[Show abstract][Hide abstract] ABSTRACT: Mesenchymal stem cells (MSCs) are pluripotent cells that primarily differentiate into osteocytes, chondrocytes, and adipocytes. Recent studies indicate that MSCs can also be induced to generate tenocyte-like cells; moreover, MSCs have been suggested to have great therapeutic potential for tendon pathologies. Yet the precise molecular cascades governing tenogenic differentiation of MSCs remain unclear. We demonstrate scleraxis, a transcription factor critically involved in embryonic tendon development and formation, plays a pivotal role in the fate determination of MSC towards tenocyte differentiation. Using murine C3H10T1/2 pluripotent stem cells as a model system, we show scleraxis is extensively expressed in the early phase of bone morphogenetic protein (BMP)-12-triggered tenocytic differentiation. Once induced, scleraxis directly transactivates tendon lineage-related genes such as tenomodulin and suppresses osteogenic, chondrogenic, and adipogenic capabilities, thus committing C3H10T1/2 cells to differentiate into the specific tenocyte-like lineage, while eliminating plasticity for other lineages. We also reveal that mechanical loading-mediated tenocytic differentiation follows a similar pathway and that BMP-12 and cyclic uniaxial strain act in an additive fashion to augment the maximal response by activating signal transducer Smad8. These results provide critical insights into the determination of multipotent stem cells to the tenocyte lineage induced by both chemical and physical signals.
[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. DOI:10.1371/journal.pone.0017531 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Expression profiling of selected matrix remodeling genes was conducted to evaluate differences in molecular response to low-cycle (100) and high-cycle (7,200) sub-failure-fatigue loading of patellar tendons. Using our previously developed in vivo patellar tendon model, tendons were loaded for 100 or 7,200 cycles and expression of selected metalloproteinases (MMPs), tissue inhibitors of metalloproteinases (TIMPs), and collagens were quantified by real-time RT-PCR at 1- and 7-day post-loading. Expression profiles were also obtained from lacerated tendons as an acute injury model. The high-cycle group showed upregulation of TIMP-1, -2, Col3a1, and Col5a1, and downregulation TIMP-4 at both time points, upregulation of MMP-2 at 7-day post-loading and downregulation of MMP-13 and -14 at 1-day post-loading, suggesting overall repair/remodeling. In contrast, the low-cycle loaded group showed upregulation of MMP-2, -3, -13, and Col12a1 at both time points, upregulation of TIMP-1, -2, -3, Col3a1, and integrin β1 and downregulation of integrin α11 at 1-day post-loading and upregulation of Col1a1 at 7-day post-loading, consistent with a hypertrophic (adaptive) pattern. Lacerated tendons showed a typical acute wound response with upregulation of all examined remodeling genes. Differences found in tendon response to high- and low-cycle loading are suggestive of the underlying mechanisms associated with a healthy or damaging response.
Journal of Orthopaedic Research 10/2010; 28(10):1380-6. DOI:10.1002/jor.21132 · 2.99 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. DOI:10.1016/j.matbio.2010.02.004 · 5.07 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. DOI:10.1016/j.jbiomech.2009.08.039 · 2.75 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Mechanical overloading is a major causative factor of tendinopathy; however, its underlying mechanisms are unclear. We hypothesized mechanical overloading would damage tendons and alter genes associated with tendinopathy in a load-dependent manner. To test this hypothesis, we fatigue loaded rat patellar tendons in vivo and measured expression of the matrix-degrading enzyme MMP-13 and the inflammatory cytokine IL-1beta. We also examined these responses in cultured tenocytes exposed to intermittent hydrostatic pressure in vitro. Additionally, we hypothesized load-induced changes in tenocyte MMP-13 expression would be dependent on expression of IL-1beta. In vivo fatigue loading at 1.7% strain caused overt microstructural damage and upregulated expression of MMP-13 and IL-1beta, while 0.6% strain produced only minor changes in matrix microstructure and downregulated expression of both MMP-13 and IL-1beta. Loading of cultured tenocytes at 2.5 and 7.5 MPa produced comparable changes in expression to those of in vivo tendon loading. Blocking IL-1beta expression with siRNA suppressed load-induced both MMP-13 mRNA expression and activity. The data suggest fatigue loading alters expression of MMP-13 and IL-1beta in tendons in vivo and tenocytes in vitro in a load-dependent manner. The data also suggest MMP-13 is regulated by both IL-1beta-dependent and IL-1beta-independent pathways.
Clinical Orthopaedics and Related Research 08/2008; 466(7):1555-61. DOI:10.1007/s11999-008-0278-4 · 2.77 Impact Factor