Characterization of the Developing Lacunocanalicular Network During Fracture Repair

Abstract

Fracture repair is a normal physiological response to bone injury. During the process of bony callus formation, a lacuno-canalicular network (LCN) is formed de novo that evolves with callus remodeling. Our aim was the longitudinal assessment of the development and evolution of the LCN during fracture repair. To this end, 45 adult wild type C57BL/6 mice underwent closed tibial fracture surgery. Fractured and intact contralateral tibiae were harvested after 2, 3, and 6 weeks of bone healing (n=15/group). High-resolution micro-computed tomography (µCT) and deconvolution microscopy (DV) approaches were applied to quantify lacunar number density from the calluses and intact bone. On histological sections, Goldner's trichrome staining was used to assess lacunar occupancy, fluorescein isothiocyanate staining to visualize the canalicular network, This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as

Individualized cyclic mechanical loading improves callus properties during the remodelling phase of fracture healing in mice as assessed from time-lapsed in vivo imaging

“…In the in vivo setting osteocytes reside in lacunae within the lacunocanalicular network (LCN). A recent study by Casanova et al 42 longitudinally assessed the development and evolution of the LCN during fracture healing indicating a progressive increase in the complexity of the LCN, which they attributed to factors expressed by osteocytes such as matrix metalloproteinases, Sclerostin and RANKL. To capture potential underlying molecular targets of the spatio-temporal loading effects seen in the current study, we performed immunohistochemistry of Sclerostin (inhibitor of the mechano-responsive and osteoanabolic Wnt signalling pathway) and RANKL (negatively-regulated target gene of Wnt signalling) which have both previously been associated with the mechanical regulation of bone adaptation and healing 16 , 29 – 33 .…”

Detailing the influence of PEO-coated biodegradable Mg-based implants on the lacuno-canalicular network in sheep bone: A pilot study

“…As a result, bone remodelled around the PEO-coated material might be more resistant to micro-damage and micro-cracks as investigated by Liu et al [ 34 ]. Furthermore, smooth ellipse lacunar shapes were determined to be more prominent in mature bone post-operations [ 60 ]. With regards to the influence of time, both coated and uncoated bone samples near the implant begin to increase in smoothness as time persists.…”

“…In conjunction, the Gain Factor of bone further from the implant was determined to be lower than bone located closer to the implant surface. Casanova et al have described a progressive increase in connectivity of the LCN during fracture healing in mice during early stages of repair [ 60 ], which does not seem to be present at the bone samples further from the implant surface. The lower transportation efficiency and network connectivity observed in distant bone analysed in this investigation may be inherent to undisturbed LCN.…”

Contributions of Resin Cast Etching to Visualising the Osteocyte Lacuno-Canalicular Network Architecture in Bone Biology and Tissue Engineering

“…Fluorescent dyes [126] Excellent lateral (x-y plane) resolution Poor axial (z-direction) resolution. Need for fluorescent staining .…”

“…Metal implant (if present) must be removed [ 106 – 110 ] FIB lift-out [ 75 , 111 , 112 ] Site-specific sample preparation Nanoscale characterisation of intact bone-biomaterial interface Very small areas analysed (~ 20 × ~ 8 μm 2 ) CLSM Fluorescent dyes [ 16 , 36 , 115 – 117 ] Volumetric/depth imaging Photobleaching Immunostaining [ 118 – 122 ] Volumetric/depth imaging. Labelling of cytoskeleton, cell membrane, nucleus, gap junctions Photobleaching Multiphoton microscopy 3PEF [ 115 , 125 ] Intravital imaging of osteocytes [ 123 ] Staining with fluorescent dyes Photobleaching THG [ 115 , 125 ] Label-free imaging of porosity (osteocytes, canaliculi, blood vessels) Resin embedding reduces the contrast of canaliculi Deconvolution microscopy Fluorescent dyes [ 126 ] Excellent lateral (x–y plane) resolution Poor axial (z-direction) resolution. Need for fluorescent staining .…”