Mechanosensitive miR-100 coordinates TGFß and Wnt signaling in osteocytes during fluid shear stress.

Organism scale mechanical forces elicit cellular scale changes through coordinated regulation of multiple signaling pathways. The mechanisms by which cells integrate signaling to generate a unified biological response remains a major question in mechanobiology. For example, the mechanosensitive response of bone and other tissues requires coordinated signaling by the transforming growth factor beta (TGFß) and Wnt pathways through mechanisms that are not well-defined. Here we report a new microRNA-dependent mechanism that mediates mechanosensitive crosstalk between TGFß and Wnt signaling in osteocytes exposed to fluid shear stress (FSS). From 60 mechanosensitive microRNA (miRs) identified by small-RNAseq, miR100 expression is suppressed by in vivo hindlimb loading in the murine tibia and by cellular scale FSS in OCY454 cells. Though FSS activates both TGFß and Wnt signaling in osteocytes, only TGFß represses miR-100 expression. miR-100, in turn, antagonizes Wnt signaling by targeting and inhibiting expression of Frizzled receptors (FZD5/FZD8). Accordingly, miR-100 inhibition blunts FSS- and TGFß-inducible Wnt signaling. Therefore, our results identify FSS-responsive miRNAs in osteocytes, including one that integrates the mechanosensitive function of two essential signaling pathways in the osteoanabolic response of bone to mechanical load.

Fluid shear stress generates a unique signaling response by activating multiple TGFß family type I receptors in osteocytes.

Bone is a dynamic tissue that constantly adapts to changing mechanical demands. The transforming growth factor beta (TGFß) signaling pathway plays several important roles in maintaining skeletal homeostasis by both coupling the bone-forming and bone-resorbing activities of osteoblasts and osteoclasts and by playing a causal role in the anabolic response of bone to applied loads. However, the extent to which the TGFß signaling pathway in osteocytes is directly regulated by fluid shear stress (FSS) is unknown, despite work suggesting that fluid flow along canaliculi is a dominant physical cue sensed by osteocytes following bone compression. To investigate the effects of FSS on TGFß signaling in osteocytes, we stimulated osteocytic OCY454 cells cultured within a microfluidic platform with FSS. We find that FSS rapidly upregulates Smad2/3 phosphorylation and TGFß target gene expression, even in the absence of added TGFß. Indeed, relative to treatment with TGFß, FSS induced a larger increase in levels of pSmad2/3 and Serpine1 that persisted even in the presence of a TGFß receptor type I inhibitor. Our results show that FSS stimulation rapidly induces phosphorylation of multiple TGFß family R-Smads by stimulating multimerization and concurrently activating several TGFß and BMP type I receptors, in a manner that requires the activity of the corresponding ligand. While the individual roles of the TGFß and BMP signaling pathways in bone mechanotransduction remain unclear, these results implicate that FSS activates both pathways to generate a downstream response that differs from that achieved by either ligand alone.

Osteocyte-Intrinsic TGF-ß Signaling Regulates Bone Quality through Perilacunar/Canalicular Remodeling.

Poor bone quality contributes to bone fragility in diabetes, aging, and osteogenesis imperfecta. However, the mechanisms controlling bone quality are not well understood, contributing to the current lack of strategies to diagnose or treat bone quality deficits. Transforming growth factor beta (TGF-ß) signaling is a crucial mechanism known to regulate the material quality of bone, but its cellular target in this regulation is unknown. Studies showing that osteocytes directly remodel their perilacunar/canalicular matrix led us to hypothesize that TGF-ß controls bone quality through perilacunar/canalicular remodeling (PLR). Using inhibitors and mice with an osteocyte-intrinsic defect in TGF-ß signaling (TßRIIocy-/-), we show that TGF-ß regulates PLR in a cell-intrinsic manner to control bone quality. Altogether, this study emphasizes that osteocytes are key in executing the biological control of bone quality through PLR, thereby highlighting the fundamental role of osteocyte-mediated PLR in bone homeostasis and fragility.

Phage-Based Structural Color Sensors and Their Pattern Recognition Sensing System.

The mammalian olfactory system provides great inspiration for the design of intelligent sensors. To this end, we have developed a bioinspired phage nanostructure-based color sensor array and a smartphone-based sensing network system. Using a M13 bacteriophage (phage) as a basic building block, we created structural color matrices that are composed of liquid-crystalline bundled nanofibers from self-assembled phages. The phages were engineered to express cross-responsive receptors on their major coat protein (pVIII), leading to rapid, detectable color changes upon exposure to various target chemicals, resulting in chemical- and concentration-dependent color fingerprints. Using these sensors, we have successfully detected 5-90% relative humidity with 0.2% sensitivity. In addition, after modification with aromatic receptors, we were able to distinguish between various structurally similar toxic chemicals including benzene, toluene, xylene, and aniline. Furthermore, we have developed a method of interpreting and disseminating results from these sensors using smartphones to establish a wireless system. Our phage-based sensor system has the potential to be very useful in improving national security and monitoring the environment and human health.

Mechanobiology of TGFß signaling in the skeleton.

Physical and biochemical cues play fundamental roles in the skeleton at both the tissue and cellular levels. The precise coordination of these cues is essential for skeletal development and homeostasis, and disruption of this coordination can drive disease progression. The growth factor TGFß is involved in both the regulation of and cellular response to the physical microenvironment. It is essential to summarize the current findings regarding the mechanisms by which skeletal cells integrate physical and biochemical cues so that we can identify and address remaining gaps that could ultimately improve skeletal health. In this review, we describe the role of TGFß in mechanobiological signaling in bone and cartilage at the tissue and cellular levels. We provide detail on how static and dynamic physical cues at the macro-level are transmitted to the micro-level, ultimately leading to regulation at each level of the TGFß pathway and to cell differentiation. The continued integration of engineering and biological approaches is needed to answer many remaining questions, such as the mechanisms by which cells generate a coordinated response to physical and biochemical cues. We propose one such mechanism, through which the combination of TGFß and an optimal physical microenvironment leads to synergistic induction of downstream TGFß signaling.

Discrete spatial organization of TGFß receptors couples receptor multimerization and signaling to cellular tension.

Cell surface receptors are central to the cell's ability to generate coordinated responses to the multitude of biochemical and physical cues in the microenvironment. However, the mechanisms by which receptors enable this concerted cellular response remain unclear. To investigate the effect of cellular tension on cell surface receptors, we combined novel high-resolution imaging and single particle tracking with established biochemical assays to examine TGFß signaling. We find that TGFß receptors are discretely organized to segregated spatial domains at the cell surface. Integrin-rich focal adhesions organize TßRII around TßRI, limiting the integration of TßRII while sequestering TßRI at these sites. Disruption of cellular tension leads to a collapse of this spatial organization and drives formation of heteromeric TßRI/TßRII complexes and Smad activation. This work details a novel mechanism by which cellular tension regulates TGFß receptor organization, multimerization, and function, providing new insight into the mechanisms that integrate biochemical and physical cues.

Inkjet printed antibiotic- and calcium-eluting bioresorbable nanocomposite micropatterns for orthopedic implants.

Inkjet printing of antibiotic- and calcium-eluting micropatterns was explored as a novel means of preventing the formation of biofilm colonies and facilitating osteogenic cell development on orthopedic implant surfaces. The micropatterns consisted of a periodic array of ∼50 µm circular dots separated by ∼150 µm. The composition of the micropatterns was controlled by formulating inks with rifampicin (RFP) and poly(D,L-lactic-co-glycolic) acid (PLGA) dissolved in an organic solvent with ∼100 nm biphasic calcium phosphate (BCP) nanoparticles suspended in the solution. During printing RFP and PLGA co-precipitated to form a nanocomposite structure with ∼10-100 nm RFP and the BCP particles dispersed in the PLGA matrix. The rate of RFP release was strongly influenced by the RFP loading in the micropattern, particularly on the first day. The RFP-containing micropatterns effectively prevented the formation of Staphylococcus epidermidis biofilm colonies due to their ability to kill bacteria prior to forming colonies on the patterned surfaces. The BCP-containing micropatterns printed on the surface of the alloy TiAl6V4 significantly accelerated osteoblast cell differentiation, as measured by alkaline phosphatase expression and calcium deposition, without compromising cell proliferation.