Mechanical and mineral properties of osteogenesis imperfecta human bones at the tissue level

Osteogenesis imperfecta (OI) is a genetic disorder characterized by an increase in bone fragility on the macroscopic scale, but few data are available to describe the mechanisms involved on the tissue scale and the possible correlations between these scales. To better understand the effects of OI on the properties of human bone, we studied the mechanical and chemical properties of eight bone samples from children suffering from OI and compared them to the properties of three controls. High-resolution computed tomography, nanoindentation and Raman microspectroscopy were used to assess those properties. A higher tissue mineral density was found for OI bone (1.131 gHA/cm³ vs. 1.032 gHA/cm³, p = 0.032), along with a lower Young's modulus (17.6 GPa vs. 20.5 GPa, p = 0.024). Obviously, the mutation-induced collagen defects alter the collagen matrix, thereby affecting the mineralization. Raman spectroscopy showed that the mineral-to-matrix ratio was higher in the OI samples, while the crystallinity was lower, suggesting that the mineral crystals were smaller but more abundant in the case of OI. This change in crystal size, distribution and composition contributes to the observed decrease in mechanical strength.     

Short-Term Effects of High-Dose Zoledronic Acid Treatment on Bone Mineralization Density Distribution After Orthotopic Liver Transplantation

Patients with “hepatic” bone disease exhibit increased fracture incidence. The effects on bone material properties, their changes due to orthotopic liver transplantation (OLT), as well as zolendronate (ZOL) treatment have not yet been investigated. We studied bone mineralization density distribution (BMDD) in paired transiliacal biopsies (at and 6 months after OLT) from patients (control CON n = 18, treatment group ZOL n = 21, the latter treated with i.v. ZOL at doses of 4 mg/month) for how bone at the material level was affected by the “hepatic” disease in general, as well as by OLT and ZOL in particular. (1) BMDD parameters at baseline reflected disturbed bone matrix mineralization in “hepatic” bone disease combined with low turnover. Trabecular bone displayed a decrease in mean and most frequent calcium concentration (Ca_MEAN −2.9% and Ca_PEAK −2.8%, respectively; both P < 0.001), increased heterogeneity of mineralization (Ca_WIDTH +12.2%, P = 0.01), and increased percentage of bone areas with low mineralization (Ca_LOW +32.4%, P = 0.02) compared to normal; however, there were no differences compared to cortical bone. (2) Six months after OLT, ZOL-treated trabecular bone displayed reduced Ca_LOW (−32.0%, P = 0.047), cortical bone increased Ca_MEAN (+4.2%, P = 0.009), increased Ca_PEAK (+3.3%, P = 0.040), and decreased Ca_LOW (−55.7, P = 0.038) compared to CON and increased Ca_MEAN compared to baseline (+1.9, P = 0.032) without any signs of hyper- or defective mineralization. These changes as consequence of the antiresorptive action of ZOL visible already after 6 months result in beneficial effects on bone matrix mineralization, likely contributing to the significant decrease in fracture incidence observed in these patients 2 years post transplantation. B. M. Misof and M. Bodingbauer contributed equally.     

Hypermineralization and High Osteocyte Lacunar Density in Osteogenesis Imperfecta Type V Bone Indicate Exuberant Primary Bone Formation

In contrast to “classical” forms of osteogenesis imperfecta (OI) types I to IV, caused by a mutation in COL1A1/A2, OI type V is due to a gain‐of‐function mutation in the IFITM5 gene, encoding the interferon‐induced transmembrane protein 5, or bone‐restricted interferon‐inducible transmembrane (IFITM)‐like protein (BRIL). Its phenotype distinctly differs from OI types I to IV by absence of blue sclerae and dentinogenesis imperfecta, by the occurrence of ossification disorders such as hyperplastic callus and forearm interosseous membrane ossification. Little is known about the impact of the mutation on bone tissue/material level in untreated and bisphosphonate‐treated patients. Therefore, investigations of transiliac bone biopsy samples from a cohort of OI type V children (n = 15, 8.7 ± 4 years old) untreated at baseline and a subset (n = 8) after pamidronate treatment (2.6 years in average) were performed. Quantitative backscattered electron imaging (qBEI) was used to determine bone mineralization density distribution (BMDD) as well as osteocyte lacunar density. The BMDD of type V OI bone was distinctly shifted toward a higher degree of mineralization. The most frequently occurring calcium concentration (CaPeak) in cortical (Ct) and cancellous (Cn) bone was markedly increased (+11.5%, +10.4%, respectively, p < 0.0001) compared to healthy reference values. Treatment with pamidronate resulted in only a slight enhancement of mineralization. The osteocyte lacunar density derived from sectioned bone area was elevated in OI type V Ct and Cn bone (+171%, p < 0.0001; +183.3%, p < 0.01; respectively) versus controls. The high osteocyte density was associated with an overall immature primary bone structure (“mesh‐like”) as visualized by polarized light microscopy. In summary, the bone material from OI type V patients is hypermineralized, similar to other forms of OI. The elevated osteocyte lacunar density in connection with lack of regular bone lamellation points to an exuberant primary bone formation and an alteration of the bone remodeling process in OI type V. © 2017 American Society for Bone and Mineral Research.     

Bone Mineral Density and Fracture Rate in Response to Intravenous and Oral Bisphosphonates in Adult Osteogenesis Imperfecta

The effect of bisphosphonate treatment on bone mineral density (BMD) and fracture rates was assessed in adults with osteogenesis imperfecta (OI). This observational nonrandomized study included 90 OI adults treated with intravenous pamidronate (n = 28), oral alendronate (n = 10), or oral residronate (n = 17) or not treated (n = 35). There were 63 type I, 15 type III, and 12 type IV OI patients. BMD results were observed for up to 161 months and an average of 52 months of treatment. For type I and grouped type III/IV patients, treatment with pamidronate showed an increasing rate in L1–L4 BMD from baseline (0.006 [P = 0.03] and 0.016 [P < 0.001] gm/cm²/year, respectively); oral bisphosphonate treatment showed a significant increasing rate in L1–L4 BMD (0.004 gm/cm²/year [P = 0.047]) for type I patients. Pamidronate-treated type III/IV and oral bisphosphonate-treated type I patients showed significant increases in total-hip BMD (0.006 [P = 0.003] and 0.011 [P = 0.046] gm/cm²/year, respectively). Bisphosphonate effect on fracture rate was assessed for 5-year periods before and after treatment in 51 treated and 22 nontreated individuals matched for age at which bisphosphonate was first administered to the treated group. Bisphosphonate treatment did not decrease fracture rate in type I OI patients. Fracture rate decreased in type III/IV patients following pamidronate but not following oral bisphosphonate treatment. These results underscore a need to consider whether bisphosphonate treatment is appropriate for all adults with OI.     

Are Changes in Composition in Response to Treatment of a Mouse Model of Osteogenesis Imperfecta Sex-dependent?

Background

Osteogenesis imperfecta (OI) is a genetic disease characterized by skeletal fragility and deformity. There is extensive debate regarding treatment options in adults with OI. Antiresorptive treatment reduces the number of fractures in growing oim/oim mice, an animal model that reproducibly mimics the moderate-to-severe form of OI in humans. Effects of long-term treatments with antiresorptive agents, considered for treatment of older patients with OI with similar presentation (moderate-to-severe OI) are, to date, unknown.

Questions/purposes

Fourier transform infrared (FTIR) imaging, which produces a map of the spatial variation in chemical composition in thin sections of bone, was used to address the following questions: (1) do oim/oim mice show a sex dependence in compositional properties at 6.5 months of age; (2) is there a sex-dependent response to treatment with antiresorptive agents used in the treatment of OI in humans; and (3) are any compositional parameters in oim/oim mice corrected to wild-type (WT) values after treatment?

Methods

FTIR imaging data were collected from femurs from four to five mice per sex per genotype per treatment. Treatments were 24 weeks of saline, alendronate, or RANK-Fc; and 12 weeks of saline + 12 weeks RANK-Fc and 12 weeks of alendronate + RANK-Fc. FTIR imaging compositional parameters measured in cortical and cancellous bones were mineral-to-matrix ratio, carbonate-to-mineral ratio, crystal size/perfection, acid phosphate substitution, collagen maturity, and their respective distributions (heterogeneities). Because of the small sample size, nonparametric statistics (Mann-Whitney U- and Kruskal-Wallis tests with Bonferroni correction) were used to compare saline-treated male and female mice of different genotypes and treatment effects by sex and genotype, respectively. Statistical significance was defined as p < 0.05.

Results

At 6.5 months, saline-treated male cortical oim/oim bone had increased mineral-to-matrix ratio (p = 0.016), increased acid phosphate substitution (p = 0.032), and decreased carbonate-to-mineral ratio (p = 0.016) relative to WT. Cancellous bone in male oim/oim also had increased mineral-to-matrix ratio (p = 0.016) relative to male WT. Female oim/oim mouse bone composition for all cortical and cancellous bone parameters was comparable to WT (p > 0.05). Only the female WT mice showed a response of mean compositional properties to treatment, increasing mineral-to-matrix after RANK-Fc treatment in cancellous bone (p = 0.036) compared with saline-treated mice. Male oim/oim increased mineral-to-matrix cortical and cancellous bone heterogeneity in response to all long-term treatments except for saline + RANK-Fc (p < 0.04); female oim/oim cortical mineral-to-matrix bone heterogeneity increased with ALN + RANK-Fc and all treatments increased cancellous female oim/oim bone acid phosphate substitution heterogeneity (p < 0.04).

Conclusions

Both oim/oim and WT mice, which demonstrate sex-dependent differences in composition with saline treatment, showed few responses to long-term treatment with antiresorptive agents. Female WT mice appeared to be more responsive; male oim/oim mice showed more changes in compositional heterogeneity. Changes in bone composition caused by these agents may contribute to improved bone quality in oim/oim mice, because the treatments are known to reduce fracture incidence.

Clinical Relevance

The optimal drug therapy for long-term treatment of patients with moderate-to-severe OI is unknown. Based on bone compositional changes in mice, antiresorptive treatments are useful for continued treatment in OI. There is a reported sexual dimorphism in fracture incidence in adults with OI, but to date, no one has reported differences in response to pharmaceutical intervention. This study suggests that such an investigation is warranted.     

Mineral particle size in children with osteogenesis imperfecta type I is not increased independently of specific collagen mutations

Osteogenesis imperfecta (OI) type I represents the mildest form of OI and is usually caused by two classes of autosomal dominant mutations in collagen type I: haploinsufficiency leading to a reduced quantity of structurally normal collagen (quantitative mutation), or sequence abnormalities generating structurally aberrant collagen chains (qualitative mutation). An abnormally high bone matrix mineralization has been observed in all OI cases investigated so far, independently of mutation type. This raises the question whether the increased amount of mineral is due to mineral particles growing to larger sizes or to a higher number of more densely packed particles. For this reason, we revisit the problem by investigating the mineral particle size in cancellous bone from two subsets of the previously analyzed biopsies (patient's age: 2–4.2 and 7.6–11 years) comparing OI quantitative mutations (n = 5), OI qualitative mutations (n = 5) and controls (n = 6). We used a combined small-angle X-ray scattering (SAXS) and wide-angle X-ray diffraction (WAXD) setup with a beam diameter of 10 μm of synchrotron radiation, which allows the determination of mineral particle characteristics in 10 μm thick sections at the same positions where the matrix mineralization density was previously determined. The thickness parameter of mineral particles (T) was obtained from SAXS data and the mineral volume fraction was calculated from the mean calcium content of the bone matrix determined by quantitative back-scattered electron imaging (qBEI). The combination of these two quantities allowed calculating the true particle width (W) of the plate-like mineral crystals.T was larger in the older than in the younger age-group independently of genotype (p < 0.004) and was larger in the controls than in each OI group. The qBEI results showed that the mineral volume fraction increased from 32.45wt.% in controls to 36.44wt.% in both OI groups (corresponding to a 12% increase in relative terms). Combining these data, we find that also W was larger in the older than in the younger age-group (p < 0.002), but stayed equal or smaller in both OI genotypes (controls: 2.3 nm ± 0.04, OI qualitative: 2.2 ± 0.05; OI quantitative 2.3 ± 0.04, mean ± SEM). A linear regression analysis even suggests a slower increase of W in qualitative OI as compared to quantitative OI and controls, where the particle sizes stayed similar at all ages. We thus conclude that the high mineral density in human OI is not due to increased particle size but rather to increased particle packing density. The lack of an observed difference between the two classes of mutations suggests the occurrence of a bone cell defect downstream of the collagen mutation.     

Differing Lumbar Vertebral Mineralization Rates in Ambulatory Pediatric Patients with Osteogenesis Imperfecta

The purpose of this study was to evaluate serial changes in bone mineral density (BMD) of the lumbar spine in individual children and adolescents with untreated osteogenesis imperfecta (OI) using dual X-ray absorptiometry (DXA). Twenty-seven pediatric patients with OI who had no historical or radiographic evidence of lumbar fracture, required no assistive device for mobility, and were taking no medications known to affect skeletal mineralization during the study period comprised the investigational group. Absolute BMD and age- and gender-matched BMD (Z-scores) were assessed relative to standard parameters of growth (height, weight, age, height adjusted for age and gender and body surface area) and severity of disease (lifetime fracture rate). The spinal mineralization rate (SMR) between examinations for 15 patients with more than one measurement (n= 20 intervals) was expressed as the magnitude of the change in BMD Z-score per year. Both BMD and BMD Z-score were closely correlated with height, height Z-score, weight and body surface area and were inversely related to fracture rate (P < 0.001 for all comparisons). BMD was also highly correlated with patient age (P < 0.001). Stepwise regression analysis showed that together height Z-score and lifetime fracture rate improved the prediction of BMD Z-score (r= 0.71; P < 0.001). SMRs ranged from −0.5 to 3.5. The average change in SMR between sequential measurements was 168% for the five children who had more than two DXA examinations. Linear regression showed a significant negative correlation between SMR and height Z-score (r=−0.79, P < 0.001). We conclude that vertebral body size is a critical determinant of BMD and BMD Z-score in OI because DXA results are expressed per unit area, not per unit volume. Pediatric patients with OI mineralize their lumbar vertebrae at rates similar to healthy children but tend to lag behind in overall mineralization. The rate of mineralization at any age appears to be related to the patient's height (adjusted for age- and gender-matched controls) and inversely related to the patient's lifetime rate of fractures. Our data suggest that vertebral mineralization in children with OI is related primarily to rapid increases in vertebral volume and only secondarily to increases in vertebral mineral density. Received: 2 March 1997 / Accepted: 5 June 1997     

Soy protein diet and exercise training increase relative bone volume and enhance bone microarchitecture in a mouse model of uremia

Soy protein consumption and exercise training have been widely studied for their effects on the vasculature and bone in healthy populations, but little is known about the effectiveness of these interventions in chronic kidney disease (CKD). Cardiovascular disease and bone fracture risk are significantly elevated in CKD, and current pharmacological interventions have been unsuccessful in treating these conditions simultaneously. The purpose of this study was to compare the effects of a soy protein diet and endurance exercise training, alone or in combination, on cardiovascular and bone health in a mouse model of renal insufficiency. At 8 weeks of age, 60 female apolipoprotein E^−/− mice underwent a two-step surgical procedure to induce uremia. These mice were then randomized at 12 weeks of age to one of four treatment groups for the 16-week intervention period: sedentary, control diet (n = 16); sedentary, soy protein diet (n = 18); exercise, control diet (n = 14); and exercise, soy protein diet (n = 12). There were no significant treatment effects on atherosclerotic lesion areas or aortic calcium deposits. We demonstrated a significant main effect of both diet and exercise on relative bone volume, trabecular number, trabecular separation, and trabecular connective density in the proximal femur as measured by microcomputed tomography. There were no treatment effects on trabecular thickness. We also showed a main effect of diet on plasma urea levels. These data suggest that soy protein intake and exercise training exert beneficial effects on properties of bone and plasma urea levels in mice with surgically induced renal impairment.     

Impaired bone microarchitecture and strength in patients with tumor-induced osteomalacia

Some studies based on bone biopsy have demonstrated that in patients with tumor-induced osteomalacia (TIO) the mineralization process of the bone matrix is profoundly disturbed. However, the interrelationship between clinical and biochemical features and bone microarchitecture in this disease needs further analysis. With this purpose in mind, we set out three objectives: (i) to determine bone microarchitecture and estimated bone strength in a group of patients with tumor-induced osteomalacia using high-resolution peripheral quantitative computed tomography (HR-pQCT) and finite element analysis (FEA), (ii) to investigate correlations between duration of disease, biochemical features, bone density, HR-pQCT and FEA parameters, and (iii) to compare HR-pQCT and FEA parameters with a healthy control group. Ten patients with TIO were included. All patients had non-resolved disease. At the distal radius, all bone microarchitecture parameters were significantly affected in patients with TIO in comparison with healthy controls. At the distal tibia, all parameters were significantly impaired, except for trabecular thickness. All the parameters were more affected in the distal tibia than in the distal radius. Women with TIO (n = 7) had significantly lower bone strength parameters than healthy controls. In men (n = 3), bone strength parameters were significantly lower than in the control group at the distal tibia. Alkaline phosphatase levels exhibited a negative correlation with microarchitecture parameters, failure load, and stiffness. Higher levels of parathyroid hormone correlated with poorer microarchitecture parameters. We believe that in TIO, hormonal disturbances and the lack of mechanical stimulus specially converge to generate an extremely harmful combination for bone health. © 2021 American Society for Bone and Mineral Research (ASBMR).     

Kinetics of peripheral blood lymphocyte subpopulations predicts the occurrence of opportunistic infection after kidney transplantation

Serial monitoring of peripheral blood lymphocyte subpopulations (PBLSs) counts might be useful in predicting post‐transplant opportunistic infection (OI) after kidney transplantation (KT). PBLSs were prospectively measured in 304 KT recipients at baseline and post‐transplant months 1 and 6. Areas under receiver operating characteristic curves were used to evaluate the accuracy of different subpopulations in predicting the occurrence of overall OI and, specifically, cytomegalovirus (CMV) disease. We separately analyzed patients not receiving (n = 164) or receiving (n = 140) antithymocyte globulin (ATG) as induction therapy. In the non‐ATG group, a CD8⁺ T‐cell count at month 1 <0.100 × 10³ cells/μl had negative predictive values of 0.84 and 0.86 for the subsequent occurrence of overall OI and CMV disease, respectively. In the multivariate Cox model, a CD8⁺ T‐cell count <0.100 × 10³ cells/μl was an independent risk factor for OI (adjusted hazard ratio: 3.55; P‐value = 0.002). In the ATG group, a CD4⁺ T‐cell count at month 1 <0.050 × 10³ cells/μl showed negative predictive values of 0.92 for the subsequent occurrence of overall OI and CMV disease. PBLSs monitoring effectively identify KT recipients at low risk of OI, providing an opportunity for individualizing post‐transplant prophylaxis practices.     

Evidence for a Role for Nanoporosity and Pyridinoline Content in Human Mild Osteogenesis Imperfecta

Osteogenesis imperfecta (OI) is a clinically and genetically heterogeneous connective tissue disorder characterized by bone fragility that arises from decreased bone mass and abnormalities in bone material quality. OI type I represents the milder form of the disease and according to the original Sillence classification is characterized by minimal skeletal deformities and near‐normal stature. Raman microspectroscopy is a vibrational spectroscopic technique that allows the determination of bone material properties in bone biopsy blocks with a spatial resolution of ～1 µm, as a function of tissue age. In the present study, we used Raman microspectroscopy to evaluate bone material quality in transiliac bone biopsies from children with a mild form of OI, either attributable to collagen haploinsufficiency OI type I (OI‐Quant; n = 11) or aberrant collagen structure (OI‐Qual; n = 5), as a function of tissue age, and compared it against the previously published values established in a cohort of biopsies from healthy children (n = 54, ages 1 to 23 years). The results indicated significant differences in bone material compositional characteristics between OI‐Quant patients and healthy controls, whereas fewer were evident in the OI‐Qual patients. Differences in both subgroups of OI compared with healthy children were evident for nanoporosity, mineral maturity/crystallinity as determined by maxima of the v₁PO₄ Raman band, and pyridinoline (albeit in different direction) content. These alterations in bone material compositional properties most likely contribute to the bone fragility characterizing this disease. © 2016 American Society for Bone and Mineral Research.     

The functional muscle–bone unit in patients with osteogenesis imperfecta type I

ContextOsteogenesis imperfecta (OI) type I is a heritable bone fragility disorder that is caused by mutations affecting collagen type I. We recently showed that patients with OI type I frequently have muscle weakness. As muscle force and bone mass are usually closely related, we hypothesized that muscle weakness in OI type I could contribute to increase bone mass deficit in the lower extremities.ObjectiveTo assess the muscle–bone relationship in the lower extremities of children and adolescents with OI type I.SettingThe study was carried out in the outpatients department of a pediatric orthopedic hospital.Patients and other participantsThirty children and adolescents with OI type I (20 females; mean age [SD]: 11.2 years [3.9]) were compared with 30 healthy age- and sex-matched controls (mean age [SD]: 11.1 years [4.5]).Main outcome measuresTibia bone mineral content (BMC; mg/mm) was measured by peripheral quantitative computed tomography to estimate bone strength at the 4% and 14% sites. Lower extremity peak force (kN) was measured by mechanography using the multiple two-legged hopping test.ResultsCompared with age- and sex-matched controls, patients with OI type I had 17% lower peak force (1.3 kN vs. 1.7 kN; p = 0.002) as well as a 22% lower BMC (128 mg/mm vs. 165 mg/mm; p < 0.001). Stepwise regression analysis showed that muscle force and tibia length were positively related to bone strength (r² = 0.90, p < 0.001) whereas there was no effect of the disease status (OI vs. control).ConclusionsThese results suggest that the muscle–bone relationship is similar between children and adolescents with OI type I and healthy age and sex-matched controls. It also suggests that muscle weakness may contribute to decreased bone strength in individuals with OI type I.     

Duration-Dependent Increase of Human Bone Matrix Mineralization in Long-Term Bisphosphonate Users with Atypical Femur Fracture

Bisphosphonates (BPs) are the most widely used drugs for the treatment of osteoporosis but prolonged use of BPs might increase the risk of atypical femur fracture (AFF). There are only a few studies that address the bone material quality in patients on long-term BP treatment with or without AFFs. We analyzed 52 trans-iliac bone biopsies from patients on long-term BP therapy with (n = 26) and without (n = 26) AFF. At the microscopic level, the degree of mineralization of bone (DMB) was assessed on whole bone by X-ray digitized microradiography while microhardness by Vickers microindentation, and bone matrix characteristics by Fourier transform infrared microspectroscopy (FTIRM) (mineral/organic ratio, mineral maturity and crystallinity, and collagen maturity) were measured at random focal areas. The AFF patients were treated longer than non-AFF patients (9.7 ± 3.3 years versus 7.9 ± 2.7 years). As expected, bone remodeling was low in both groups, without difference between them. The AFF group had significantly higher DMB in cortical bone (+2.9%, p = .001), which remained so after adjusting for treatment duration (p = .007), and showed a trend in cancellous bone (+1.6%, p = .05). Consistent with higher DMB, heterogeneity index (HI) was lower in the AFF than in the non-AFF group, illustrating lower heterogeneity of mineralization in the AFF group. A significant positive correlation between the duration of treatment and DMB in cortical bone was found in AFF, and not in the non-AFF group. Microhardness and bone matrix characteristics were similar between groups. We conclude that the AFF group had a duration-dependent increase in DMB leading to a significantly higher DMB than the non-AFF. Because BPs have high affinity to bone mineral and lining the walls of the osteocyte lacunae, the accumulation of matrix-bound BPs in AFF could lead to inhibition of the osteocyte cytoskeleton blunting their response to mechanical strains, a hypothesis to be further investigated. © 2021 American Society for Bone and Mineral Research (ASBMR).     

Strontium does not affect the intrinsic bone quality at tissue and BSU levels in iliac samples from Macaca fascicularis monkeys

Our purpose was to evaluate the impact of strontium ranelate (SrRan) on bone mineral quality at both tissue and bone structural unit (BSU) levels. Thirty iliac bone samples (dehydrated then embedded) were taken from monkeys who received 0 (controls), 200, 500 or 1250 mg/kg/day of SrRan for 52 weeks and were sacrificed either at the end of administration (treated animals, n = 16) or 10 weeks later (reverse animals, n = 14). Degree of mineralization (DMB), heterogeneity index of mineralization (HI), Vickers microhardness (Hv) and focal bone strontium content (BSC) were measured globally at tissue level and focally on the same 923 BSUs. Mineral and collagen characteristics, as well as chemometric analyses were performed on younger and older tissues in cortical bone and cancellous bone in 737 other BSUs. At tissue level, SrRan preserved material properties. At BSU level, BSC increased (significant) dose dependently in treated and reverse animals. DMB and Hv were greater in older than in younger bone in controls and treated animals. In treated animals, DMB was positively correlated with Hv and inversely correlated with the BSC. Thus, younger BSUs were less mineralized and less hard than older BSUs independently from the presence of strontium. Mineral maturity, crystallinity index, mineralization index, carbonation and collagen maturity were not modified by SrRan. Chemometry confirmed the absence of a direct effect of strontium on mineralization. Thus, surrogates of micro- and nano-structural mineral properties were not altered by SrRan and remained at a physiological level.     

Short-term menatetrenone therapy increases gamma-carboxylation of osteocalcin with a moderate increase of bone turnover in postmenopausal osteoporosis: a randomized prospective study

The effect of vitamin K₂ (menatetrenone) on bone turnover was investigated in postmenopausal patients with osteoporosis. A 6-month open-label, randomized prospective study was conducted in 109 patients. The control group (n = 53) received calcium aspartate (133.8 mg of elemental calcium daily), while the menatetrenone group (n = 56) received 45 mg of menatetrenone daily for 6 months. Serum and urinary levels of bone turnover markers were monitored. The serum level of undercarboxylated osteocalcin (uc-OC) was significantly lower (P < 0.001) in the menatetrenone group than in the control group (at 1 month), while there was a higher level of osteocalcin containing gamma-carboxylated glutamic acid (Gla-OC) in the menatetrenone group than the control group (P = 0.018). Significant differences of uc-OC and Gla-OC between the two groups were observed from 1 month onward. In addition, a higher level of intact osteocalcin was found in the menatetrenone group compared with the control group after 6 months (P = 0.006). Assessment of bone resorption markers showed that menatetrenone therapy was associated with significantly higher urinary N-telopeptide of type I collagen (NTX) excretion compared with the control group after 6 months, while there was no significant difference of urinary deoxypyridinoline excretion between the two groups. In conclusion, one month of menatetrenone therapy enhanced the secretion and gamma-carboxylation of osteocalcin, while urinary NTX excretion was increased after 6 months of treatment. Further investigations are required to determine whether the effects of menatetrenone on bone turnover are associated with fracture prevention.     

Influence of creatine supplementation on bone quality in the ovariectomized rat model: an FT-Raman spectroscopy study

The influence of creatine (Cr) supplementation on cortical and trabecular bone from ovariectomized rats was studied using FT-Raman spectroscopy. The intensity of organic-phase Raman bands was compared to mineral phase ones. Twenty-one female Wistar rats aged 3 months were divided into three groups (n = 7 per group): ovariectomized (OVX), ovariectomized treated with creatine (CRE) and sham-operated (SHAM) groups. Creatine supplementation (300 mg kg⁻¹ day⁻¹) was provided for 8 weeks, starting 12 weeks after ovariectomy. FT-Raman spectroscopy was performed on the right medial femoral mid-shaft (cortical bone) and third lumbar vertebral body (trabecular bone). The integrated intensities of mineral phase (phosphate and carbonate bands at 959 and 1,071 cm⁻¹, respectively) and organic phase (amide I band at 1,665 cm⁻¹) Raman bands were analyzed. The mineral-to-matrix (phosphate/amide I), carbonate-to-phosphate, and carbonate-to-amide I ratios were analyzed to assess bone quality. The phosphate content on trabecular bone was higher in the CRE group than the OVX group (p < 0.05). No significant changes in mineral or organic phases on cortical bone were observed. A radiographic assessment of bone density was encouraging as the same findings were showed by Raman intensity of phosphate from cortical (r ²  = 0.8037) and trabecular bones (r ² = 0.915). Severe ovariectomy-induced bone loss was confirmed by FT-Raman spectroscopy. The results suggest that the chemical composition of trabecular bone tissue may be positively influenced by Cr supplementation after ovariectomy.     

Etiopathogenesis of Hepatic Osteodystrophy in Wistar Rats with Cholestatic Liver Disease

The pathophysiology of hepatic osteodystrophy (HO) remains poorly understood. Our aim was to evaluate bone histomorphometry, biomechanical properties, and the role of the growth hormone (GH)/insulin-like growth factor-I (IGF-I) system in the onset of this disorder. Forty-six male Wistar rats were divided into two groups: sham-operated (SO, n = 23) and bile duct–ligated (BDL, n = 23). Rats were killed on day 30 postoperatively. Immunohistochemical expression of IGF-I and GH receptor was determined in liver tissue and in the proximal growth plate cartilage of the left tibia. Histomorphometric analysis was performed in the right tibia, and the right femur was used for biomechanical analysis. The maximal force at fracture and the stiffness of the mid-shaft femur were, respectively, 53% and 24% lower in BDL compared to SO. Histomorphometric measurements showed low cancellous bone volume and decreased cancellous bone connectivity in BDL, compatible with osteoporosis. This group also showed increased mineralization lag time, indicating disturbance in bone mineralization. Serum levels of IGF-I were lower in BDL (basal 1,816 ± 336 vs. 30 days 1,062 ± 191 ng/ml, P < 0.0001). BDL also showed higher IGF-I expression in the liver tissue but lower IGF-I and GH receptor expression in growth plate cartilage than SO. Osteoporosis is the most important feature of HO; BDL rats show striking signs of reduced bone volume and decreased bone strength, as early as after 1 month of cholestasis. The endocrine and autocrine–paracrine IGF-I systems are deeply affected by cholestasis. Further studies will be necessary to establish their role in the pathogenesis of HO.     

The effects of weekly alendronate therapy in Taiwanese males with osteoporosis

The aim of this study was to evaluate the efficacy, safety, and tolerability of weekly alendronate administration on male osteoporosis in Taiwan. This 6-month, randomized, open-label controlled trial enrolled 46 men with osteoporosis who were randomized to either 70 mg alendronate once weekly (n = 23) or control (n = 23). Bone mineral density (BMD) of lumbar spine and hip and biochemical bone turnover markers were measured; adverse events and tolerability were assessed. Subjects treated with alendronate showed a significant increase in BMD of 5.5% (vs. 2% in control group) at the lumbar spine and 2.7% (vs. 0.7%) at the femoral neck (P < 0.05) at 6 months, respectively. There were also significant decreases in serum level of bone formation marker (bone-specific alkaline phosphatase) and urinary excretion of bone resorption marker (deoxypyridinoline) at 3 and 6 months. Thus, alendronate showed anti-osteoporotic effects by increasing BMD and decreasing the concentrations of bone markers. The adverse events were mild and showed no significant difference between the two groups on safety assessments.     

Bone microarchitecture in patients with autoimmune hepatitis

In patients with autoimmune hepatitis (AIH), osteoporosis represents a common extrahepatic complication, which we recently showed by an assessment of areal bone mineral density (aBMD) via dual-energy x-ray absorptiometry (DXA). However, it is well established that bone quality and fracture risk does not solely depend on aBMD, but also on bone microarchitecture. It is currently not known whether AIH patients exhibit a site-specific or compartment-specific deterioration in the skeletal microarchitecture. In order to assess potential geometric, volumetric, and microarchitectural changes, high-resolution peripheral quantitative computed tomography (HR-pQCT) measurements were performed at the distal radius and distal tibia in female patients with AIH (n = 51) and compared to age-matched female healthy controls (n = 32) as well as to female patients with AIH/primary biliary cholangitis (PBC) overlap syndrome (n = 25) and female patients with PBC alone (PBC, n = 36). DXA at the lumbar spine and hip, clinical characteristics, transient elastography (FibroScan) and laboratory analyses were also included in this analysis. AIH patients showed a predominant reduction of cortical thickness (Ct.Th) in the distal radius and tibia compared to healthy controls (p < .0001 and p = .003, respectively). In contrast, trabecular parameters such as bone volume fraction (BV/TV) did not differ significantly at the distal radius (p = .453) or tibia (p = .508). Linear regression models revealed significant negative associations between age and Ct.Th (95% confidence interval [CI], −14 to −5 μm/year, p < .0001), but not between liver stiffness, cumulative prednisolone dose (even after an adjustment for age), or disease duration with bone microarchitecture. The duration of high-dose prednisolone (≥7.5 mg) was negatively associated with trabecular thickness (Tb.Th) at the distal radius. No differences in bone microarchitecture parameters between AIH, AIH/PBC, and PBC could be detected. In conclusion, AIH patients showed a severe age-dependent deterioration of the cortical bone microarchitecture, which is most likely the major contribution to the observed increased fracture risk in these patients. © 2021 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).     

Trabecular packet-level lamellar density patterns differ by fracture status and bone formation rate in white females

Spatial patterns of mineralization for human iliac crest cancellous bone were measured from images obtained by quantitative backscattered electron microscopy. Biopsies collected from vertebral fracture patients and healthy individuals with high or low bone formation rate (BFR_s) were examined (fracture/low BFR_s: N = 12, fracture/high BFR_s: N = 10, normal/low BFR_s: N = 12, normal/high BFR_s: N = 15). 20 by 20 pixel square areas or smaller were sampled from superficial and deep remodeling packets. Mean (Z_mean) and standard deviation (SD) of mineralization were measured, and coefficients of variation (CV = SD / Z_mean) were calculated. Fast Fourier transform analysis was used to quantify the distribution of the mineral in the packets. “FFT_ratio” was defined as the ratio magnitude of the principal spatial frequency to the average atomic number density. A higher FFT_ratio occurred in specimens with more pronounced alternating layers of light and dark as visible in the backscattered electron image, which was defined as lamellar patterning. Two-way ANOVA revealed that the coefficients of variation of mineralization for both superficial and deep packets were significantly lower in fracture patients than in normal individuals. However, the interaction between turnover rate and group (fracture/non-fracture) indicated that the difference in packet CV occurred among the low turnover individuals and not among those with high turnover. Mean mineralization levels and CV between deep and superficial packets were highly correlated. Regressions of packet CV of mineralization and FFT_ratio were highly significant (p < 0.001) for all packets pooled and for packets divided by group (fracture/normal). However, analyses of packet CV and FFT_ratio by individual were variable (R² from 0.00338 to 0.700).Packet-level mineralization variability may be associated with fracture toughness, and fracture patients had less variable packet-level mineralization. The result that the packet CV varied significantly between fracture and non-fracture individuals with low turnover suggests that for low turnover subjects without fracture, high variability in mineralization may have a protective effect. In high turnover patients, the accelerated turnover may prevent the lamellar variability from developing over time. Strong correlations between CV and Z_mean for both superficial and deep packets imply that newly formed bone is created similarly to older bone within an individual. Fourier transform results show that the mineralization variability found within packets is associated with lamellar patterning. Lamellar structure has been hypothesized to guide microcrack propagation in order to optimize bone strength and toughness. Osteoporotics with fracture had less pronounced lamellation than healthy normals and may be more prone to fracture.