Age-related changes in the collagen network and toughness of bone

The hypothesis of this study is that the mechanical integrity of the collagen network in bone deteriorates with age, and such adverse changes correlate with the decreased toughness of aged bone. To test the hypothesis, 30 human cadaveric femurs from donors ranging from 19 to 89 years of age were tested to determine the age-related changes in the mechanical properties of demineralized bone and fresh bone samples. Along with bone porosity, bone density, and weight fractions of the mineral and organic phases, collagen denaturation and concentrations of collagen cross-links (HP, hydroxylysylpyridinoline; LP, lysylpyridinoline; PE, pentosidine) were determined for these bone specimens as a function age. Analysis of variance (ANOVA) showed that age-dependent changes were reflected in the decreased strength, work to fracture, and fracture toughness of bone; in the decreased strength, elastic modulus, and work to fracture of the collagen network; as well as in the increased concentration of pentosidine (a marker of nonenzymatic glycation) and increased bone porosity. Regression analyses of the measured parameters showed that the age-related decrease in work to fracture of bone (especially its postyield portion) correlated significantly with deterioration in the mechanical integrity of the collagen network. The results of this study indicate that the adverse changes in the collagen network occur as people age and such changes may lead to the decreased toughness of bone. Also, the results suggest that nonenzymatic glycation may be an important contributing factor causing changes in collagen and, consequently, leading to the age-related deterioration of bone quality.     

Bone Morphometry Strongly Predicts Cortical Bone Stiffness and Strength,but Not Toughness,in Inbred Mouse Models of High and Low Bone Mass

Inbred strains of mice make useful models to study bone properties. Our aim was to compare bone competence and cortical morphometric parameters of two inbred strains to better determine the role of bone structure and geometry in the process of bone failure. Morphometric analysis was performed on 20 murine femora with a low bone mass (C57BL/6J; B6) and 20 murine femora with a high bone mass (C3H/HeJ; C3H) using desktop μCT. The bones were tested under three‐point bending to measure their mechanical properties. Results showed that the C3H strain is a more reproducible model regarding bone morphometric and mechanical phenotypes than the B6 strain. Bone strength, stiffness, yield force, yield displacement, and toughness, as well as morphometric traits, were all significantly different between the two strains, whereas postyield displacement was not. It was found that bone volume, cortical thickness, and cross‐sectional area predicted almost 80% (p < 0.05) of bone stiffness, strength, and yield force. Nevertheless, cortical bone postyield properties such as bone toughness could not be explained by morphometry, but postyield whitening was observed in that phase. In conclusion, we found that morphometric parameters are strong predictors of preyield but not postyield properties. The lack of morphometric influence on bone competence in the postyield phase in combination with the observed postyield whitening confirmed the important contribution of ultrastructure and microdamage in the process of overall bone failure behavior, especially in the postyield phase.     

Influence of nonenzymatic glycation on biomechanical properties of cortical bone

In this study, the influence of nonenzymatic glycation (NEG) on the mechanical properties of bone and bone collagen were investigated. Bovine cortical bone specimens were incubated in ribose to cause collagen cross-links in vitro, and nondestructive mechanical testing was used to determine tensile and compressive elastic modulus as a function of incubation time. Mechanical properties associated with yield, postyield, and final fracture of bone were determined at the end of the incubation period. The stiffness of the collagen network was measured using stress relaxation tests of demineralized bone cylinders extracted periodically throughout the incubation period. It was found that accumulation of nonenzymatic glycation end-products in cortical bone caused stiffening of the type I collagen network in bone (r2 = 0.92; p < 0.001) but did not significantly affect the overall stiffness of the mineralized bone (p = 0.98). The ribosylated group had significantly more NEG products and higher yield stress and strain than the control group (p < 0.05). Postyield properties including postyield strain and strain energy were lower in the ribosylated group but were not significantly different from the control group (p = 0.24). Compared with the control group, the ribosylated group was characterized by significantly higher secant modulus and lower damage fraction (p < 0.05). Taken together, the results of this study suggest that collagen in bone is susceptible to the same NEG-mediated changes as collagen in other connective tissues and that an increased stiffness of the collagen network in bone due to NEG may explain some of the age-related increase in skeletal fragility and fracture risk.     

Infrared imaging of calcified tissue in bone biopsies from adults with osteomalacia

Osteomalacia is a pathological bone condition in which there is deficient primary mineralization of the matrix, leading to an accumulation of osteoid tissue and reduced bone mechanical strength. The hypothesis that there are no qualitative or quantitative differences in osteomalacic bone mineral or matrix compared to disease-free bones was tested by examining unstained sections of polymethyl methacrylate (PMMA) embedded iliac crest biopsies using Fourier transform infrared imaging (FTIRI) at approximately 6-microm spatial resolution. Controls were seven female subjects, aged 36-57, without apparent bone disease. The experimental group consisted of 11 patients aged 22-72, diagnosed with osteomalacia. The spectroscopic parameters analyzed in each data set were previously established as sensitive to bone quality: phosphate/amide I band area ratio (mineral content), 1660/1690 cm(-1) peak ratio (collagen cross-links), and the 1030/1020 cm(-1) peak ratio (mineral crystallinity). The correspondence between spectroscopic mineral content (phosphate/amide I ratio) and ash weight was validated for apatite crystals of different composition and crystallite size. The FTIRI results from the biopsies expressed as color-coded images and pixel population means were compared with the nonparametric Mann-Whitney U test. There were no significant differences in the cortical parameters. Significant difference was found in the mineral content of the trabecular regions with a lower mean value in osteomalacia (P = 0.01) than in controls. Mineral crystallinity tended to be decreased in the trabecular bone (P = 0.09). This study supports the hypothesis that, in osteomalacia, the quality of the organic matrix and of mineral in the center of bone does not change, while less-than-optimal mineralization occurs at the bone surface. This study provides the first spectroscopic evaluation of whole bone mineral and matrix properties in osteomalacia, demonstrating that there are few differences in collagen cross-links between biopsies from patients with osteomalacia and from individuals without histological evidence of bone disease.     

Age-related factors affecting the postyield energy dissipation of human cortical bone.

The risk of bone fracture depends in part on tissue quality, not just the size and mass. This study assessed the postyield energy dissipation of cortical bone in tension as a function of age and composition. Specimens were prepared from tibiae of human cadavers in which male and female donors were divided into two age groups: middle aged (51 to 56 years, n = 9) and elderly (72 to 90 years, n = 8). By loading, unloading, and reloading a specimen with rest periods inserted in between, tensile properties at incremental strain levels were assessed. In addition, postyield toughness was estimated and partitioned as plastic strain energy related to permanent deformation, released elastic strain energy related to stiffness loss, and hysteresis energy related to viscous behavior. Porosity, mineral and collagen content, and collagen crosslinks of each specimen were also measured to determine the micro- and ultrastructural properties of the tissue. Age affected all the energy terms plus strength but not elastic stiffness. The postyield energy terms were correlated with porosity, pentosidine (a marker of nonenzymatic crosslinks), and collagen content, all of which varied significantly with age. General linear models suggested that pentosidine concentration and collagen content provided the best explanation of the age-related decrease in the postyield energy dissipation. Among them, pentosidine concentration had the greatest contribution to plastic strain energy and was the best explanatory variable of damage accumulation.     

The role of collagen in determining bone mechanical properties.

The hypothesis of this study was that collagen denaturation would lead to a significant decrease in the toughness of bone, but has little effect on the stiffness of bone. Using a heating model, effects of collagen denaturation on the biomechanical properties of human cadaveric bone were examined. Prior to testing, bone specimens were heat treated at varied temperatures (37-200 degrees C) to induce different degrees of collagen denaturation. Collagen denaturation and mechanical properties of bone were determined using a selective digestion technique and three-point bending tests, respectively. The densities and weight fractions of the mineral and organic phases in bone also were determined. A repeated measures analysis of variance showed that heating had a significant effect on the biomechanical integrity of bone, corresponding to the degree of collagen denaturation. The results of this study indicate that the toughness and strength of bone decreases significantly with increasing collagen denaturation, whereas the elastic modulus of bone is almost constant irrespective of collagen denaturation. These results suggest that the collagen network plays an important role in the toughness of bone, but has little effect on the stiffness of bone, thereby supporting the hypothesis of this study.     

Spectroscopic markers of bone quality in alendronate-treated postmenopausal women

Summary  Comparison of infrared spectroscopic images of sections from biopsies of placebo-treated post-menopausal women and women treated for 3 years with 10 mg/day alendronate demonstrated significant increases in cortical bone mineral content, no alterations in other spectroscopic markers of “bone quality,” but a decrease in tissue heterogeneity. Methods  The material properties of thick sections from iliac crest biopsies of seven alendronate-treated women were compared to those from ten comparably aged post-menopausal women without bone disease, using infrared spectroscopic imaging at ∼7 μm spatial resolution. Parameters evaluated were mineral/matrix ratio, crystallinity, carbonate/amide I ratio, and collagen maturity. The line widths at half maximum of the pixel histograms for each parameter were used as measures of heterogeneity. Results  The mineral content (mineral/matrix ratio) in the cortical bone of the treated women’s biopsies was higher than that in the untreated control women. Crystallinity, carbonate/protein, and collagen maturity indices were not significantly altered; however, the pixel distribution was significantly narrowed for all cortical and trabecular parameters with the exception of collagen maturity in the alendronate treatment group. Conclusions  The increases in mineral density and decreased fracture risk associated with bisphosphonate treatment may be counterbalanced by a decrease in tissue heterogeneity, which could impair tissue mechanical properties. These consistent data suggest that alendronate treatment, while increasing the bone mass, decreases the tissue heterogeneity.     

On the increasing fragility of human teeth with age: a deep-UV resonance Raman study.

UV resonance Raman spectroscopy (UVRRS) using 244-nm excitation was used to study the impact of aging on human dentin. The intensity of a spectroscopic feature from the peptide bonds in the collagen increases with tissue age, similar to a finding reported previously for human cortical bone. INTRODUCTION: The structural changes that lead to compromised mechanical properties with age in dentin and bone are under intense study. However, in situ analyses of the content and distribution of the mineral phase are more highly developed at present than equivalent probes of the organic phase. MATERIALS AND METHODS: Thirty-five human molars were divided into three groups: young/normal (23.3 +/- 3.8 years); aged/transparent (74.3 +/- 6.0 years), which had become transparent because of filling of the tubule lumens with mineral deposits; and aged/nontransparent (73.3 +/- 5.7 years). Control experiments were performed by demineralizing normal dentin. RESULTS: Spectral features caused by both the amide backbone and resonance-enhanced side-chain vibrations were observed. This finding contrasts with reported Raman spectra of proteins in solution excited with similar UV wavelengths, where side chain vibrations, but not strong amide features, are observed. The strong intensity of the amide features observed from dentin is attributed to broadening of the resonance profile for the amide pi --> pi* transition caused by the environment of the collagen molecules in dentin. With increasing age, the height of one specific amide vibration (amide I) becomes significantly higher when comparing teeth from donors with an average age of 23 years to those of 73 years (p < 0.001). This trend of increasing amide I peak height with age is similar to that previously reported for human cortical bone. The amide I feature also increased in dentin that had been demineralized and dehydrated. CONCLUSIONS: The similar trend of increasing amide I peak height with age in the UVRR spectra of both teeth and bone is surprising, given that only bone undergoes remodeling. However, by considering those observations together with this study of demineralized/dehydrated dentin and our prior work on dentin dehydrated with polar solvents, a consistent relationship between changes in the UVRR spectra and the collagen environment in the tissue can be developed.     

Decreased collagen organization and content are associated with reduced strength of demineralized and intact bone in the SAMP6 mouse.

To examine the link between bone material properties and skeletal fragility, we analyzed the mechanical, histological, biochemical, and spectroscopic properties of bones from a murine model of skeletal fragility (SAMP6). Intact bones from SAMP6 mice are weak and brittle compared with SAMR1 controls, a defect attributed to reduced strength of the bone matrix. The matrix weakness is attributed primarily to poorer organization of collagen fibers and reduced collagen content. INTRODUCTION: The contribution of age-related changes in tissue material properties to skeletal fragility is poorly understood. We previously reported that bones from SAMP6 mice are weak and brittle versus age-matched controls. Our present objectives were to use the SAMP6 mouse to assess bone material properties in a model of skeletal fragility and to relate defects in the mechanical properties of bone to the properties of demineralized bone and to the structure and organization of collagen and mineral. MATERIALS AND METHODS: Femora from 4- and 12-month-old SAMR1 (control) and SAMP6 mice were analyzed using bending and torsional mechanical testing of intact bones, tensile testing of demineralized bone, quantitative histology (including collagen fiber orientation), collagen cross-links biochemistry, and Raman spectroscopic analysis of mineral and collagen. RESULTS: Intact bones from SAMP6 mice have normal elastic properties but inferior failure properties, with 60% lower fracture energy versus SAMR1 controls. The strength defect in SAMP6 bones was associated with a 23% reduction in demineralized bone strength, which in turn was associated with poorer collagen fiber organization, lower collagen content, and higher hydroxylysine levels. However, SAMP6 have normal levels of collagen cross-links and normal apatite mineral structure. CONCLUSIONS: Bones from SAMP6 osteoporotic mice are weak and brittle because of a defect in the strength of the bone matrix. This defect is attributed primarily to poorer organization of collagen fibers and reduced collagen content. These findings highlight the role of the collagen component of the bone matrix in influencing skeletal fragility.     

Effects of collagen unwinding and cleavage on the mechanical integrity of the collagen network in bone

The objective of this study was to investigate how molecular level changes in the collagen network affect its mechanical integrity. Our hypothesis is that the cleavage and unwinding of triple helices of collagen molecules significantly reduce the mechanical integrity of the collagen network in bone, whereas collagen crosslinks play a major role in sustaining the structural integrity of the collagen network. To test this hypothesis, the collagen molecular structure was altered in demineralized human cadaveric bone samples in the following two ways: heat induced unwinding and pancreas elastase induced cleavage of collagen molecules. Along with control specimens, the treated specimens were mechanically tested in tension to determine their strength, elastic modulus, toughness, and strain to failure. Also, the percentage of denatured collagen molecules and amounts of two major collagen crosslinks (hydroxylysylpyridinoline and lysylpyridinoline) were determined using high-performance liquid chromatography techniques. It was found that unwinding of collagen molecules may cause more reduction in stiffness (E) but less strain to failure (ef) than cleavage. Both collagen denaturation types cause similar changes in the strength (ss) and work to fracture (Wf) of the collagen network with no significant changes in hydroxylysylpyridinoline and lysylpyridinoline crosslinks. The results of this study indicate that the integrity of collagen molecules significantly affect the mechanical properties of the collagen network in bone, and that collagen crosslinks may play an important role in maintaining the mechanical integrity of the collagen network after collagen denaturation occurs.     

Bone brittleness varies with genetic background in A/J and C57BL/6J inbred mice.

The contribution of genetic and environmental factors to variations in bone quality are understood poorly. We tested whether bone brittleness varies with genetic background using the A/J and C57BL/6J inbred mouse strains. Whole bone four-point bending tests revealed a 70% decrease in postyield deflection of A/J femurs compared with C57BL/6J, indicating that A/J femurs failed in a significantly more brittle manner. Cyclic loading studies indicated that A/J femurs accumulated damage differently than C57BL/6J femurs, consistent with their increased brittleness. Differences in matrix composition also were observed between the two mouse strains. A/J femurs had a 4.5% increase in ash content and an 11.8% decrease in collagen content. Interestingly, a reciprocal relationship was observed between femoral geometry and material stiffness; this relationship may have contributed to the brittle phenotype of A/J femurs. A/J femurs are more slender than those of C57BL/6J femurs; however, their 47% smaller moment of inertia appeared to be compensated by an increased tissue stiffness at the expense of altered tissue damageability. Importantly, these differences in whole bone mechanical properties between A/J and C57BL/6J femurs could not have been predicted from bone mass or density measures alone. The results indicated that bone brittleness is a genetically influenced trait and that it is associated with genetically determined differences in whole bone architecture, bone matrix composition, and mechanisms of cyclical damage accumulation.     

Novel assessment of bone using time-resolved transcutaneous Raman spectroscopy.

With fragility fractures increasing as the population ages, there is a need for improved means to estimate risk of fracture. We recorded Raman spectra of both the mineral and organic phases of bone transcutaneously, a technology with potential to enhance bone quality and fracture risk assessment. INTRODUCTION: The current "gold standard" assessment of bone quality is BMD determined by DXA. However, this accounts for only 60-70% of bone strength. X-rays are absorbed by the mineral phase of bone, whereas the organic phase remains essentially invisible; however, bone strength is critically dependent on both phases. We report, for the first time, a Raman spectroscopic technique that analyses both phases of bone beneath unbroken skin by eliminating spectral components of overlying tissues. MATERIALS AND METHODS: We used an 800-nm laser (1-kHz, 1-ps pulses) with a synchronized 4-ps Kerr gate with variable picosecond delay that effectively shuttered out photons from overlying tissues. We measured bone Raman spectra at a point 2 mm above the carpus from two mouse genotypes with extreme differences in bone matrix quality: wildtype and oim/oim (matched for age, sex, and weight). Typical depth was 1.1 mm. We repeated the measurements with overlying tissues removed down to bone. Oim/oim mice produce only homotrimeric collagen, which results in poorly mineralized bone tissue. RESULTS: The main spectral features were present from both bone phases. The spectral bands were in similar ratios when measured through the skin or directly from bone (in both genotypes). The band of the mineral phase (phosphate nu1) was smaller in oim/oim mice when measured directly from bone and through skin. The band associated with a particular vibrational mode of organic phase collagen (CH2 wag) showed a frequency shift between the genotypes. CONCLUSIONS: This novel technique allowed us, for the first time, to make objective transcutaneous spectral measurements of both the mineral and the organic phases of bones and distinguish between normal and unhealthy bone tissue. After further optimization, this technology may help improve fracture risk assessments and open opportunities for screening in anticipation of the predicted increase in fragility fractures.     

Spectroscopic characterization of collagen cross-links in bone.

Collagen is the most abundant protein of the organic matrix in mineralizing tissues. One of its most critical properties is its cross-linking pattern. The intermolecular cross-linking provides the fibrillar matrices with mechanical properties such as tensile strength and viscoelasticity. In this study, Fourier transform infrared (FTIR) spectroscopy and FTIR imaging (FTIRI) analyses were performed in a series of biochemically characterized samples including purified collagen cross-linked peptides, demineralized bovine bone collagen from animals of different ages, collagen from vitamin B6-deficient chick homogenized bone and their age- and sex-matched controls, and histologically stained thin sections from normal human iliac crest biopsy specimens. One region of the FTIR spectrum of particular interest (the amide I spectral region) was resolved into its underlying components. Of these components, the relative percent area ratio of two subbands at approximately 1660 cm(-1) and approximately 1690 cm(-1) was related to collagen cross-links that are abundant in mineralized tissues (i.e., pyridinoline [Pyr] and dehydrodihydroxylysinonorleucine [deH-DHLNL]). This study shows that it is feasible to monitor Pyr and DHLNL collagen cross-links spatial distribution in mineralized tissues. The spectroscopic parameter established in this study may be used in FTIRI analyses, thus enabling the calculation of relative Pyr/DHLNL amounts in thin (approximately 5 microm) calcified tissue sections with a spatial resolution of approximately 7 microm.     

Infrared spectroscopy indicates altered bone turnover and remodeling activity in renal osteodystrophy

Renal osteodystrophy alters metabolic activity and remodeling rate of bone and also may lead to different bone composition. The objective of this study was to characterize the composition of bone in high‐turnover renal osteodystrophy patients by means of Fourier transform infrared spectroscopic imaging (FTIRI). Iliac crest biopsies from healthy bone (n = 11) and patients with renal osteodystrophy (ROD, n = 11) were used in this study. The ROD samples were from patients with hyperparathyroid disease. By using FTIRI, phosphate‐to‐amide I ratio (mineral‐to‐matrix ratio), carbonate‐to‐phosphate ratio, and carbonate‐to‐amide I ratio (turnover rate/remodeling activity), as well as the collagen cross‐link ratio (collagen maturity), were quantified. Histomorphometric analyses were conducted for comparison. The ROD samples showed significantly lower carbonate‐to‐phosphate (p < .01) and carbonate‐to‐amide I (p < .001) ratios. The spatial variation across the trabeculae highlighted a significantly lower degree of mineralization (p < .05) at the edges of the trabeculae in the ROD samples than in normal bone. Statistically significant linear correlations were found between histomorphometric parameters related to bone‐remodeling activity and number of bone cells and FTIRI‐calculated parameters based on carbonate‐to‐phosphate and carbonate‐to‐amide I ratios. Hence the results suggested that FTIRI parameters related to carbonate may be indicative of turnover and remodeling rate of bone. © 2010 American Society for Bone and Mineral Research     

Altered Tissue Composition,Microarchitecture, and Mechanical Performance in Cancellous Bone From Men With Type 2 Diabetes Mellitus

People with type 2 diabetes mellitus (T2DM) have normal-to-high BMDs, but, counterintuitively, have greater fracture risks than people without T2DM, even after accounting for potential confounders like BMI and falls. Therefore, T2DM may alter aspects of bone quality, including material properties or microarchitecture, that increase fragility independently of bone mass. Our objective was to elucidate the factors that influence fragility in T2DM by comparing the material properties, microarchitecture, and mechanical performance of cancellous bone in a clinical population of men with and without T2DM. Cancellous specimens from the femoral neck were collected during total hip arthroplasty (T2DM: n = 31, age = 65 ± 8 years, HbA1c = 7.1 ± 0.9%; non-DM: n = 34, age = 62 ± 9 years, HbA1c = 5.5 ± 0.4%). The T2DM specimens had greater concentrations of the advanced glycation endproduct pentosidine (+ 36%, P < 0.05) and sugars bound to the collagen matrix (+ 42%, P < 0.05) than the non-DM specimens. The T2DM specimens trended toward a greater bone volume fraction (BV/TV) (+ 24%, NS, P = 0.13) and had greater mineral content (+ 7%, P < 0.05) than the non-DM specimens. Regression modeling of the mechanical outcomes revealed competing effects of T2DM on bone mechanical behavior. The trend of higher BV/TV values and the greater mineral content observed in the T2DM specimens increased strength, whereas the greater values of pentosidine in the T2DM group decreased postyield strain and toughness. The long-term medical management and presence of osteoarthritis in these patients may influence these outcomes. Nevertheless, our data indicate a beneficial effect of T2DM on cancellous microarchitecture, but a deleterious effect of T2DM on the collagen matrix. These data suggest that high concentrations of advanced glycation endproducts can increase fragility by reducing the ability of bone to absorb energy before failure, especially for the subset of T2DM patients with low BV/TV. © 2019 American Society for Bone and Mineral Research.     

Relationship between bone morphology and bone quality in male tibias: implications for stress fracture risk.

Biomechanical properties were assessed from the tibias of 17 adult males 17-46 years of age. Tissue-level mechanical properties varied with bone size. Narrower tibias were comprised of tissue that was more brittle and more prone to accumulating damage compared with tissue from wider tibias. INTRODUCTION: A better understanding of the factors contributing to stress fractures is needed to identify new prevention strategies that will reduce fracture incidence. Having a narrow (i.e., more slender) tibia relative to body mass has been shown to be a major predictor of stress fracture risk and fragility in male military recruits and male athletes. The intriguing possibility that slender bones, like those shown in animal models, may be composed of more damageable material has not been considered in the human skeleton. MATERIALS AND METHODS: Polar moment of inertia, section modulus, and antero-posterior (AP) and medial-lateral (ML) widths were determined for tibial diaphyses from 17 male donors 17-46 years of age. A slenderness index was defined as the inverse ratio of the section modulus to tibia length and body weight. Eight prismatic cortical bone samples were generated from each tibia, and tissue-level mechanical properties including modulus, strength, total energy, postyield strain, and tissue damageability were measured by four-point bending from monotonic (n = 4/tibia) and damage accumulation (n = 4/tibia) test methods. Partial correlation coefficients were determined between each geometrical parameter and each tissue-level mechanical property while taking age into consideration. RESULTS: Significant correlations were observed between tibial morphology and the mechanical properties that characterized tissue brittleness and damageability. Positive correlations were observed between measures of bone size (AP width) and measures of tissue ductility (postyield strain, total energy), and negative correlations were observed between bone size (moment of inertia, section modulus) and tissue modulus. CONCLUSIONS: The correlation analysis suggested that bone morphology could be used as a predictor of tissue fragility and stress fracture risk. The average mechanical properties of cortical tissue varied as a function of the overall size of the bone. Therefore, under extreme loading conditions (e.g., military training), variation in bone quality parameters related to damageability may be a contributing factor to the increased risk of stress fracture for individuals with more slender bones.     

Osteolytic and mixed cancer metastasis modulates collagen and mineral parameters within rat vertebral bone matrix

Metastatic involvement in vertebral bone diminishes the mechanical integrity of the spine; however minimal data exist on the potential impact of metastases on the intrinsic material characteristics of the bone matrix. Thirty‐four (34) female athymic rats were inoculated with HeLa (N = 17) or Ace‐1 (N = 17) cancer cells lines producing osteolytic or mixed (osteolytic and osteoblastic) metastases, respectively. A maximum of 21 days was allowed between inoculation and rat sacrifice for vertebrae extraction. High performance liquid chromatography (HPLC) was utilized to determine modifications in collagen‐I parameters such as proline hydroxylation and the formation of specific enzymatic and non‐enzymatic (pentosidine) cross‐links. Raman spectroscopy was used to determine relative changes in mineral crystallinity, mineral carbonation, mineral/collagen matrix ratio, collagen quality ratio, and proline hydroxylation. HPLC results showed significant increase in the formation of pentosidine and decrease in the formation of the enzymatic cross‐link deoxy‐pryridinoline within osteolytic bone compared to mixed bone. Raman results showed decreased crystallinity, increased carbonation, and collagen quality (aka 1660/1690 sub‐band) ratio with osteolytic bone compared to mixed bone and healthy controls along with an observed increase in proline hydroxylation with metastatic involvement. The mineral/matrix ratio decreased in both osteolytic and mixed bone compared to healthy controls. Quantifying modifications within the intrinsic characteristics of bone tissue will provide a foundation to assess the impact of current therapies on the material behavior of bone tissue in the metastatic spine and highlight targets for the development of new therapeutics and approaches for treatment. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:2126–2136, 2016.     

Cortical bone mass,composition, and mechanical properties in female rats in relation to age,long-term ovariectomy,and estrogen substitution

Summary The study comprised 12 groups of female rats: 6 groups of intact rats killed at 2, 6, 9, 12, 15, and 24 months of age, 4 groups of rats ovariectomized at 6 months and killed together with the intact rats at 9, 12, 15, and 24 months of age, and 2 groups of rats (one intact and one ovariectomized) treated with estrogen (2 g estradiol valerate/rat/week s.c.) for 8 months before they were killed at 24 months of age. The composition, dimensions, and mechanical strength of intact bone and bone collagen from femoral diaphyses were investigated in relation to age, ovariectomy, and estrogen administration. Up to 6–9 months of age, the axial length, percentage ash, density, and compressive mechanical stress increased, whereas percentage collagen decreased. An age-related increase in bone mass, crosssectional area, and wall thickness and a decrease in mechanical quality of bone collagen were apparent from 2 to 24 months of age. An age-related periosteal bone formation and the absence of endosteal bone resorption were demonstrated in intact rats. Compared with intact rats, ovariectomy was followed by an increase in body weight, a tendency to reduced percentage ash and a depressed bone mass, crosssectional area, and wall thickness of femoral diaphyses. The compressive mechanical stress of intact bone and the mechanical quality of bone collagen were unaffected by ovariectomy. Ovariectomy did not influence the periosteal bone formation but induced an endosteal bone resorption not present in the intact rats. The estrogen treatment of the ovariectomized rats normalized the body weight of the rats and brought to an end the endosteal bone resorption induced by ovariectomy. Estrogen treatment of both ovariectomized and intact rats tended to reduced the rate of periosteal bone formation.     

Mechanical Competence and Bone Quality Develop During Skeletal Growth

Bone fracture risk is influenced by bone quality, which encompasses bone's composition as well as its multiscale organization and architecture. Aging and disease deteriorate bone quality, leading to reduced mechanical properties and higher fracture incidence. Largely unexplored is how bone quality and mechanical competence progress during longitudinal bone growth. Human femoral cortical bone was acquired from fetal (n = 1), infantile (n = 3), and 2- to 14-year-old cases (n = 4) at the mid-diaphysis. Bone quality was assessed in terms of bone structure, osteocyte characteristics, mineralization, and collagen orientation. The mechanical properties were investigated by measuring tensile deformation at multiple length scales via synchrotron X-ray diffraction. We find dramatic differences in mechanical resistance with age. Specifically, cortical bone in 2- to 14-year-old cases exhibits a 160% greater stiffness and 83% higher strength than fetal/infantile cases. The higher mechanical resistance of the 2- to 14-year-old cases is associated with advantageous bone quality, specifically higher bone volume fraction, better micronscale organization (woven versus lamellar), and higher mean mineralization compared with fetal/infantile cases. Our study reveals that bone quality is superior after remodeling/modeling processes convert the primary woven bone structure to lamellar bone. In this cohort of female children, the microstructural differences at the femoral diaphysis were apparent between the 1- to 2-year-old cases. Indeed, the lamellar bone in 2- to 14-year-old cases had a superior structural organization (collagen and osteocyte characteristics) and composition for resisting deformation and fracture than fetal/infantile bone. Mechanistically, the changes in bone quality during longitudinal bone growth lead to higher fracture resistance because collagen fibrils are better aligned to resist tensile forces, while elevated mean mineralization reinforces the collagen scaffold. Thus, our results reveal inherent weaknesses of the fetal/infantile skeleton signifying its inferior bone quality. These results have implications for pediatric fracture risk, as bone produced at ossification centers during children's longitudinal bone growth could display similarly weak points. © 2019 American Society for Bone and Mineral Research.     

Bone embrittlement and collagen modifications due to high-dose gamma-irradiation sterilization

Bone allografts are often used in orthopedic reconstruction of skeletal defects resulting from trauma, bone cancer or revision of joint arthroplasty. γ-Irradiation sterilization is a widely-used biological safety measure; however it is known to embrittle bone. Irradiation has been shown to affect the post-yield properties, which are attributed to the collagen component of bone. In order to find a solution to the loss of toughness in irradiated bone allografts, it is important to fully understand the effects of irradiation on bone collagen. The objective of this study was to evaluate changes in the structure and integrity of bone collagen as a result of γ-irradiation, with the hypothesis that irradiation fragments collagen molecules leading to a loss of collagen network connectivity and therefore loss of toughness.Using cortical bone from bovine tibiae, sample beams irradiated at 33 kGy on dry ice were compared to native bone beams (paired controls). All beams were subjected to three-point bend testing to failure followed by characterization of the decalcified bone collagen, using differential scanning calorimetry (DSC), hydrothermal isometric tension testing (HIT), high performance liquid chromatography (HPLC) and gel electrophoresis (SDS-PAGE). The carbonyl content of demineralized bone collagen was also measured chemically to assess oxidative damage. Barium sulfate staining after single edge notch bending (SEN(B)) fracture testing was also performed on bovine tibia bone beams with a machined and sharpened notch to evaluate the fracture toughness and ability of irradiated bone to form micro-damage during fracture.Irradiation resulted in a 62% loss of work-to-fracture (p ≤ 0.001). There was significantly less micro-damage formed during fracture propagation in the irradiated bone. HPLC showed no significant effect on pentosidine, pyridinoline, or hydroxypyridinoline levels suggesting that the loss of toughness is not due to changes in these stable crosslinks. For DSC, there was a 20% decrease in thermal stability (p < 0.001) with a 100% increase (p < 0.001) in enthalpy of denaturation (melting). HIT testing also showed a decrease in thermal stability (20% lower denaturation temperature, p < 0.001) and greatly reduced measures of collagen network connectivity (p < 0.001). Interestingly, the increase in enthalpy of denaturation suggests that irradiated collagen requires more energy to denature (melt), perhaps a result of alterations in the hydrogen bonding sites (increased carbonyl content detected in the insoluble collagen) on the irradiated bone collagen.Altogether, this new data strongly indicates that a large loss of overall collagen connectivity due to collagen fragmentation resulting from γ-irradiation sterilization leads to inferior cortical bone toughness. In addition, notable changes in the thermal denaturation of the bone collagen along with chemical indicators of oxidative modification of the bone collagen indicate that the embrittlement may be a function not only of collagen fragmentation but also of changes in bonding.