore, we discovered strong correlations between Lc. Sr from trabecular regions with the measured BV/TV (R=-0.90, p<0.05). The findings and comprehensive lacunar dataset we present here will be a crucial foundation for future investigations of the relationship between osteocyte lacunar morphology and disease.

Effect of vitamin D sterols on bone histology in pre-dialysis patients: Aprospective controlled study.

Abnormalities related to mineral and bone metabolism are a common finding in chronic kidney disease (CKD). Vitamin D compounds are often prescribed to CKD patients with the purpose to control secondary hyperparathyroidism and reduce the risk of high-turnover bone disease. However, data on the effect of vitamin D sterols on bone histology in non-dialysis CKD is limited. A prospective controlled study was conducted on a cohort of 56 patients with CKD stages 3 and 4. 19 patients on calcitriol and 12 patients on cholecalciferol were compared to a group of 25 age- and sex-matched controls. Participants underwent a tetracycline double-labelled transiliac bone biopsy before starting therapy and again 12 months later. Changes from baseline in circulating biomarkers and bone histomorphometric parameters were analyzed. Low-turnover bone disease was the most common pattern of renal osteodystrophy on the initial biopsy. There was no difference in biochemical or histomorphometric values between the three study groups at baseline. Serum intact parathormone (iPTH) and bone formation rate decreased significantly in calcitriol-treated patients, with prevalence of low-turnover bone disease doubling from baseline. In contrast, no significant changes were noted in cholecalciferol-treated and control subjects. Calcitriol was effective in preventing secondary hyperparathyroidism and high-turnover bone disease. However, it was associated with an increased risk of developing or aggravating low-turnover bone disease. In the absence of a bone biopsy, calcitriol use in pre-dialysis CKD should be reserved for patients with a progressive rise in iPTH levels, in whom high-turnover bone disease is suspected.

Bone Turnover, Mineralization, and Volume Estimated by 18F-Sodium Fluoride PET/CT and Biomarkers in Chronic Kidney Disease: Mineral and Bone Disorder Compared with Bone Biopsy

Introduction: Invasive bone biopsy to assess bone metabolism in patients with chronic kidney disease-mineral and bone disorder may be replaced by the noninvasive ¹⁸F-NaF PET/CT and biomarkers of bone metabolism. We aimed to compare parameters of bone turnover, mineralization, and volume assessed by bone biopsies with results derived from ¹⁸F-NaF PET/CT and biomarkers (bone-specific alkaline phosphatase, osteocalcin, fibroblast growth factor 23, and osteoprotegerin). Methods: A cross-sectional study included 17 dialysis patients, and results from ¹⁸F-NaF PET/CT scans and the biomarkers were directly compared with the results of histomorphometric analyses of tetracycline double-labeled trans-iliac bone biopsies. Results: Bone biopsies showed 40% high, 20% normal, and 40% low bone turnover. No biopsies had generalized abnormal mineralization, and the bone volume/total tissue volume was low in 80% and high in 7%. The pelvic skeletal plasma clearance (K_i) from ¹⁸F-NaF PET/CT correlated with bone turnover parameters obtained by bone biopsy (activation frequency: r = 0.82, p < 0.01; bone formation rate/bone surface: r = 0.81, p < 0.01), and K_i defined low turnover with high sensitivity (83%) and specificity (100%). CT-derived radiodensity correlated with bone volume, r = 0.82, p < 0.01. Of the biomarkers, only osteocalcin showed a correlation with turnover assessed by histomorphometry. Conclusion: In conclusion, ¹⁸F-NaF PET/CT may be applicable for noninvasive assessment of bone turnover and volume in CKD-MBD.