[Show abstract][Hide abstract] ABSTRACT: Approximately half of all cardiovascular deaths associated with acute coronary syndrome occur when the thin fibrous cap tissue overlying the necrotic core in a coronary vessel is torn, ripped or fissured under the action of high blood pressure. From a biomechanics point of view, the rupture of an atheroma is due to increased mechanical stresses in the lesion, in which the ultimate stress (i.e. peak circumferential stress (PCS) at failure) of the tissue is exceeded. Several factors including the cap thickness, morphology, residual stresses and tissue composition of the atheroma have been shown to affect the PCS. Also important, we recently demonstrated that microcalcifications (μCalcs>5µm are a common feature in human atheroma caps, which behave as local stress concentrators, increasing the local tissue stress by at least a factor of two surpassing the ultimate stress threshold for cap tissue rupture. In the present study, we used both idealized µCalcs with spherical shape and actual µCalcs from human coronary atherosclerotic caps, to determine their effect on increasing the circumferential stress in the fibroatheroma cap using different hyperelastic constitutive models. We have found that the stress concentration factor (SCF) produced by μCalcs in the fibroatheroma cap is affected by the material tissue properties, μCalcs spacing, aspect ratio and their alignment relative to the tensile axis of the cap.
Journal of biomechanics 01/2014; · 2.66 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Using 2.1-µm high-resolution microcomputed tomography, we have examined the spatial distribution, clustering, and shape of nearly 35,000 microcalcifications (µCalcs) ≥ 5 µm in the fibrous caps of 22 nonruptured human atherosclerotic plaques. The vast majority of these µCalcs were <15 µm and invisible at the previously used 6.7-µm resolution. A greatly simplified 3D finite element analysis has made it possible to quickly analyze which of these thousands of minute inclusions are potentially dangerous. We show that the enhancement of the local tissue stress caused by particle clustering increases rapidly for gap between particle pairs (h)/particle diameter (D) < 0.4 if particles are oriented along the tensile axis of the cap. Of the thousands of µCalcs observed, there were 193 particle pairs with h/D ≤ 2 (tissue stress factor > 2), but only 3 of these pairs had h/D ≤ 0.4, where the local tissue stress could increase a factor > 5. Using nondecalcified histology, we also show that nearly all caps have µCalcs between 0.5 and 5 µm and that the µCalcs ≥ 5 µm observed in high-resolution microcomputed tomography are agglomerations of smaller calcified matrix vesicles. µCalcs < 5 µm are predicted to be not harmful, because the tiny voids associated with these very small particles will not explosively grow under tensile forces because of their large surface energy. These observations strongly support the hypothesis that nearly all fibrous caps have µCalcs, but only a small subset has the potential for rupture.
Proceedings of the National Academy of Sciences 06/2013; · 9.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Diabetes and low back pain are debilitating diseases and modern epidemics. Diabetes and obesity are also highly correlated with intervertebral disc (IVD) degeneration and back pain. Advanced-glycation-end-products (AGEs) increase reactive-oxygen-species (ROS) and inflammation, and are one cause for early development of diabetes mellitus. We hypothesize that diabetes results in accumulation of AGEs in spines and associated spinal pathology via increased catabolism. We present a mouse model showing that: 1) diabetes induces pathological changes to structure and composition of IVDs and vertebrae; 2) diabetes is associated with accumulation of AGEs, TNFα, and increased catabolism spinal structures; and 3) oral-treatments with a combination of anti-inflammatory and anti-AGE drugs mitigate these diabetes-induced degenerative changes to the spine.
Three age-matched groups of ROP-Os mice were compared: non-diabetic, diabetic (streptozotocin (STZ)-induced), or diabetic mice treated with pentosan-polysulfate (anti-inflammatory) and pyridoxamine (AGE-inhibitor). Mice were euthanized and vertebra-IVD segments were analyzed by μCT, histology and Immunohistochemistry.
Diabetic mice exhibited several pathological changes including loss in IVD height, decreased vertebral bone mass, decreased glycosaminoglycan content and morphologically altered IVDs with focal deposition of tissues highly expressing TNFα, MMP-13 and ADAMTS-5. Accumulation of larger amounts of methylglyoxal suggested that AGE accumulation was associated with these diabetic degenerative changes. However, treatment prevented or reduced these pathological effects on vertebrae and IVD.
This is the first study to demonstrate specific degenerative changes to nucleus pulposus (NP) morphology and their association with AGE accumulation in a diabetic mouse model. Furthermore, this is the first study to demonstrate that oral-treatments can inhibit AGE-induced ROS and inflammation in spinal structures and provide a potential treatment to slow progression of degenerative spine changes in diabetes. Since diabetes, IVD degeneration, and accumulation of AGEs are frequent consequences of aging, early treatments to reduce AGE-induced ROS and Inflammation may have broad public-health implications.
PLoS ONE 05/2013; 8(5):e64302. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: This study examines the effect of TNFα on whole bovine intervertebral discs in organ culture and its association with changes characteristic of intervertebral disc degeneration (IDD) in order to inform future treatments to mitigate the chronic inflammatory state commonly found with painful IDD. Pro-inflammatory cytokines such as TNFα contribute to disc pathology and are implicated in the catabolic phenotype associated with painful IDD. Whole bovine discs were cultured to examine cellular (anabolic/catabolic gene expression, cell viability and senescence using β-galactosidase) and structural (histology and aggrecan degradation) changes in response to TNFα treatment. Control or TNFα cultures were assessed at 7 and 21days; the 21day group also included a recovery group with 7days TNFα followed by 14days in basal media. TNFα induced catabolic and anti-anabolic shifts in the nucleus pulposus (NP) and annulus fibrosus (AF) at 7days and this persisted until 21days however cell viability was not affected. Data indicates that TNFα increased aggrecan degradation products and suggests increased β-galactosidase staining at 21days without any recovery. TNFα treatment of whole bovine discs for 7days induced changes similar to the degeneration processes that occur in human IDD: aggrecan degradation, increased catabolism, pro-inflammatory cytokines and nerve growth factor expression. TNFα significantly reduced anabolism in cultured IVDs and a possible mechanism may be associated with cell senescence. Results therefore suggest that successful treatments must promote anabolism and cell proliferation in addition to limiting inflammation.
Biochemical and Biophysical Research Communications 02/2013; · 2.28 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: STUDY DESIGN:: Cells isolated from moderately and severely degenerated human intervertebral discs (IVDs) cultured in an alginate scaffold. OBJECTIVE:: To compare the regenerative potential of moderately vs. severely degenerated cells using three pro-anabolic stimulants. SUMMARY OF BACKGROUND DATA:: Injection of soluble cell signaling factors has potential to slow the progression of IVD degeneration. While degenerative grade is thought to be an important factor in targeting therapeutic interventions it remains unknown whether cells in severely degenerated IVDs have impaired metabolic functions compared to lesser degenerative levels or if they are primarily influenced by the altered microenvironment. METHODS:: NP cells were cultured in alginate for 21 days and treated with three different pro-anabolic stimulants: a growth factor/anti-inflammatory combination of TGFβ3+Dex, or matricellular proteins CTGF or Link-N. They were assayed for metabolic activity, DNA content, glycosaminoglycan (GAG), and qRT-PCR gene profiling. RESULTS:: Moderately degenerated cells responded to stimulation with increased proliferation, decreased IL-1β, MMP9 and COL1A1 expression, and upregulated HAS1 as compared to severely degenerated cells. TGFβR1 (ALK5) receptors were expressed at greater levels in moderately than severely degenerated cells. TGFβ3+Dex had a notable stimulatory effect on moderately degenerated NP cells with increased anabolic gene expression, and decreased COL1A1 and ADAMTS5 gene expression. Link-N and CTGF had similar responses in all assays, and both treatments up-regulated IL-1β expression and had a more catabolic response than TGFβ3+Dex, particularly in the more severely degenerated group. All groups, including different degenerative grades, produced similar amounts of GAG. CONCLUSION:: Pro-anabolic stimulants alone had limited capacity to overcome the catabolic and pro-inflammatory cytokine expression of severely degenerated NP cells and likely require additional anti-inflammatory treatments. Moderately degenerated NP cells had greater TGFβ receptor 1 expression and better responded to anabolic stimulation.
[Show abstract][Hide abstract] ABSTRACT: The role of microcalcifications (μCalcs) in the biomechanics of vulnerable plaque rupture is examined. Our laboratory previously proposed (Ref. 44), using a very limited tissue sample, that μCalcs embedded in the fibrous cap proper could significantly increase cap instability. This study has been greatly expanded. Ninety-two human coronary arteries containing 62 fibroatheroma were examined using high-resolution microcomputed tomography at 6.7-μm resolution and undecalcified histology with special emphasis on calcified particles <50 μm in diameter. Our results reveal the presence of thousands of μCalcs, the vast majority in lipid pools where they are not dangerous. However, 81 μCalcs were also observed in the fibrous caps of nine of the fibroatheroma. All 81 of these μCalcs were analyzed using three-dimensional finite-element analysis, and the results were used to develop important new clinical criteria for cap stability. These criteria include variation of the Young's modulus of the μCalc and surrounding tissue, μCalc size, and clustering. We found that local tissue stress could be increased fivefold when μCalcs were closely spaced, and the peak circumferential stress in the thinnest nonruptured cap (66 μm) if no μCalcs were present was only 107 kPa, far less than the proposed minimum rupture threshold of 300 kPa. These results and histology suggest that there are numerous μCalcs < 15 μm in the caps, not visible at 6.7-μm resolution, and that our failure to find any nonruptured caps between 30 and 66 μm is a strong indication that many of these caps contained μCalcs.
[Show abstract][Hide abstract] ABSTRACT: Recent developments on high resolution micro computed tomography (μCT) allow imaging of soft tissues in small animal joints. Nevertheless, μCT images cannot distinguish soft tissues from synovial fluid due to their similar mass density, limiting the 3D assessment of soft tissues volume and thickness. This study aimed to evaluate a lead chromate contrast agent for μCΤ arthrography of rat knee joints ex vivo.
Intact tibiofemoral rat joints were injected with the contrast agent at different concentrations and imaged using a μCT at 2.7 μm isotropic voxel size. Cartilage thickness was measured using an automated procedure, validated against histological measurements, and analyzed as a function of μCT image resolution. Changes in hard and soft tissues were also analyzed in tibiofemoral joints 4 weeks after surgical destabilization of the medial meniscus (DMM).
The contrast agent diffused well throughout the whole knee cavity without penetrating the tissues, therefore providing high contrast at the boundaries between soft tissues and synovial fluid space. Thickness analysis of cartilage demonstrated a high similarity between histology and μ-arthrography approaches (R(2) = 0.90). Four weeks after surgical DMM, the development of osteophytes (Oph) and cartilage ulcerations was recognizable with μCT, as well as a slight increase in trabecular bone porosity, and decrease in trabecular thickness.
A lead chromate-based contrast agent allowed discriminating the synovial fluid from soft tissues of intact knee joints, and thus made possible both qualitative and quantitative assessment of hard and soft tissues in both intact and DMM tibiofemoral joints using high resolution μCT.
Osteoarthritis and Cartilage 05/2012; 20(9):1011-9. · 4.26 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Introduction: Stretch injuries in peripheral nerves can cause pain, paralysis, and loss of sensation. Although optimal treatment depends on the degree of injury, it is difficult to determine the severity of induced nerve damage. Methods: The load-deformation curves of rat median nerves were generated from monotonic load-to-failure experiments to determine low, medium, and high strain levels. Additional excised median nerves were then elongated to induce damage at low (4%), medium (10% and 12%), and high (14% and 20%) tensile strains and the resulting structural damage was evaluated using second harmonic generation (SHG) imaging and light microscopy. Results: No substantial structural changes occurred at 4% strain, but higher strain values resulted in disruption of the normal collagen architecture. Conclusions: The results demonstrate a spectrum of structural damage that can be monitored using SHG, a non-destructive imaging modality, and that the pattern of damage may correspond to functional deficit. Muscle Nerve, 2012.
[Show abstract][Hide abstract] ABSTRACT: Osteocyte apoptosis is required to initiate osteoclastic bone resorption following fatigue-induced microdamage in vivo; however, it is unclear whether apoptotic osteocytes also produce the signals that induce osteoclast differentiation. We determined the spatial and temporal patterns of osteocyte apoptosis and expression of pro-osteoclastogenic signaling molecules in vivo. Ulnae from female Sprague-Dawley rats (16-18weeks old) were cyclically loaded to a single fatigue level, and tissues were analyzed 3 and 7days later (prior to the first appearance of osteoclasts). Expression of genes associated with osteoclastogenesis (RANKL, OPG, VEGF) and apoptosis (caspase-3) were assessed by qPCR using RNA isolated from 6mm segments of ulnar mid-diaphysis, with confirmation and spatial localization of gene expression performed by immunohistochemistry. A novel double staining immunohistochemistry method permitted simultaneous localization of apoptotic osteocytes and osteocytes expressing pro-osteoclastogenic signals relative to microdamage sites. Osteocyte staining for caspase-3 and osteoclast regulatory signals exhibited different spatial distributions, with apoptotic (caspase 3-positive) cells highest in the damage region and declining to control levels within several hundred microns of the microdamage focus. Cells expressing RANKL or VEGF peaked between 100 and 300μm from the damage site, then returned to control levels beyond this distance. Conversely, osteocytes in non-fatigued control bones expressed OPG. However, OPG staining was reduced markedly in osteocytes immediately surrounding microdamage. These results demonstrate that while osteocyte apoptosis triggers the bone remodeling response to microdamage, the neighboring non-apoptotic osteocytes are the major source of pro-osteoclastogenic signals. Moreover, both the apoptotic and osteoclast-signaling osteocyte populations are localized in a spatially and temporally restricted pattern consistent with the targeted nature of this remodeling response.
[Show abstract][Hide abstract] ABSTRACT: We characterized the differentiation of rat bone marrow-derived mesenchymal stem cells (BM-MSCs) into tenocyte-like cells in response to bone morphogenetic protein-12 (BMP-12). BM-MSCs were prepared from Sprague-Dawley rats and cultured as monolayers. Recombinant BMP-12 treatment (10 ng/ml) of BM-MSCs for 12 hours in vitro markedly increased expression of the tenocyte lineage markers scleraxis (Scx) and tenomodulin (Tnmd) over 14 days. Treatment with BMP-12 for a further 12-hour period had no additional effect. Colony formation assays revealed that ~80% of treated cells and their progeny were Scx- and Tnmd-positive. BM-MSCs seeded in collagen scaffolds and similarly treated with a single dose of BMP-12 also expressed high levels of Scx and Tnmd, as well as type I collagen and tenascin-c. Furthermore, when the treated BM-MSC-seeded scaffolds were implanted into surgically created tendon defects in vivo, robust formation of tendon-like tissue was observed after 21 days as evidenced by increased cell number, elongation and alignment along the tensile axis, greater matrix deposition and the elevated expression of tendon markers. These results indicate that brief stimulation with BMP-12 in vitro is sufficient to induce BM-MSC differentiation into tenocytes, and that this phenotype is sustained in vivo. This strategy of pretreating BM-MSCs with BMP-12 prior to in vivo transplantation may be useful in MSC-based tendon reconstruction or tissue engineering.
PLoS ONE 03/2011; 6(3):e17531. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Both overuse and disuse of joints up-regulate matrix metalloproteinases (MMPs) in articular cartilage and cause tissue degradation; however, moderate (physiological) loading maintains cartilage integrity. Here, we test whether CBP/p300-interacting transactivator with ED-rich tail 2 (CITED2), a mechanosensitive transcriptional coregulator, mediates this chondroprotective effect of moderate mechanical loading. In vivo, hind-limb immobilization of Sprague-Dawley rats up-regulates MMP-1 and causes rapid, histologically detectable articular cartilage degradation. One hour of daily passive joint motion prevents these changes and up-regulates articular cartilage CITED2. In vitro, moderate (2.5 MPa, 1 Hz) intermittent hydrostatic pressure (IHP) treatment suppresses basal MMP-1 expression and up-regulates CITED2 in human chondrocytes, whereas high IHP (10 MPa) down-regulates CITED2 and increases MMP-1. Competitive binding and transcription assays demonstrate that CITED2 suppresses MMP-1 expression by competing with MMP transactivator, Ets-1 for its coactivator p300. Furthermore, CITED2 up-regulation in vitro requires the p38δ isoform, which is specifically phosphorylated by moderate IHP. Together, these studies identify a novel regulatory pathway involving CITED2 and p38δ, which may be critical for the maintenance of articular cartilage integrity under normal physical activity levels.
The FASEB Journal 01/2011; 25(1):182-91. · 5.48 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Conventional histologic methods provide valuable information regarding the physical nature of damage in fatigue-loaded tendons, limited to thin, two-dimensional sections. We introduce an imaging method that characterizes tendon microstructure three-dimensionally and develop quantitative, spatial measures of damage formation within tendons. Rat patellar tendons were fatigue loaded in vivo to low, moderate, and high damage levels. Tendon microstructure was characterized using multiphoton microscopy by capturing second harmonic generation signals. Image stacks were analyzed using Fourier transform-derived computations to assess frequency-based properties of damage. Results showed 3D microstructure with progressively increased density and variety of damage patterns, characterized by kinked deformations at low, fiber dissociation at moderate, and fiber thinning and out-of-plane discontinuities at high damage levels. Image analysis generated radial distributions of power spectral gradients, establishing a "fingerprint" of tendon damage. Additionally, matrix damage was mapped using local, discretized orientation vectors. The frequency distribution of vector angles, a measure of damage content, differed from one damage level to the next. This study established an objective 3D imaging and analysis method for tendon microstructure, which characterizes directionality and anisotropy of the tendon microstructure and quantitative measures of damage that will advance investigations of the microstructural basis of degradation that precedes overuse injuries.
Annals of Biomedical Engineering 03/2010; 38(5):1741-51. · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Both underloading and overloading of joints can lead to articular cartilage degradation, a process mediated in part by matrix metalloproteinases (MMPs). Here we examine the effects of reduced loading of rat hindlimbs on articular cartilage expression of MMP-3, which not only digests matrix components but also activates other proteolytic enzymes. We show that hindlimb immobilization resulted in elevated MMP-3 mRNA expression at 6h that was sustained throughout the 21day immobilization period. MMP-3 upregulation was higher in the medial condyle than the lateral, and was greatest in the superficial cartilage zone, followed by middle and deep zones. These areas also showed decreases in safranin O staining, consistent with reduced cartilage proteoglycan content, as early as 7days after immobilization. One hour of daily moderate mechanical loading, applied as passive joint motion, reduced the MMP-3 and ADAMTS-5 increases that resulted from immobilization, and also prevented changes in safranin O staining. Intra-articular injections of an MMP-3 inhibitor, N-isobutyl-N-(4-methoxyphenylsulfonyl)-glycylhydroxamic acid (NNGH), dampened the catabolic effects of a 7day immobilization period, indicating a likely requirement for MMP-3 in the regulation of proteoglycan levels through ADAMTS-5. These results suggest that biomechanical forces have the potential to combat cartilage destruction and can be critical in developing effective therapeutic strategies.
Matrix biology: journal of the International Society for Matrix Biology 02/2010; 29(5):420-6. · 3.56 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: This study describes the development and application of a novel rat patellar tendon model of mechanical fatigue for investigating the early in vivo response to tendon subfailure injury. Patellar tendons of adult female Sprague-Dawley rats were fatigue loaded between 1-35N using a custom-designed loading apparatus. Patellar tendons were subjected to Low-, Moderate- or High-level fatigue damage, defined by grip-to-grip strain measurement. Molecular response was compared with that of a laceration-repair injury. Histological analyses showed that progression of tendon fatigue involves formation of localized kinked fiber deformations at Low damage, which increased in density with presence of fiber delaminations at Moderate damage, and fiber angulation and discontinuities at High damage levels. RT-PCR analysis performed at 1- and 3-day post-fatigue showed variable changes in type I, III and V collagen mRNA expression at Low and Moderate damage levels, consistent with clinical findings of tendon pathology and were modest compared with those observed at High damage levels, in which expression of all collagens evaluated were increased markedly. In contrast, only type I collagen expression was elevated at the same time points post-laceration. Findings suggest that cumulative fatigue in tendon invokes a different molecular response than laceration. Further, structural repair may not be initiated until reaching end-stage fatigue life, where the repair response may unable to restore the damaged tendon to its pre-fatigue architecture.
Journal of biomechanics 11/2009; 43(2):274-9. · 2.66 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Osteocyte apoptosis is spatially and temporally linked to bone fatigue-induced microdamage and to subsequent intracortical remodeling. Specifically, osteocytes surrounding fatigue microcracks in bone undergo apoptosis, and those regions containing apoptotic osteocytes co-localize exactly with areas subsequently resorbed by osteoclasts. Here we tested the hypothesis that osteocyte apoptosis is a key controlling step in the activation and/or targeting of osteoclastic resorption after bone fatigue. We carried out in vivo fatigue loading of ulna from 4- to 5-mo-old Sprague-Dawley rats treated with an apoptosis inhibitor (the pan-caspase inhibitor Q-VD-OPh) or with vehicle. Intracortical bone remodeling and osteocyte apoptosis were quantitatively assessed by standard histomorphometric techniques on day 14 after fatigue. Continuous exposure to Q-VD-OPh completely blocked both fatigue-induced apoptosis and the activation of osteoclastic resorption, whereas short-term caspase inhibition during only the first 2 days after fatigue resulted in >50% reductions in both osteocyte apoptosis and bone resorption. These results (1) show that osteocyte apoptosis is necessary to initiate intracortical bone remodeling in response to fatigue microdamage, (2) indicate a possible dose-response relationship between the two processes, and (3) suggest that early apoptotic events after fatigue-induced microdamage may play a substantial role in determining the subsequent course of tissue remodeling.
Journal of bone and mineral research: the official journal of the American Society for Bone and Mineral Research 04/2009; 24(4):597-605. · 6.04 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The mechanical and microstructural bases of tendon fatigue, by which damage accumulates and contributes to degradation, are poorly understood. To investigate the tendon fatigue process, rat flexor digitorum longus tendons were cyclically loaded (1-16 N) until reaching one of three levels of fatigue damage, defined as peak clamp-to-clamp strain magnitudes representing key intervals in the fatigue life: i) Low (6.0%-7.0%); ii) Moderate (8.5%-9.5%); and iii) High (11.0%-12.0%). Stiffness, hysteresis, and clamp-to-clamp strain were assessed diagnostically (by cyclic loading at 1-8 N) before and after fatigue loading and following an unloaded recovery period to identify mechanical parameters as measures of damage. Results showed that tendon clamp-to-clamp strain increased from pre- to post-fatigue loading significantly and progressively with the fatigue damage level (p <or= 0.010). In contrast, changes in both stiffness and hysteresis were significant only at the High fatigue level (p <or= 0.043). Correlative microstructural analyses showed that Low level of fatigue was characterized by isolated, transverse patterns of kinked fiber deformations. At higher fatigue levels, tendons exhibited fiber dissociation and localized ruptures of the fibers. Histomorphometric analysis showed that damage area fraction increased significantly with fatigue level (p <or= 0.048). The current findings characterized the sequential, microstructural events that underlie the tendon fatigue process and indicate that tendon deformation can be used to accurately assess the progression of damage accumulation in tendons.
Journal of Orthopaedic Research 09/2008; 27(2):264-73. · 2.88 Impact Factor