Damien M. Laudier

Osteocyte apoptosis controls activation of intracortical resorption in response to bone fatigue.

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.

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.

A mechanistic analysis of the role of microcalcifications in atherosclerotic plaque stability: potential implications for plaque rupture.

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 Youngs 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.

Revised microcalcification hypothesis for fibrous cap rupture in human coronary arteries

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.

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.

Subrupture Tendon Fatigue Damage

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 ≤ 0.010). In contrast, changes in both stiffness and hysteresis were significant only at the High fatigue level (p ≤ 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 ≤ 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.

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.

A role for TNFα in intervertebral disc degeneration: A non-recoverable catabolic shift

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 21 days; the 21 day group also included a recovery group with 7 days TNFα followed by 14 days in basal media. TNFα induced catabolic and anti-anabolic shifts in the nucleus pulposus (NP) and annulus fibrosus (AF) at 7 days and this persisted until 21 days however cell viability was not affected. Data indicates that TNFα increased aggrecan degradation products and suggests increased β-galactosidase staining at 21 days without any recovery. TNFα treatment of whole bovine discs for 7 days 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.

Long-Term Disuse Osteoporosis Seems Less Sensitive to Bisphosphonate Treatment Than Other Osteoporosis†

We sought to determine whether risedronate can preserve cortical bone mass and mechanical properties during long‐term disuse in dogs, assessed by histomorphometry and biomechanics on metacarpal diaphyses. Risedronate slowed cortical thinning and partially preserved mechanical properties, but it was unable to suppress bone loss to the degree seen in other osteoporoses.

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