Hui B. Sun

Activation of resorption in fatigue-loaded bone involves both apoptosis and active pro-osteoclastogenic signaling by distinct osteocyte populations.

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.

BMP-12 treatment of adult mesenchymal stem cells in vitro augments tendon-like tissue formation and defect repair in vivo.

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.

Tendon‐derived stem/progenitor cell aging: defective self‐renewal and altered fate

Aging is a major risk factor for tendon injury and impaired tendon healing, but the basis for these relationships remains poorly understood. Here we show that rat tendon‐derived stem/progenitor cells (TSPCs) differ in both self‐renewal and differentiation capability with age. The frequency of TSPCs in tendon tissues of aged animals is markedly reduced based on colony formation assays. Proliferation rate is decreased, cell cycle progression is delayed and cell fate patterns are also altered in aged TSPCs. In particular, expression of tendon lineage marker genes is reduced while adipocytic differentiation increased. Cited2, a multi‐stimuli responsive transactivator involved in cell growth and senescence, is also downregulated in aged TSPCs while CD44, a matrix assembling and organizing protein implicated in tendon healing, is upregulated, suggesting that these genes participate in the control of TSPC function.

Early response to tendon fatigue damage accumulation in a novel in vivo model

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.

Osteoblast Responses One Hour After Load-Induced Fluid Flow in a Three-Dimensional Porous Matrix

When bone is loaded, substrate strain is generated by the external force and this strain induces fluid flow that creates fluid shear stress on bone cells. Our current understanding of load-driven gene regulation of osteoblasts is based primarily on in vitro studies on planer two-dimensional tissue culture substrates. However, differences between a flat layer of cells and cells in 3-dimensional (3D) ECM are being recognized for signal transduction. Proliferation and differentiation of osteoblasts are affected by substrate geometry. Here we developed a novel 3D culture system that would mimic physiologically relevant substrate strain as well as strain-induced fluid flow in a 3D porous collagen matrix. The system allowed us to evaluate the responses of osteoblasts in a 3D stress-strain environment similar to a mechanical field to which bone is exposed. Using MC3T3-E1 osteoblasts grown in the 3D collagen matrix with and without hydroxyapatite deposition, we tested the role of strain and the strain-induced fluid flow in the expression of the load-responsive genes such as c-fos, egr1, cox2, osteopontin, and mmp1B involved in transcriptional regulation, osteogenesis, and rearrangement of ECM. Strain-induced fluid flow was visualized with a microspheres ~3 μm in diameter in real time, and three viscoelastic parameters were determined. The results obtained by semi-quantitative PCR, immunoblot assay, enzymatic activity assays for collagenase and gelatinase, and mechanical characterization of collagen matrices supported the dominant role of strain-induced fluid flow in expression of the selected genes one hour after the mechanical treatment.

Physiological loading of joints prevents cartilage degradation through CITED2

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 transacti‐vator with ED‐rich tail 2 (CITED2), a mechanosensitive transcriptional coregulator, mediates this chondropro‐tective 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 chondro‐cytes, 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.—Leong, D. J., Li, Y. H., Gu, X. I., Sun, L., Zhou, Z., Nasser, P., Laudier, D. M., Iqbal, J., Majeska, R. J., Schaffler, M. B., Goldring, M. B., Cardoso, L., Zaidi, M., Sun, H. B. Physiological loading of joints prevents cartilage degradation through CITED2. FASEB J. 25, 182–191 (2011). www.fasebj.org

Mechanical Loading: Bone Remodeling and Cartilage Maintenance

Bone remodeling and cartilage maintenance are strongly influenced by biomechanical signals generated by mechanical loading. Although moderate loading is required to maintain bone mass and cartilage homeostasis, loading can cause deleterious effects such as bone fracture and cartilage degradation. Because a tight coupling exists between cartilage and bone, alterations in one tissue can affect the other. Bone marrow lesions are often associated with an increased risk of developing cartilage defects, and changes in the articular cartilage integrity are linked to remodeling responses in the underlying bone. Although mechanisms regulating the maintenance of these two tissues are different, compelling evidence indicates that the signal pathways crosstalk, particularly with the Wnt pathway. A better understanding of the complex tempero-spatial interplay between bone remodeling and cartilage degeneration will help develop a therapeutic loading strategy that prevents bone loss and cartilage degeneration.

Matrix metalloproteinase-3 in articular cartilage is upregulated by joint immobilization and suppressed by passive joint motion

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.

The Role of Scleraxis in Fate Determination of Mesenchymal Stem Cells for Tenocyte Differentiation

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.

Molecular response of the patellar tendon to fatigue loading explained in the context of the initial induced damage and number of fatigue loading cycles

Accumulation of sub‐rupture fatigue damage has been implicated in the development of tendinopathy. We previously developed an in vivo model of damage accumulation using the rat patellar tendon. Our model allows us to control the input loading parameters to induce fatigue damage in the tendon. Despite this precise control, the resulting induced damage could vary among animals because of differences in size or strength among their patellar tendons. In this study, we used number of applied cycles and initial (day‐0) parameters that are indicative of induced damage to assess the molecular response 7 days after fatigue loading. We hypothesized that day‐0 hysteresis, elongation, and stiffness of the loading and unloading load–displacement curves would be predictive of the 7‐day molecular response. Results showed correlations between the 7‐day molecular response and both day‐0 elongation and unloading stiffness. Additionally, loading resulted in upregulation of several extracellular matrix genes that suggest adaptation; however, several of these genes (Col‐I, ‐XII, MMP 2, and TIMP 3) shut down after a high level of damage was induced. We showed that evaluating the 7‐day molecular profile in light of day‐0 elongation provides important insight that is lost from comparing number of fatigue loading cycles only. Our data showed that loading generally results in an adaptive response. However, the tendons ability to effectively respond deteriorates as greater damage is induced.