This work was supported by National Institutes of Health (NIH) R01 DK121327 to R.A.E and R01 AR071968 to F.K.

<ol>
	<li>R&#252;egsegger, P., Koller, B., M&#252;ller, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+microtomographic+system+for+the+nondestructive+evaluation+of+bone+architecture.">A microtomographic system for the nondestructive evaluation of bone architecture.</a> Calcified Tissue International. 58 (1), 24-29 (1996).</li><li>M&#252;ller, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphometric+analysis+of+human+bone+biopsies:+a+quantitative+structural+comparison+of+histological+sections+and+micro-computed+tomography.">Morphometric analysis of human bone biopsies: a quantitative structural comparison of histological sections and micro-computed tomography.</a> Bone. 23 (1), 59-66 (1998).</li><li>Waarsing, J. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detecting+and+tracking+local+changes+in+the+tibiae+of+individual+rats:+a+novel+method+to+analyse+longitudinal+in+vivo+micro-CT+data.">Detecting and tracking local changes in the tibiae of individual rats: a novel method to analyse longitudinal in vivo micro-CT data.</a> Bone. 34 (1), 163-169 (2004).</li><li>Boyd, S. K., Davison, P., M&#252;ller, R., Gasser, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+individual+morphological+changes+over+time+in+ovariectomized+rats+by+in+vivo+micro-computed+tomography.">Monitoring individual morphological changes over time in ovariectomized rats by in vivo micro-computed tomography.</a> Bone. 39 (4), 854-862 (2006).</li><li>Christiansen, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+micro-computed+tomography+voxel+size+and+segmentation+method+on+trabecular+bone+microstructure+measures+in+mice.">Effect of micro-computed tomography voxel size and segmentation method on trabecular bone microstructure measures in mice.</a> Bone Reports. 5, 136-140 (2016).</li><li>Holdsworth, D. W., Thornton, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micro-CT+in+small+animal+and+specimen+imaging.">Micro-CT in small animal and specimen imaging.</a> Trends in Biotechnology. 20 (8), 34-39 (2002).</li><li>Schambach, S. J., Bag, S., Schilling, L., Groden, C., Brockmann, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+micro-CT+in+small+animal+imaging.">Application of micro-CT in small animal imaging.</a> Methods. 50 (1), 2-13 (2010).</li><li>Bouxsein, M. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Guidelines+for+assessment+of+bone+microstructure+in+rodents+using+micro-computed+tomography.">Guidelines for assessment of bone microstructure in rodents using micro-computed tomography.</a> Journal of Bone and Mineral Research. 25 (7), 1468-1486 (2010).</li><li>Morgan, E. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micro-computed+tomography+assessment+of+fracture+healing:+Relationships+among+callus+structure,+composition,+and+mechanical+function.">Micro-computed tomography assessment of fracture healing: Relationships among callus structure, composition, and mechanical function.</a> Bone. 44 (2), 335-344 (2009).</li><li>O'Neill, K. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micro-computed+tomography+assessment+of+the+progression+of+fracture+healing+in+mice.">Micro-computed tomography assessment of the progression of fracture healing in mice.</a> Bone. 50 (6), 1357-1367 (2012).</li><li>Bissinger, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fully+automated+segmentation+of+callus+by+micro-CT+compared+to+biomechanics.">Fully automated segmentation of callus by micro-CT compared to biomechanics.</a> Journal of Orthopaedic Surgery and Research. 12 (1), 108 (2017).</li><li>Brown, M. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Delayed+fracture+healing+and+increased+callus+adiposity+in+a+C57BL/6J+murine+model+of+obesity-associated+type+2+diabetes+mellitus.">Delayed fracture healing and increased callus adiposity in a C57BL/6J murine model of obesity-associated type 2 diabetes mellitus.</a> PLOS One. 9 (6), 99656 (2014).</li><li>Khajuria, D. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aberrant+structure+of+fibrillar+collagen+and+elevated+levels+of+advanced+glycation+end+products+typify+delayed+fracture+healing+in+the+diet-induced+obesity+mouse+model.">Aberrant structure of fibrillar collagen and elevated levels of advanced glycation end products typify delayed fracture healing in the diet-induced obesity mouse model.</a> Bone. 137, 115436 (2020).</li><li>Sigurdsen, U., Reikeras, O., Hoiseth, A., Utvag, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correlations+between+strength+and+quantitative+computed+tomography+measurement+of+callus+mineralization+in+experimental+tibial+fractures.">Correlations between strength and quantitative computed tomography measurement of callus mineralization in experimental tibial fractures.</a> Clinical Biomechanics. 26 (1), 95-100 (2011).</li><li>Duvall, C. L., Taylor, W. R., Weiss, D., Wojtowicz, A. M., Guldberg, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impaired+angiogenesis,+early+callus+formation,+and+late+stage+remodeling+in+fracture+healing+of+osteopontin-deficient+mice.">Impaired angiogenesis, early callus formation, and late stage remodeling in fracture healing of osteopontin-deficient mice.</a> Journal of Bone and Mineral Research. 22 (2), 286-297 (2007).</li><li>Gerstenfeld, L. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+effects+of+the+bisphosphonate+alendronate+versus+the+RANKL+inhibitor+denosumab+on+murine+fracture+healing.">Comparison of effects of the bisphosphonate alendronate versus the RANKL inhibitor denosumab on murine fracture healing.</a> Journal of Bone and Mineral Research. 24 (2), 196-208 (2009).</li><li>Alentado, V. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Validation+of+the+modified+radiographic+union+score+for+tibia+fractures+(mRUST)+in+murine+femoral+fractures.">Validation of the modified radiographic union score for tibia fractures (mRUST) in murine femoral fractures.</a> Frontiers in Endocrinology. 13, 911058 (2022).</li><li>Yu, K. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhancement+of+impaired+MRSA-infected+fracture+healing+by+combinatorial+antibiotics+and+modulation+of+sustained+inflammation.">Enhancement of impaired MRSA-infected fracture healing by combinatorial antibiotics and modulation of sustained inflammation.</a> Journal of Bone and Mineral Research. 37 (1), 1352-1365 (2022).</li><li>Nyman, J. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+measures+of+femoral+fracture+repair+in+rats+derived+by+micro-computed+tomography.">Quantitative measures of femoral fracture repair in rats derived by micro-computed tomography.</a> Journal of Biomechanics. 42 (7), 891-897 (2009).</li><li>Fiset, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+validation+of+the+radiographic+union+score+for+tibial+fractures+(RUST)+using+micro-computed+tomography+scanning+and+biomechanical+testing+in+an+in-vivo+rat+model.">Experimental validation of the radiographic union score for tibial fractures (RUST) using micro-computed tomography scanning and biomechanical testing in an in-vivo rat model.</a> The Journal of Bone and Joint Surgery. 100 (21), 1871-1878 (2018).</li><li>Shefelbine, S. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prediction+of+fracture+callus+mechanical+properties+using+micro-CT+images+and+voxel-based+finite+element+analysis.">Prediction of fracture callus mechanical properties using micro-CT images and voxel-based finite element analysis.</a> Bone. 36 (3), 480-488 (2005).</li><li>Liu, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glucocorticoid-induced+delayed+fracture+healing+and+impaired+bone+biomechanical+properties+in+mice.">Glucocorticoid-induced delayed fracture healing and impaired bone biomechanical properties in mice.</a> Clinical Interventions in Aging. 13, 1465-1474 (2018).</li><li>Watson, P. J., Fitton, L. C., Meloro, C., Fagan, M. J., Gr&#246;ning, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+adaptation+of+trabecular+bone+morphology+in+the+mammalian+mandible.">Mechanical adaptation of trabecular bone morphology in the mammalian mandible.</a> Scientific Reports. 8 (1), 7277 (2018).</li><li>Nie, C., Wang, Z., Liu, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+depression+on+fracture+healing+and+osteoblast+differentiation+in+rats.">The effect of depression on fracture healing and osteoblast differentiation in rats.</a> Neuropsychiatric Disease and Treatment. 14, 1705-1713 (2018).</li></ol>

The authors have no conflicts of interest to disclose.

The purpose of this study is to describe a detailed protocol for &#181;CT analysis with the goal of accurate quantification of the 3D mineralized callus structure, which is often fundamental in bone and fracture healing studies. The protocol utilizes a general-purpose state-of-the-art 3D image analysis software platform which facilitates image visualization, segmentation/labelling, and measurements ranging from simple to complex.
The most time-consuming task in the protocol is semi-automated segmentation of the callus, with exclusion of the cortical bone and medullary canal. This region has also been excluded in many previous studies9,16,17,18. Some studies have included the native cortical bone and canal regions in their analyses19,21, while in other studies the approach was not clear. Including the native cortices avoids the difficulty and potential subjectivity in contouring comminuted regions of fractured cortices but inflates callus mineralization measures.
The protocol focuses on obtaining output measures including total callus volume, mineralized volume, bone volume fraction, and bone mineral density. These parameters are readily interpreted and are commonly reported in the literature. Mineralized volume and bone volume fraction are dependent on the selected threshold for differentiating mineralized versus unmineralized, whereas bone mineral density is not. Tissue mineral density may also be computed based on only the tissue labeled as mineralized, instead of bone mineral density based on both mineralized and unmineralized callus. Tissue mineral density has been reported to be associated with torsional strength and rigidity9; however, these measures are more likely affected by partial volume effects and imaging resolution than bone mineral density.
Investigators have reported good correlation between quantified 3D cortical bridging and callus strength and stiffness (cortical bridging assessed on 2D radiographs is commonly assessed clinically in human patients)20. Additional 3D callus properties reported in the literature include moments of inertia10,15,19, which characterize the geometric distribution of the callus (i.e., how spread out the tissue is). Polar moment of inertia theoretically relates to torsional resistance and bending moment of inertia relates to bending resistance. Although these properties could be computed based on the segmented callus data described in this study, their correlation with measured biomechanical properties has been reported to be inconsistent9,19,21. Other previously reported callus properties include connectivity density, trabecular thickness, and structure model index11,17,,22. These parameters are often used to characterize trabecular bone and are readily computed by &#181;CT scanner software; however, their relationship to fracture healing quality is not as clear. The software utilized in this protocol is a general-purpose program, not specific to bone. Thus, if certain bone parameters such as trabecular thickness are calculated outside of this protocol, segmented data may be exported to other programs for further analysis (e.g., as in Watson et al.23).
This protocol provides detailed workflows for complex callus structure characterization and quality control from a single software environment as compared to other methods in which multiple programs are required for analysis24. Therefore, time saving is a potential advantage of this protocol. The software enables a variety of flexible, sophisticated 3D visualization methods that help ensure accurate analysis and also allow for parallel tabulation of all results.
The &#181;CT analysis protocol can be adapted to different fracture models in mice as well as rats; for other applications, optimization of some of the critical steps is recommended to ensure minimizing result variation. Specifically, investigating the impact of changing the size of the VOI or the number of contoured slices within the VOI on the reproducibility of the results should be considered. Also, using digital realignment as described in step 3.4 is recommended, but if different software is used for analysis, then assessing the necessity of this step, by comparing data generated with and without digital realignment, might be required.
In this protocol, a semi-automated segmentation approach was used for the identification and separation of the callus from the cortical bone and marrow. In cases like comminuted fractures, where the structure of the callus is extremely complex, contouring the callus and the periosteal surface of the cortex becomes challenging. It is advisable in these cases to perform the contouring with multiple experimenters to assess and attempt to limit subjectivity.
Limitations exist with this protocol. The protocol requires the conversion and export of DICOM images so that images can be subsequently analyzed in additional software; this step takes some additional time and may necessitate use of a calibration phantom within the image. As automated segmentation techniques continue to evolve, including ones based on machine learning, it may be advantageous to replace the manual contouring portions of the protocol with these new techniques. Overall, the detailed protocol described here for the analysis of bone healing callus in rodents may especially benefit labs without substantial &#181;CT analysis experience and may help establish a more consistent and standardized approach across the field.

Micro-computed tomography (&#181;CT) imaging has been widely used in preclinical bone research, providing noninvasive, high-resolution images to evaluate the microstructure of bones1,2,3,4,5. &#181;CT involves a large number of X-ray images, obtained from a rotating sample or by using a rotating X-ray source and detector. Algorithms are used to reconstruct 3D volumetric data in the form of a stack of image slices. Clinical CT is the gold standard for 3D imaging of human bones, and &#181;CT is a commonly used technique for evaluating bone healing efficiency in experimental animals1,2,3,4,6,7. Mineralized bone has excellent contrast to X-ray, while soft tissues have relatively poor contrast unless a contrast agent is used. In the assessment of fracture healing, &#181;CT generates images that provide detailed information about the 3D structure and density of the mineralized callus. In vivo &#181;CT scanning can also be used for longitudinal, time-course assessment of fracture healing.
The quantification of intact cortical and trabecular bone using &#181;CT is generally well-established and standardized8. Although preclinical studies use a variety of quantification methodologies to analyze fracture healing9,10,11, a detailed protocol of &#181;CT image analysis for callus quantification has not been published yet.Therefore, the aim of this study is to provide a detailed step-by-step protocol for &#181;CT imaging and analysis of bone healing callus.

<table><tbody><tr><td>10% neutral buffered formalin&amp;nbsp;</td><td>Fisher chemical</td><td>SF100-20</td><td>Used for bone tissue fixation</td></tr><tr><td>Avizo</td><td>Thermo Scientific</td><td></td><td>Image processing and analysis software</td></tr><tr><td>Hydroxyapatite phantom&amp;nbsp;</td><td>Micro-CT HA D4.5, QRM</td><td>QRM-70128</td><td></td></tr><tr><td>Image Processing Language</td><td>Scanco</td><td></td><td>Used to convert raw images to DICOM images</td></tr><tr><td>Micro-Mosquito Straight Hemostatic Forceps</td><td>Medline</td><td></td><td>Used to remove the intramedullary pin&amp;nbsp;</td></tr><tr><td>Microsoft Excel</td><td>Microsoft</td><td></td><td>Spreadsheet software</td></tr><tr><td>Scanco mCT system (vivaCT 40)</td><td>Scanco</td><td></td><td>Used for &amp;micro;CT imaging&amp;nbsp;</td></tr></tbody></table>

assessment of bone fracture healing using micro-computed tomography

Micro-computed tomography (&#181;CT) is the most common imaging modality to characterize the three-dimensional (3D) morphology of bone and newly formed bone during fracture healing in translational science investigations. Studies of long bone fracture healing in rodents typically involve secondary healing and the formation of a mineralized callus. The shape of the callus formed and the density of the newly formed bone may vary substantially between timepoints and treatments. Whereas standard methodologies for quantifying parameters of intact cortical and trabecular bone are widely used and embedded in commercially available software, there is a lack of consensus on procedures for analyzing the healing callus. The purpose of this work is to describe a standardized protocol that quantitates bone volume fraction and callus mineral density in the healing callus. The protocol describes different parameters that should be considered during imaging and analysis, including sample alignment during imaging, the size of the volume of interest, and the number of slices that are contoured to define the callus.

To monitor bone formation during fracture healing, a mid-diaphyseal open tibial fracture was induced in adult, male C75BL/6J mice. The fracture was stabilized using an intramedullary nail, an established model of secondary healing13. Callus tissues were harvested at days 14, 21, and 28 post-fracture12. These timepoints represent different phases of healing. Endochondral bone formation during secondary bone healing proceeds via initial formation of a fibro-cartilaginous (soft) callus, which mineralizes at later stages to reduce micromotion at the fracture gap, allowing the formation of new blood vessels across the fracture line13. Day 14 post-fracture in the murine fracture model used in this study represents the stage of mineralized soft callus. As healing proceeds from day 14 to day 21, the mineralized soft callus is completely replaced by newly formed woven bone, resulting in bony bridging of the fracture gap13. Between days 21 and 28, the callus undergoes resorption and remodeling to re-establish the characteristic structure of cortical bone12.
&#181;CT images were acquired and analyzed at three timepoints using the protocol described above. A minimum of 10 samples were analyzed at each timepoint. For each sample, bone volume fraction and BMD were calculated. Bone volume fraction was calculated by dividing the volume of mineralized callus (BV) by the total callus volume (TV). Results demonstrated substantial formation of mineralized callus at day 14 (Figure 7A,B) and incremental increases in bone fraction volume and BMD as healing proceeded from day 14 to days 21 and 28 (Figure 7A,B), consistent with bony bridging of the fracture gap. As expected, the callus underwent resorption/remodeling between days 21 and 28 as evidenced by a decline in total callus volume (Figure 7A,B). Cortical bridging of the callus was more evident at day 28 than any preceding timepoint (Figure 7A). These results indicate that the provided &#181;CT protocol allows monitoring of bone formation and callus structure during different phases of bone healing.
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/64262/64262fig07.jpg" /> 
Figure 7: Monitoring bone healing using &#181;CT. (A) 2D (sagittal, left panel) and 3D (right panel) images of the healing callus generated by &#181;CT at the indicated post-fracture timepoints. (B) BMD, bone volume fraction (BV/TV), and total callus volume calculated from images shown in A. Results show healing progression through the late repair and remodeling phases. N = 10-12. The dots on the line plot&#160;represent average &#177; SEM. (*) p &#60; 0.05 using one-way ANOVA followed by Tukey&#39;s post-hoc test. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/64262/64262fig07large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Assessment of Bone Fracture Healing Using Micro-Computed Tomography at JoVE.com

Assessment of Bone Fracture Healing Using Micro-Computed Tomography

Micro-computed tomography (&#181;CT) is a non-destructive imaging tool that is instrumental in assessing bone structure in preclinical studies, however there is a lack of consensus on &#181;CT procedures for analyzing the bone healing callus. This study provides a step-by-step &#181;CT protocol that allows the monitoring of fracture healing.

Micro-computed tomography (&#181;CT) is the most common imaging modality to characterize the three-dimensional (3D) morphology of bone and newly formed ...

assessment-of-bone-fracture-healing-using-micro-computed-tomography

Universidad San Sebastian

Research

JoVE Journal

Biology

1.7K Views.  The Pennsylvania State University College of Medicine. Micro-computed tomography (µCT) is a non-destructive imaging tool that is instrumental in assessing bone structure in preclinical studies, however there is a lack of consensus on µCT procedures for analyzing the bone healing callus. This study provides a step-by-step µCT protocol that allows the monitoring of fracture healing. 

Video: Assessment of Bone Fracture Healing Using Micro-Computed Tomography

Sequential In vivo Imaging of Osteogenic Stem/Progenitor Cells During Fracture Repair

Bone turns over continuously and is highly regenerative following injury. Osteogenic stem/progenitor cells have long been hypothesized to exist, but in vivo demonstration of such cells has only recently been attained. Here, in vivo imaging techniques to investigate the role of endogenous osteogenic stem/progenitor cells (OSPCs) and their progeny in bone repair are provided. Using osteo-lineage cell tracing models and intravital imaging of induced microfractures in calvarial bone, OSPCs can be directly observed during the first few days after injury, in which critical events in the early repair process occur. Injury sites can be sequentially imaged revealing that OSPCs relocate to the injury, increase in number and differentiate into bone forming osteoblasts. These methods offer a means of investigating the role of stem cell-intrinsic and extrinsic molecular regulators for bone regeneration and repair.

Sequential In vivo Imaging of Osteogenic Stem/Progenitor Cells During Fracture Repair

Quantitative measurement of bone progenitor function in fracture healing requires high resolution serial imaging technology. Here, protocols are provided for using intravital microscopy and osteo-lineage tracking to sequentially image and quantify the migration, proliferation and differentiation of endogenous osteogenic stem/progenitor cells in the process of repairing bone fracture.

Bone turns over continuously and is highly regenerative following injury. Osteogenic stem/progenitor cells have long been hypothesized to exist, but in ...

Medicine

Computed Tomography and Optical Imaging of Osteogenesis-angiogenesis Coupling to Assess Integration of Cranial Bone Autografts and Allografts

A major parameter determining the success of a bone-grafting procedure is vascularization of the area surrounding the graft. We hypothesized that implantation of a bone autograft would induce greater bone regeneration by abundant blood vessel formation. To investigate the effect of the graft on neovascularization at the defect site, we developed a micro&#8211;computed tomography (&#181;CT) approach to characterize newly forming blood vessels, which involves systemic perfusion of the animal with a polymerizing contrast agent. This method enables detailed vascular analysis of an organ in its entirety. Additionally, blood perfusion was assessed using fluorescence imaging (FLI) of a blood-borne fluorescent agent. Bone formation was quantified by FLI using a hydroxyapatite-targeted probe and &#181;CT analysis. Stem cell recruitment was monitored by bioluminescence imaging (BLI) of transgenic mice that express luciferase under the control of the osteocalcin promoter. Here we describe and demonstrate preparation of the allograft, calvarial defect surgery, &#181;CT scanning protocols for the neovascularization study and bone formation analysis (including the in vivo perfusion of contrast agent), and the protocol for data analysis.
The 3D high-resolution analysis of vasculature demonstrated significantly greater angiogenesis in animals with implanted autografts, especially with respect to arteriole formation. Accordingly, blood perfusion was significantly higher in the autograft group by the 7th day after surgery. We observed superior bone mineralization and measured greater bone formation in animals that received autografts. Autograft implantation induced resident stem cell recruitment to the graft-host bone suture, where the cells differentiated into bone-forming cells between the 7th and 10th postoperative day. This finding means that enhanced bone formation may be attributed to the augmented vascular feeding that characterizes autograft implantation. The methods depicted may serve as an optimal tool to study bone regeneration in terms of tightly bounded bone formation and neovascularization.

Implantation of autologous and allogeneic bone grafts constitute accepted approaches to treat major craniofacial bone loss. Yet the effect of graft composition on the interplay among neovascularization, cell differentiation and bone formation is unclear. We present a multimodal imaging protocol aimed to elucidate the angiogenesis-osteogenesis interdependence at the graft proximity.

A major parameter determining the success of a bone-grafting procedure is vascularization of the area surrounding the graft. We hypothesized that ...

Bioengineering

Real-Time Imaging of CCL5-Induced Migration of Periosteal Skeletal Stem Cells in Mice

Periosteal skeletal stem cells (P-SSCs) are essential for lifelong bone maintenance and repair, making them an ideal focus for the development of therapies to enhance fracture healing.&#160; Periosteal cells rapidly migrate to an injury to supply new chondrocytes and osteoblasts for fracture healing. Traditionally, the efficacy of a cytokine to induce cell migration has only been conducted in vitro by performing a transwell or scratch assay. With advancements in intravital microscopy using multiphoton excitation, it was recently discovered that 1) P-SSCs express the migratory gene CCR5 and 2) treatment with the CCR5 ligand known as CCL5 improves fracture healing and the migration of P-SSCs in response to CCL5. These results have been captured in real-time. Described here is a protocol to visualize P-SSC migration from the calvarial suture skeletal stem cell (SSC) niche towards an injury after treatment with CCL5. The protocol details the construction of a mouse restraint and imaging mount, surgical preparation of the mouse calvaria, induction of a calvaria defect, and acquisition of time-lapse imaging.

This protocol describes the detection of CCL5-mediated periosteal skeletal stem cell migration in real-time using live animal intravital microscopy.

Periosteal skeletal stem cells (P-SSCs) are essential for lifelong bone maintenance and repair, making them an ideal focus for the development of ...

Developmental Biology

A Simple Critical-sized Femoral Defect Model in Mice

While bone has a remarkable capacity for regeneration, serious bone trauma often results in damage that does not properly heal. In fact, one tenth of all limb bone fractures fail to heal completely due to the extent of the trauma, disease, or age of the patient. Our ability to improve bone regenerative strategies is critically dependent on the ability to mimic serious bone trauma in test animals, but the generation and stabilization of large bone lesions is technically challenging. In most cases, serious long bone trauma is mimicked experimentally by establishing a defect that will not naturally heal. This is achieved by complete removal of a bone segment that is larger than 1.5 times the diameter of the bone cross-section. The bone is then stabilized with a metal implant to maintain proper orientation of the fracture edges and allow for mobility. 
Due to their small size and the fragility of their long bones, establishment of such lesions in mice are beyond the capabilities of most research groups. As such, long bone defect models are confined to rats and larger animals. Nevertheless, mice afford significant research advantages in that they can be genetically modified and bred as immune-compromised strains that do not reject human cells and tissue.
Herein, we demonstrate a technique that facilitates the generation of a segmental defect in mouse femora using standard laboratory and veterinary equipment. With practice, fabrication of the fixation device and surgical implantation is feasible for the majority of trained veterinarians and animal research personnel. Using example data, we also provide methodologies for the quantitative analysis of bone healing for the model.

Animal models are frequently employed to mimic serious bone injury in biomedical research. Due to their small size, establishment of stabilized bone lesions in mice are beyond the capabilities of most research groups. Herein, we describe a simple method for establishing and analyzing experimental femoral defects in mice.

While bone has a remarkable capacity for regeneration, serious bone trauma often results in damage that does not properly heal. In fact, one tenth of all ...

Imaging of the Microstructural Failure Mechanism in the Human Hip

Imaging the bone microstructure under progressively increasing loads allows for observing the microstructural failure behavior of bone. Here, we describe a protocol for obtaining a sequence of three-dimensional microstructural images of the entire proximal femur under progressively increasing deformation, causing clinically relevant fractures of the femoral neck. The protocol is demonstrated using four femora from female donors aged 66-80 years at the lower end of bone mineral density in the population (T-score range = &#8722;2.09 to &#8722;4.75). A radio-transparent compressive stage was designed for loading the specimens replicating a one-leg stance, while recording the applied load during&#160;micro-computed tomography (micro-CT) imaging. The field of view was 146 mm wide and 132 mm high, and the isotropic pixel size was 0.03 mm. The force increment was based on finite-element predictions of the fracture load. The compressive stage was used to apply the displacement to the specimen and enact the prescribed force increments. Sub-capital fractures due to opening and shear of the femoral neck occurred after four to five load increments. The micro-CT&#160;images and the reaction force measurements were processed to study the bone strain and energy absorption capacity. Instability of the cortex appeared at the early loading steps. The subchondral bone in the femoral head displayed large deformations reaching 16% before&#160;fracture, and a progressive increase in the support capacity up to&#160;fracture. The deformation energy linearly increased with the displacement up to&#160;fracture, while the stiffness decreased to near-zero values immediately before&#160;fracture. Three-fourths of the fracture energy was taken by the specimen during the final 25% force increment. In conclusion, the protocol developed revealed a remarkable energy absorption capacity, or damage tolerance, and a synergic interaction between the cortical and trabecular bone at an advanced donor age.

The protocol enables the measurement of the deformation of the bone microstructure in the entire proximal human femur and its toughness by combining large-volume micro-CT scanning, a custom-made compressive stage, and advanced image processing tools.

Imaging the bone microstructure under progressively increasing loads allows for observing the microstructural failure behavior of bone. Here, we describe ...

Engineering

Outer-Boundary Assisted Segmentation and Quantification of Trabecular Bones by an Imagej Plugin

Micro-computed tomography (micro-CT) is routinely used to assess bone quantity and trabecular microstructural properties in small animals under different bone loss conditions. However, the standard approach for trabecular analysis of micro-CT images is slice-by-slice semi-automatic hand-contouring, which is labor intensive and error prone. Described here is an efficient method for automatic segmentation of trabecular bones according to the bone's outer boundaries, where trabecular bones can be identified and segmented automatically with accuracy with less operator bias when appropriate segmentation parameters are set. To profile satisfactory segmentation parameters, an image stack of segmentation results is displayed, where all possible combinations of the segmentation parameters are changed one by one in sequence, and segmentation results with associated parameters can easily be visually checked. As a quality-control feature of the plugin, simulated standard objects are quantified where the measured quantities can be compared with theoretical values. Layer-by-layer quantification of trabecular properties and trabecular thicknesses are reported by such a plugin, and the distributions of such properties within the selected regions can be profiled easily. Although layer-by-layer quantification retains more information about trabecular bones and facilitates further statistical analysis of structural changes, such measures are unavailable from the output of current commercial software, where only a single quantified value for each parameter is reported for each sample. Therefore, the described workflows are better approaches for analyzing trabecular bones with accuracy and efficiency.

We present a workflow for segmenting and quantifying trabecular bones for 2D and 3D images based on the bone&#39;s outer boundary using an ImageJ plugin. This approach is more efficient and accurate than the current manual hand-contouring approach, and provides layer-by-layer quantifications, which are not available in current commercial software.

Micro-computed tomography (micro-CT) is routinely used to assess bone quantity and trabecular microstructural properties in small animals under different ...

In Vivo Evaluation of Fracture Callus Development During Bone Healing in Mice Using an MRI-compatible Osteosynthesis Device for the Mouse Femur

Endochondral fracture healing is a complex process involving the development of fibrous, cartilaginous, and osseous tissue in the fracture callus. The amount of the different tissues in the callus provides important information on the fracture healing progress. Available in vivo techniques to longitudinally monitor the callus tissue development in preclinical fracture-healing studies using small animals include digital radiography and &#181;CT imaging. However, both techniques are only able to distinguish between mineralized and non-mineralized tissue. Consequently, it is impossible to discriminate cartilage from fibrous tissue. In contrast, magnetic resonance imaging (MRI) visualizes anatomical structures based on their water content and might therefore be able to noninvasively identify soft tissue and cartilage in the fracture callus. Here, we report the use of an MRI-compatible external fixator for the mouse femur to allow MRI scans during bone regeneration in mice. The experiments demonstrated that the fixator and a custom-made mounting device allow repetitive MRI scans, thus enabling longitudinal analysis of fracture-callus tissue development.

In Vivo Evaluation of Fracture Callus Development During Bone Healing in Mice Using an MRI-compatible Osteosynthesis Device for the Mouse Femur

The evaluation of tissue development in the fracture callus during endochondral bone healing is essential to monitor the healing process. Here, we report the use of a magnetic resonance imaging (MRI)-compatible external fixator for the mouse femur to allow MRI scans during bone regeneration in mice.

Endochondral fracture healing is a complex process involving the development of fibrous, cartilaginous, and osseous tissue in the fracture callus. The ...

A Minimally Invasive Model to Analyze Endochondral Fracture Healing in Mice Under Standardized Biomechanical Conditions

Bone healing models are necessary to analyze the complex mechanisms of fracture healing to improve clinical fracture treatment. During the last decade, an increased use of mouse models in orthopedic research was noted, most probably because mouse models offer a large number of genetically-modified strains and special antibodies for the analysis of molecular mechanisms of fracture healing. To control the biomechanical conditions, well-characterized osteosynthesis techniques are mandatory, also in mice. Here, we report on the design and use of a closed bone healing model to stabilize femur fractures in mice. The intramedullary screw, made of medical-grade stainless steel, provides through fracture compression an axial and rotational stability compared to the mostly used simple intramedullary pins, which show a complete lack of axial and rotational stability. The stability achieved by the intramedullary screw allows the analysis of endochondral healing. A large amount of callus tissue, received after stabilization with the screw, offers ideal conditions to harvest tissue for biochemical and molecular analyses. A further advantage of the use of the screw is the fact that the screw can be inserted into the femur with a minimally invasive technique without inducing damage to the soft tissue. In conclusion, the screw is a unique implant that can ideally be used in closed fracture healing models offering standardized biomechanical conditions.

This protocol describes a minimally invasive osteosynthesis technique using an intramedullary screw for standardized stabilization of femur fractures, which can be used to analyze endochondral bone healing in mice.

Bone healing models are necessary to analyze the complex mechanisms of fracture healing to improve clinical fracture treatment. During the last decade, an ...

The Generation of Closed Femoral Fractures in Mice: A Model to Study Bone Healing

Bone fractures impose a tremendous socio-economic burden on patients, in addition to significantly affecting their quality of life. Therapeutic strategies that promote efficient bone healing are non-existent and in high demand. Effective and reproducible animal models of fractures healing are needed to understand the complex biological processes associated with bone regeneration. Many animal models of fracture healing have been generated over the years; however, murine fracture models have recently emerged as powerful tools to study bone healing. A variety of open and closed models have been developed, but the closed femoral fracture model stands out as a simple method for generating rapid and reproducible results in a physiologically relevant manner. The goal of this surgical protocol is to generate unilateral closed femoral fractures in mice and facilitate a post-fracture stabilization of the femur by inserting an intramedullary steel rod. Although devices such as a nail or a screw offer greater axial and rotational stability, the use of an intramedullary rod provides a sufficient stabilization for consistent healing outcomes without producing new defects in the bone tissue or damaging nearby soft tissue. Radiographic imaging is used to monitor the progression of callus formation, bony union, and subsequent remodeling of the bony callus. Bone healing outcomes are typically associated with the strength of the healed bone and measured with torsional testing. Still, understanding the early cellular and molecular events associated with fracture repair is critical in the study of bone tissue regeneration. The closed femoral fracture model in mice with intramedullary fixation serves as an attractive platform to study bone fracture healing and evaluate therapeutic strategies to accelerate healing.

The murine closed femoral fracture model is a powerful platform to study fracture healing and novel therapeutic strategies to accelerate bone regeneration. The goal of this surgical protocol is to generate unilateral closed femoral fractures in mice using an intramedullary steel rod to stabilize the femur.

Bone fractures impose a tremendous socio-economic burden on patients, in addition to significantly affecting their quality of life. Therapeutic strategies ...

HE Staining and Micro-CT to Evaluate Murine Trabecular Bone Microarchitecture

Trabecular Bone Microarchitecture Evaluation in an Osteoporosis Mouse Model

Bone microstructure refers to the arrangement and quality of bone tissue at the microscopic level. Understanding the bone microstructure of the skeleton is crucial for gaining insight into the pathophysiology of osteoporosis and improving its treatment. However, handling bone samples can be complex due to their hard and dense properties. Secondly, specialized software makes image processing and analysis difficult. In this protocol, we present a cost-effective and easy-to-use solution for trabecular bone microstructure analysis. Detailed steps and precautions are provided. Micro-CT is a non-destructive three-dimensional (3D) imaging technique that provides high-resolution images of trabecular bone structure. It allows for the objective and quantitative evaluation of bone quality, which is why it is widely regarded as the gold standard method for bone quality assessment. However, histomorphometry remains indispensable as it offers crucial cellular-level parameters, bridging the gap between two-dimensional (2D) and 3D assessments of bone specimens. As for the histologic techniques, we chose to decalcify the bone tissue and then perform traditional paraffin embedding. In summary, combining these two methods can provide more comprehensive and accurate information on bone microstructure.

Author Spotlight: An Economic and Efficient Method for Quantitative Evaluation of Bone Microarchitecture in a Murine Osteoporosis Model 

This protocol presents an economical and efficient method for quantitative evaluation of bone microarchitecture in a mouse model of osteoporosis by combining Hematoxylin-Eosin (HE) staining and micro-computed tomography (Micro-CT) techniques.

Bone microstructure refers to the arrangement and quality of bone tissue at the microscopic level. Understanding the bone microstructure of the skeleton ...

Assessment of Bone Fracture Healing Using Micro-Computed Tomography

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Sequential In vivo Imaging of Osteogenic Stem/Progenitor Cells During Fracture Repair

Computed Tomography and Optical Imaging of Osteogenesis-angiogenesis Coupling to Assess Integration of Cranial Bone Autografts and Allografts

Real-Time Imaging of CCL5-Induced Migration of Periosteal Skeletal Stem Cells in Mice

A Simple Critical-sized Femoral Defect Model in Mice

Imaging of the Microstructural Failure Mechanism in the Human Hip

Outer-Boundary Assisted Segmentation and Quantification of Trabecular Bones by an Imagej Plugin

In Vivo Evaluation of Fracture Callus Development During Bone Healing in Mice Using an MRI-compatible Osteosynthesis Device for the Mouse Femur

A Minimally Invasive Model to Analyze Endochondral Fracture Healing in Mice Under Standardized Biomechanical Conditions

The Generation of Closed Femoral Fractures in Mice: A Model to Study Bone Healing

Trabecular Bone Microarchitecture Evaluation in an Osteoporosis Mouse Model