Characterization of the Developing Lacunocanalicular Network During Fracture Repair.

Michele Casanova,Lucinda Lee,Aaron Schindeler,Philipp Schneider,David G Little,Ralph Müller,Lauren Peacock

doi:10.1002/jbm4.10525

Abstract

ABSTRACTFracture repair is a normal physiological response to bone injury. During the process of bony callus formation, a lacunocanalicular 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 tibias 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, and terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate‐biotin nick end labeling (TUNEL) staining to examine osteocyte apoptosis. Analysis of μCT scans showed progressive decreases in mean lacuna volume over time (−27% 2–3 weeks; −13% 3–6 weeks). Lacunar number density increased considerably between 2 and 3 weeks (+156%). Correlation analysis was performed, showing a positive linear relationship between canalicular number density and trabecular thickness (R 2 = 0.56, p < 0.001) and an inverse relationship between mean lacuna volume and trabecular thickness (R 2 = 0.57, p < 0.001). Histology showed increases in canalicular number density over time (+22% 2–3 weeks, +51% 3–6 weeks). Lacunar occupancy in new bone of the callus was high (>90%), but the old cortical bone within the fracture site appeared necrotic as it underwent resorption. In conclusion, our data shows a progressive increase in the complexity of the LCN over time during fracture healing and demonstrates that this network is initiated during the early stages of repair. Further studies are needed to address the functional importance of osteocytes in bone healing, particularly in detecting and translating the effects of micromotion in the fracture. © 2021 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Highlights

Osteocytes are bone cells buried within the bone matrix that act as mechanotransducers[1] and orchestrate bone remodeling.[2]. They communicate with each other via the lacunocanalicular network (LCN), which is critical for regulating bone homeostasis.[2]. The LCN has been recently shown to play a major role in the spatial distribution of mass density in bone[3,4] and acts to modulate bone mineral based on paracrine and hormonal factors.[5,6]
Multiple studies have shown that osteocyte number density can be positively correlated with bone biomechanical properties and its resistance to fracture.[7,8,9,10] both lacunar number density and mean lacuna volume are correlated with the propagation of microcracks.[10,11,12] It is established that lacunae can act as force concentrators,(13) and can lead to bone fragility in poorly ordered bone structures.[14] changes in the organization of 1 of 9 n canaliculi have been suggested to affect the mechanical properties of bone.[15,16,17]
The importance of the forming LCN within bone healing has been a subject for recent discussion,(18) and may have a central role in determining callus size.[19,20] Micromotion has been shown to be important for fracture healing, whereas overly rigid fixation can lead to stress shielding and insufficient new bone formation, which may be monitored by the nascent LCN

Summary

Introduction

Osteocytes are bone cells buried within the bone matrix that act as mechanotransducers[1] and orchestrate bone remodeling.[2]. The importance of the forming LCN within bone healing has been a subject for recent discussion,(18) and may have a central role in determining callus size.[19,20] Micromotion has been shown to be important for fracture healing, whereas overly rigid fixation can lead to stress shielding and insufficient new bone formation, which may be monitored by the nascent LCN. In this preclinical study, we aimed to investigate how lacunar measures and canalicular number density evolve during fracture repair. The presence of apoptotic osteocytes could possibly initiate perilacunar osteolysis and localized bone destruction.[22,25]

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