Original Full Length ArticleElevated cross-talk between subchondral bone and cartilage in osteoarthritic joints
Highlights
► Bone-cartilage cross-talk examined in two models (surgical DMM and ageing) of OA. ► Decreased (but not significant) tissue permeability in mineralized ECM of OA joints. ► Increased vessels invasion in aged (+100%) and DMM (+50%) joints vs. controls. ► 60% thinning of thickness in aged joints and no changes in DMM joints vs. controls. ► Bone-cartilage cross-talk elevated in the two OA models via different mechanisms.
Introduction
Osteoarthritis (OA), one of the leading causes of chronic disability in the United States and across the world [1], is a degenerative joint disease characterized by the loss of articular cartilage and abnormal changes in the surrounding soft and hard tissues of the joint, including bone. Currently, no effective treatment is available to cure OA due to its complex etiology and a lack of effective pharmaceutical targets [1]. Although many risk factors such as genetics, age, obesity, and altered joint loading have been identified in OA patients [1], the mechanisms for the initiation and progression of OA are not well understood. There is a growing consensus that OA is a whole-joint disease [2], [3]. In particular, an increased turnover of subchondral bone in OA patients and animal models [4], [5] has led to the hypothesis that the various cytokines and growth factors released during subchondral bone turnover may reach the overlying articular cartilage and initiate a positive feedback loop between the attempted cartilage and bone repair processes that eventually leads to OA progression [6]. Some recent experimental data support this hypothesis. For example, administration of osteoprotegerin prevented not only trabecular bone loss but also cartilage degradation in a surgically induced OA model [7]. The aggrecanase-2 deficient (ADAMTS5−/−) mice, which were found to be protected from cartilage degeneration after joint instability, showed less severe subchondral bone changes compared to wild type controls [8]. At the cellular level, osteoblasts from OA patients induced hypertrophic differentiation and matrix mineralization of normal chondrocytes in vitro [9]. Despite this indirect evidence, direct cross-talk between the subchondral bone and articular cartilage has yet to be established in OA joints.
Recently we demonstrated the potential for cross-talk between subchondral bone and articular cartilage in normal mature joints [10]. By using an advanced imaging approach based on fluorescence loss induced by photobleaching (FLIP), we quantified the permeability of calcified cartilage and the osteochondral interface to a small molecular-weight tracer (sodium fluorescein, mol. wt. 376 Da) in the knee joints of adult mice. Our results, of measurable solute transport across the joints, challenged the long held view that an impermeable calcified cartilage separates the subchondral bone and articular cartilage in adult joints [11], [12], [13]. With OA, alterations in the structural and material properties of joint tissues have been reported [8], [14], [15], [16], [17]. In addition, vascular channels [18], [19] or microcracks [20], [21], which could act as transport conduits, have been reported in OA joints. How the matrix property and structural alterations of the OA joints translate to functional changes in bone–cartilage cross-talk is not clear.
In the present study, we quantified matrix permeability, vessel invasion, and overall joint morphology in two well-established OA models: age-related spontaneous OA [22], and altered loading (surgery) induced OA [23]. Our hypothesis was that the communication potential between subchondral bone and articular cartilage increases in OA. In this study, we first used FLIP to quantify the permeability of the calcified cartilage and the osteochondral interface, the main barrier for transport of molecular signals between the joint tissues (permeability on the order of 0.1–0.5 μm2/s [10], two to three orders of magnitude smaller than those of bone and articular cartilage [24], [25]). We then measured the overall joint morphology and the number density of invading vessels using confocal microscopy. When compared to controls it was observed that OA was not associated with significant changes in tissue matrix permeability in either OA model. However, OA did result in an increase in the blood vessels that invaded (and perforated) the calcified cartilage in both models and also led to a thinning of the subchondral bone and calcified cartilage layers in the aged joints. Together, these results suggested that the capacity for cross-talk between subchondral bone and articular cartilage could be elevated in OA due to morphological alterations in the joint.
Section snippets
Experimental groups
Two murine OA models were utilized within this study. The first was the age-related spontaneous OA model in which C57BL/6J mice (n = 13) were aged until 20 to 24 months of age, at which time mild-to-moderate bi-lateral knee OA is observed as previously described in the literature [22]. The second model was the surgical destabilization of the medical meniscus (DMM) OA model [23]. In this portion of the study, male C57BL/6J mice (n = 14) were subjected to DMM surgery at the age of three-months as
Validation of the OA models
Compared with the normal age controls (n = 6), higher scores were seen in most quadrants of the aged joints (except for lateral tibia, n = 5) and in all four quadrants of the DMM joints (n = 6, * indicates p < 0.05 in Fig. 2). Overall, the damage was mild (OA score less than 1.2 out of a full range of 6) for both the aged and the DMM joints (Fig. 2). The left unoperated joints in the DMM mice displayed negligible cartilage damage similar to the normal age controls (data not shown), and thus were used
Discussion
Recent evidence suggests that subchondral bone may be involved in OA development and that bone and cartilage are functionally coupled through either the distribution of joint loads or by exchanging signaling molecules [2], [15], [17], [27]. However, the transport pathways for the cross-talk between these two tissues are not fully understood. Combining animal models, advanced imaging, and mathematical modeling, the present work demonstrated that the capacity for communicating biochemical signals
Conclusions
This investigation provided quantitative measurements of transport characteristics of the joints from the aged, surgically destabilized, and normal age control mice. Elevated molecular transport was found in the OA joints, and various mechanisms (altered local matrix permeability, vessel invasion, and thinning of the transport resistant layers) were identified. Our data support further studies on subchondral bone, its role in OA development, and potential for OA treatments.
Acknowledgments
We thank Dr. Sonya Glasson for demonstrating the DMM surgery, Dr. Weidong (William) Yang for initiating the DMM operations, Mr. Frank Warren for animal care, and Dr. Chaoying Ni for providing the cryostat. Author contributions are the following: hypothesis formulation (LW), experimental design (LW, JP), microsurgery (WL), data collection (JP, BW,TS, YY, and CP), data analysis (JP, BW, ZZ, CP, and LW), data interpretation (LW, JP, and CP), and manuscript preparation and revision (LW, JP, CP, and
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