Automated trabecular and cortical bone selection

Transcription

1 Automated trabecular and cortical bone Method note Page 1 of 18

2 2 Introduction In practice, quantitative analysis of long bone morphology is subdivided into analysis of the trabecular bone and analysis of the cortical bone. To do so, one has to adequately set a region of interest corresponding to either the trabecular bone or the cortical bone. This is very time-consuming as it has to be done manually for every individual bone. The goal of this application note is to demonstrate how this can be done in an automated way using custom processing, which is much faster and more accurate than the manual way, increasing reproducibility of the results and lowering variation. Part 1: Regions of interest (ROI) page Before opening the dataset in CTAn, make sure the long axis of the bone is aligned along the Z-axis. If not, one can reorient the dataset using dataviewer and save the dataset in the right orientation. Upon opening of the dataset in CTAn, browse to the Regions of interest page. The first and only manual step is to save 2 new datasets: a subset of slices in the long bone metaphysis to run the trabecular analysis ( VOI trab ), and a subset of slices in the long bone diaphysis to run the cortical analysis ( VOI cort ). Typically, one will select the growth plate as a reference point and define a specific offset and height to select the appropriate region (described in detail in the method note mouse bone analysis method ). Yet, and in contrast to what used to be done, no specific ROI has to be created on the slices themselves to separate the trabecular bone from the cortical bone. Slice of metapyseal ROI Slice of diapyseal ROI Page 2 of 18

3 3 Part 2: Custom processing page Starting from the ROI datasets that have been saved, one used to manually draw a ROI separating the trabecular bone from the cortical bone in order not to contaminate the trabecular analysis with (parts of the) cortical bone and the other way around. However, using custom processing, it is possible to run an automated task list to do so. First a protocol will be set up to automatically select the trabecular part of the bone (the bone marrow cavity in principal), and a few more steps will be added to this protocol in case a cortical is required. A. Trabecular After opening the VOI trab dataset, one first has to browse to the binary page to select an appropriate threshold to segment the bone from the soft tissue and background. In this case, a threshold of 80 was selected. Once the correct threshold values are selected, proceed to the Custom processing page. A copy of the dataset will be loaded. Three very important buttons are on the right side of the plug-ins bar: image view image inside ROI view ROI view As you have just loaded a copy of the dataset into the custom processing page, the initial settings are: Page 3 of 18

4 4 The idea behind an automated trabecular is in fact delineating the bone marrow cavity, including all trabecular bone and excluding any cortical bone. Therefore a region of interest has to be selected corresponding to the bone crosssectional area (step 1). To do so, a (global) threshold will be applied to adequately segment the bone tissue from the soft tissue and background. Other parts of the bone besides the tibia or femur metaphysis or other segmented structures (such as the fibula or noise speckles) can be included in the binary. These can easily be removed by running a sweep function in 3D removing all except the largest object (step 2). Page 4 of 18

5 5 In the next step, a ROI shrink-wrap function is applied in 2D to limit the ROI to the outside borders of the cortical bone (step 3). The stretch over holes function is activated to ignore pores running through the cortical bone towards the bone marrow. If not, one might risk to exclude the bone marrow from the ROI at slices where the bone marrow is connected to the space outside of the bone through pores in the cortical bone. Page 5 of 18

6 6 Once the ROI is set to the outside of the bone, one can proceed with the of the bone marrow cavity. To do so, run a bitwise operation setting image = NOT image to invert the image (step 4). In the image view, one can see that not only the bone marrow, but also the outside background is selected by the invert function. Therefore, apply a bitwise operation image = image AND region of interest to limit the image to the region of interest and exclude the outside background from the (step 5). Page 6 of 18

7 7 Due to the ROI shrink wrap stretch over holes function, several white speckles appear at the periosteal surface of the bone which have to be excluded from the. Several despeckling functions can be applied to do so, although a small opening procedure is more effective in this specific case (step 6). Page 7 of 18

8 8 Optionally, remaining white speckles, if any are still present, can be removed by applying a sweep function in 3D, removing all except the largest object (step7). Page 8 of 18

9 9 At this point, the bone marrow is selected but the trabecular bone is not included in this and appears as black speckles or gaps inside the bone marrow. These gaps can be removed by applying a closing function in 2D with a large radius (step 8). Setting the radius too small will not include trabecula in the that have a thickness larger that the closing radius. Following the closing procedure, the bone marrow cavity including the trabecular bone is selected. However, at slides showing a pore running through the cortical bone, the might include these pore spaces. To limit the bone marrow cavity to the endosteal surface, an opening function can be applied in 2D with a rather large radius which will result in removal of these spikes and smoothing of the bone marrow cavity (step 9). Page 9 of 18

10 10 To make sure the bone marrow cavity doesn t include any parts of thye cortical bone, a small erosion function is applied, resulting in a bone marrow cavity starting only a few pixels away from the endocortical surface (step 10). Page 10 of 18

11 11 In order to be able to save this bone marrow cavity as a region of interest delineating the trabecular bone, as well as a new dataset from this, one has to copy the bone marrow cavity to the region of interest by running a bitwise operation region of interest = COPY image (step 11). The second last step of the trabecular tasklist involves reloading the image in orde to be able to save a new dataset from the region of the ROI (step 12). Page 11 of 18

12 12 The last step is to save a new dataset containing the bone marrow cavity only, as well as the bone marrow cavity dataset as a region of interest. Therefore, save the image inside the region of interest, as well as the region of interest dataset as a binary (step 13). Page 12 of 18

13 13 Two new folders will be created by default in the VOI trab dataset folder, the first one being the first dataset saved, the second one being the second dataset saved. The binary ROI dataset can be loaded as a region of interest in the regions of interest tab in CTan, selecting bitmap files to be loaded instead of a.roi file, either on the VOI trab dataset or the newly saved bone marrow cavity dataset. Original 'VOI trab' dataset with ROI applied New trabecular dataset with ROI applied Page 13 of 18

14 14 B. Cortical For the cortical, open the VOI cort dataset and browse to the custom processing page. First part of the cortical tasklist is basically a recapitulation of the trabecular tasklist. Repeat steps 1 to 10 from the trabecular tasklist (A), to end up with a region of interest corresponding to the bone cross-sectional area and an image view being the bone marrow cavity. Selection of the cortical area now involves dilating the region of interest with a few pixels to be sure all of the cortical bone is included in the region of interest, followed by substraction of the bone marrow cavity to exclude the trabecular from the cortical region of interest. First, the region of interest is dilated with a few pixels in 2D to ensure all cortical bone being included in the region of interest. Page 14 of 18

15 15 Following the dilation, the trabecular is excluded fom the cortical region of interest by applying a bitwise operation region of interest = region of interest SUB image. As such, a donut-like region of interest is created which contains the cortical bone only. Page 15 of 18

16 16 In analogy with the trabecular seletion tasklist, the image is now reloaded to allow saving the region of interest as a binary, as well as a new dataset of this region of interest for further analysis. Again, the final step is to save a new dataset containing the cortical only, as well as the cortical dataset as a region of interest. Therefore, save the image inside the region of interest, as well as the region of interest dataset as a binary. Page 16 of 18

17 17 This binary ROI dataset can be loaded as a region of interest in the regions of interest tab in CTan, selecting bitmap files to be loaded instead of a.roi file, either on the VOI cort dataset or the newly saved cortical dataset. Original 'VOI cort' dataset with ROI applied New cortical dataset with ROI applied Page 17 of 18

18 18 Part 3: Run the tasklist on multiple datasets using Batch Manager For high throughput analysis, one can make trabecular and cortical regions of interest automatically by running the corresponding tasklist to a batch of bones. To do so, select the Batch manager icon in the custom processing tab. The different steps of the protocol (thresholding, despeckle, bitwise operations,.) can be saved in a task list. Therefore each plug-in needs to be added to the task list by clicking the + button (custom processing tab) or add button (top level Batch manager). You can apply the task list to several datasets using batch manager, by adding the datasets into the bottom part of the batch manager window. Both the task list and the sample list can be exported if wanted. Page 18 of 18