Lacunocanalicular Flow in Osseous Repair and Regeneration

Introduction: Understanding of the molecular events leading to osseous regeneration in response to mechanical tension is fundamental to evolution of therapeutic interventions designed to accelerate distraction osteogenesis (DO) and the development of tissue-engineering strategies. Our hypothesis is that the tension stress of activation increases lacunocanalicular flow and upregulates the mechanotransductive-osteogenic pathway. Furthermore, we hypothesize improving vascularization by endothelial progenitor cell (EPC) mobilization enhances lacunocanalicular flow in the consolidation period, maintaining upregulation of the mechanotransductive pathway to accelerate osteogenesis. We develop strategies to manipulate this mechanism to recapitulate lacunocanalicular flow and optimize the biomimicry of tissue-engineered implants.

Methods: Sprague-Dawley rats were subjected to mandibular osteotomy alone or distraction. Subjects were injected with fluorescent reactive red lacunocanalicular tracer. FAK (cascade regulator), SUN, lamin, nesprins (cytoskeletal mediators), sox 9, osterix, runx2, BMP 2,4, Smads 1,5,8 (osteogenic markers) were measured with western blotting. Additional animals underwent latency, activation and consolidation, receiving daily AMD3100 (EPC mobilizer) or saline. Reactive red tracing and western blotting for mechanotransductive proteins were performed. Bone regeneration was assessed (µ-CT, DEXA, immunohistochemistry, mechanical testing). In ongoing investigations, we develop a novel vacuum-assisted lacunocanalicular flow system to increase lacunocanalicular flow through a cell-seeded matrix and upregulate the mechanotransductive-osteogenic pathway to create an implant that fully recapitulates native bone.

Results: Fluorescent intensity was 6.5±1.4% following mechanical activation vs 1.6±0.4% following osteotomy. Mechanotransductive mediators in the activated group were upregulated eg FAK (13.9x105 vs 8.9x105), SUN (12.1x105 vs 6.6x105)etc Fluorescent intensity was 1.9% with AMD3100 vs 0.1% with saline. Upregulation of mechanotransductive mediators was seen with EPC mobilization eg FAK (3.9x106 vs 1.4x106), SUN (3.7x106 vs 1.2x106), Nesprin (11.4x105 vs 6.5x105)etc. DEXA, µ-CT and immunohistochemistry demonstrated AMD3100 treatment increased bone formation (8.93 vs 3.94%). Greater force was required to break AMD3100-supplemented bone (98 vs. 60N).

Conclusion: This is the first description of lacunocanalicular flow as the transducer of mechanical tension stress to osteogenesis in distraction and introduces a novel mechanism to accelerate osteogenesis. This may provide new therapies to allow DO to be used situations known to have impaired bony healing, and lead to more rapid distraction protocols. Furthermore this mechanism may be manipulated to improve efficacy of bioenigneered implants, to enhance bony healing and prevent bone loss in low gravity, and may explain high bone formation in highly vascularized tumours.