[Show abstract][Hide abstract] ABSTRACT: Methods: Total fluorescent advanced glycation end-products (AGEs) were measured in 96 human cortical bone samples from 83 donors. Resorption pit density, average resorption pit area, and percent resorption area were quantified in samples from 48 common donors with AGE measurements. Linear microcrack density and diffuse damage were measured in 21 common donors with AGE and resorption measurements. Correlation analyses were performed between all measured variables to establish the relationships among them and their variation with age.
Full-text · Article · Oct 2014 · Osteoporosis International
[Show abstract][Hide abstract] ABSTRACT: Toughening in hierarchically structured materials like bone arises from the arrangement of constituent material elements and their interactions. Unlike microcracking, which entails micrometer-level separation, there is no known evidence of fracture at the level of bone's nanostructure. Here, we show that the initiation of fracture occurs in bone at the nanometer scale by dilatational bands. Through fatigue and indentation tests and laser confocal, scanning electron, and atomic force microscopies on human and bovine bone specimens, we established that dilatational bands of the order of 100 nm form as ellipsoidal voids in between fused mineral aggregates and two adjacent proteins, osteocalcin (OC) and osteopontin (OPN). Laser microdissection and ELISA of bone microdamage support our claim that OC and OPN colocalize with dilatational bands. Fracture tests on bones from OC and/or OPN knockout mice (OC(-/-), OPN(-/-), OC-OPN(-/-;-/-)) confirm that these two proteins regulate dilatational band formation and bone matrix toughness. On the basis of these observations, we propose molecular deformation and fracture mechanics models, illustrating the role of OC and OPN in dilatational band formation, and predict that the nanometer scale of tissue organization, associated with dilatational bands, affects fracture at higher scales and determines fracture toughness of bone.
Full-text · Article · Nov 2012 · Proceedings of the National Academy of Sciences
[Show abstract][Hide abstract] ABSTRACT: The use of tissue grafting for the repair of large bone defects has numerous limitations including donor site morbidity and the risk of disease transmission. These limitations have prompted research efforts to investigate the effects of combining biomaterial scaffolds with biochemical cues to augment bone repair. The goal of this study was to use a critically-sized rat femoral segmental defect model to investigate the efficacy of a delivery system consisting of an electrospun polycaprolactone (PCL) nanofiber mesh tube with a silk fibroin hydrogel for local recombinant bone morphogenetic protein 2 (BMP-2) delivery. Bilateral 8 mm segmental femoral defects were formed in 13-week-old Sprague Dawley rats. Perforated electrospun PCL nanofiber mesh tubes were fitted into the adjacent native bone such that the lumen of the tubes contained the defect (Kolambkar et al., 2011b). Silk hydrogels with or without BMP-2 were injected into the defect. Bone regeneration was longitudinally assessed using 2D X-ray radiography and 3D microcomputed topography (μCT). Following sacrifice at 12 weeks after surgery, the extracted femurs were either subjected to biomechanical testing or assigned for histology. The results demonstrated that silk was an effective carrier for BMP-2. Compared to the delivery system without BMP-2, the delivery system that contained BMP-2 resulted in more bone formation (p<0.05) at 4, 8, 12 weeks after surgery. Biomechanical properties were also significantly improved in the presence of BMP-2 (p<0.05) and were comparable to age-matched intact femurs. Histological evaluation of the defect region indicated that the silk hydrogel has been completely degraded by the end of the study. Based on these results, we conclude that a BMP-2 delivery system consisting of an electrospun PCL nanofiber mesh tube with a silk hydrogel presents an effective strategy for functional repair of large bone defects.
[Show abstract][Hide abstract] ABSTRACT: The effects of a 3-year alendronate treatment on trabecular stresses/strains associated with microdamage initiation were investigated using finite element modeling (FEM). Severely damaged trabeculae in the low-dose treatment group were associated with increased stresses compared with the high-dose treatment group (p = 0.006) and approached significance in the control group (p = 0.02).
Alendronate, a commonly prescribed anti-remodeling agent, decreases fracture risk in the vertebrae, hip, and wrist of osteoporotic individuals. However, evaluation of microdamage accumulation in animal and human studies shows increased microdamage density relative to controls. Microstructural von Mises stresses associated with severe and linear damage have been found to decrease after 1 year of alendronate treatment. In the present study, stresses/strains associated with damage were assessed after 3 years of treatment to determine whether they continued to decrease with increased treatment duration.
Microdamaged trabeculae visualized with fluorescent microscopy were associated with stresses and strains obtained using image-based FEM. Stresses/strains associated with severe, diffuse, and linearly damaged and undamaged trabeculae were compared among groups treated for 3 years with an osteoporotic treatment dose of alendronate, a Paget's disease treatment dose of alendronate, or saline control. Architectural characteristics and mineralization were also analyzed from three-dimensional microcomputed tomography reconstructed images.
Severely damaged trabeculae in the osteoporotic treatment dose group were associated with increased stress compared with the Paget's disease treatment dose group (p = 0.006) and approached significance compared to the control group (p = 0.02). Trabecular mineralization in severely damaged trabeculae of the low-dose treatment group was significantly greater compared to severely damaged trabeculae in the high-dose treatment and control group, suggesting that changes at the tissue level may play a role in these findings.
Trabecular level stresses associated with microdamage do not continue to decrease with prolonged alendronate treatment. Changes in mineralization may account for these findings.
No preview · Article · Jan 2012 · Osteoporosis International
[Show abstract][Hide abstract] ABSTRACT: Microdamage density has been shown to increase with age in trabecular bone and is associated with decreased fracture toughness. Numerous studies of crack propagation in cortical bone have been conducted, but data in trabecular bone is lacking. In this study, propagation of severe, linear, and diffuse damage was examined in trabecular bone cores from the femoral head of younger (61.3±3.1 years) and older (75.0±3.9 years) men and women. Using a two-step mechanical testing protocol, damage was first initiated with static uniaxial compression to 0.8% strain then propagated at a normalized stress level of 0.005 to a strain endpoint of 0.8%. Coupling mechanical testing with a dual-fluorescent staining technique, the number and length/area of propagating cracks were quantified. It was found that the number of cycles to the test endpoint was substantially decreased in older compared to younger samples (younger: 77,372±15,984 cycles; older: 34,944±11,964 cycles, p=0.06). This corresponded with a greater number of severely damaged trabeculae expanding in area during the fatigue test in the older group. In the younger group, diffusely damaged trabeculae had a greater damage area, which illustrates an efficient energy dissipation mechanism. These results suggest that age-related differences in fatigue life of human trabecular bone may be due to differences in propagated microdamage morphology.
Full-text · Article · Aug 2011 · Journal of Biomechanics
[Show abstract][Hide abstract] ABSTRACT: Accumulation of microdamage in aging and disease can cause skeletal fragility and is one of several factors contributing to osteoporotic fractures. To better understand the role of microdamage in fragility fracture, the mechanisms of bone failure must be elucidated on a tissue-level scale where interactions between bone matrix properties, the local biomechanical environment, and bone architecture are concurrently examined for their contributions to microdamage formation. A technique combining histological damage assessment of individual trabeculae with linear finite element solutions of trabecular von Mises and principal stress and strain was used to compare the damage initiation threshold between pre-menopausal (32-37 years, n=3 donors) and post-menopausal (71-80 years, n=3 donors) femoral cadaveric bone. Strong associations between damage morphology and stress and strain parameters were observed in both groups, and an age-related decrease in undamaged trabecular von Mises stress was detected. In trabeculae from younger donors, the 95% CI for von Mises stress on undamaged regions ranged from 50.7-67.9MPa, whereas in trabeculae from older donors, stresses were significantly lower (38.7-50.2, p<0.01). Local microarchitectural analysis indicated that thinner, rod-like trabeculae oriented along the loading axis are more susceptible to severe microdamage formation in older individuals, while only rod-like architecture was associated with severe damage in younger individuals. This study therefore provides insight into how damage initiation and morphology relate to local trabecular microstructure and the associated stresses and strains under loading. Furthermore, by comparison of samples from pre- and post-menopausal women, the results suggest that trabeculae from younger individuals can sustain higher stresses prior to microdamage initiation.
Full-text · Article · Jul 2011 · Journal of Biomechanics
[Show abstract][Hide abstract] ABSTRACT: Biomaterial scaffolds functionalized to stimulate endogenous repair mechanisms via the incorporation of osteogenic cues offer a potential alternative to bone grafting for the treatment of large bone defects. We first quantified the ability of a self-complementary adeno-associated viral vector encoding bone morphogenetic protein 2 (scAAV2.5-BMP2) to enhance human stem cell osteogenic differentiation in vitro. In two-dimensional culture, scAAV2.5-BMP2-transduced human mesenchymal stem cells (hMSCs) displayed significant increases in BMP2 production and alkaline phosphatase activity compared with controls. hMSCs and human amniotic-fluid-derived stem cells (hAFS cells) seeded on scAAV2.5-BMP2-coated three-dimensional porous polymer Poly(ε-caprolactone) (PCL) scaffolds also displayed significant increases in BMP2 production compared with controls during 12 weeks of culture, although only hMSC-seeded scaffolds displayed significantly increased mineral formation. PCL scaffolds coated with scAAV2.5-BMP2 were implanted into critically sized immunocompromised rat femoral defects, both with or without pre-seeding of hMSCs, representing ex vivo and in vivo gene therapy treatments, respectively. After 12 weeks, defects treated with acellular scAAV2.5-BMP2-coated scaffolds displayed increased bony bridging and had significantly higher bone ingrowth and mechanical properties compared with controls, whereas defects treated with scAAV2.5-BMP2 scaffolds pre-seeded with hMSCs failed to display significant differences relative to controls. When pooled, defect treatment with scAAV2.5-BMP2-coated scaffolds, both with or without inclusion of pre-seeded hMSCs, led to significant increases in defect mineral formation at all time points and increased mechanical properties compared with controls. This study thus presents a novel acellular bone-graft-free endogenous repair therapy for orthotopic tissue-engineered bone regeneration.
Full-text · Article · Jun 2011 · Cell and Tissue Research
[Show abstract][Hide abstract] ABSTRACT: Raloxifene (RAL) and alendronate (ALN) improve the biomechanical properties of bone by different mechanisms. The goal here was to investigate the effects of combination treatment of RAL and ALN on the biomechanical properties of vertebral bone. Six-month-old Sprague-Dawley rats (n = 80) were randomized into five experimental groups (sham, OVX, OVX + RAL, OVX + ALN, and OVX + RAL + ALN; n = 16/group). Following euthanization, structural and derived material biomechanical properties of vertebral bodies were assessed. Density and dynamic histomorphometric measurements were made on cancellous bone. The results demonstrate that the structural biomechanical properties of vertebral bone are improved with the combination treatment. Stiffness and ultimate load of the OVX + RAL and OVX + ALN groups were significantly lower than those of sham animals, but the combination treatment with RAL + ALN was not significantly different from sham. Furthermore, the OVX + RAL + ALN group was the only agent-treated group in which the ultimate load was significantly higher than that in OVX animals (p < .05). Cancellous bone fractional volume (BV/TV(canc)) and bone mineral density (aBMD) also were improved with the combination treatment. BV/TV(canc) of the OVX + RAL + ALN group was 6.7% and 8.7% greater than that of the OVX + RAL (p < .05) and OVX + ALN (p < .05) groups, respectively. Areal BMD of the OVX + RAL or OVX + ALN groups was not significantly different from that in OVX animals, but the value in animals undergoing combination treatment was significantly higher than that in OVX or OVX + RAL animals alone and not significantly different from that in sham-operated animals. Turnover rates of both the RAL + ALN and ALN alone groups were lower than in the RAL-treated alone group (p < .05). We conclude that the combination treatment of raloxifene and alendronate has beneficial effects on bone volume, resulting in improvement in the structural properties of vertebral bone.
Full-text · Article · Feb 2011 · Journal of bone and mineral research: the official journal of the American Society for Bone and Mineral Research
[Show abstract][Hide abstract] ABSTRACT: Alendronate, an anti-remodeling agent, is commonly used to treat patients suffering from osteoporosis by increasing bone mineral density. Though fracture risk is lowered, an increase in microdamage accumulation has been documented in patients receiving alendronate, leading to questions about the potentially detrimental effects of remodeling suppression on the local tissue (material) properties. In this study, trabecular bone cores from the distal femur of beagle dogs treated for one year with alendronate, at doses scaled by weight to approximate osteoporotic and Paget's disease treatment doses in humans, were subjected to uniaxial compression to induce microdamage. Tissue level von Mises stresses were computed for alendronate-treated and non-treated controls using finite element analysis and correlated to microdamage morphology. Using a modified version of the Moore and Gibson classification for damage morphology, we determined that the von Mises stress for trabeculae exhibiting severe and linear microcrack patterns was decreased by approximately 25% in samples treated with alendronate compared with non-treated controls (p<0.01), whereas there was no reduction in the von Mises stress state for diffuse microdamage formation. Furthermore, an examination of the architectural and structural characteristics of damaged trabeculae demonstrated that severely damaged trabeculae were thinner, more aligned with the loading axis, and less mineralized than undamaged trabeculae in alendronate-treated samples (p<0.01). Similar relationships with damage morphology were found only with trabecular orientation in vehicle-treated control dogs. These results indicate that changes in bone's architecture and matrix properties associated with one year of alendronate administration reduce trabecular bone's ability to resist the formation of loading-induced severe and linear microcracks, both of which dissipate less energy prior to fracture than does diffuse damage.
[Show abstract][Hide abstract] ABSTRACT: Bisphosphonate (BP) treatment used to prevent bone loss in postmenopausal osteoporosis has recently been implicated in an apparent increase in subtrochanteric femoral fractures. Previous work showed that BPs can reduce the energy to fracture of cancellous bone, but limited data exist on material-level mechanical properties of compact bone from the long bones. This study examined intrinsic mechanical properties of the femoral diaphysis of a canine model treated for 1 or 3 years with alendronate at two different doses. Seventy-two dogs were treated orally with 0.2 mg/kg/day alendronate or 1.0 mg/kg/day alendronate; a control group was administered saline. Prismatic beam specimens were tested in four-point bending under displacement control, and the intrinsic mechanical properties were calculated. No significant differences were found among groups in any mechanical property at either 1 or 3 years of treatment. We conclude that the material properties of the femoral diaphysis are not degraded following 1 to 3 years treatment with alendronate, even at high doses. Longer periods of treatment have not been studied using clinical doses of alendronate, but such studies need to be carried out to confirm a lack of effect of alendronate on mechanical properties of cortical bone in the subtrochanteric region of the femur.
Full-text · Article · Oct 2009 · Journal of Orthopaedic Research
[Show abstract][Hide abstract] ABSTRACT: We aimed to determine whether trabecular bone in sites that have different surface-based remodeling rates, the femoral neck and vertebra, are differently affected by alendronate treatment. Alendronate treatment resulted in similar levels of turnover in both sites, suggesting that a lower limit of bone turnover suppression with alendronate may exist.
Bone turnover suppression in sites that already have a low surface-based remodeling rate may lead to oversuppression that could have negative effects on the biomechanical properties of bone. The goal was to determine how alendronate suppresses bone turnover at sites with different surface-based remodeling rates.
Dynamic histomorphometric parameters were assessed in trabecular bone of the femoral neck and lumbar vertebrae obtained from skeletally mature beagles treated with saline (1 ml/kg/day) or alendronate (ALN 0.2 or 1.0 mg/kg/day). The ALN0.2 and ALN1.0 doses approximate, on a milligram per kilogram basis, the clinical doses used for the treatment of postmenopausal osteoporosis and Paget's disease, respectively.
Alendronate treatment resulted in similar absolute levels of bone turnover in the femoral neck and vertebrae, although the femoral neck had 33% lower pre-treatment surface-based remodeling rate than the vertebra (p < 0.05). Additionally, the high dose of alendronate (ALN 1.0) suppressed bone turnover to similar absolute levels as the low dose of alendronate (ALN 0.2) in both sites.
Alendronate treatment may result in a lower limit of trabecular bone turnover suppression, suggesting that sites of low pre-treatment remodeling rate are not more susceptible to oversuppression than those of high pre-treatment remodeling rate.
Full-text · Article · Sep 2008 · Osteoporosis International
[Show abstract][Hide abstract] ABSTRACT: At the tissue level it is well established that the rate of remodeling is related to the degree of mineralization. However, it is unknown how long it takes for an individual bone structural unit (BSU) to become fully mineralized during secondary mineralization. Using synchrotron Fourier transform infrared microspectroscopy (FTIRM) we examined the time required for newly formed bone matrix to reach a physiological mineralization limit. Twenty-six, four-month old female New Zealand white rabbits were administered up to four different fluorochrome labels at specific time points to evaluate the chemical composition of labeled osteons from the tibial diaphysis that had mineralized for 1, 8, 18, 35, 70, 105, 140, 175, 210, 245, 280, 315, 350, and 385 days. Interstitial bone from 505 day old rabbits was used as a reference value for the physiological limit to which bone mineralizes. Using synchrotron FTIRM, area integrations were carried out on protein (Amide I: 1688-1623 cm(-1)), carbonate (v(2)CO(3)(2-): 905-825 cm(-1)), and phosphate (v(4)PO(4)(3-): 650-500 cm(-1)) IR bands. IR spectral data are presented as ratios of phosphate/protein (overall matrix mineralization) and carbonate/protein. The rate of mineralization of osteonal bone proceeded rapidly between day 1 and 18, reaching 67% of interstitial bone levels. This was followed by a slower, more progressive accumulation of mineral up to day 350. By 350 days the rate of increase plateaued. The ratio of carbonate/protein also increased rapidly during the first 18 days, reaching 73% of interstitial bone levels. The ratio of carbonate/protein plateaued by day 315, reaching levels not significantly different to interstitial bone levels. In conclusion, our data demonstrate that bone accumulates mineral rapidly during the first 18 days (primary mineralization), followed by a more gradual increase in the accumulation of mineral (secondary mineralization) which we found to be completed in 350 days.
[Show abstract][Hide abstract] ABSTRACT: In vivo, microdamage occurs in the form of linear microcracks and diffuse damage. However, it is unknown whether the age-related changes in bone quality predispose bone to form one type of damage morphology over the other during in vivo loading. In this study, histological and histomorphometrical analyses were conducted on transverse cross sections, obtained from the tibiae of aging human bone (age 19 to 89), to investigate the in vivo accumulation and localization of damage morphologies. The results demonstrate that old donor bone (83+/-3 years) contains more linear microcracks than younger donor bone in the cortices predominantly subjected to compressive (p<0.01) and tensile loading (p<0.01). In contrast, young donor bone (40+/-10 years) contains more diffuse damage than older donor bone in the cortex predominantly subjected to tensile loading (p<0.01). The formation of damage morphology showed no correlation with bone geometry parameters and exhibited distinct preferences with bone microstructure. Linear microcracks formed in the interstitial bone (p<0.01) and were either trapped or arrested by the microstructural interfaces (cement line and lamellar interface) (p<0.05). Areas of diffuse damage, however, were preferentially associated with secondary osteonal bone (p<0.01) and had no relationship with the microstructural interfaces (p<0.01). Based upon these findings, we conclude that age-related changes in bone microstructure, but not bone geometry, play a key role in the propensity of old donors to form linear microcrack over diffuse damage under in vivo loading conditions.
[Show abstract][Hide abstract] ABSTRACT: Application of cyclic loading results in the formation of distinct strain-dependent microdamage morphologies. It is still unknown; however, how the morphology of microdamage affects age-related increase in bone fragility. In this study, four-point bending fatigue tests were conducted on aging human bone (age 26 to 89) in conjunction with histological evaluation of the resultant tensile (diffuse damage) and compressive (linear microcracks) damage to identify the damage morphologies associated with an increase in age-related bone fragility. The results demonstrate that young donors (38 +/- 9 years) had a longer fatigue life (P < 0.05) and formed more diffuse damage than the older donors (82 +/- 5 years) (P < 0.05). In contrast, old donors had a shorter fatigue life and formed more linear microcracks than the younger donors (P < 0.05). Linear microcracks were longer in older than in younger donors (P < 0.05) and were associated with weak lamellar interfaces. Areas of diffuse damage were, however, larger in younger than in older donors (P < 0.05), and these showed no relationship with the lamellar arrangement of bone. These findings show, for the first time, that the propensity of bone to form a particular damage morphology is subject to change with age and that the propensity of young donors to form diffuse damage over interlamellae linear microcracks plays a critical role in the ability of bone to dissipate energy and resist a catastrophic fracture. Age-related changes in damage morphology may therefore be an important contributor to the increased bone fragility in the elderly.
[Show abstract][Hide abstract] ABSTRACT: Despite a general understanding that bone microdamage has distinct strain dependent morphologies, very little information exists on how different damage morphologies develop and participate in bone fracture. In this study, cortical bone beams were subjected to the primary or tertiary phases of bending fatigue followed by either post-hoc fracture toughness tests or microdamage analysis to determine the sequence in which linear microcracks and diffuse damage form during bending fatigue and how they affect the propensity of bone to fracture. The results demonstrate that, following the primary phase, linear microcracks and diffuse damage are formed on the compressive and tensile sides, respectively (p<0.05). Furthermore, this mode of damage formation results in a greater toughness loss if a fracture crack initiates from the tensile side rather than the compressive side (p<0.05). Continued loading of bone specimens to the tertiary phase, however, leads to further accumulation of damage only on the compressive side (p<0.05), and this mode of damage formation results in a further toughness loss if a fracture crack initiates from the compressive side rather than the tensile side (p<0.05). Thus, cortical bone compartmentalizes the damage morphologies in different regions and the sequence of damage production in different phases of cyclic loading to dissipate energy and resist a catastrophic fracture.
[Show abstract][Hide abstract] ABSTRACT: Despite a general understanding that bone quality contributes to skeletal fragility, very little information exits on the age-dependent fatigue behavior of human bone. In this study four-point bending fatigue tests were conducted on aging bone in conjunction with the analysis of stiffness loss and preliminary investigation of nanoindentation based measurements of local tissue stiffness and histological evaluation of resultant tensile and compressive damage to identify the damage mechanism responsible for the increase in age-related bone fragility. The results obtained show that there is an exponential decrease in fatigue life with age, and old bone exhibits different modulus degradation profiles than young bone. In addition, this study provides preliminary evidence indicating that during fatigue loading, younger bone formed diffuse damage, lost local tissue stiffness on the tensile side. Older bone, in contrast, formed linear microcracks lost local tissue stiffness on the compressive side. Thus, the propensity of aging human bone to form more linear microcracks than diffuse damage may be a significant contributor to bone quality, and age related fragility in bone.
No preview · Article · Apr 2005 · European Journal of Morphology