Contrast-Enhanced Computed Tomography for Quantitative Assessment of Multiple Stages of Fracture Healing

1de Bakker, CM; 1Mroszczyk, KA; 2Pittman, J; 3McDonald, G; 1Hayward, LN; 1, 4Grinstaff, M; 2Gerstenfeld, LC; 3, 1, 2Morgan, EF

1Boston University, Boston, MA, USA, 2Boston University School of Medicine, Boston, MA, USA, 3Boston University, Boston, MA, USA, 4Boston University, Boston, MA, USA

Introduction: Quantitative tools that allow rapid, non-destructive evaluation of the progression of healing of a bone injury would benefit pre-clinical screening of candidate interventions for enhancement of bone repair and improve study of the mechanisms of healing. A key pre-requisite for such evaluation, particularly in the early to intermediate stages of healing, is visualization of the cartilaginous “soft” callus that forms initially at the injury site in the vast majority of fractures [1]. Building on the use of computed tomography (CT) to assess formation of mineralized tissue in fracture healing [2,3], recent studies have demonstrated and validated use of contrast-enhanced computed tomography (CECT) to provide complementary evaluation of the cartilaginous portions of the callus [4,5]. The goal of this study was to extend the CECT approach to examine the spectrum of different tissues—cartilage, non-cartilaginous soft tissues, mineralized cartilage, and bone—across the timespan of healing.

Methods: Specimen Preparation. All protocols were approved by our institution’s Animal Care and Use Committee. Closed, stabilized fractures [6] were created in the femora of male C57BL/6 mice, 8-12 weeks of age. Calluses were harvested on post-operative days 7 (n=7), 14 (n=9), and 21 (n=8) and were scanned at a resolution of 12 μm/voxel (μCT40, Scanco Medical, Brüttisellen, Switzerland) before and after eight hours of incubation in a cationic contrast agent, CA4+ [7]. Image Processing. Pre- and post-incubation images were aligned by global, rigid registration, and the pre-incubation images were subtracted from the post-incubation images (Amira 5.2.2, Visage Imaging, Andover, MA) [3]. Intensity thresholds that identified cartilage in the subtracted images, and partially mineralized tissue in the pre-incubation images, were determined by analyses of the intensity histograms, as validated previously [5]. Well mineralized tissue, exclusive of the cortex, was identified using a threshold equal to 45% of the cortex attenuation [2]. Mineralized cartilage was defined at days 14 and 21 as voxels labeled as both cartilage and partially mineralized tissue [5]. The callus boundaries were defined using a semi-automated process (Scanco Medical), which excluded the cortex and medullary canal. From these analyses, the volumes of cartilage, partially mineralized tissue, well mineralized tissue, mineralized cartilage, and total callus were computed, as were the mineral content, mean and standard deviation of the tissue mineral density (considering only the well mineralized tissue), and mean intensity of the contrast-labeled cartilage. The distribution of cartilage along the length of the callus was quantified by measurements of callus cross-sectional area and cartilage area in 15 transverse cross-sections spaced 250 µm apart [6]. Statistical Analysis. Measures were compared among time-points using analyses of variance (ANOVA) with Tukey post hoc tests (JMP 10, SAS, Inc., Cary, NC).

Results: Over time, the percentage of the callus volume consisting of well mineralized tissue, mean and standard deviation of the tissue mineral density, mineral content, and volume of well mineralized tissue steadily increased, while that of cartilage decreased (Figure 1A). No change in the percentage of the callus volume consisting of mineralized cartilage was observed from day 14 to day 21; however, the percentage of cartilage that was mineralized increased over this time interval (Figure 1B). The mean intensity of the contrast-labeled cartilage also increased over time (Figure 1C). Qualitatively, mineralized cartilage was concentrated at the edges of cartilage clusters, between regions of non-mineralized cartilage and well mineralized tissue (Figure 1B). The distribution of cartilage along the callus length changed over time from a broad distribution along the periosteal surface at day 7 to a single peak at the fracture line at days 14 and 21 (Figure 2).

Discussion: The results indicate that CECT can provide non-destructive assessment of the formation of multiple, key tissues at the fracture site over a wide timespan of healing. Consistent with histological observations [6,8], but revealed in 3-D, diffuse cartilage formation throughout the callus at day 7 gave way to more focal and, subsequently, smaller cartilage regions located near the fracture line at days 14 and 21. Concurrent examination of mineralized cartilage and well mineralized tissue indicated that endochondral ossification was heavily, though not exclusively, involved in these changes in cartilage volume and location. Based on earlier studies using CA4+ in CECT imaging of articular cartilage [7], the lower mean intensity of contrast-labeled cartilage at day 7 vs. later time-points may indicate a lower concentration of GAGs in the comparatively immature cartilage present at the early time-point. Pending complete validation by paired comparisons to histological sections [5] at each time-point, the full CECT method presented here can provide higher throughput evaluations of healing than are currently available via histological analysis and can allow subsequent mechanical testing for direct comparisons of callus structure and composition to strength.

Significance: Non-destructive evaluation of the formation of cartilage and mineralized cartilage in the early and intermediate stages of fracture healing would allow for more rapid assessment of healing and of candidate interventions for impaired healing.

Acknowledgements: Funding was provided by Acceleron Pharma, Inc. Lauren Mangano and Jon Freedman provided technical assistance.

References: [1] Einhorn, TA; Clin Orthop, 1998; 1S; S7-S21. [2] Morgan, EF et al.; Bone, 2009; 44; 335-44. [3] Nyman, JS et al.; Bone, 2012; 50; 1357-67. [4] Hayward, LN et al.; Microsc Res Tech, 2012; 75; 7-14. [5] Hayward, LN et al.; JOR, in review. [6] Gerstenfeld, LC et al.; J Histochem Cytochem, 2006; 54; 1215-28. [7] Joshi, NS et al.; J Am Chem Soc, 2009; 37; 13234-5. [8] Zhang, M et al.; JBMR, 2011; 26; 792-802.

Figure 1. Quantitative CECT results: bar height = mean value. Error bars = ±1 standard deviation. * ≠ day 21 (p<0.05). # ≠ day 14 (p<0.05). Panel B also shows representative 3-D renderings of cortex (gray), cartilage (blue), mineralized cartilage (red) at days 14 (left) and 21 (right).
Figure 2. (A) Representative 3-D rendering at each time-point showing well mineralized tissue (blue) and cartilage (red); (B) Distribution of cartilage along the length of the callus at each time-point (error bars = +1 standard deviation)