Limbostomy: Longitudinal Intravital Microendoscopy in Murine Osteotomies.

Bone healing involves the interplay of immune cells, mesenchymal cells, and vasculature over the time course of regeneration. Approaches to quantify the spatiotemporal aspects of bone healing at cellular resolution during long bone healing do not yet exist. Here, a novel technique termed Limbostomy is presented, which combines intravital microendoscopy with an osteotomy. This design allows a modular combination of an internal fixator plate with a gradient refractive index (GRIN) lens at various depths in the bone marrow and can be combined with a surgical osteotomy procedure. The field of view (FOV) covers a significant area of the fracture gap and allows monitoring cellular processes in vivo. The GRIN lens causes intrinsic optical aberrations which have to be corrected. The optical system was characterized and a postprocessing algorithm was developed. It corrects for wave front aberration-induced image plane deformation and for background and noise signals, enabling us to observe subcellular processes. Exemplarily, we quantitatively and qualitatively analyze angiogenesis in bone regeneration. We make use of a transgenic reporter mouse strain with nucleargreen fluorescent protein and membrane-bound tdTomato under the Cadherin-5 promoter. We observe two phases of vascularization. First, rapid vessel sprouting pervades the FOV within 3-4 days after osteotomy. Second, the vessel network continues to be dynamically remodeled until the end of our observation time, 14 days after surgery. Limbostomy opens a unique set of opportunities and allows further insight on spatiotemporal aspects of bone marrow biology, for example, hematopoiesis, analysis of cellular niches, immunological memory, and vascularization in the bone marrow during health and disease. © 2020 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry.

Longitudinal intravital imaging of the femoral bone marrow reveals plasticity within marrow vasculature.

The bone marrow is a central organ of the immune system, which hosts complex interactions of bone and immune compartments critical for hematopoiesis, immunological memory, and bone regeneration. Although these processes take place over months, most existing imaging techniques allow us to follow snapshots of only a few hours, at subcellular resolution. Here, we develop a microendoscopic multi-photon imaging approach called LIMB (longitudinal intravital imaging of the bone marrow) to analyze cellular dynamics within the deep marrow. The approach consists of a biocompatible plate surgically fixated to the mouse femur containing a gradient refractive index lens. This microendoscope allows highly resolved imaging, repeatedly at the same regions within marrow tissue, over months. LIMB reveals extensive vascular plasticity during bone healing and steady-state homeostasis. To our knowledge, this vascular plasticity is unique among mammalian tissues, and we expect this insight will decisively change our understanding of essential phenomena occurring within the bone marrow.

Biomechanical comparison of pin and tension-band wire fixation with a prototype locking plate fixation in a transverse canine patellar fracture model.

OBJECTIVE: To compare a locking plate (LP) with pin and tension-band wire (pin/TBW) for fixation of mid-patellar transverse fractures. MATERIALS AND METHODS: Cadaveric canine stifle joints from 10 adult mixed breed dogs (23-36 kg) were used. Mid-patellar transverse osteotomies were randomly stabilized (in pairs) with either pin/TBW or a prototype LP. Cyclic loads (1 Hz, 500 cycles) at 100% body weight (90°-135° stifle joint extension), were applied. Survival or failure of constructs was defined as <2 mm fracture gap distraction at 500 cycles, or ≥2 mm fracture gap distraction at the number of cycles sustained, respectively. Number of cycles at failure and distraction gap were compared with a paired Student's t-test, and a survival analysis performed with a Mantel-Cox test. All constructs that survived cyclic testing were tested in single cycle load to failure (1.0 mm/sec; 110° stifle joint extension); yield strength was compared with a Wilcoxon rank sum test. Significance was set at p <0.05. RESULTS: All 10/10 LP and three out of 10 pin/TBW fixations survived cyclic testing. Survival analysis, number of cycles at failure, and distraction gap all were significantly different between the two groups (p = 0.0011, p = 0.0013, and p <0.0001, respectively). Construct yield strength was not significantly different (p = 0.1273). CONCLUSIONS: The failure mode with pin/TBW was consistently similar to failures observed clinically. The LP demonstrated consistent, reliable and stable fixation.