Diabetes is associated with impaired tendon homeostasis and subsequent tendon dysfunction, but the mechanisms underlying these associations is unclear. Advanced glycation end-products (AGEs) accumulate with diabetes and have been suggested to alter tendon function. In vivo imaging in humans has suggested collagen disorganization is more frequent in individuals with diabetes, which could also impair tendon mechanical function. The purpose of this study was to examine relationships between tendon tensile mechanics in human Achilles tendon with accumulation of advanced glycation end-products and collagen disorganization. Achilles tendon specimens (n = 16) were collected from individuals undergoing lower extremity amputation or from autopsy. Tendons were tensile tested with simultaneous quantitative polarized light imaging to assess collagen organization, after which AGEs content was assessed using a fluorescence assay. Moderate to strong relationships were observed between measures of collagen organization and tendon tensile mechanics (range of correlation coefficients: 0.570–0.727), whereas no statistically significant relationships were observed between AGEs content and mechanical parameters (range of correlation coefficients: 0.020–0.210). Results suggest that the relationship between AGEs content and tendon tensile mechanics may be masked by multifactorial collagen disorganization at larger length scales (i.e., the fascicle level).
Mouse models are often used for studies of intervertebral disc (IVD) homeostasis and degeneration, yet the relatively small size of the IVD poses challenges for noninvasive, longitudinal imaging modalities. The recently developed contrast‐enhanced microCT (CEμCT) using Ioversol has been successful in detecting degenerative changes in the murine IVD ex vivo at the micrometer scale. Further leveraging the superior biocompatibility of Ioversol as a contrast agent, we demonstrate the in vivo use of this CEμCT technique to examine IVDs at multiple spinal sites. Ioversol was administered via tail vein injection (TVI) in growing and adult male FVB/NJ mice (n = 5 /group). The animals were anesthetized and underwent in vivo micro‐computed tomographic (microCT) at the coccygeal (CC5/CC6), lumbar (L5/6), and thoracic (T12/T13) IVDs. TVI of Ioversol was well‐tolerated by all animals. As Ioversol filtered through the kidneys and accumulated in the bladder, the attenuations of the mouse bladder and kidneys increased due to the high molecular weight of Ioversol, confirming that the Ioversol is biological available. Average IVD attenuations increased 3%‐15% following TVI (ANOVA; P < .01). The presence of Ioversol in the IVD combined with high‐resolution microCT allow for nondestructive visualization of structural features of the IVD. These results demonstrate CEμCT with Ioversol as a viable strategy for the in vivo monitoring of multiple mouse IVDs during degeneration, disease, and injury.
Introduction: Diabetes has long been implicated as a major risk factor for intervertebral disc (IVD) degeneration, interfering with molecular signaling and matrix biochemistry, which ultimately aggravates the progression of the disease. Glucose content has been previously shown to influence structural and compositional changes in engineered discs in vitro, impeding fiber formation and mechanical stability.Methods: In this study, we investigated the impact of diabetic hyperglycemia on young IVDs by assessing biochemical composition, collagen fiber architecture, and mechanical behavior of discs harvested from 3-to 4-month-old db/db mouse caudal spines.Results: We found that discs taken from diabetic mice with elevated blood glucose levels demonstrated an increase in total glycosaminoglycan and collagen content, but comparable advanced glycation end products (AGE) levels to wild-type discs. Diabetic discs also contained ill-defined boundaries between the nucleus pulposus and annulus fibrosus, with the latter showing a disorganized and unaligned collagen fiber network at this same boundary.Conclusions: These compositional and structural changes had a detrimental effect on function, as the diabetic discs were twice as stiff as their wild-type counterparts and demonstrated a significant resistance to deformation. These results indicate that diabetes may predispose the young disc to DDD later in life by altering patterns of extracellular matrix deposition, fiber formation, and motion segment mechanics independently of AGE accumulation.
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.
BACKGROUND AND PURPOSE: When one uses T2 relaxometry to classify lumbar intervertebral discs as degenerated, it is unclear whether the normative data should be based on other intervertebral discs from the same individual or from a pool of extraneous controls. This study aimed to explore the extent of intra-versus intersubject variation in the T2 times of healthy intervertebral discs. MATERIALS AND METHODS:Using prospectively acquired T2-relaxometry data from 606 intervertebral discs in 101 volunteers without back pain (47 men, 54 women) in a narrow age range (25-35 years), we calculated intra-and intersubject variation in T2 times of intervertebral discs graded by 2 neuroradiologists on the Pfirrmann scale. Intrasubject variation of intervertebral discs was assessed relative to other healthy intervertebral discs (Pfirrmann grade, #2) in the same individual. Multiple intersubject variability measures were calculated using healthy extraneous references ranging from a single randomly selected intervertebral disc to all healthy extraneous intervertebral discs, without and with segmental stratification. These variability measures were compared for healthy and degenerated (Pfirrmann grade $3) intervertebral discs. RESULTS:The mean T2 values of healthy (493/606, 81.3%) and degenerated intervertebral discs were 121.1 6 22.5 ms and 91.5 6 18.6 ms, respectively (P , .001). The mean intrasubject variability for healthy intervertebral discs was 9.8 6 10.7 ms, lower than all intersubject variability measures (P , .001), and provided the most pronounced separation for healthy and degenerated intervertebral discs. Among intersubject variability measures, using all segment-matched healthy discs as references provided the lowest variability (P , .001). CONCLUSIONS:Normative measures based on the T2 times of healthy intervertebral discs from the same individual are likely to provide the most discriminating means of identifying degenerated intervertebral discs on the basis of T2 relaxometry. ABBREVIATIONS: I, X, Xs, Xc, Xcs ¼ various measures of variability in T2 times of intervertebral discs; IVD ¼ intervertebral disc; np ¼ used as a suffix to indicate measures assumed to represent nucleus pulposus L oss of signal intensity of the central part of intervertebral discs (IVDs) on T2-weighted MR images is a well-established indicator of underlying degenerative changes. [1][2][3][4] Visual recognition of the extent of this loss of signal intensity also forms the basis of grading the extent of disc degeneration on frequently used grading schemes such as one proposed by Pfirrmann et al. 5 To overcome the subjectivity of visual assessment, T2 relaxometry provides a reliable, objective, and
Study Design: Preclinical animal study. Objective: Evaluation of the degenerative progression resulting from either a partial- or full- width injury to the mouse lumbar intervertebral disc (IVD) using contrast-enhanced micro-computed tomography and histological analyses. We utilized a lateral-retroperitoneal surgical approach to access the lumbar IVD, and the injuries to the IVD were induced by either incising one side of the annulus fibrosus or puncturing both sides of the annulus fibrosus. The full-width injury caused dramatic reduction in nucleus pulposus hydration and significant degeneration. A partial-width injury produces localized deterioration around the annulus fibrosus site that resulted in local tissue remodeling without gross degeneration to the IVD. Methods: Female C57BL/6J mice of 3-4 months age were used in this study. They were divided into three groups to undergo a partial-width, full-width, or sham injuries. The L5/L6 and L6/S1 lumbar IVDs were surgically exposed using a lateral-retroperitoneal approach. The L6/S1 IVDs were injured using either a surgical scalpel (partial-width) or a 33G needle (full-width), with the L5/L6 serving as an internal control. These animals were allowed to recover and then sacrificed at 2-, 4-, or 8- weeks post-surgery. The IVDs were assessed for degeneration using contrast-enhanced microCT (CEμCT) and histological analysis. Results: The high-resolution 3D evaluation of the IVD confirmed that the respective injuries localized within one side of the annulus fibrosus or spanned the full width of the IVD. The full-width injury caused deteriorations in the nucleus pulposus after 2 weeks that culminated in significant degeneration at 8 weeks, while the partial width injury caused localized disruptions that remained limited to the annulus fibrosus. Conclusion: The use of CEμCT revealed distinct IVD degeneration profiles resulting from partial- and full- width injuries. The partial width injury may serve as a better model for IVD degeneration resulting from localized annulus fibrosus injuries in humans.
Category: Diabetes Introduction/Purpose: Advanced glycation endproducts (AGEs) accumulate in tendon tissue in individuals with diabetes mellitus (DM). Although AGEs have been shown to impact tendon function by decreasing collagen sliding, this relationship has not been explored in humans with diabetes. Despite the prevalence of foot deformity in this population and implications of posterior tibialis dysfunction, the mechanical behavior of the posterior tibialis tendon has only been reported in a small (n=5), cadaveric study that did not report DM status. Therefore, the purpose of this study is to determine the effects of DM-associated AGEs accumulation on the mechanical properties of the posterior tibialis tendon. Methods: Posterior tibialis tendons were collected from individuals with and without DM undergoing lower extremity amputation. A 1-2 mm tendon transection was used for AGEs quantification. AGEs were quantified via fluorescence following papain digestion and hydrolyzation as described previously. Fluorescence was compared to a quinine standard to calculate AGEs content, which was normalized to sample wet weight. Tensile mechanical testing was completed with the remaining specimen (˜25 mm long). Tendon cross-sectional area was measured with a non-contact laser scanning device. Specimens were preloaded to 10 N and preconditioned for 10 cycles at 6% strain, subjected to stress-relaxation at 6% strain for 10 minutes, and loaded with a triangular waveform to a maximum of 10% strain at a rate of 1% strain per second. Individual values and group descriptive statistics are reported for AGEs content and mechanical testing. Relationships between AGEs content and various mechanical testing parameters were evaluated using Spearman correlation. Results: Six individuals (5 with DM, 4 male, mean(SD) age: 56(5)years) were included. AGEs content was increased in DM tendon (DM: 20.5(5.1), non-DM: 9.5 ng quinine/mg wet weight). Compared to non-DM tendon, DM tendons had larger cross-sectional area (DM: 44.3(4.9), non-DM: 11mm2). From stress relaxation, DM tendons had smaller peak (DM: 0.41(0.25), non-DM: 1.16 MPa) and equilibrium stress (DM: 0.23(0.13), non-DM: 0.83 MPa), and larger percent relaxation (DM: 46(6)%, non-DM: 29%)(Figure 1-A). DM tendons had decreased maximum stress at 10% strain (DM: 0.63(0.45), non-DM: 1.75 MPa), increased linear stiffness (DM: 35.2(27.6), non-DM: 19.2N/mm), and decreased linear modulus (DM: 8.5(7.0), non-DM: 20.1 MPa)(Figure 1-B, C) compared to non- DM tendon. Hysteresis (i.e., energy loss upon unloading) was higher in DM tendons (DM: 0.35(0.05), non-DM: 0.22), and positively correlated to AGEs (rho=0.943, p=0.005, Figure 1-D). Conclusion: Posterior tibialis tendons with DM exhibited increased AGEs content and altered mechanical properties. DM tendons were less stiff when accounting for cross-sectional area but had 2-4x the cross-sectional area of non-DM tendon, with inconsistent patterns in total tendon stiffness potentially attributable to several factors. DM tendons showed impaired energy storage and return, which was most strongly associated with AGEs. Non-DM samples were limited and the linear modulus was smaller than previously reported, however, all but one DM tendon had a modulus less than 50% of the non-DM sample. Future work will explore the mechanisms of AGEs-associated DM tendon impairments.
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