Christian E. Gonzalez

Background: Impact microindentation (IMI) is a minimally invasive technique for assessing bone material properties that can be performed clinically. In this study, we aimed to determine how measurements obtained by IMI (bone material strength index [BMSi]) relate to bone strength, fracture resistance, and susceptibility to fracture. Questions/purposes: (1) Is BMSi associated with bone strength of the femur, radius, and vertebrae, and does incorporating BMSi alongside bone mineral density (BMD) improve the estimation of strength? (2) Does BMSi evaluate fracture toughness and mechanical properties of the bone tissue? (3) Can BMSi discriminate fracture susceptibility of the hip and wrist? Methods: This single-site laboratory study included the tibiae, femurs, radii, and L4 vertebrae from fresh-frozen cadavers (n = 67; mean ± SD age 71 ± 13 years). To answer the first question, IMI was performed on the tibia midshaft, dual-energy x-ray absorptiometry (DXA) was used to assess hip and spine BMD, and bone strength (ultimate fracture force) was mechanically determined for each site. To address the second question, stress intensity factor (K _C ) was quantified from notched cortical bone beam specimens from tibiae using the fracture toughness test. Bone mechanical behavior (ultimate stress, elastic modulus, work-to-fracture), microstructure (cortical porosity, tissue mineral density), and organic matrix quality (percentage of collagen denaturation, concentration of advanced glycation end products) were quantified from beam specimens from the femoral midshaft. To address the last question, hip and wrist fracture susceptibility was defined using the fall-load to fracture-load ratio, where values ≥ 1 indicate susceptibility. Receiver operating characteristic (ROC) curve analyses evaluated the ability of IMI and DXA to discriminate fracture susceptibility. Primary outcomes included evaluating the association between BMSi and BMD with bone strength and ROC analysis for discriminating fracture susceptibility. Results: Higher BMSi values were positively associated with higher bone strength, showing moderate association at the femur (r = 0.62 [95% confidence interval (CI) 0.49 to 0.72]; p < 0.001) and radius (r = 0.52 [95% CI 0.34 to 0.72]; p < 0.001) and weaker association at the L4 vertebrae (r = 0.31 [95% CI 0.03 to 0.53]; p = 0.02). At the femur, BMSi was more strongly associated with strength than total hip BMD (TH-BMD) (r = 0.45 [95% CI 0.3 to 0.62]; p < 0.001), and the inclusion of BMSi in multivariate analyses improved the explained variance in femur strength from 28% to 52% (ΔR ² = 0.24; p < 0.001). At the radius, both BMSi and TH-BMD (r = 0.50 [95% CI 0.31 to 0.71]; p < 0.01) showed moderate association with radius strength, with the combined model improving the explained variance in radius strength from 28% to 39% (ΔR ² = 0.11; p < 0.001). At the vertebrae, strength was weakly associated with BMSi (r = 0.31 [95% CI 0.03 to 0.53]; p = 0.02) but not with BMD. For the hip and wrist of each donor, BMSi discriminated hip fracture susceptibility with moderate accuracy (area under the curve [AUC] 0.75 [95% CI 0.63 to 0.88]; p < 0.001), whereas TH-BMD could not discriminate hip fracture susceptibility. Both BMSi (AUC 0.77 [95% CI 0.63 to 0.90]; p = 0.003) and TH-BMD (AUC 0.78 [95% CI 0.64 to 0.90]; p = 0.002) classified wrist fracture susceptibility with modest discriminatory performance. Conclusion: Our study demonstrates that BMSi provides information related to bone strength and discriminates fracture susceptibility of the hip and wrist. Clinical Relevance: IMI can be performed at the point of care using an FDA-cleared device and used to evaluate fragility in patients in whom fracture risk or bone strength is uncertain based on BMD alone. Prospective clinical studies are needed to determine whether IMI improves fracture risk prediction and informs patient treatment.

Background: Chronic low back pain, commonly associated with intervertebral disc (IVD) degeneration, is highly prevalent in patients with Alzheimer's disease (AD), and the severity of the pain is correlated with the severity of the dementia. While incidences of both afflictions increase dramatically in the elderly population, it is unknown whether AD exacerbates the health of the IVD. Aims: The aim of this study is to examine how AD‐related mutations affect the structure and mechanics of the IVD. Materials and Methods: Utilizing one‐year‐old male and female 5xFAD mice that constitutively express human APP and PSEN1 transgenes with five AD‐linked mutations, we measured the lumbar IVD's extracellular matrix composition, the three‐dimensional structure, histopathological degeneration, and mechanical behaviour. Results: The amount of collagen, glycosaminoglycans, and advanced glycation end‐products in the IVD did not differ in the 5xFAD animals. Likewise, the 5xFAD IVDs were not histopathologically degenerated. However, the IVD volume, measured by contrast‐enhanced microCT, was larger in the 5xFAD animals ( p = 0.046; 1–β = 0.53; Cohen's d = 1.19). Morphologically, the 5xFAD IVD heights were homogeneous and flatter than the WT IVDs ( p = 0.043; 1–β = 0.60; Cohen's d = 1.41). Finally, dynamic micro compression revealed that 5xFAD IVDs have higher loss tangent ( p = 0.0062; 1–β = 0.85; Cohen's d = 1.60) and greater energy dissipation ( p = 0.0379; 1–β = 0.55; Cohen's d = 1.21). Discussion: Shifts in viscoelastic behaviour and disc morphology are associated with early tissue degradation and affect the IVD's ability to efficiently recover from mechanical loading, predisposing the IVD to eventual degeneration. Conclusion: This work suggests that IVD's structure, morphology, and mechanics are affected by AD. Future work will focus on defining the molecular mechanisms of these changes in the IVD and their consequences in individuals with AD.

The chronic inflammation observed during type 2 diabetes (T2D) is associated with spinal pathologies, including intervertebral disc (IVD) degeneration and chronic spine pain. Despite confounding factors, such as obesity, studies show that, after adjusting for age, body mass index and genetics (e.g. twins), patients with T2D experience disproportionate severity of IVD degeneration and/or back pain than individuals without T2D. We hypothesized that chronic T2D fosters a proinflammatory microenvironment within the IVD that promotes degeneration and disrupts homeostasis. To test this, we evaluated two common mouse models of T2D – leptin-receptor deficient (db/db) mice and mice with a chronic high-fat diet and impaired β-cell function (STZ-HFD). IVDs of STZ-HFD mice exhibited more severe degeneration and elevated chemokine expression than controls. RNA sequencing further revealed extensive transcriptional dysregulation in STZ-HFD IVDs not observed in db/db IVDs. STZ-HFD IVDs expressed enzymes that enhance advanced glycation end product precursors, impaired non-AGE DAMP pathways and suppressed RAGE turnover. These results suggest that, under controlled genetic and environmental conditions, the STZ-HFD model more accurately reflects the multifactorial inflammatory milieu characteristic of T2D-induced IVD degeneration.

The chronic inflammation observed during type 2 diabetes (T2D) is associated with spinal pathologies, including intervertebral disc (IVD) degeneration and chronic spine pain. Despite the presence of confounding factors, such as obesity, studies have shown that after adjusting for age, body mass index, and genetics (e.g. twins), patients with T2D suffer from disproportionately more IVD degeneration and/or back pain. We hypothesize that chronic T2D fosters a proinflammatory microenvironment within the IVD that promotes degeneration and disrupts IVD homeostasis. To test this hypothesis, we evaluated two commonly used mouse models of T2D – the leptin-receptor deficient mouse (db/db) and the chronic high-fat diet in mice with impaired beta-cell function (STZ-HFD). Compared to their genetic controls—C57BL/6 wild-type mice for STZ-HFD and heterozygous littermates for db/db—STZ-HFD IVDs exhibited more severe degeneration and elevated chemokine expression profiles. RNA-seq further revealed extensive transcriptional dysregulation in STZ-HFD IVDs that was not observed in the db/db model. The STZ-HFD IVDs also expressed enzymes that enhanced production of glycolytic AGE precursors, impaired non-AGE DAMP pathways, and reduced suppressors of RAGE turnover. These results suggest that, under controlled genetic and environmental conditions, the STZ-HFD model more accurately reflect the multifactorial inflammatory milieu characteristic of T2D-induced IVD degeneration.

Inflammatory cytokine production and de novo neurovascularization have been identified in painful, degenerated intervertebral discs (IVDs). However, the temporal trajectories of these key pathoanatomical features, including the cascade of inflammatory chemokines and neo-vessel and neurite infiltration, and their associations with IVD degeneration, remain relatively unknown. Investigating this process in the caudal mouse IVD enables the opportunity to study the tissue-specific response without confounding inflammatory signaling from neighboring structures. Thus this study aims to define the progression of chemokine production and neurovascular invasion during the IVD degeneration initiated by injury in the caudal spine 3-month-old C57BL6/J mice. Forty-nine IVD-secreted chemokines and matrix metalloproteinases (MMPs) was measured using multiplex ELISA, and the intradiscal infiltrating vessels (endomucin) and nerves (protein-gene-product 9.5) was quantified in the tissue volume using immunohistochemistry. Injury provoked the increase secretion of IL6, CCL2, CCL12, CCL17, CCL20, CCL21, CCL22, CXCL2 and MMP2 proteins. The centrality and structure of inflammatory networks in IVDs evolved over the 12 post-injury weeks, highlighting distinct responses between the acute and chronic phases. Neurites propagated rapidly within 2-weeks post-injury and remained relatively constant until 12-weeks. Vascular vessel length was observed to peak at 4-weeks post-injury and it regressed by 12-weeks. These findings identified the temporal flux of inflammatory chemokines and pain-associated pathoanatomy in a model of IVD degeneration using the mouse caudal spine.

Non-specific lower back pain (LBP) is a world-wide public health problem that affects people of all ages. Despite the high prevalence of non-specific LBP and the associated economic burdens, the pathoanatomical mechanisms for the development and course of the condition remain unclear. While intervertebral disc degeneration (IDD) is associated with LBP, there is overlapping occurrence of IDD in symptomatic and asymptomatic individuals, suggesting that degeneration alone cannot identify LBP populations. Previous work has been done trying to relate linear measurements of compression obtained from Magnetic Resonance Imaging (MRI) to pain unsuccessfully. To bridge this gap, we propose to use advanced non-Euclidean statistical shape analysis methods to develop biomarkers that can help identify symptomatic and asymptomatic adults who might be susceptible to standing-induced LBP. We scanned 4 male and 7 female participants who exhibited lower back pain after prolonged standing using an Open Upright MRI. Supine and standing MRIs were obtained for each participant. Patients reported their pain intensity every fifteen minutes within a period of 2 h. Using our proposed geodesic logistic regression, we related the structure of their lower spine to pain and computed a regression model that can delineate lower spine structures using reported pain intensities. These results indicate the feasibility of identifying individuals who may suffer from lower back pain solely based on their spinal anatomy. Our proposed spinal shape analysis methodology have the potential to provide powerful information to the clinicians so they can make better treatment decisions. Keywords geodesic logistic regression intervertebral disc degeneration longitudinal shape analysis

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.

Two current hypotheses of O2 sensing in the carotid body (CB) chemoreceptors suggest participation of oxygen reactive (ROS) species, but they are mechanistically opposed. One postulates that hypoxia decreases ROS levels; the other that hypoxia increases them. Yet, both propose that the ensuing alteration in the cellular redox environment is the key signal triggering hypoxic chemoreception. Since the glutathione redox pair is the main cellular buffer for ROS and the main determinant of the general redox environment of the cells, a way to test whether ROS participate in chemoreception is to determine glutathione levels and to correlate them with the activity of CB chemoreceptor cells. We found that hypoxia does not alter the glutathione reduction potential but that it activates chemoreceptor cell neurosecretion. Incubation of tissues with reduced glutathione increases the glutathione-reducing potential but does not activate chemoreceptor cells in normoxia nor does it modify hypoxic activation. Like reduced glutathione, N-acetylcysteine promoted a general reducing environment in the cells without alteration of chemoreceptor cell activity. N-(mercaptopropionyl)-glycine, like the two previous agents, increases the reduction potential of glutathione. In contrast, the compound activated chemoreceptor cells in normoxia, promoting a dose- and Ca(2+)-dependent neurosecretion and a potentiation of the hypoxic responses. The existence of multiple relationships between glutathione reduction potential in the cells and their activity indicates that the general cellular redox environment is not a factor determining chemoreceptor cell activation. It cannot be excluded that the local redox environments of restricted microdomain(s) in the cells with specific regulating mechanisms are important signals for chemoreceptor cell activity.