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Background Most diagnostic imaging of the spine is performed in supine, a relatively unloaded position. Although the spine is subjected to functional loading that changes the spinal alignment and intervertebral disc geometry, little data exists on how healthy spines adapt to standing. This study seeks to quantify the changes of the lumbar spine from supine to standing in young, back-healthy individuals using a positional magnetic resonance imaging system. Methods This is an observational study that examined the changes in the lumbar spine alignment and intervertebral disc geometry between supine and standing of forty participants (19 males/21 females) without a history of low back pain. The regional lumbar spinal alignment was measured by the sagittal Cobb angle. Segmental intervertebral disc measurements included the segmental Cobb angle, anterior-to-posterior height ratio, and intervertebral disc width measured at L1/L2 - L5/S1 levels. Intra-class correlation was performed for intra- and inter-observer measurements. Findings The intra-observer intra-class correlation consistency model ranged from 0.76 to 0.98 with the inter-observer correlation ranging from 0.68 to 0.99. The Cobb angle decreased in standing. The L5/S1 segmental Cobb angle decreased in standing. The L2/L3 and L3/L4 anterior-to-posterior height ratios increased and the L5/S1 anterior-to-posterior height ratio decreased in standing. No difference in intervertebral disc widths was observed from supine to standing. Interpretations We established normative data for a back-healthy population, using a positional magnetic resonance imaging system, that could inform future investigations that examine the standing-induced adaptations of the lumbar spine in individuals with spinal or intervertebral disc pathologies.

Successful fracture fixation depends critically on the stability of the screw-bone interface. Maximum achievable screw torque reflects the competence of this interface, but it cannot be quantified prior to screw stripping. Typically, the surgeon relies on the patients' bone mineral density and radiographs, along with experience and tactile feedback to assess whether sufficient compression can be generated by the screw and bone. However, the local bone quality would also critically influence the strength of the bone-screw interface. We investigated whether Reference Point Indentation can provide quantitative local bone quality measures that can inform subsequent screw-bone competence.We examined the associations between the maximum screw torque that can be achieved using 3.5 mm, 4.5 mm, and 6.5 mm diameter stainless steel screws at the distal femoral metaphysis and mid-diaphysis from 20 cadavers, with the femoral neck bone mineral density and the local measures of bone quality using Reference Point Indentation.Indentation Distance Increase, a measure of bone's resistance to microfracture, correlated with the maximum screw stripping torque for the 3.5 mm (p 0.01; R = 0.56) and 4.5 mm diameter stainless steel screws (p 0.01; R = 0.57) at the femoral diaphysis. At the femoral metaphysis, femoral neck bone mineral density significantly correlated with the maximum screw stripping torque achieved by the 3.5 mm (p 0.01; R = 0.61), 4.5 mm (p 0.01; R = 0.51), and 6.5 mm diameter stainless steel screws (p 0.01; R = 0.56).Reference Point Indentation can provide localized measurements of bone quality that may better inform surgeons of the competence of the bone-implant interface and improve effectiveness of fixation strategies particularly in patients with compromised bone quality.

Aging and many disease conditions, most notably diabetes, are associated with the accumulation of non-enzymatic cross-links in the bone matrix. The non-enzymatic crosslinks, also known as advanced glycation end products (AGEs), occur at the collagen tissue level, where they are associated with reduced plasticity and increased fracture risk. In this study, Fourier-transform infrared (FTIR) imaging was used to detect spectroscopic changes associated with the formation of non-enzymatic cross-links in human bone collagen. Here, the non-enzymatic cross-link profile was investigated in one cohort with an in vitro ribose treatment as well as another cohort with an in vivo bisphosphonate treatment. With FTIR imaging, the two-dimensional (2D) spatial distribution of collagen quality associated with non-enzymatic cross-links was measured through the area ratio of the 1678/1692 cm−1 subbands within the amide I peak, termed the non-enzymatic crosslink-ratio (NE-xLR). The NE-xLR increased by 35% in the ribation treatment group in comparison to controls (p< 0.005), with interstitial bone tissue being more susceptible to the formation of non-enzymatic cross-links. Ultra high performance liquid chromatography, fluorescence microscopy, and fluorometric assay confirm a correlation between the non-enzymatic cross-link content and the NE-xLR ratio in the control and ribated groups. High resolution FTIR imaging of the 2D bone microstructure revealed enhanced accumulation of non-enzymatic cross-links in bone regions with higher tissue age (i.e., interstitial bone). This non-enzymatic cross-link ratio (NE-xLR) enables researchers to study not only the overall content of AGEs in the bone but also its spatial distribution, which varies with skeletal aging and diabetes mellitus and provides an additional measure of bone's propensity to fracture.

Purpose To test the effectiveness of a novel locking pin cap to attach a K-wire rigidly to a volar locking plate and resist fracture displacement compared with commercially available alternatives. Methods Two different methods of fracture fixation were tested on a total of 12 Sawbones models with volar shear distal radius fracture (6/group). The fragments were fixed with either 2 commercially available pin plates (industry standard) or a volar plate with 2 locking screws fixing the scaphoid facet and 2 pins locked to the plate with a novel locking pin cap in the lunate facet. Axial load conditioning was performed followed by sinusoidal loading to 250 N at 50 mm/s. A motion capture system was used to assess the relative movement of the fracture fragments relative to the intact shaft. The strength of the fixation construct was quantified by (1) the force required to achieve a 2-mm gap between the shaft and fracture fragments and (2) ultimate load to failure. Results One industry standard pin plate demonstrated disassociation of the pin from the plate after fatigue conditioning. This did not occur in the locking pin cap group. The locking pin cap construct group was able to sustain a significantly higher load compared with the industry standard when the construct was displaced to the 2-mm gap. The locking pin cap also significantly increased the ultimate load to failure compared with the industry standard. Conclusions The novel locking pin cap creates a fixed-angle attachment of a K-wire to an existing locking screw hole in a plate. This fixed-angle K-wire is significantly stronger in preventing gap formation and resisting ultimate failure than commercially available plates that use bent K-wires. Clinical relevance The development of novel techniques to secure small articular fragments may ultimately improve clinical outcomes.

Disc degeneration is one of the leading factors that contribute to low back pain. Thus, the further understanding of the mechanisms contributing to degeneration of the intervertebral disc degeneration is critical for the development of therapies and strategies for treating low back pain. Rodent models are attractive for conducting mechanistic studies particularly because of the availability of genetically modified animals. However, current imaging technologies such as magnetic resonance imaging, do not have the ability to resolve spatial features at the tens- to single- micrometer scale. We propose here a contrast-enhanced microCT technique to conduct high-resolution imaging of the rodent intervertebral discs at 10µm spatial resolution. Based on the iodinated-hydrophilic contrast agent Ioversol, we are able to conduct high resolution imaging on rat and mouse intervertebral discs. Leveraging the hydrophilic characteristic of the contrast agent, we are able to discriminate the annulus fibrosus from the water-rich nucleus pulposus. Moreover, this technique allows for the quantitative measurement of disc morphologies and volumes, and we demonstrate the versatility of this technique on cultured live intervertebral discs. Coupled with our semi-automated segmentation technique, we are able to quantify the intervertebral disc volumes with a high degree of reproducibility. The contrast-enhanced microCT images were qualitatively and quantitatively indistinguishable from the traditional histological assessment of the same sample. Furthermore, stereological measures compared well between histology and microCT images. Taken together, the results reveal that rat and mouse intervertebral discs can be imaged longitudinally in vitro at high resolutions, with no adverse effects on viability and features of the intervertebral disc.

The assessment of fracture risk often relies primarily on measuring bone mineral density, thereby accounting for only a single pathology: the loss of bone mass. However, bone’s ability to resist fracture is a result of its biphasic composition and hierarchical structure that imbue it with high strength and toughness. Reference Point Indentation (RPI) testing is designed to directly probe bone mechanical behavior at the microscale in situ, although it remains unclear which aspects of bone composition and structure influence the results at this scale. Therefore, our goal in this study was to investigate factors that contribute to bone mechanical behavior measured by cyclic reference point indentation, impact reference point indentation, and three-point bending. Twenty-eight female cadavers (age 57–97) were subjected to cyclic and impact RPI in parallel at the unmodified tibia mid-diaphysis. After RPI, the middiaphyseal tibiae were removed, scanned using micro-CT to obtain cortical porosity (Ct.Po.) and tissue mineral density (TMD), then tested using three-point bending, and lastly assayed for the accumulation of advanced glycation end-products (AGEs). Both the indentation distance increase from cyclic RPI (IDI) and bone material strength index from impact RPI (BMSi) were significantly correlated with TMD (r = −0.390, p = 0.006; r = 0.430, p = 0.002; respectively). Accumulation of AGEs was significantly correlated with IDI (r = 0.281, p = 0.046), creep indentation distance (CID, r = 0.396, p = 0.004), and BMSi (r = −0.613, p < 0.001). There were no significant relationships between tissue TMD or AGEs accumulation with the quasi-static material properties. Toughness decreased with increasing tissue Ct. Po. (r = −0.621, p < 0.001). Other three-point bending measures also correlated with tissue Ct. Po. including the bending modulus (r = −0.50, p < 0.001) and ultimate stress (r = −0.56, p < 0.001). The effects of Ct.Po. on indentation were less pronounced with IDI (r = 0.290, p = 0.043) and BMSi (r = −0.299, p = 0.037) correlated modestly with tissue Ct. Po. These results suggest that RPI may be sensitive to bone quality changes relating to collagen.

article i nfo Bisphosphonates are widely used to treat osteoporosis, but have been associated with atypical femoral fractures (AFFs) in the long term, which raises a critical health problem for the aging population. Several clinical studies havesuggested that theoccurrence of AFFs may berelated tothe bisphosphonate-induced changes of bone turn- over, but large discrepancies in the results of these studies indicate that the salient mechanisms responsible for any loss in fracture resistance are still unclear. Here the role of bisphosphonates is examined in terms of the po- tential deterioration in fracture resistance resulting from both intrinsic (plasticity) and extrinsic (shielding) toughening mechanisms, which operate over a wide range of length-scales. Specifically, we compare the me- chanical properties of two groups of humeri from healthy beagles, one control group comprising eight females (oral doses of saline vehicle, 1 mL/kg/day, 3 years) and one treated group comprising nine females (oral doses of alendronate used to treat osteoporosis, 0.2 mg/kg/day, 3 years). Our data demonstrate treatment-specifi cr e- organization of bone tissue identified at multiple length-scales mainly through advanced synchrotron x-ray ex- periments. We confirm that bisphosphonate treatments can increase non-enzymatic collagen cross-linking at molecular scales, which critically restricts plasticity associated with fibrillar sliding, and hence intrinsic toughen- ing, at nanoscales. We also observe changes in the intracortical architecture of treated bone at microscales, with partial fillingof the Haversiancanalsandreductionof osteon number.We hypothesize thatthereducedplasticity associated with BP treatmentsmay induce an increaseinmicrocrack accumulation and growth undercyclic daily loadings, and potentially increase the susceptibility of cortical bone to atypical (fatigue-like) fractures.

LBP symptoms can be induced in back-healthy individuals in prolonged standing and are relieved upon exiting standing [1]. These individuals who develop LBP (pain developers -PDs) are 3 times more likely to develop a clinical episode of LBP within two years than their non-pain developer (NPD) counterparts [2]. Yet the specific anatomical changes that provoke these LBP symptoms remain unknown. We hypothesized that PDs have distinct adaptations in each intervertebral disc (IVD) level compared to NPDs. We investigated the changes in spinal alignment (Cobb angle) and local IVD structure over time in PDs. Forty human subjects (age 18-30, BMI

Adhesive spider glues are required to perform a variety of tasks, including web construction, prey capture, and locomotion. To date, little is known regarding the molecular and structural features of spider glue proteins, in particular bioadhesives that interconnect dragline or scaffolding silks during three-dimensional web construction. Here we use biochemical and structural approaches to identify and characterize two aggregate gland specific gene products, AgSF1 and AgSF2, and demonstrate that these proteins co-localize to the connection joints of both webs and wrapping silks spun from the black widow spider, Latrodectus hesperus. Protein architectures are markedly divergent between AgSF1 and AgSF2, as well as traditional spider silk fibroin family members, suggesting connection joints consist of a complex proteinaceous network. AgSF2 represents a nonglycosylated 40-kDa protein that has novel internal amino acid block repeats with the consensus sequence NVNVN embedded in a glycine-rich matrix. Analysis of the amino acid sequence of AgSF1 reveals pentameric QPGSG iterations that are similar to conserved modular elements within mammalian elastin, a rubber-like elastomeric protein that interfaces with collagen. Wet-spinning methodology using purified recombinant proteins show AgSF1 has the potential to self-assemble into fibers. X-ray fiber diffraction studies performed on these synthetic fibers reveal the presence of noncrystalline domains that resemble classical rubber networks. Collectively, these data support that the aggregate gland serves to extrude a protein mixture that contains substances that allow for the self-assembly of fiber-like structures that interface with dragline silks to mediate prey capture.

Bone comprises a complex structure of primarily collagen, hydroxyapatite and water, where each hierarchical structural level contributes to its strength, ductility and toughness. These properties, however, are degraded by irradiation, arising from medical therapy or bone-allograft sterilization. We provide here a mechanistic framework for how irradiation affects the nature and properties of human cortical bone over a range of characteristic (nano to macro) length-scales, following x-ray exposures up to 630 kGy. Macroscopically, bone strength, ductility and fracture resistance are seen to be progressively degraded with increasing irradiation levels. At the micron-scale, fracture properties, evaluated using insitu scanning electron microscopy and synchrotron x-ray computed micro-tomography, provide mechanistic information on how cracks interact with the bone-matrix structure. At sub-micron scales, strength properties are evaluated with insitu tensile tests in the synchrotron using small-/wide-angle x-ray scattering/diffraction, where strains are simultaneously measured in the macroscopic tissue, collagen fibrils and mineral. Compared to healthy bone, results show that the fibrillar strain is decreased by ~40% following 70 kGy exposures, consistent with significant stiffening and degradation of the collagen. We attribute the irradiation-induced deterioration in mechanical properties to mechanisms at multiple length-scales, including changes in crack paths at micron-scales, loss of plasticity from suppressed fibrillar sliding at sub-micron scales, and the loss and damage of collagen at the nano-scales, the latter being assessed using Raman and Fourier Transform Infrared spectroscopy and a fluorometric assay.

The risk of fracture increases with age due to the decline of bone mass and bone quality. One of the age-related changes in bone quality occurs through the formation and accumulation of advanced glycation end-products (AGEs) due to non-enzymatic glycation (NEG). However as a number of other changes including increased porosity occur with age and affect bone fragility, the relative contribution of AGEs on the fracture resistance of aging bone is unknown. Using a high-resolution nonlinear finite element model that incorporate cohesive elements and micro-computed tomography-based 3d meshes, we investigated the contribution of AGEs and cortical porosity on the fracture toughness of human bone. The results show that NEG caused a 52% reduction in propagation fracture toughness (R-curve slope). The combined effects of porosity and AGEs resulted in an 88% reduction in propagation toughness. These findings are consistent with previous experimental results. The model captured the age-related changes in the R-curve toughening by incorporating bone quantity and bone quality changes, and these simulations demonstrate the ability of the cohesive models to account for the irreversible dynamic crack growth processes affected by the changes in post-yield material behavior. By decoupling the matrix-level effects due to NEG and intracortical porosity, we are able to directly determine the effects of NEG on fracture toughness. The outcome of this study suggests that it may be important to include the age-related changes in the material level properties by using finite element analysis towards the prediction of fracture risk.

An understanding of the mechanical behavior of polymers is critical towards the design, implementation, and quality control of such materials. Yet experiments and method for the characterization of material properties of polymers remain challenging due the need to reconcile constitutive assumptions with experimental conditions. Well-established modes of mechanical testing, such as unconfined compression or uniaxial tension, require samples with specific geometries and carefully controlled orientations. Moreover, producing specimens that conform to such specifications often requires a considerable amount of sample material. In this study we validate a micromechanical indentation device, the Tissue Diagnostic Instrument (TDI), which implements a cyclic indentation method to determine the material properties of polymers and elastomeric materials. Measurements using the TDI require little or no sample preparation, and they allow the testing of sample materials in situ . In order to validate the use of the TDI, we compared measurements of modulus determined by the TDI to those obtained by unconfined compression tests and by uniaxial tension tests within the limit of small stresses and strains. The results show that the TDI measurements were significantly correlated with both unconfined compression ( p r 2 = 0.92) and uniaxial tension tests ( p r 2 = 0.87). Moreover, the measurements across all three modes of testing were statistically indistinguishable from each other ( p = 0.92; ANOVA) and demonstrate that TDI measurements can provide a surrogate for the conventional methods of mechanical characterization.

An accurate analysis and quantification of microdamage is critical to understand how microdamage affects the mechanics and biology of bone fragility. In this study we demonstrate the development and validation of a novel in vitro micro-computed tomography (microCT) method that employs lead-uranyl acetate as a radio-opaque contrast agent for automated quantification of microdamage in trabecular bone. Human trabecular bone cores were extracted from the femoral neck, scanned via microCT, loaded in unconfined compression to a range of apparent strains (0.5% to 2.25%), stained in lead-uranyl acetate, and subsequently re-scanned via microCT. An investigation of the regions containing microdamage using the backscatter mode of a scanning electron microscope (BSEM) showed that the lead-uranyl sulfide complex was an effective contrast agent for microdamage in bone. Damaged volume fraction (DV/BV), as determined by microCT, increased exponentially with respect to applied strains and proportionately to mechanically determined modulus reduction (p

In their recent publication, Chuang et al examined the effects of genipin-induced collagen crosslinking on the mechanical behavior of lumbar intervertebral discs [1]. Based on their experimental results, Chuang et al concluded, “crosslink augmentation may in fact increase fatigue resistance, and modestly stiffen tissue without a loss of joint range of motion.” While this is an interesting result, their conclusions should be reconsidered in light of several caveats: Specifically, this manuscript has mis-characterized some of the experimental results in the cited literature and inappropriately generalized the role of crosslinks in the biomechanical behavior of collagenous tissues. Crosslinking in collagen can result from a number of different pathways, such as enzymatic maturation by lysyl oxidase [2], non-enzymatic glycation by extracellular sugars [3], or even in vitro crosslinking by genipin [4]. These pathways yield molecularly distinct crosslinks, and these crosslinks may contribute differently towards overall tissue biomechanical function. As a biopolymer, collagen commonly provides structural support for load-bearing tissues. Because the maturation and mechanical competence of collagen is contingent on the formation of enzymatic crosslinks, pathologies can arise when there is inadequate formation of mature crosslinks. The process of collagen crosslinking is initiated by the conversion of telopeptidyl lysine and hydroxylysine residue to aldehyde through the action of lysyl oxidase, a copper metalloenzyme. The hydroxylysine–aldehyde pathway predominates in the telopeptides of bone, cartilage, ligament, tendon, and most major internal connective tissues [5]. Inadequate enzymatic crosslink formation, for example, such as in the abnormal inhibition of lysyl oxidase in Lathyrus odorathus, results in increased fragility of collagenous tissues [6]. Thus, enzymatic crosslinks play a crucial role in mechanical competence of adult skeletal system. It is important to note, however, the accumulation of enzymatic crosslinks plateaus with skeletal maturity [7], and does not show a direct correlation with the age-related fragility of connective tissues [7, 8]. On the other hand, non-enzymatic glycation (NEG)-induced crosslinks known as Advanced Glycation End-products (AGEs), which occurs spontaneously in the presence of extracellular sugars, are known to accumulate with age in collagenous tissues [7]. The reactive sugars form Schiffs’ bases with free amino groups in lysine, hydrosylysine, or arginine residues on the collagen molecule, and the subsequent structure then undergoes Amadori rearrangement that ultimately results in a family of molecules known AGEs [3]. There are numerous studies, including those cited by the authors, which show the deleterious effects of AGEs accumulation on fracture resistance and energy dissipation of the afflicted tissues [8–12]. The authors cited several works, which “lacked direct evidence to support the hypothesis” of “age increasing crosslinks causes tissue brittleness and loss of fatigue resistance in load supporting tissues.” Despite their claims, two of the cited manuscripts presented direct experimental evidence of increased crosslinking, mediated by NEG, results in significant tissue stiffening and increased fragility of articular cartilage in both tension [10] and compression [9]. A previous study by Wagner et al specifically tested the effects of crosslinking by NEG on the mechanical performance of the intervertebral disc and found that increased crosslinking significantly correlated with the deterioration of strain energy function and subsequent decreased energy dissipation [13]. There is also a growing body of literature in other collagen-containing tissues such as bone [8,11,12] that show the age-related accumulation of NEG crosslinks are adventitious to the overall mechanical performance of the tissue matrix. In each of the studies cited above, the degree of crosslinking was quantified and inversely related to decreased tissue mechanics. Tissue maturation in vivo produces a degree of collagen crosslinking that is optimal for mechanical function, and additional post-translational modifications, such as the age-related accumulation crosslinks caused by non-enzymatic glycation, result in reduced matrix strength. The study conducted by Chuang et al provides a provocative clinical motivation, but its conclusions may be flawed. Given that the typical bovine animal reaches skeletal maturity at two years, the selection of 4-month old bovine specimens may have resulted in experimentally testing skeletally immature tissue, and the model presented here may not represent an ideal animal model for age-related disc degeneration. In the absence of sufficient enzymatic crosslink accumulation due to incomplete skeletal development, it is thus not surprising that the augmentation of genipin-induced crosslinks enhances the overall tissue mechanics of the intervertebral disc. However, because the crosslinking profiles of the samples were not disclosed, it is not possible to attribute the alterations in tissue mechanics specifically to the type and degree of crosslinking.