Aging impairs the osteocytic regulation of collagen integrity and bone quality

Poor bone quality is a major factor in skeletal fragility in elderly individuals.The molecular mechanisms that establish and maintain bone quality,independent of bone mass,are unknown but are thought to be primarily determined by osteocytes.We hypothesize that the age-related decline in bone quality results from the suppression of osteocyte perilacunar/canalicular remodeling(PLR),which maintains bone material properties.We examined bones from young and aged mice with osteocyte-intrinsic repression of TGFβsignaling(TβRII^(ocy−/−))that suppresses PLR.The control aged bone displayed decreased TGFβsignaling and PLR,but aging did not worsen the existing PLR suppression in male TβRII^(ocy−/−)bone.This relationship impacted the behavior of collagen material at the nanoscale and tissue scale in macromechanical tests.The effects of age on bone mass,density,and mineral material behavior were independent of osteocytic TGFβ.We determined that the decline in bone quality with age arises from the loss of osteocyte function and the loss of TGFβ-dependent maintenance of collagen integrity.

Osteocyte dysfunction promotes osteoarthritis through MMP13-dependent suppression of subchondral bone homeostasis

Osteoarthritis(OA),long considered a primary disorder of articular cartilage,is commonly associated with subchondral bone sclerosis.However,the cellular mechanisms responsible for changes to subchondral bone in OA,and the extent to which these changes are drivers of or a secondary reaction to cartilage degeneration,remain unclear.In knee joints from human patients with end-stage OA,we found evidence of profound defects in osteocyte function.Suppression of osteocyte perilacunar/canalicular remodeling(PLR)was most severe in the medial compartment of OA subchondral bone,with lower protease expression,diminished canalicular networks,and disorganized and hypermineralized extracellular matrix.As a step toward evaluating the causality of PLR suppression in OA,we ablated the PLR enzyme MMP13 in osteocytes while leaving chondrocytic MMP13 intact,using Cre recombinase driven by the 9.6-kb DMP1 promoter.Not only did osteocytic MMP13 deficiency suppress PLR in cortical and subchondral bone,but it also compromised cartilage.Even in the absence of injury,osteocytic MMP13 deficiency was sufficient to reduce cartilage proteoglycan content,change chondrocyte production of collagen II,aggrecan,and MMP13,and increase the incidence of cartilage lesions,consistent with early OA.Thus,in humans and mice,defects in PLR coincide with cartilage defects.Osteocyte-derived MMP13 emerges as a critical regulator of cartilage homeostasis,likely via its effects on PLR.Together,these findings implicate osteocytes in bone-cartilage crosstalk in the joint and suggest a causal role for suppressed perilacunar/canalicular remodeling in osteoarthritis.

Miniscope3D:optimized single-shot miniature 3D fluorescence microscopy

Miniature fluorescence microscopes are a standard tool in systems biology.However,widefield miniature microscopes capture only 2D information,and modifications that enable 3D capabilities increase the size and weight and have poor resolution outside a narrow depth range.Here,we achieve the 3D capability by replacing the tube lens of a conventional 2D Miniscope with an optimized multifocal phase mask at the objective’s aperture stop.Placing the phase mask at the aperture stop significantly reduces the size of the device,and varying the focal lengths enables a uniform resolution across a wide depth range.The phase mask encodes the 3D fluorescence intensity into a single 2D measurement,and the 3D volume is recovered by solving a sparsity-constrained inverse problem.We provide methods for designing and fabricating the phase mask and an efficient forward model that accounts for the fieldvarying aberrations in miniature objectives.We demonstrate a prototype that is 17mm tall and weighs 2.5 grams,achieving 2.76μm lateral,and 15μm axial resolution across most of the 900×700×390μm^(3) volume at 40 volumes per second.The performance is validated experimentally on resolution targets,dynamic biological samples,and mouse brain tissue.Compared with existing miniature single-shot volume-capture implementations,our system is smaller and lighter and achieves a more than 2×better lateral and axial resolution throughout a 10×larger usable depth range.Our microscope design provides single-shot 3D imaging for applications where a compact platform matters,such as volumetric neural imaging in freely moving animals and 3D motion studies of dynamic samples in incubators and lab-on-a-chip devices.

A micromirror array with annular partitioning for high-speed random-access axial focusing

Dynamic axial focusing functionality has recently experienced widespread incorporation in microscopy,augmented/virtual reality(AR/VR),adaptive optics and material processing.However,the limitations of existing varifocal tools continue to beset the performance capabilities and operating overhead of the optical systems that mobilize such functionality.The varifocal tools that are the least burdensome to operate(e.g.liquid crystal,elastomeric or optofluidic lenses)suffer from low(≈100 Hz)refresh rates.Conversely,the fastest devices sacrifice either critical capabilities such as their dwelling capacity(e.g.acoustic gradient lenses or monolithic micromechanical mirrors)or low operating overhead(e.g.deformable mirrors).Here,we present a general-purpose random-access axial focusing device that bridges these previously conflicting features of high speed,dwelling capacity and lightweight drive by employing lowrigidity micromirrors that exploit the robustness of defocusing phase profiles.Geometrically,the device consists of an 8.2mm diameter array of piston-motion and 48-μm-pitch micromirror pixels that provide 2πphase shifting for wavelengths shorter than 1100 nm with 10-90% settling in 64.8μs(i.e.,15.44 kHz refresh rate).The pixels are electrically partitioned into 32 rings for a driving scheme that enables phase-wrapped operation with circular symmetry and requires<30 V per channel.Optical experiments demonstrated the array’s wide focusing range with a measured ability to target 29 distinct resolvable depth planes.Overall,the features of the proposed array offer the potential for compact,straightforward methods of tackling bottlenecked applications,including high-throughput single-cell targeting in neurobiology and the delivery of dense 3D visual information in AR/VR.

Subcellular western blotting of single cells

Although immunoassays are the de facto standard for determining subcellular protein localization in individual cells,antibody probe cross-reactivity and fixation artifacts remain confounding factors.To enhance selectivity while providing single-cell resolution,we introduce a subcellular western blotting technique capable of separately assaying proteins in the 14 pL cytoplasm and 2 pL nucleus of individual cells.To confer precision fluidic control,we describe a passive multilayer microdevice that leverages the rapid transport times afforded by miniaturization.After isolating single cells in microwells,we apply single-cell differential detergent fractionation to lyse and western blot the cytoplasmic lysate,whereas the nucleus remains intact in the microwell.Subsequently,we lyse the intact nucleus and western blot the nuclear lysate.To index each protein analysis to the originating subcellular compartment,we utilize bi-directional electrophoresis,a multidimensional separation that assays the lysate from each compartment in a distinct region of the separation axis.Single-cell bi-directional electrophoresis eliminates the need for semi-subjective image segmentation algorithms required in immunocytochemistry.The subcellular,single-cell western blot is demonstrated for six targets per cell,and successfully localizes spliceosome-associated proteins solubilized from large protein and RNA complexes,even for closely sized proteins(a 7 kDa difference).Measurement of NF-κB translocation dynamics in unfixed cells at 15-min intervals demonstrates reduced technical variance compared with immunofluorescence.This chemical cytometry assay directly measures the nucleocytoplasmic protein distribution in individual unfixed cells,thus providing insight into protein signaling in heterogeneous cell populations.