-
Daniel J Leong,
Yong H Li,
Xiang I Gu,
Li Sun,
Zuping Zhou,
Philip Nasser, Damien M Laudier,
Jameel Iqbal,
Robert J Majeska,
Mitchell B Schaffler,
Mary B Goldring,
Luis Cardoso,
Mone Zaidi,
Hui B Sun
-
[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.
Muscle & Nerve 05/2012; · 2.37 Impact Factor
-
[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.
Bone 02/2012; 50(5):1115-22. · 4.02 Impact Factor
-
Jonathan Y Lee,
Zuping Zhou,
Peter J Taub,
Melissa Ramcharan,
Yonghui Li,
Takintope Akinbiyi,
Edward R Maharam,
Daniel J Leong, Damien M Laudier,
Takuya Ruike,
Phillip J Torina,
Mone Zaidi,
Robert J Majeska,
Mitchell B Schaffler,
Evan L Flatow,
Hui B Sun
[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 01/2011; 6(3):e17531. · 4.09 Impact Factor
-
Daniel J Leong,
Yong H Li,
Xiang I Gu,
Li Sun,
Zuping Zhou,
Philip Nasser, Damien M Laudier,
Jameel Iqbal,
Robert J Majeska,
Mitchell B Schaffler,
Mary B Goldring,
Luis Cardoso,
Mone Zaidi,
Hui B Sun
[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. · 5.71 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. · 2.41 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: 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. · 2.81 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Disuse induces rapid and severe bone loss in larger mammals as a result of greatly elevated osteoclastic resorption. In this study, we tested whether risedronate (RIS), a potent inhibitor of osteoclastic activity, would effectively prevent cancellous bone loss in female beagles (5-7 years old, N = 28) subjected to single forelimb immobilization (IM) for 12 months. Age-matched, non-IM dogs served as controls (Con). Half the animals from each group received RIS 1 mg/kg p.o. daily (Con + RIS, IM + RIS). Remaining dogs received sterile water (Con, IM). Histomorphometry showed that IM caused a dramatic reduction in cancellous bone mass (-71%) of distal 2nd metacarpals, characterized by marked decreases in trabecular width (-51%) and number (-41%), and 4-fold increases in the indices of bone resorption (eroded surface, osteoclast number, and surface). Bone formation indices (calcein-labeled surface, osteoid surface, and bone formation rate) were also significantly higher in IM than in controls. Activation frequency in IM increased about 4-fold beyond control level. RIS treatment reduced, but did not abolish cancellous bone loss due to immobilization. IM animals treated with RIS lost nearly 50% of cancellous bone mass, while trabecular width and number were reduced by 31% and 25%, respectively. In both RIS-treated control and IM animals, overall bone formation parameters (mineralized bone surface fraction and bone formation rate) remained roughly at intact control levels; however, mineral apposition rate relative to intact control was reduced 40% in RIS-treated control and 86% in RIS-treated IM animals. These results indicate that high-dose RIS treatment might suppress osteoblastic function, especially under long-term disuse. Interestingly, bone resorption parameters in RIS-treated IM animals reached levels even higher than in vehicle-treated IM animals; values for eroded surface, osteoclast number, and surface were 84%, 53%, and 83% above vehicle-treated IM values, respectively. Our data indicate that risedronate treatment is partially effective in preventing cancellous bone loss during long-term disuse. Moreover, our results suggest that bisphosphonates can impair the ability of mature osteoclasts to resorb bone, but cannot overcome the strong stimulus for osteoclast recruitment caused by long-term disuse.
Bone 10/2005; 37(3):287-95. · 4.02 Impact Factor
