Increased tissue modulus and hardness in the TallyHO mouse model of early onset type 2 diabetes mellitus.

Individuals with type 2 diabetes mellitus (T2DM) have a higher fracture risk compared to those without T2DM despite having higher bone mineral density (BMD). Thus, T2DM may alter other aspects of resistance to fracture beyond BMD such as bone geometry, microarchitecture, and tissue material properties. We characterized the skeletal phenotype and assessed the effects of hyperglycemia on bone tissue mechanical and compositional properties in the TallyHO mouse model of early-onset T2DM using nanoindentation and Raman spectroscopy. Femurs and tibias were harvested from male TallyHO and C57Bl/6J mice at 26 weeks of age. The minimum moment of inertia assessed by micro-computed tomography was smaller (-26%) and cortical porosity was greater (+490%) in TallyHO femora compared to controls. In three-point bending tests to failure, the femoral ultimate moment and stiffness did not differ but post-yield displacement was lower (-35%) in the TallyHO mice relative to that in C57Bl/6J age-matched controls after adjusting for body mass. The cortical bone in the tibia of TallyHO mice was stiffer and harder, as indicated by greater mean tissue nanoindentation modulus (+22%) and hardness (+22%) compared to controls. Raman spectroscopic mineral:matrix ratio and crystallinity were greater in TallyHO tibiae than in C57Bl/6J tibiae (mineral:matrix +10%, p < 0.05; crystallinity +0.41%, p < 0.10). Our regression model indicated that greater values of crystallinity and collagen maturity were associated with reduced ductility observed in the femora of the TallyHO mice. The maintenance of structural stiffness and strength of TallyHO mouse femora despite reduced geometric resistance to bending could potentially be explained by increased tissue modulus and hardness, as observed at the tibia. Finally, with worsening glycemic control, tissue hardness and crystallinity increased, and bone ductility decreased in TallyHO mice. Our study suggests that these material factors may be sentinels of bone embrittlement in adolescents with T2DM.

An integrated experimental-computational framework to assess the influence of microstructure and material properties on fracture toughness in clinical specimens of human femoral cortical bone.

Microstructural and compositional changes that occur due to aging, pathological conditions, or pharmacological treatments alter cortical bone fracture resistance. However, the relative importance of these changes to the fracture resistance of cortical bone has not been quantified in detail. In this technical note, we developed an integrated experimental-computational framework utilizing human femoral cortical bone biopsies to advance the understanding of how fracture resistance of cortical bone is modulated due to modifications in its microstructure and material properties. Four human biopsy samples from individuals with varying fragility fracture history and osteoporosis treatment status were converted to finite element models incorporating specimen-specific material properties and were analyzed using fracture mechanics-based modeling. The results showed that cement line density and osteonal volume had a significant effect on crack volume. The removal of cement lines substantially increased the crack volume in the osteons and interstitial bone, representing straight crack growth, compared to models with cement lines due to the lack of crack deflection in the models without cement lines. Crack volume in the osteons and interstitial bone increased when mean elastic modulus and ultimate strength increased and mean fracture toughness decreased. Crack volume in the osteons and interstitial bone was reduced when material property heterogeneity was incorporated in the models. Although both the microstructure and the heterogeneity of the material properties of the cortical bone independently increased the fracture toughness, the relative contribution of the microstructure was more significant. The integrated experimental-computational framework developed here can identify the most critical microscale features of cortical bone modulated by pathological processes or pharmacological treatments that drive changes in fracture resistance and improve our understanding of the relative influence of microstructure and material properties on fracture resistance of cortical bone.

Increased Advanced Glycation Endproducts, Stiffness, and Hardness in Iliac Crest Bone From Postmenopausal Women With Type 2 Diabetes Mellitus on Insulin.

Individuals with type 2 diabetes mellitus (T2DM) have a greater risk of bone fracture compared with those with normal glucose tolerance (NGT). In contrast, individuals with impaired glucose tolerance (IGT) have a lower or similar risk of fracture. Our objective was to understand how progressive glycemic derangement affects advanced glycation endproduct (AGE) content, composition, and mechanical properties of iliac bone from postmenopausal women with NGT (n = 35, age = 65 ± 7 years, HbA1c = 5.8% ± 0.3%), IGT (n = 26, age = 64 ± 5 years, HbA1c = 6.0% ± 0.4%), and T2DM on insulin (n = 25, age = 64 ± 6 years, HbA1c = 9.1% ± 2.2%). AGEs were assessed in all samples using high-performance liquid chromatography to measure pentosidine and in NGT/T2DM samples using multiphoton microscopy to spatially resolve the density of fluorescent AGEs (fAGEs). A subset of samples (n = 14 NGT, n = 14 T2DM) was analyzed with nanoindentation and Raman microscopy. Bone tissue from the T2DM group had greater concentrations of (i) pentosidine versus IGT (cortical +24%, p = 0.087; trabecular +35%, p = 0.007) and versus NGT (cortical +40%, p = 0.003; trabecular +35%, p = 0.004) and (ii) fAGE cross-link density versus NGT (cortical +71%, p < 0.001; trabecular +44%, p < 0.001). Bone pentosidine content in the IGT group was lower than in the T2DM group and did not differ from the NGT group, indicating that the greater AGE content observed in T2DM occurs with progressive diabetes. Individuals with T2DM on metformin had lower cortical bone pentosidine compared with individuals not on metformin (-35%, p = 0.017). Cortical bone from the T2DM group was stiffer (+9%, p = 0.021) and harder (+8%, p = 0.039) versus the NGT group. Bone tissue AGEs, which embrittle bone, increased with worsening glycemic control assessed by HbA1c (Pen: R2  = 0.28, p < 0.001; fAGE density: R2  = 0.30, p < 0.001). These relationships suggest a potential mechanism by which bone fragility may increase despite greater tissue stiffness and hardness in individuals with T2DM; our results suggest that it occurs in the transition from IGT to overt T2DM. © 2022 American Society for Bone and Mineral Research (ASBMR).

Components of the Gut Microbiome That Influence Bone Tissue-Level Strength.

Modifications to the constituents of the gut microbiome influence bone density and tissue-level strength, but the specific microbial components that influence tissue-level strength in bone are not known. Here, we selectively modify constituents of the gut microbiota using narrow-spectrum antibiotics to identify components of the microbiome associated with changes in bone mechanical and material properties. Male C57BL/6J mice (4 weeks) were divided into seven groups (n = 7-10/group) and had taxa within the gut microbiome removed through dosing with: (i) ampicillin; (ii) neomycin; (iii) vancomycin; (iv) metronidazole; (v) a cocktail of all four antibiotics together (with zero-calorie sweetener to ensure intake); (vi) zero-calorie sweetener only; or (vii) no additive (untreated) for 12 weeks. Individual antibiotics remove only some taxa from the gut, while the cocktail of all four removes almost all microbes. After accounting for differences in geometry, whole bone strength was reduced in animals with gut microbiome modified by neomycin (-28%, p = 0.002) and was increased in the group in which the gut microbiome was altered by sweetener alone (+39%, p < 0.001). Analysis of the fecal microbiota detected seven lower-ranked taxa differentially abundant in animals with impaired tissue-level strength and 14 differentially abundant taxa associated with increased tissue-level strength. Histological and serum markers of bone turnover and trabecular bone volume per tissue volume (BV/TV) did not differ among groups. These findings demonstrate that modifications to the taxonomic components of the gut microbiome have the potential to decrease or increase tissue-level strength of bone independent of bone quantity and without noticeable changes in bone turnover. © 2021 American Society for Bone and Mineral Research (ASBMR).

Measures of Bone Mineral Carbonate Content and Mineral Maturity/Crystallinity for FT-IR and Raman Spectroscopic Imaging Differentially Relate to Physical-Chemical Properties of Carbonate-Substituted Hydroxyapatite.

Bone mineral carbonate content assessed by vibrational spectroscopy relates to fracture incidence, and mineral maturity/ crystallinity (MMC) relates to tissue age. As FT-IR and Raman spectroscopy become more widely used to characterize the chemical composition of bone in pre-clinical and translational studies, their bone mineral outcomes require improved validation to inform interpretation of spectroscopic data. In this study, our objectives were (1) to relate Raman and FT-IR carbonate:phosphate ratios calculated through direct integration of peaks to gold-standard analytical measures of carbonate content and underlying subband ratios; (2) to relate Raman and FT-IR MMC measures to gold-standard analytical measures of crystal size in chemical standards and native bone powders. Raman and FT-IR direct integration carbonate:phosphate ratios increased with carbonate content (Raman: p < 0.01, R2 = 0.87; FT-IR: p < 0.01, R2 = 0.96) and Raman was more sensitive to carbonate content than the FT-IR (Raman slope + 95% vs FT-IR slope, p < 0.01). MMC increased with crystal size for both Raman and FT-IR (Raman: p < 0.01, R2 = 0.76; FT-IR p < 0.01, R2 = 0.73) and FT-IR was more sensitive to crystal size than Raman (c-axis length: slope FT-IR MMC + 111% vs Raman MMC, p < 0.01). Additionally, FT-IR but not Raman spectroscopy detected differences in the relationship between MMC and crystal size of carbonated hydroxyapatite (CHA) vs poorly crystalline hydroxyapatites (HA) (slope CHA + 87% vs HA, p < 0.01). Combined, these results contribute to the ability of future studies to elucidate the relationships between carbonate content and fracture and provide insight to the strengths and limitations of FT-IR and Raman spectroscopy of native bone mineral.

Bone Tissue Composition in Postmenopausal Women Varies With Glycemic Control From Normal Glucose Tolerance to Type 2 Diabetes Mellitus.

The risk of fragility fracture increases for people with type 2 diabetes mellitus (T2DM), even after controlling for bone mineral density, body mass index, visual impairment, and falls. We hypothesize that progressive glycemic derangement alters microscale bone tissue composition. We used Fourier-transform infrared (FTIR) imaging to analyze the composition of iliac crest biopsies from cohorts of postmenopausal women characterized by oral glucose tolerance testing: normal glucose tolerance (NGT; n = 35, age = 65 ± 7 years, HbA1c = 5.8 ± 0.3%), impaired glucose tolerance (IGT; n = 26, age = 64 ± 5 years, HbA1c = 6.0 ± 0.4%), and overt T2DM on insulin (n = 25, age = 64 ± 6 years, HbA1c = 9.13 ± 0.6). The distributions of cortical bone mineral content had greater mean values (+7%) and were narrower (-10%) in T2DM versus NGT groups (p < 0.05). The distributions of acid phosphate, an indicator of new mineral, were narrower in cortical T2DM versus NGT and IGT groups (-14% and -14%, respectively) and in trabecular NGT and IGT versus T2DM groups (-11% and -10%, respectively) (all p < 0.05). The distributions of crystallinity were wider in cortical NGT versus T2DM groups (+16%) and in trabecular NGT versus T2DM groups (+14%) (all p < 0.05). Additionally, bone turnover was lower in T2DM versus NGT groups (P1NP: -25%, CTx: -30%, ucOC: -24%). Serum pentosidine was similar across groups. The FTIR compositional and biochemical marker values of the IGT group typically fell between the NGT and T2DM group values, although the differences were not always statistically significant. In summary, worsening glycemic control was associated with greater mineral content and narrower distributions of acid phosphate, an indicator of new mineral, which together are consistent with observations of lower turnover; however, wider distributions of mineral crystallinity were also observed. A more mineralized, less heterogeneous tissue may affect tissue-level mechanical properties and in turn degrade macroscale skeletal integrity. In conclusion, these data are the first evidence of progressive alteration of bone tissue composition with worsening glycemic control in humans. © 2020 American Society for Bone and Mineral Research (ASBMR).

Raman and Fourier transform infrared imaging for characterization of bone material properties.

As the application of Raman spectroscopy to study bone has grown over the past decade, making it a peer technology to FTIR spectroscopy, it has become critical to understand their complimentary roles. Recent technological advancements have allowed these techniques to collect grids of spectra in a spatially resolved fashion to generate compositional images. The advantage of imaging with these techniques is that it allows the heterogenous bone tissue composition to be resolved and quantified. In this review we compare, for non-experts in the field of vibrational spectroscopy, the instrumentation and underlying physical principles of FTIR imaging (FTIRI) and Raman imaging. Additionally, we discuss the strengths and limitations of FTIR and Raman spectroscopy, address sample preparation, and discuss outcomes to provide researchers insight into which techniques are best suited for a given research question. We then briefly discuss previous applications of FTIRI and Raman imaging to characterize bone tissue composition and relationships of compositional outcomes with mechanical performance. Finally, we discuss emerging technical developments in FTIRI and Raman imaging which provide new opportunities to identify changes in bone tissue composition with disease, age, and drug treatment.

Sequential Treatment of Estrogen Deficient, Osteopenic Rats with Alendronate, Parathyroid Hormone (1-34), or Raloxifene Alters Cortical Bone Mineral and Matrix Composition.

Anti-resorptive and anabolic treatments can be used sequentially to treat osteoporosis, but their effects on bone composition are incompletely understood. Osteocytes may influence bone tissue composition with sequential therapies because bisphosphonates diffuse into the canalicular network and anabolic treatments increase osteocyte lacunar size. Cortical bone composition of osteopenic, ovariectomized (OVX) rats was compared to that of Sham-operated rats and OVX rats given monotherapy or sequential regimens of single approved anti-osteoporosis medications. Adult female Sprague-Dawley rats were OVX (N = 37) or Sham-OVXd (N = 6). After 2 months, seven groups of OVX rats were given three consecutive 3-month periods of treatment with vehicle (V), h-PTH (1-34) (P), alendronate (A), or raloxifene (R), using the following orders: VVV, PVV, RRR, RPR, AAA, AVA, and APA. Compositional properties around osteocyte lacunae of the left tibial cortex were assessed from Raman spectra in perilacunar and non-perilacunar bone matrix regions. Sequential treatments involving parathyroid hormone (PTH) caused lower mean collagen maturity relative to monotherapies. Mean mineral:matrix ratio was 2.2% greater, mean collagen maturity was 1.4% greater, and mean carbonate:phosphate ratio was 2.2% lower in the perilacunar than in the non-perilacunar bone matrix region (all P < 0.05). These data demonstrate cortical bone tissue composition differences around osteocytes caused by sequential treatment with anti-osteoporosis medications. We speculate that the region-specific differences demonstrate the ability of osteocytes to alter bone tissue composition adjacent to lacunae.

Effects of Diabetes on Bone Material Properties.

PURPOSE OF REVIEW: Individuals with type 1 and type 2 diabetes mellitus (T1DM, T2DM) have an increased risk of bone fracture compared to non-diabetic controls that is not explained by differences in BMD, BMI, or falls. Thus, bone tissue fracture resistance may be reduced in individuals with DM. The purpose of this review is to summarize work that analyzes the effects of T1DM and T2DM on bone tissue compositional and mechanical properties. RECENT FINDINGS: Studies of clinical T2DM specimens revealed increased mineralization and advanced glycation endproduct (AGE) concentrations and significant relationships between mechanical performance and composition of cancellous bone. Specifically, in femoral cancellous tissue, compressive stiffness and strength increased with mineral content; and post-yield properties decreased with AGE concentration. In addition, cortical resistance to in vivo indentation (bone material strength index) was lower in patients with T2DM vs. age-matched non-diabetic controls, and this resistance decreased with worsening glycemic control. Recent studies on patients with T1DM and history of a prior fragility fracture found greater mineral content and concentrations of AGEs in iliac trabecular bone and correspondingly stiffer, harder bone at the nanosacle. Recent observational data showed greater AGE and mineral content in surgically retrieved bone from patients with T2DM vs. non-DM controls, consistent with reduced bone remodeling. Limited data on human T1DM bone tissue also showed higher mineral and AGE content in patients with prior fragility fractures compared to non-DM and non-fracture controls.

Raman and Fourier Transform Infrared (FT-IR) Mineral to Matrix Ratios Correlate with Physical Chemical Properties of Model Compounds and Native Bone Tissue.

Raman and Fourier transform infrared (FT-IR) spectroscopic imaging techniques can be used to characterize bone composition. In this study, our objective was to validate the Raman mineral:matrix ratios (ν1 PO4:amide III, ν1 PO4:amide I, ν1 PO4:Proline + hydroxyproline, ν1 PO4:Phenylalanine, ν1 PO4:δ CH2 peak area ratios) by correlating them to ash fraction and the IR mineral:matrix ratio (ν3 PO4:amide I peak area ratio) in chemical standards and native bone tissue. Chemical standards consisting of varying ratios of synthetic hydroxyapatite (HA) and collagen, as well as bone tissue from humans, sheep, and mice, were characterized with confocal Raman spectroscopy and FT-IR spectroscopy and gravimetric analysis. Raman and IR mineral:matrix ratio values from chemical standards increased reciprocally with ash fraction (Raman ν1 PO4/Amide III: P < 0.01, R2 = 0.966; Raman ν1 PO4/Amide I: P < 0.01, R2 = 0.919; Raman ν1 PO4/Proline + Hydroxyproline: P < 0.01, R2 = 0.976; Raman ν1 PO4/Phenylalanine: P < 0.01, R2 = 0.911; Raman ν1 PO4/δ CH2: P < 0.01, R2 = 0.894; IR P < 0.01, R2 = 0.91). Fourier transform infrared mineral:matrix ratio values from native bone tissue were also similar to theoretical mineral:matrix ratio values for a given ash fraction. Raman and IR mineral:matrix ratio values were strongly correlated ( P < 0.01, R2 = 0.82). These results were confirmed by calculating the mineral:matrix ratio for theoretical IR spectra, developed by applying the Beer-Lambert law to calculate the relative extinction coefficients of HA and collagen over the same range of wavenumbers (800-1800 cm-1). The results confirm that the Raman mineral:matrix bone composition parameter correlates strongly to ash fraction and to its IR counterpart. Finally, the mineral:matrix ratio values of the native bone tissue are similar to those of both chemical standards and theoretical values, confirming the biological relevance of the chemical standards and the characterization techniques.