Perlecan-Containing Pericellular Matrix Regulates Solute Transport and Mechanosensing Within the Osteocyte Lacunar-Canalicular System

Abstract

The pericellular matrix (PCM), a thin “coating” surrounding nearly all mammalian cells, plays a critical role in many cell-surface phenomena. In osteocytes, the PCM is believed to control both “outside-in” (mechanosensing) and “inside-out” (signaling molecule transport) processes. However, the osteocytic PCM is challenging to study in situ because it is thin (~100nm) and enclosed in mineralized matrix. To this end, we recently developed a novel tracer velocimetry approach that combined fluorescence recovery after photobleaching (FRAP) imaging with hydrodynamic modeling to quantify the osteocytic PCM in young murine bone (Wang et al., J Bone Miner Res. 2013; 28:1075–86). In this study, we applied the technique to older mice expressing or deficient for perlecan/HSPG2, a large heparan-sulfate proteoglycan normally secreted in osteocytic PCM. The objectives were to i) characterize transport within an altered PCM; ii) to test the sensitivity of our approach in detecting the PCM alterations; and iii) to dissect the roles of the PCM in osteocyte mechanosensing. We found that i) solute transport increases in the perlecan-deficient (hypomorphic: Hypo) mice compared with control mice; ii) PCM fiber density decreases with aging and perlecan deficiency; iii) the osteocytes in the Hypo bones are predicted to experience higher shear stress (+34%), but decreased fluid drag force (−35%) under 3N peak tibial loading, and iv) when subjected to tibial loading in a preliminary in vivo experiment, the Hypo mice did not respond to the anabolic stimuli as CTL mice. These findings support the hypothesis that the PCM fibers act as osteocyte’s sensing antennae, regulating load-induced cellular stimulations and thus bone’s sensitivity and in vivo bone adaptation. If this hypothesis is further confirmed, osteocytic PCM could be new targets to develop osteoporosis treatments by modulating bone’s intrinsic sensitivity to mechanical loading and be used to design patient-specific exercise regimens to promote bone formation.

Single molecule force measurements of perlecan/HSPG2: A key component of the osteocyte pericellular matrix

“…This result supports our previous hypothesis that perlecan possesses the physical properties to serve as a transverse tether in the LCS to sense fluid drag caused by mechanical loading [16]. We measured the unstressed end-to-end length of the perlecan core protein (~170 nm) to demonstrate that perlecan was of sufficient size to transverse the annular gap (~100 nm) and tether the osteocyte cell body to the canalicular wall [37].…”

“…In 2011, we confirmed perlecan to be an important component of the osteocyte PCM, where reduced expression of perlecan results in fewer tethering elements within the pericellular space and narrower canalicular channels [15]. Furthermore, we demonstrated a 30% reduction of the PCM fiber density and the lack of anabolic responses to in vivo mechanical loading using a perlecan deficient mouse [16]. Based on these in vivo results and the known properties of perlecan, we hypothesized that the perlecan-containing PCM tethers serve as flow sensors in the bone LCS and the fluid drag forces experienced by the PCM tethers were predicted to be at piconewton levels under physiological loading conditions [16].…”

“…Recent studies demonstrated that the perlecan-rich pericellular matrix (PCM) surrounding chondrocytes in articular cartilage exhibits distinct mechanical properties from the territorial ECM and modulates chondrocytes responses to mechanical stimuli via binding and release of FGF-2 [12–14]. More recently, our group identified the presence of perlecan in another functional interface, i.e., osteocyte lacunar-canalicular system (LCS) and its involvement in osteocyte-mediated mechanosensing in response to load-induced fluid flow [15,16]. …”

“…Furthermore, we demonstrated a 30% reduction of the PCM fiber density and the lack of anabolic responses to in vivo mechanical loading using a perlecan deficient mouse [16]. Based on these in vivo results and the known properties of perlecan, we hypothesized that the perlecan-containing PCM tethers serve as flow sensors in the bone LCS and the fluid drag forces experienced by the PCM tethers were predicted to be at piconewton levels under physiological loading conditions [16]. The question remains as to whether perlecan molecule can withstand the predicted fluid drag forces in the bone LCS.…”

Bone strain magnitude is correlated with bone strain rate in tetrapods: implications for models of mechanotransduction

“…Theoretical mechanotransduction models suggest that fluid flow in this matrix generates high drag on the tethering elements [28,29], which generates radial (hoop) strains in the osteocyte processes' cell membranes [29]. These strains have further been proposed to relate directly to the tissue-level modelling response [30]. In this way, models predict that interstitial fluid flow through a bone's lacunar-canalicular porosity is able to generate more than five times greater strain at the cell membrane than is required to elicit an intracellular response [29].…”