TRPV4 differentially controls inflammatory cytokine networks during static and dynamic compression of the intervertebral disc.

Background: The ion channel transient receptor potential vanilloid 4 (TRPV4) critically transduces mechanical forces in the IVD, and its inhibition can prevent IVD degeneration due to static overloading. However, it remains unknown whether different modes of loading signals through TRPV4 to regulate the expression of inflammatory cytokines. We hypothesized that TRPV4 signaling is essential during static and dynamic loading to mediate homeostasis and mechanotransduction. Methods: Mouse functional spine units were isolated and either cyclically compressed for 5 days (1 Hz, 1 h, 10% strain) or statically compressed (24 h, 0.2 MPa). Conditioned media were monitored at 6 h, 24 h, 2 days, and 5 days, with and without TRPV4 inhibition. Effects of TRPV4 activation was also evaluated without loading. The media was analyzed for a panel of 44 cytokines using a microbead array and then a correlative network was constructed to explore the regulatory relationships during loading and TRPV4 inhibition. After the loading regimen, the IVDs were evaluated histologically for degeneration. Results: Activation of TRPV4 led to an increase interleukin-6 (IL-6) family of cytokines (IL-6, IL-11, IL-16, and leukemia inhibitory factor [LIF]) and decreased the T-cell (CCL3, CCL4, CCL17, CCL20, CCL22, and CXCL10) and monocyte (CCL2 and CCL12) recruiting chemokines by the IVD. Dynamic and static loading each provoked unique chemokine correlation networks. The inhibition of TRPV4 during dynamic loading dysregulated the relationship between LIF and other cytokines, while the inhibition of TRPV4 during static loading disrupted the connectivity of IL-16 and VEGFA. Conclusions: We demonstrated that TRPV4 critically mediates the cytokine production following dynamic and static loading. The activation of TRPV4 upregulated a diverse set of cytokines that may suppress the chemotaxis of T-cells and monocytes, implicating the role of TRPV4 in maintaining the immune privilege of healthy IVD.

Relative Effects of Radiation-Induced Changes in Bone Mass, Structure, and Tissue Material on Vertebral Strength in a Rat Model.

The observed increased risk of fracture after cancer radiation therapy is presumably due to a radiation-induced reduction in whole-bone strength. However, the mechanisms for impaired strength remain unclear, as the increased fracture risk is not fully explained by changes in bone mass. To provide insight, a small animal model was used to determine how much of this whole-bone weakening effect for the spine is attributable to changes in bone mass, structure, and material properties of the bone tissue and their relative effects. Further, because women have a greater risk of fracture after radiation therapy than men, we investigated if sex had a significant influence on bone's response to irradiation. Fractionated in vivo irradiation (10 × 3 Gy) or sham irradiation (0 Gy) was administered daily to the lumbar spine in twenty-seven 17-week-old Sprague-Dawley rats (n = 6-7/sex/group). Twelve weeks after final treatment, animals were euthanized, and lumbar vertebrae (L4 and L5 ) were isolated. Using a combination of biomechanical testing, micro-CT-based finite element analysis, and statistical regression analysis, we separated out the effect of mass, structural, and tissue material changes on vertebral strength. Compared with the sham group (mean ± SD strength = 420 ± 88 N), the mean strength of the irradiated group was lower by 28% (117 N/420 N, p < 0.0001). Overall, the response of treatment did not differ with sex. By combining results from both general linear regression and finite element analyses, we calculated that mean changes in bone mass, structure, and material properties of the bone tissue accounted for 56% (66 N/117 N), 20% (23 N/117 N), and 24% (28 N/117 N), respectively, of the overall change in strength. As such, these results provide insight into why an elevated clinical fracture risk for patients undergoing radiation therapy is not well explained by changes in bone mass alone. © 2023 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

Bullying, harassment, and discrimination of musculoskeletal researchers and the impact of the COVID-19 pandemic: an international study.

PURPOSE: Bullying, harassment, and discrimination (BHD) are prevalent in academic, scientific, and clinical departments, particularly orthopedic surgery, and can have lasting effects on victims. As it is unclear how BHD affects musculoskeletal (MSK) researchers, the following study assessed BHD in the MSK research community and whether the COVID-19 pandemic, which caused hardships in other industries, had an impact. METHODS: A web-based anonymous survey was developed in English by ORS Spine Section members to assess the impact of COVID-19 on MSK researchers in North America, Europe, and Asia, which included questions to evaluate the personal experience of researchers regarding BHD. RESULTS: 116 MSK researchers completed the survey. Of respondents, 34.5% (n = 40) focused on spine, 30.2% (n = 35) had multiple areas of interest, and 35.3% (n = 41) represented other areas of MSK research. BHD was observed by 26.7% (n = 31) of respondents and personally experienced by 11.2% (n = 13), with mid-career faculty both observing and experiencing the most BHD. Most who experienced BHD (53.8%, n = 7) experienced multiple forms. 32.8% (n = 38) of respondents were not able to speak out about BHD without fear of repercussions, with 13.8% (n = 16) being unsure about this. Of those who observed BHD, 54.8% (n = 17) noted that the COVID-19 pandemic had no impact on their observations. CONCLUSIONS: To our knowledge, this is the first study to address the prevalence and determinants of BHD among MSK researchers. MSK researchers experienced and observed BHD, while many were not comfortable reporting and discussing violations to their institution. The COVID-19 pandemic had mixed-effects on BHD. Awareness and proactive policy changes may be warranted to reduce/eliminate the occurrence of BHD in this community.

Modulation of TRPV4 protects against degeneration induced by sustained loading and promotes matrix synthesis in the intervertebral disc.

While it is well known that mechanical signals can either promote or disrupt intervertebral disc (IVD) homeostasis, the molecular mechanisms for transducing mechanical stimuli are not fully understood. The transient receptor potential vanilloid 4 (TRPV4) ion channel activated in isolated IVD cells initiates extracellular matrix (ECM) gene expression, while TRPV4 ablation reduces cytokine production in response to circumferential stretching. However, the role of TRPV4 on ECM maintenance during tissue-level mechanical loading remains unknown. Using an organ culture model, we modulated TRPV4 function over both short- (hours) and long-term (days) and evaluated the IVDs' response. Activating TRPV4 with the agonist GSK101 resulted in a Ca2+ flux propagating across the cells within the IVD. Nuclear factor (NF)-κB signaling in the IVD peaked at 6 h following TRPV4 activation that subsequently resulted in higher interleukin (IL)-6 production at 7 days. These cellular responses were concomitant with the accumulation of glycosaminoglycans and increased hydration in the nucleus pulposus that culminated in higher stiffness of the IVD. Sustained compressive loading of the IVD resulted in elevated NF-κB activity, IL-6 and vascular endothelial growth factor A (VEGFA) production, and degenerative changes to the ECM. TRPV4 inhibition using GSK205 during loading mitigated the changes in inflammatory cytokines, protected against IVD degeneration, but could not prevent ECM disorganization due to mechanical damage in the annulus fibrosus. These results indicate TRPV4 plays an important role in both short- and long-term adaptations of the IVD to mechanical loading. The modulation of TRPV4 may be a possible therapeutic for preventing load-induced IVD degeneration.

Single Cell RNA-Sequence Analyses Reveal Uniquely Expressed Genes and Heterogeneous Immune Cell Involvement in the Rat Model of Intervertebral Disc Degeneration.

Intervertebral disc (IVD) degeneration is characterized by a loss of cellularity, and changes in cell-mediated activity that drives anatomic changes to IVD structure. In this study, we used single-cell RNA-sequencing analysis of degenerating tissues of the rat IVD following lumbar disc puncture. Two control, uninjured IVDs (L2-3, L3-4) and two degenerated, injured IVDs (L4-5, L5-6) from each animal were examined either at the two- or eight-week post-operative time points. The cells from these IVDs were extracted and transcriptionally profiled at the single-cell resolution. Unsupervised cluster analysis revealed the presence of four known cell types in both non-degenerative and degenerated IVDs based on previously established gene markers: IVD cells, endothelial cells, myeloid cells, and lymphoid cells. As a majority of cells were associated with the IVD cell cluster, sub-clustering was used to further identify the cell populations of the nucleus pulposus, inner and outer annulus fibrosus. The most notable difference between control and degenerated IVDs was the increase of myeloid and lymphoid cells in degenerated samples at two- and eight-weeks post-surgery. Differential gene expression analysis revealed multiple distinct cell types from the myeloid and lymphoid lineages, most notably macrophages and B lymphocytes, and demonstrated a high degree of immune specificity during degeneration. In addition to the heterogenous infiltrating immune cell populations in the degenerating IVD, the increased number of cells in the AF sub-cluster expressing Ngf and Ngfr, encoding for p75NTR, suggest that NGF signaling may be one of the key mediators of the IVD crosstalk between immune and neuronal cell populations. These findings provide the basis for future work to understand the involvement of select subsets of non-resident cells in IVD degeneration.

Contrast-enhanced microCT evaluation of degeneration following partial and full width injuries to the mouse lumbar intervertebral disc.

A targeted injury to the mouse intervertebral disc (IVD) is often used to recapitulate the degenerative cascade of the human pathology. Since injuries can vary in magnitude and localization, it is critical to examine the effects of different injuries on IVD degeneration. We thus evaluated the degenerative progression resulting from either a partial- or full-width injury to the mouse lumbar IVD using contrast-enhanced micro-computed tomography and histological analyses. A lateral-retroperitoneal surgical approach was used to access the lumbar IVD, and the injuries to the IVD were produced by either incising one side of the annulus fibrosus or puncturing both sides of the annulus fibrosus. Female C57BL/6J mice of 3-4 months age were used in this study. They were divided into three groups to undergo partial-width, full-width, or sham injuries. The L5/6 and L6/S1 lumbar IVDs were surgically exposed, and then the L6/S1 IVDs were injured using either a surgical scalpel (partial-width) or a 33G needle (full-width), with the L5/6 serving as an internal control. These animals recovered and then euthanized at either 2-, 4-, or 8-weeks after surgery for evaluation. The IVDs were assessed for degeneration using contrast-enhanced microCT (CEµCT) and histological analysis. The high-resolution 3D CEµCT evaluation of the IVD confirmed that the respective injuries were localized within one side of the annulus fibrosus or spanned the full width of the IVD. The full-width injury caused significant deteriorations in the nucleus pulposus, annulus fibrous and at the interfaces after 2 weeks, which was sustained through the 8 weeks, while the partial width injury caused localized disruptions that remained limited to the annulus fibrosus. The use of CEµCT revealed distinct IVD degeneration profiles resulting from partial- and full-width injuries. The partial width injury may serve as an alternative model for IVD degeneration resulting from localized annulus fibrosus injuries.

Relations Between Bone Quantity, Microarchitecture, and Collagen Cross-links on Mechanics Following In Vivo Irradiation in Mice.

Humans are exposed to ionizing radiation via spaceflight or cancer radiotherapy, and exposure from radiotherapy is known to increase risk of skeletal fractures. Although irradiation can reduce trabecular bone mass, alter trabecular microarchitecture, and increase collagen cross-linking, the relative contributions of these effects to any loss of mechanical integrity remain unclear. To provide insight, while addressing both the monotonic strength and cyclic-loading fatigue life, we conducted total-body, acute, gamma-irradiation experiments on skeletally mature (17-week-old) C57BL/6J male mice (n = 84). Mice were administered doses of either 0 Gy (sham), 1 Gy (motivated by cumulative exposures from a Mars mission), or 5 Gy (motivated by clinical therapy regimens) with retrieval of the lumbar vertebrae at either a short-term (11-day) or long-term (12-week) time point after exposure. Micro-computed tomography was used to assess trabecular and cortical quantity and architecture, biochemical composition assays were used to assess collagen quality, and mechanical testing was performed to evaluate vertebral compressive strength and fatigue life. At 11 days post-exposure, 5 Gy irradiation significantly reduced trabecular mass (p < 0.001), altered microarchitecture (eg, connectivity density p < 0.001), and increased collagen cross-links (p < 0.001). Despite these changes, vertebral strength (p = 0.745) and fatigue life (p = 0.332) remained unaltered. At 12 weeks after 5 Gy exposure, the trends in trabecular bone persisted; in addition, regardless of irradiation, cortical thickness (p < 0.01) and fatigue life (p < 0.01) decreased. These results demonstrate that the highly significant effects of 5 Gy total-body irradiation on the trabecular bone morphology and collagen cross-links did not translate into detectable effects on vertebral mechanics. The only mechanical deficits observed were associated with aging. Together, these vertebral results suggest that for spaceflight, irradiation alone will likely not alter failure properties, and for radiotherapy, more investigations that include post-exposure time as a positive control and testing of both failure modalities are needed to determine the cause of increased fracture risk. © 2021 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research. This article has been contributed to by US Government employees and their work is in the public domain in the USA.

Sclerostin Regulation, Microarchitecture, and Advanced Glycation End-Products in the Bone of Elderly Women With Type 2 Diabetes.

Increased circulating sclerostin and accumulation of advanced glycation end-products (AGEs) are two potential mechanisms underlying low bone turnover and increased fracture risk in type 2 diabetes (T2D). Whether the expression of the sclerostin-encoding SOST gene is altered in T2D, and whether it is associated with AGEs accumulation or regulation of other bone formation-related genes is unknown. We hypothesized that AGEs accumulate and SOST gene expression is upregulated in bones from subjects with T2D, leading to downregulation of bone forming genes (RUNX2 and osteocalcin) and impaired bone microarchitecture and strength. We obtained bone tissue from femoral heads of 19 T2D postmenopausal women (mean glycated hemoglobin [HbA1c] 6.5%) and 73 age- and BMI-comparable nondiabetic women undergoing hip replacement surgery. Despite similar bone mineral density (BMD) and biomechanical properties, we found a significantly higher SOST (p = .006) and a parallel lower RUNX2 (p = .025) expression in T2D compared with non-diabetic subjects. Osteocalcin gene expression did not differ between T2D and non-diabetic subjects, as well as circulating osteocalcin and sclerostin levels. We found a 1.5-fold increase in total bone AGEs content in T2D compared with non-diabetic women (364.8 ± 78.2 versus 209.9 ± 34.4 µg quinine/g collagen, respectively; p < .001). AGEs bone content correlated with worse bone microarchitecture, including lower volumetric BMD (r = -0.633; p = .02), BV/TV (r = -0.59; p = .033) and increased trabecular separation/spacing (r = 0.624; p = .023). In conclusion, our data show that even in patients with good glycemic control, T2D affects the expression of genes controlling bone formation (SOST and RUNX2). We also found that accumulation of AGEs is associated with impaired bone microarchitecture. We provide novel insights that may help understand the mechanisms underlying bone fragility in T2D. © 2020 American Society for Bone and Mineral Research (ASBMR).

Rapid determination of internal strains in soft tissues using an experimentally calibrated finite element model derived from magnetic resonance imaging.

BACKGROUND: Finite element models (FEMs) of medical images can provide information about the underlying tissue that cannot be obtained from the original images. Preforming an accurate simulation requires the careful experimental calibration of boundary conditions. Here we describe a method for deriving a geometric mesh for soft biological materials using a magnetic resonance imaging (MRI) system, and an experimental workflow for calibrating the boundary conditions and optimizing the mesh density in these simulations. METHODS: A three-dimensional image stack of a ballistic sphere gel, a bovine caudal intervertebral disc (IVD), and a human lumbar IVD were generated using a positional MRI system. These images were then segmented using a semi-automated process, converted to a tetrahedral mesh, and then modeled as a linear elastic solid. The mesh density was optimized based on simulation time and convergence with the experimental results. The modulus of the ballistic gel was determined experimentally, while the material properties for the nucleus pulposus (NP) and the annulus fibrosus (AF) within the bovine and human IVDs were assigned from literature. The simulation for the spherical gel and the bovine IVD matched the reaction forces determined experimentally in compression. We then simulated a 0.3 MPa compressive load on the human lumbar IVD at the optimal mesh density and material properties determined from the bovine model and then examined the resultant internal strains. RESULTS: The scaled mesh density demonstrated excellent correspondence with the experimental results, confirming that accuracy was not compromised. Both the ballistic gel and the IVD samples exhibited a wide range of internal strains. The NP of the IVD underwent greater deformation than the AF under loading. CONCLUSIONS: This study validated a strategy for mesh optimization and FEM of soft biological materials from data generated from MRI scans. This calibrated approach allows for the rapid examination of internal strain distributions medical images that can be performed on the order of minutes.

Determination of the Depth- and Time- Dependent Mechanical Behavior of Mouse Articular Cartilage Using Cyclic Reference Point Indentation.

Mouse models of osteoarthritis and cartilage degeneration are important and powerful tools for investigating the molecular mechanisms of the disease pathology. Because of the vast number of genetically modified mouse models that are available for research, the ability to use these models is particularly attractive for the mechanobiologic interactions in the pathogenesis of osteoarthritis. However, the very small scale of mouse articular cartilage, where the healthy tissue is only 80 µm in thickness, poses challenges in quantifying mechanical characteristics of the tissue. We introduce here a novel approach that combines experimental and analytical methods to quantify the nuanced mechanical changes during cartilage degeneration at this scale. Cyclic reference point indentation is used to directly test the murine articular cartilage to obtain the force-deformation and the phase-shift characteristics of the tissue. The cartilage zonal thicknesses are confirmed from histology. These data are then fitted to a parallel spring model to determine the depth-dependent tissue stiffness and modulus. Using this approach, we investigated the effects of trypsin degradation on the zonal mechanical behavior of mouse articular cartilage. We observe a decline of the superficial zone stiffness coupled with the loss of the superficial layer. Subsequent degradation by trypsin allowed the identification of middle- and deep- zone properties. Taken together, this approach can be a useful tool for understanding the disease mechanisms of cartilage homeostasis and degeneration, and for monitoring of therapies for osteoarthritis.

The age-dependent effect of high-dose X-ray radiation on NFκB signaling, structure, and mechanical behavior of the intervertebral disc.

Purpose: Ionizing radiation damages tissue and provokes inflammatory responses in multiple organ systems. We investigated the effects of high-dose X-ray radiation on the molecular inflammation and mechanical function of the intervertebral disc (IVD).Methods: Functional spine units (FSUs) containing the vertebrae-IVDs-vertebrae structure extracted from 1-month, 6-month, and 16-month-old NFκB-luciferase reporter mice and from 6-month-old myeloid differentiation factor 88 (MyD88)-null mice. After a preconditioning period in culture, the FSUs were subjected a single dose of ionizing X-ray radiation at 20 Gys, and then NFκB expression was monitored. The IVDs were then subjected to mechanical testing using dynamic compression, glycosaminoglycan (GAG) quantification, and histological analyses.Results: In the 1-month-old FSUs, the NFκB-driven luciferase activity was significantly elevated for 1 day following the exposure to radiation. The 6-month-old FSUs showed increased NFκB activity for 3 days, while the 16-month-old FSUs sustained elevated levels of NFκB activity throughout the 10-day culture period. All irradiated groups showed significant loss of disc height, GAG content, mechanical function and changes in structure. Ablation of MyD88 blunted the radiation-mediated NFκB signaling, and preserved GAG content, and the IVDs' structure and mechanical performance.Conclusions: These results suggest that high-dose radiation affects the IVDs' NFκB-dependent inflammatory processes that subsequently lead to functional deterioration. Blocking the transactivation potential of NFκB via MyD88 ablation preserved the structure and mechanical function of the FSUs. The long-term effects of radiation on IVD homeostasis should be considered in individuals susceptible to occupational and medical exposure.

Effects of ex vivo ionizing radiation on collagen structure and whole-bone mechanical properties of mouse vertebrae.

Bone can become brittle when exposed to ionizing radiation across a wide range of clinically relevant doses that span from radiotherapy (accumulative 50 Gy) to sterilization (~35,000 Gy). While irradiation-induced embrittlement has been attributed to changes in the collagen molecular structure, the relative role of collagen fragmentation versus non-enzymatic collagen crosslinking remains unclear. To better understand the effects of radiation on the bone material without cellular activity, we conducted an ex vivo x-ray radiation experiment on excised mouse lumbar vertebrae. Spinal tissue from twenty-week old, female, C57BL/6J mice were randomly assigned to a single x-ray radiation dose of either 0 (control), 50, 1000, 17,000, or 35,000 Gy. Measurements were made for collagen fragmentation, non-enzymatic collagen crosslinking, and both monotonic and cyclic-loading compressive mechanical properties. We found that the group differences for mechanical properties were more consistent with those for collagen fragmentation than for non-enzymatic collagen crosslinking. Monotonic strength at 17,000 and 35,000 Gy was lower than that of the control by 50% and 73% respectively, (p < 0.001) but at 50 and 1000 Gy was not different than the control. Consistent with those trends, collagen fragmentation only occurred at 17,000 and 35,000 Gy. By contrast, non-enzymatic collagen crosslinking was greater than control for all radiation doses (p < 0.001). All results were consistent both for monotonic and cyclic loading conditions. We conclude that the reductions in bone compressive monotonic strength and fatigue life due to ex vivo ionizing radiation are more likely caused by fragmentation of the collagen backbone than any increases in non-enzymatic collagen crosslinks.

T1ρ-based fibril-reinforced poroviscoelastic constitutive relation of human articular cartilage using inverse finite element technology.

BACKGROUND: Mapping of T1ρ relaxation time is a quantitative magnetic resonance (MR) method and is frequently used for analyzing microstructural and compositional changes in cartilage tissues. However, there is still a lack of study investigating the link between T1ρ relaxation time and a feasible constitutive relation of cartilage which can be used to model complicated mechanical behaviors of cartilage accurately and properly. METHODS: Three-dimensional finite element (FE) models of ten in vitro human tibial cartilage samples were reconstructed such that each element was assigned by material-level parameters, which were determined by a corresponding T1ρ value from MR maps. A T1ρ-based fibril-reinforced poroviscoelastic (FRPE) constitutive relation for human cartilage was developed through an inverse FE optimization technique between the experimental and simulated indentations. RESULTS: A two-parameter exponential relationship was obtained between the T1ρ and the volume fraction of the hydrated solid matrix in the T1ρ-based FRPE constitutive relation. Compared with the common FRPE constitutive relation (i.e., without T1ρ), the T1ρ-based FRPE constitutive relation indicated similar indentation depth results but revealed some different local changes of the stress distribution in cartilages. CONCLUSIONS: Our results suggested that the T1ρ-based FRPE constitutive relation may improve the detection of changes in the heterogeneous, anisotropic, and nonlinear mechanical properties of human cartilage tissues associated with joint pathologies such as osteoarthritis (OA). Incorporating T1ρ relaxation time will provide a more precise assessment of human cartilage based on the individual in vivo MR quantification.

Novel Augmentation Technique for Patellar Tendon Repair Improves Strength and Decreases Gap Formation: A Cadaveric Study.

BACKGROUND: Patellar tendon ruptures commonly are repaired using transosseous patellar drill tunnels with modified-Krackow sutures in the patellar tendon. This simple suture technique has been associated with failure rates and poor clinical outcomes in a modest proportion of patients. Failure of this repair technique can result from gap formation during loading or a single catastrophic event. Several augmentation techniques have been described to improve the integrity of the repair, but standardized biomechanical evaluation of repair strength among different techniques is lacking. QUESTIONS/PURPOSES: The purpose of this study was to describe a novel figure-of-eight suture technique to augment traditional fixation and evaluate its biomechanical performance. We hypothesized that the augmentation technique would (1) reduce gap formation during cyclic loading and (2) increase the maximum load to failure. METHODS: Ten pairs (two male, eight female) of fresh-frozen cadaveric knees free of overt disorders or patellar tendon damage were used (average donor age, 76 years; range, 65-87 years). For each pair, one specimen underwent the standard transosseous tunnel suture repair with a modified-Krackow suture technique and the second underwent the standard repair with our experimental augmentation method. Nine pairs were suitable for testing. Each specimen underwent cyclic loading while continuously measuring gap formation across the repair. At the completion of cyclic loading, load to failure testing was performed. RESULTS: A difference in gap formation and mean load to failure was seen in favor of the augmentation technique. At 250 cycles, a 68% increase in gap formation was seen for the control group (control: 5.96 ± 0.86 mm [95% CI, 5.30-6.62 mm]; augmentation: 3.55 ± 0.56 mm [95% CI, 3.12-3.98 mm]; p = 0.02). The mean load to failure was 13% greater in the augmentation group (control: 899.57 ± 96.94 N [95% CI, 825.06-974.09 N]; augmentation: 1030.70 ± 122.41 N [95% CI, 936.61-1124.79 N]; p = 0.01). CONCLUSIONS: This biomechanical study showed improved performance of a novel augmentation technique compared with the standard repair, in terms of reduced gap formation during cyclic loading and increased maximum load to failure. CLINICAL RELEVANCE: Decreased gap formation and higher load to failure may improve healing potential and minimize failure risk. This study shows a potential biomechanical advantage of the augmentation technique, providing support for future clinical investigations comparing this technique with other repair methods that are in common use such as transosseous suture repair.

Comparison of different intubation techniques performed inside a moving ambulance: a manikin study.

OBJECTIVE. Airway management and endotracheal intubation may be required urgently when a patient deteriorates in an ambulance or aircraft during interhospital transfer or in a prehospital setting. The objectives of this study were: (1) to compare the effectiveness of conventional intubation by Macintosh laryngoscope in a moving ambulance versus that in a static ambulance; and (2) to compare the effectiveness of inverse intubation and GlideScope laryngoscopy with conventional intubation inside a moving ambulance. DESIGN. Comparative experimental study. SETTING. The experiment was conducted in an ambulance provided by the Auxiliary Medical Service in Hong Kong. PARTICIPANTS. A group of 22 doctors performed endotracheal intubation on manikins with Macintosh laryngoscope in a static and moving ambulance. In addition, they performed conventional Macintosh intubation, inverse intubation with Macintosh laryngoscope, and GlideScope intubation in a moving ambulance in both normal and simulated difficult airways. MAIN OUTCOME MEASURES. The primary outcome was the rate of successful intubation. The secondary outcomes were time taken for intubation, subjective glottis visualisation grading, and eventful intubation (oesophageal intubation, intubation time >60 seconds, and incisor breakage) with different techniques or devices. RESULTS. In normal airways, conventional Macintosh intubation in a static ambulance (95.5%), conventional intubation in a moving ambulance (95.5%), as well as GlideScope intubation in a moving ambulance (95.5%) were associated with high success rates; the success rate of inverse intubation was comparatively low (54.5%; P=0.004). In difficult airways, conventional Macintosh intubation in a static ambulance (86.4%), conventional intubation in a moving ambulance (90.9%), and GlideScope intubation in a moving ambulance (100%) were associated with high success rates; the success rate of inverse intubation was comparatively lower (40.9%; P=0.034). CONCLUSIONS. En-route intubation in an ambulance by conventional Macintosh laryngoscopy is superior to inverse intubation unless the cephalad access is impossible. GlideScope laryngoscopy appears to be associated with lower rates of eventful intubation in difficult airways and has better laryngoscopic view versus inverse intubation.

Load regulates bone formation and Sclerostin expression through a TGFß-dependent mechanism.

Bone continually adapts to meet changing physical and biological demands. Osteoblasts, osteoclasts, and osteocytes cooperate to integrate these physical and biochemical cues to maintain bone homeostasis. Although TGFß acts on all three of these cell types to maintain bone homeostasis, the extent to which it participates in the adaptation of bone to mechanical load is unknown. Here, we investigated the role of the TGFß pathway in load-induced bone formation and the regulation of Sclerostin, a mechanosensitive antagonist of bone anabolism. We found that mechanical load rapidly represses the net activity of the TGFß pathway in osteocytes, resulting in reduced phosphorylation and activity of key downstream effectors, Smad2 and Smad3. Loss of TGFß sensitivity compromises the anabolic response of bone to mechanical load, demonstrating that the mechanosensitive regulation of TGFß signaling is essential for load-induced bone formation. Furthermore, sensitivity to TGFß is required for the mechanosensitive regulation of Sclerostin, which is induced by TGFß in a Smad3-dependent manner. Together, our results show that physical cues maintain bone homeostasis through the TGFß pathway to regulate Sclerostin expression and the deposition of new bone.

Characterization of the effects of x-ray irradiation on the hierarchical structure and mechanical properties of human cortical bone.

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.