Poster AA2 - Temporal And Spatial Characterization Of Critical Sized Bone Defects In A Rat Femoral Defect Model

Introduction: Critical Size Defects (CSDs) are characterized by being unable to heal on their own accord without the aid of a bone graft or an adequate substitute, even after surgical stabilization. Defect size and wound environment are unquestionably factors that contribute to failure to regenerate. Yet, little is known about the mechanisms inhibited in CSDs resulting in the impairment of bone’s innate ability to regenerate itself. Efforts to comprehend the underlying etiology of CSDs and why defects that go beyond a certain size disrupt native regenerative responses are lacking. Thus, we aim to address this gap in knowledge by investigating and tracking the temporal and spatial changes in morphology, cell presence, and bone growth in a 6 mm critical-sized femoral defect rat model.

Materials and

Methods: Animal studies: All animal studies were conducted in compliance with the Animal Welfare Act and per the principles of the Guide for the Care and Use of Laboratory Animals. Briefly, 8 skeletally mature Lewis rats (Envigo), 4 males and 4 females, were put under general anesthesia. 6 mm critical sized incisions were inflicted on the right femur and mechanically stabilized with a plastic PEEK fixator and k-wires. 4 rats were euthanized at 1 and 3 weeks and the affected femora excised.

Histology: Collected femora were fixed using 4% formaldehyde, dehydrated, embedded in paraffin, sliced into 6 μm sections, and adhered to glass slides. The sections were stained with Movat Pentachrome stain to observe changes in bone regeneration and morphology. Slides were imaged at 40x (Motic Easy Scan) and % area of cartilage, fibrin, hypertrophic chondrocytes, and bone were determined using the Trainable Weka Segmentation plug-in on ImageJ software. Each tissue section was vertically segmented into 6 um segments and analyzed individually for area of tissues of interest.

Results, Conclusions, and Discussions: Pentachrome staining of the tissues at 1 and 3 weeks demonstrated changes in morphology, as observed by representative images in Figure 1A. These morphological changes were segmented and quantified into % area of total tissue. At 1 week post-surgery, the defect consisted of cartilage, fibrin, and hypertrophic chondrocytes (Figure 1B). Furthermore, there was an increase in formation of these tissues on the proximal end of the defect (segments 3-6) compared to the distal side (segments 7-10). At 3 weeks, there was an absence of a fibrin clot and hypertrophic chondrocytes within the defect and an increase in cartilage formation evenly distributed between the distal and proximal sides (Figure 1B). Figure 1C contains representative images of [top] chondrocytes (red), unmineralized cartilage (black), mineralized cartilage (orange), [middle] osteocytes (black arrow) in bone, and [bottom] a fibrin clot. Our data shows changes in tissue presence and distribution over 1 and 3 weeks in the CSD model, and a lack of bone formation in the defect at 3 weeks which was expected. Conversely, hypertrophic chondrocytes, which are a pre-cursor for bone development, are present at 1 week. Future and current work includes investigating sub-critical defect sizes to determine differences in bone remodeling between sub-CSD and CSD.

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