Microscopic characteristics of peri- and postmortem fracture surfaces

AUTHORS

Jessica Skinner, Natalie Langley, Samuel Fahrenholtz, Yuktha Shanavas, Brian Waletzki, Robert Brown, James Herrick, Loukham Shyamsunder, Peter Goguen, Subramaniam Rajan

ABSTRACT

This study investigated if microscopic surface features captured with a scanning electron microscope (SEM) effectively discriminate fracture timing. We hypothesized that microscopic fracture characteristics, including delamination, osteon pullout, and microcracks, may vary as bone elasticity decreases, elucidating perimortem and postmortem events more reliably than macroscopic analyses. Thirty-seven unembalmed, defleshed human femoral shafts from males (n=18) and females (n=2) aged 33–81 years were fractured at experimentally simulated postmortem intervals (PMIs) ranging from 1 to 60 warm weather days (250–40,600 ADH). A gravity convection oven was used to approximate tissue decomposition at 37 C and 27 C, and the resulting heat-time unit (accumulated degree hours, or ADH) was used to examine fractures in elastic/wet versus brittle/dry bone. The bones were fractured with a drop test frame using a three-point bending setup, sensors were used to calculate fracture energy, and high-speed photography documented fracture events. The following data were collected to relate fracture appearance to the biomechanical properties of bone: PMI (postmortem interval) length in ADH, temperature, humidity, collagen percentage, water loss, bone mineral density, cortical bone thickness, fracture energy, age, sex, cause of death, and microscopic fracture feature scores. SEM micrographs were collected from the primary tension zones of each fracture surface, and three microscopic fracture characteristics were scored from a region of interest in the center of the tension zone: percentage of delaminated osteons, percent osteon pullout, and number of microcracks. Multiple linear regression showed that microscopic fracture surface features are strong predictors of ADH (adjusted R-squared=0.67 for the 0 – 40,000 ADH samples; adjusted R-squared=0.92 for the 0–16,000 ADH samples). Osteon pullout is the single best predictor of ADH. Additionally, water loss is the primary driver of bone elasticity changes in low ADH samples, while collagen fibers appear to remain intact until later in the postmortem interval (approximately 40,000 ADH in this study). The results of this study indicate microscopic fracture surface analysis detects the biomechanical effects of decreased elasticity more reliably and with greater sensitivity than macroscopic analysis.

Bone canonical Wnt signaling is downregulated in type 2 diabetes and associates with higher advanced glycation end-products (AGEs) content and reduced bone strength

AUTHORS

Giulia Leanza, Francesca Cannata, Malak Faraj, Claudio Pedone, Viola Viola, Flavia Tramontana, Niccolò Pellegrini, Gianluca Vadalà, Alessandra Piccoli, Rocky Strollo, Francesca Zalfa, Alec T Beeve, Erica L Scheller, Simon Y Tang, Roberto Civitelli, Mauro Maccarrone, Rocco Papalia, Nicola Napoli

ABSTRACT

Type 2 diabetes (T2D) is associated with higher fracture risk, despite normal or high bone mineral density. We reported that bone formation genes (SOST and RUNX2) and advanced glycation end-products (AGEs) were impaired in T2D. We investigated Wnt signaling regulation and its association with AGEs accumulation and bone strength in T2D from bone tissue of 15 T2D and 21 non-diabetic postmenopausal women undergoing hip arthroplasty. Bone histomorphometry revealed a trend of low mineralized volume in T2D (T2D 0.249% [0.156–0.366]) vs non-diabetic subjects 0.352% [0.269–0.454]; p=0.053, as well as reduced bone strength (T2D 21.60 MPa [13.46–30.10] vs non-diabetic subjects 76.24 MPa [26.81–132.9]; p=0.002). We also showed that gene expression of Wnt agonists LEF-1 (p=0.0136) and WNT10B (p=0.0302) were lower in T2D. Conversely, gene expression of WNT5A (p=0.0232), SOST (p<0.0001), and GSK3B (p=0.0456) were higher, while collagen (COL1A1) was lower in T2D (p=0.0482). AGEs content was associated with SOST and WNT5A (r=0.9231, p<0.0001; r=0.6751, p=0.0322), but inversely correlated with LEF-1 and COL1A1 (r=–0.7500, p=0.0255; r=–0.9762, p=0.0004). SOST was associated with glycemic control and disease duration (r=0.4846, p=0.0043; r=0.7107, p=0.00174), whereas WNT5A and GSK3B were only correlated with glycemic control (r=0.5589, p=0.0037; r=0.4901, p=0.0051). Finally, Young’s modulus was negatively correlated with SOST (r=−0.5675, p=0.0011), AXIN2 (r=−0.5523, p=0.0042), and SFRP5 (r=−0.4442, p=0.0437), while positively correlated with LEF-1 (r=0.4116, p=0.0295) and WNT10B (r=0.6697, p=0.0001). These findings suggest that Wnt signaling and AGEs could be the main determinants of bone fragility in T2D.

Modeling anabolic and anti-resorptive therapies for fracture healing in a mouse model of osteogenesis imperfecta

AUTHORS

Alexandra O’Donohue, Aiken Dao, Justin Bobyn, Craig F Munns, David G Little, Aaron Schindeler

ABSTRACT

Osteogenesis imperfecta (OI) is a genetic bone fragility disorder that features frequent fractures. Bone healing outcomes are contingent on a proper balance between bone formation and resorption, and drugs such as bone morphogenetic proteins (BMPs) and bisphosphonates (BPs) have shown to have utility in modulating fracture repair. While BPs are used for OI to increase BMD and reduce pain and fracture rates, there is little evidence for using BMPs as local agents for fracture healing (alone or with BPs). In this study, we examined wild type and OI mice (Col1a2+/G610C) in a murine tibial open fracture model with (i) surgery only/no treatment, (ii) local BMP-2 (10 µg), or (iii) local BMP-2 and postoperative zoledronic acid (ZA, 0.1 mg/kg total dose). MicroCT reconstructions of healing fractures indicated BMP-2 was less effective in an OI setting, however BMP-2 + ZA led to considerable increases in bone volume (+193% WT, p < 0.001; +154% OI, p < 0.001) and polar moment of inertia (+125% WT, p < 0.01; +248% OI, p < 0.05). Tissue histology revealed a thinning of the neocortex of the callus in BMP-2 treated OI bone, but considerable retention of woven bone in the healing callus with BMP + ZA specimens. These data suggest a cautious approach may be warranted with the sole application of BMP-2 in an OI surgical setting as a bone graft substitute. However, this may be overcome by off-label bisphosphonate administration.

Bone Nanomechanical Properties and Relationship to Bone Turnover and Architecture in Patients With Atypical Femur Fractures: A Prospective Nested Case-Control Study

AUTHORS

Lanny V. Griffin,Elizabeth Warner,Saroj Palnitkar,Shijing Qiu,Mahalakshmi Honasoge,Shawna G. Griffin,George Divine,Sudhaker D. Rao

ABSTRACT

Atypical femur fractures (AFFs) are well-established serious complication of long-term bisphosphonate and denosumab therapy in patients with osteopenia or osteoporosis. To elucidate underlying mechanism(s) for the development of AFF, we performed a nested case-control study to investigate bone tissue nanomechanical properties and prevailing bone microstructure and tissue-level remodeling status as assessed by bone histomorphometry. We hypothesized that there would be differences in nanomechanical properties between patients with and without AFF and that bone microstructure and remodeling would be related to nanomechanical properties. Thirty-two full-thickness transiliac bone biopsies were obtained from age- and sex-matched patients on long-term bisphosphonate therapy with (n = 16) and without an AFF (n = 16). Standard histomorphometric measurements were made in each sample on three different bone envelopes (cancellous, intracortical, and endosteal). Iliac bone wall thickness was significantly lower on all three bone surfaces in patients with AFF than in those without AFF. Surface-based bone formation rate was suppressed similarly in both groups in comparison to healthy premenopausal and postmenopausal women, with no significant difference between the two groups. Nanoindentation was used to assess material properties of cortical and cancellous bone separately. Elastic modulus was higher in cortical than in cancellous bone in patients with AFF as well as compared to the elastic modulus of cortical bone from non-AFF patients. However, the elastic modulus of the cancellous bone was not different between AFF and non-AFF groups or between cortical and cancellous bone of non-AFF patients. Resistance to plastic deformation was decreased in cortical bone in both AFF and non-AFF groups compared to cancellous bone, but to a greater extent in AFF patients. We conclude that long-term bisphosphonate therapy is associated with prolonged suppression of bone turnover resulting in altered cortical remodeling and tissue nanomechanical properties leading to AFF.

Nitric oxide modulates bone anabolism through regulation of osteoblast glycolysis and differentiation

AUTHORS

Zixue Jin, Jordan Kho, Brian Dawson, Ming-Ming Jiang, Yuqing Chen, Saima Ali, Lindsay C. Burrage, Monica Grover, Donna J. Palmer, Dustin L. Turner, Philip Ng, Sandesh C.S. Nagamani, and Brendan Lee

ABSTRACT

Previous studies have shown that nitric oxide (NO) supplements may prevent bone loss and fractures in preclinical models of estrogen deficiency. However, the mechanisms by which NO modulates bone anabolism remain largely unclear. Argininosuccinate lyase (ASL) is the only mammalian enzyme capable of synthesizing arginine, the sole precursor for nitric oxide synthase–dependent (NOS-dependent) NO synthesis. Moreover, ASL is also required for channeling extracellular arginine to NOS for NO production. ASL deficiency (ASLD) is thus a model to study cell-autonomous, NOS-dependent NO deficiency. Here, we report that loss of ASL led to decreased NO production and impairment of osteoblast differentiation. Mechanistically, the bone phenotype was at least in part driven by the loss of NO-mediated activation of the glycolysis pathway in osteoblasts that led to decreased osteoblast differentiation and function. Heterozygous deletion of caveolin 1, a negative regulator of NO synthesis, restored NO production, osteoblast differentiation, glycolysis, and bone mass in a hypomorphic mouse model of ASLD. The translational significance of these preclinical studies was further reiterated by studies conducted in induced pluripotent stem cells from an individual with ASLD. Taken together, our findings suggest that ASLD is a unique genetic model for studying NO-dependent osteoblast function and that the NO/glycolysis pathway may be a new target to modulate bone anabolism.