FluoRender is an interactive rendering tool for confocal microscopy data visualization. It combines the rendering of multi-channel volume data and polygon mesh data, where the properties of each dataset can be adjusted independently and quickly. The tool is designed especially for neurobiologists, allowing them to better visualize confocal data from fluorescently-stained brains, but it is also useful for other biological samples.
Clustering algorithms. Three clustering algorithms for segmentation are added: expectation-maximization on Gaussian mixture, DBSCAN, and k-means. Users can use these methods in the "Component" window.
Improved tracking algorithms. Tracking accuracy has been improved. We improved the algorithm for generating the track map and incorporated clustering algorithms to automatically segment during tracking. Users can also adjust a series of parameters in the "Tracking" window to fine tune the tracking.
A new 4D script for tracking sparse particles. The 4D script allows users to track selected features in a time sequence. No initial segmentation is required for it to work. It can be used to track sparse and small features conveniently.
A density setting for component generation. The density setting has been added in the basic component generation. Its concept is based on the clustering algorithm DBSCAN. However, its implementation is based on the synthetic brainbows algorithm, which uses GPU to compute segmentation of dense data sets.
Render view output enlargement. The option has been added when the render view is captured and saved as an image. Users can set an output image size larger than the render view size.
4D script list. The list of built-in 4D scripts has been added to the "Record/Export" panel. Users can easily select and switch 4D scripts without browsing to the actual files.
An issue of reading Prairie/Bruker xml formats, when the TIFF files were saved/processed by third-party tools.
An issue that certain volume property settings became unavailable when switching between MIP and normal render modes.
Support of OpenCL 1.2. Please make sure the latest graphics driver is installed. Deprecated functions from OpenCL 1.1 have been updated in FluoRender to work with OpenCL 1.2.
A new 4D script type for executing OpenCL filters. It allows application of an OpenCL filter to each file of a time sequence. Use this function for image processing of a time sequence.
A new external executable for applying component analysis settings to a time sequence. It can read settings from the new component analysis module and then apply them to an input file. Use this feature along with the 4D script for external executable to apply component analysis to a sequence of files.
New OpenCL filters. Two OpenCL filters are included for computing morphological gradient and Sobel edge detection.
An option to save the alpha channel in screenshots. You can choose to save an additional alpha channel in screenshots, so that the captured images can be used in a compositing software.
An uncertainty analysis on tracking results. You can analyze an automatic tracking result and locate uncertain tracks. Use this feature to manually fix tracking issues with good efficiency.
A mode switch for zoom ratio display. You can choose how the zoom ratio is displayed. Two modes are available. The first mode is view-based, where the percentage of the zoom ratio value represents the size of an entire data set to that of the render view. The second mode is data-based, where the zoom ratio value represents the rendered size of a data set to its 1:1 voxel-pixel state.
Many of the bug fixes, improvements, and new features became available because of the cordial support from FluoRender users. We wish to thank every user and welcome any suggestions for future releases.
The selected part of a labeled volume was not displayed correctly in the Depth channel intermixing mode.
The application crashed when first a channel had been deleted and then played back a sequence in the batch mode.
An output frame was missing when the last key only was consisted of one frame in the advanced output panel.
Tracking quality has been improved in the 2.18 release of FluoRender. A new function is added to allow automatic refinement on only one time point.
A complete set of manual tracking functions is back and reorganized in the user interface. Automatic and manual tracking can be used separately or jointly. You can use manual tracking functions to fix the issues in the result from automatic tracking, or use automatic tracking to refine partial result from manual tracking. Certain operations, such as ID assignment and linking can be automated even in full manual tracking for a streamlined workflow.
You can convert a tracked trajectory to a ruler, or assign consistent IDs to a series of tracked components. A new script is added to allow visualization of all tracked components. You can get information about selected components as well as the number of broken and multiple links in the tracking results.
When you convert a volume or its selected part into a mesh object, it reports the surface area of the resulting mesh object.
You can turn on the full screen mode so that a render view port occupies the entire display area. This is a feature only available on Microsoft Windows.
You can hold the display of the clipping planes of a volume, so that they can be viewed when you move the mouse cursor outside of the clipping plane panel area. In addition, there are five modes for displaying clipping planes.
You can select how the rotation sliders in the render view panel control view rotations. The first mode is a “steering wheel” that rotates the view continuously, where the rotation speed is controlled by the slider thumb button’s displacement. The second mode directly sets the rotation angle by the button’s displacement.
A new setting for the paint brush allows you to adjust how strong a gap between two structures is considered as an edge. When edge detection is enabled for paint brush, this new setting allows finer control of selection region.
For Windows7+, download the win64 EXE file and run the setup. You may need to allow security settings to run the installer. Please be sure to read and accept the Licence.
For OSX10.9+, download the osx DMG file. Open the archive and drag the FluoRender icon to the Applications shortcut. You may need to replace a previous version. You may also need to allow security settings to run the program (Settings, Security, Open Anyway). Please be sure to read and accept the Licence.
We are excited to announce the 2.16 release of FluoRender. For Microsoft Windows users, FluoRender 2.16 incorporates minor feature improvements and issue fixes. However, we have upgraded all graphics functions, conforming to the OpenGL core profile specifications. It allows us to have continuous support for future graphics processing units. FluoRender 2.16 requires graphics cards with a minimum support of OpenGL 3.3. Older graphics hardware need to be replaced. For Apple Mac OS X users, there is the exciting news that all features previously exclusive to Windows are now available*. It still requires relatively recent Apple hardware (2009 and on) and the operating systems.
1. The calculation of the depth attenuation has been improved, which is based on the data size from current viewing direction. A higher contrast can be achieved by increasing the attenuation factor. The depth attenuation effect now also supports mesh data.
2. It allows users to change the text and color of measurement tools, including locators and rulers.
3. It supports True-type fonts in the render view. You can change the settings for font and size in the setting dialog. You can also copy your own True-type font file to the installation directory to let FluoRender use it.
5. The documentation of tutorials was updated to include new features as well as a chapter on movie export.
Note: FluoRender 2.16 was officially announced on May-26, 2015. We had put a test version on-line earlier than this date, so that the new functions, especially on Mac OS X, could be tested. If you happen to download the 2.16 version prior to the official release, please download it again.
Last but not least, we would like to thank Ken Calderone from the University of Michigan, who suggested the improvements including modifiable text for measurement tools, font, and text size settings. We appreciate the continuous support from our users. Please let us know if you have any comments.
6. Applying paint brush strokes on a data set larger than 4 GB caused FluoRender to crash. After fixing, it performs best on the latest graphics cards with 12 GB of memory (Geforce GTX Titan X or Quadro K6000).
2. Speed of applying a paint brush stroke is significantly improved, especially for large data on slow computers.
1. A probe tool to create a two-point ruler along the viewing direction. When used in combination with the paint brush tool (via key board shortcut), it creates a cylindrical selection along the ruler.
2. A profile tool to calculate intensity profiles along rulers. This tool is best used along with the probe tool to generate an averaged intensity profile within the cylindrical mask region.
This version fixed a bug in FluoRender 2.15, which wrote a blank frame when exporting movies in QuickTime MOV format. However, we also incorporated these new features and improvements:
1. OpenCL kernel editor. New in the 2.15 release of FluoRender, an OpenCL kernel editor is added to the tool collection. The editor can be accessed from menu->Tools->OpenCL kernel editor. It allows users to create, load, edit, and save an OpenCL kernel for 3D image processing. Active kernel is applied to selected channel when the "Run" button is pressed. A list of built-in kernels are also provided for basic 3D filtering operations. See Chapter 19 of the user's manual for more details.
2. Automatic threshold estimation for paint brush. New in the 2.15 release of FluoRender, threshold for paint brush can be estimated by applying brush strokes. The estimation is based on the local histogram of the data under the first applied stroke. Its calculation is performed with an OpenCL kernel. See Chapter 14 of the user's manual for more details.
Improvements include UI updates and bug fixes. More importantly, we edited a user's manual that covers all the latest features of FluoRender in great detail.
This version has expanded to MacOSX, includes improved user interface, issue fixes, and other program improvements.
1. File loading speed for TIFF format is improved. For 4D playback, the speed increase can be 100% on certain systems. For the best speed performance, save a 4D sequence in separate, uncompressed channels.
2. When saving projects, there is an additional option for embedding datasets into the project directories. However, complete 4D sequence will not be saved using this feature.
3. Rendering contrast is increased for fine details with less opacity, when a bright background color is used.
1. The colormap legend reflects transparency settings, which is more accurate to visualized results.
3. Added interpolation option in the render view settings. It turns on/off interpolations on the current selection.
9. In the edit window, a select group option is added to the selection adjustment section. It allows painting on all channels in one group simultaneously.
3. Re-organized the property window for polygon mesh, with lighting being a feature that can be turned on/off.
1. When voxel size of XY plane is not set to 1, The 1:1 button won't zoom current dataset correctly. This is now fixed.
3. In certain situations, the workspace panel become empty after loading a project. This is now fixed.
1. Fixed a sampling rate issue when a volume is anisotropic and user sets XY spacings much greater (or smaller) than 1.
4. Improved user interactions when user chooses to use the workspace toolbar for volume painting functions.
3. Derived volumes from the above operations can be saved as multi-layer Tiffs from the Datasets panel.
1. Support of importing several manufacturer-specific confocal formats, including Olympus Image Binary (*.oib), Olympus Original Imaging Format (*.oif), and Zeiss Laser Scanning Microscope (*.lsm).
3. Equalization for equalizing uneven brightness of 3D and 4D data. It also enhances details at the same time.
4. Project files as well as volume datasets can be drag-and-drop to either the FluoRender icon or the main interface to open.
4. Data within groups can be set to depth mode, even the viewport is set to layer or composite mode.
5. Micro Blending. It can render accurate colors of multiple channels in depth mode, with an arbitrary number of channels.
2. In MIP mode, and colormapped MIP mode, some parameters are disabled, in order to keep the color accuracy.
2. 3D batch rendering for RGB multi-layer TIFF files. Switching between multi-channel datasets is easy and fast.
4. RGB multi-layer TIFF format support. RGB multi-layer TIFF files are added to the render view automatically after opened.
2. Camera position and rotation are saved before switching to Free Fly mode, and they are restored after switching back to either Perspetive or Orthogonal projections.
2. New rendering modes that calculate the gradient magnitude differently. Choose different modes in the settings dialog can balance between rendering speed, data loading speed, and memory usage.
1. When new data are added to the group with existing volumes, the voxel size of the newly added volume is set to the existing ones.
4. Color icons of the volumes in the scene view are corrected to always follow their color settings.
1. Drag & drop support in the tree of the scene view. User can now drag & drop items in the scene view to re-order and re-group them. The order of the volume data will be shown in the layered mode.
2. Groups in the tree of the scene view. Volume datasets can be grouped by creating new groups and dragging into the groups. Volume data in the same group share the same 2D adjustment parameters.
2. For movie capture, the value for reverse rotation is always considered as the absolute value, no matter what the user input is.
3. 2D adjustment channel linking is automatically calculated from the color settings. Non-zero RGB channels will be linked in the 2D adjustment settings, when user changes the color of the volume data.
1. In previous versions, the scaling of the volume datasets will change after viewport resizing. The problem is now fixed.
2. In previous versions, the program will crash sometimes when user deletes volume datasets from the data view. The problem is now fixed.
3. In previous versions, the program crashes quite often when loading volume datasets with slice resolution equal or larger than 1024x1024. Now the problem is fixed.
1. Scale bar for size reference. The length, unit and text of the scale bar can be set by the user. The length of scale bar is linked to viewport zoom. Though scale bar is available for perspective projection, it's recommended that use it in orthographic mode for accuracy.
2. Clipping planes. Clipping planes can be set for different channels independently, or they can be linked for all the channels.
1. Zoom of render view is calculated according to the relative size of the dataset bounding box to the viewport, rather than previously the projected voxel size to screen pixel size. Thus even datasets acquired with different objectives and scanning resolutions can generate comparable rendered results, with zoom being set to similar values.
5. Minor bugs exist in a few tools. Please report any issues that affect your FluoRender experience.
If you use FluoRender in work that leads to published research, we humbly ask that you add the following to the 'Acknowledgments' section of your paper:
"This work was made possible in part by software funded by the NIH: FluoRender: Visualization-Based and Interactive Analysis for Multichannel Microscopy Data, 1R01EB023947-01 and the National Institute of General Medical Sciences of the National Institutes of Health under grant number P41 GM103545-18"
First of all, FluoRender is built to run on a personal computer with good graphics processing capabilities. Although most of today's personal computers can run FluoRender, including Windows desktops, laptops, Apple MacBooks, iMacs, and MacPros, it is best to purchase one with a gaming level or professional graphics card. Wikipedia provides detailed comparisons of graphics card from both Nvidia and AMD. Generally speaking, a graphics card with higher "Processing Power GFLOPs" is also better for FluoRender. We would recommend a top-of-the-line gaming graphics card over a professional graphics card. FluoRender can take advantage of some features of a professional graphics card, such as 10-bit output and better anti-aliasing. However, the difference is subtle in most situations. Second, strong CPU processing power is generally desired. We would recommend a latest CPU with higher clock frequency over more cores. Third, if you have large data sets to process, equip as much system memory as possible. For the year 2015, the consumer level computers can be equipped with as much as 64 GB of system memory, while a professional desktop can have 512 GB or more. Last, if you want to load data quickly or play back large time sequence smoothly, you need high speed access to hard drives. We would recommend a discrete RAID controller with abundant cache. Speed of hard drives is also important for handling large data sets. We would recommend 4 or more solid state drives configured in RAID0 mode.
There are several possibilities for FluoRender to fail. First, check if the graphics driver is installed and updated to the latest version. Some old graphics card may not support OpenCL, which is required since version 2.15. If that is the case, you can download and try an earlier version of FluoRender. Since version 2.16, we require the graphics card to support at least OpenGL 3.3. If you have an old graphics card and want to use FluoRender on it, please replace your graphics card or download an old version of FluoRender. Second, make sure that your operating system supports 64-bit applications. Since version 2.15, we have dropped the support of 32-bit applications, which means FluoRender is 64-bit only. For Windows, you have to purchase the x64 version of the operating system; for Mac OSX, you have to update to the recent versions. Finally, FluoRender may not be properly installed, or some required modules have been accidentally uninstalled. Reinstalling FluoRender may solve the issue.
FluoRender can only load supported file formats, which include TIFF and some microscopy manufacturer specific formats. We write our own readers for these formats to achieve the best performance, especially for large data and time sequence data. So, we can only support "Open" and standard formats. If you have files of unsupported formats, you have to convert the format using a third-party tool. ImageJ can be a good choice in most situations.
If you can use FluoRender for the basic visualizations of volume data, but not some advanced functions, such as paint brushes, it means your system does not support all OpenGL features. This can be that you are using a virtual machine, a remote client, or a system with an integrated graphics card with limited capabilities. You may want to upgrade your system to a standalone desktop computer with the latest graphics hardware to use all features in FluoRender.
The official FluoRender releases only include a Windows version and a Mac OSX version, both 64-bit. If you want to run FluoRender on systems other than these, you can download the source code of FluoRender and build it on a different operating system. Additionally, you can send us emails and let us know your requirements.
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A prologue is an explanatory and introductory discourse, which in this case should then commence by asking "Why did I become interested in sharks, sharks' cartilages, sharks' teeth and evolution?"
It all started many years ago when, soon after landing in January 1983 in Africa from the cold shores of Milano University in North Italy, I met Tracey at the University of the Witwatersrand, Johannesburg, and with her hastened to the hot, feverish Natal North Coast, spending time in Umhlanga Rocks. There I visited the Natal Shark Board and was reminded during my tour that whilst sharks have an endoskeleton of cartilage there are some bony appendages on the integument, known as placoids or denticles (Fig. 1A). These render the integument into a formidable abrasive instrument which lacerates victims, causing bleeding, and provoking a full shark attack.1
Back at the University of the Witwatersrand, Johannesburg, I went to the library of the Medical School where, with difficulties, I started to manually sieve through the Index Books then printed and made available world-wide by the National Library of Medicine of the National Institutes of Health (NIH) in Bethesda, Maryland USA.
It was serendipitous that I was attracted by an interesting, perhaps alluring, title of a published paper on sharks: "How to swim with sharks: A primer"1. I read with mounting enthusiasm and scientific interest the essay by a then unknown author who signed himself as Voltaire Cousteau. As the Editor's foot note stated "the essay will make fascinating reading material and sets fundamental rules and principles that, if followed, will make it possible to swim, albeit with difficulties, amid sharks, whilst becoming experts through practice".2
Cousteau makes the wry comment "Actually, nobody wants to swim with sharks" and, reflecting on those who may have wrongly assumed that waters were not shark infested, "has by now doubtless lost any interest in learning how to swim with sharks!1
The essay "How to swim with sharks: A primer" further sparked my interest in shark biology, leading to my reading classic papers on the cartilaginous fishes and discovering that shark genera and species are fully evolved animals with superb differentiating pathways 3-5 that make the shark the most effective killer swimming in the waters of our planet. Sharks or Selachians are named Chon-drichthyes, fishes with an endoskeleton of cartilage thus including the jaws and the chondrocrania (a cartilaginous box containing the spinal nervous tissue of the shark).
Further reading across several different scientific disciplines revealed the concept that the cartilage forming the endoskeletons of Chondrichthyans such as Elasmobranchs is not a primitive condition. It had been thought that cartilage was the original structure of the endoskeleton of fishes and that "bone only appeared at a late date in the history of fishes to augment and replace the cartilage," 6,7 However, it had been reported at the beginning of last century that Elasmobranchs, including sharks, are placoderm descendants "which have degenerated in their skeletal structure from an ancestral condition in which bone was present."7
Romer concurred in 1963, stating "the purely cartilaginous condition (of Elasmobranchs) is not truly a primitive one" and the absence of bone "is not an ancestral character, but one due to the degeneration from bone-bearingancestors."6
This has obviously changed our understanding of the evolutionary skeletogenesis in animals up the vertebrate mammals. It is worth nothing then as Romer stated " that instead of beginning with a purely cartilaginous skeleton, and later gradually acquiring bone, the early vertebrates had a considerable degree of ossification which was followed in a majority of cases by a slump toward a cartilaginous condition"6Romer further stated that "Bone is an ancient, rather than a relatively new, skeletal material in the history of vertebrates"
That regression may explain how in modern vertebrates "internal skeletal structures are first formed in cartilage, and only as development proceeds does the transformation of these cartilages into bony elements take place"6
In mammals, the majority of the bones of the skeleton arise from a cartilage anlage that serves as a strut for the induction of bone formation, undergoing vascular invasion with capillary sprouting into the hypertrophic cartilage. This vascular invasion brings about chondrolysis, i.e. the death of the cartilage anlage, and the differentiation of the first waves of osteoblast-like cells. These differentiated cells lay down the early bone matrix, later to be mineralized to form the long bones of the mammalian skeleton (Fig. 1).
The critical role of angiogenesis in osteogenesis was described in detail in the middle of last century by the classic studies of Trueta which grandly provided the first insights into the supramolecular assembly of the extracellular matrix of bone.8 In several research experiments conducted at the Bone Research Laboratory, using either naturally-derived bone morphogenetic proteins9-11 or coral-derived macroporous constructs,12,13 the critical role of angiogenesis in osteogenesis has been characterised by defining the induction of bone formation as "osteogenesis in angiogenesis" (Fig. 2E) 10,11,14-16
Intriguingly, Aristotle (384-322BC) has been credited to have stated that forming blood vessels have a patterning function during organogenesis.16,17 This concept of "morphogenetic vessels" long predates the idea proposed by Trueta in 1963 of "osteogenetic vessels."8 The invading "organogenetic blood vessels" shape, pattern and induce the multistep cascade of the induction of bone formation, which is "osteogenesis in angiogenesis" (Fig. 2). An overview of the critical role of the vessels in bone formation was published in Science-in- Africa.18
Recently, a team of scientists has identified a specific vessel subtype in bone that links angiogenesis to osteogenesis.19 These specialized capillary sub-types, morphogenetic and osteogenet-ic vessels, provide niche signals to perivascular osteoprogenitor cells, as previously postulated by the grand insights of Aristotle and Trueta.8,11 As in mammals and as briefly discussed above, the cartilage anlage serves to initiate endochondral bone formation, that is a molecular and cellular morphogenetic event that forms the basis for the induction of endochondral bone formation, i.e. via a cartilaginous phase (Fig. 1B-D).10,11,15
Different molecular and morphogenetic events initiate the induction of bone formation via the intramembranous ossification pathway without a cartilage anlage. Angiogenesis with capillary sprouting is the three-dimensional construct for the induction of bone formation. Osteogenic precursor and other committed mes-enchymal cells aggregate and condense in selected areas of the craniofacial skeleton. The mesenchymal condensations are surfaced by multiple osteoblast-like cells and other committed precursors which embrace the invading capillaries. The blood vessels are morphogenetic since there is further differentiation of surrounding mesenchymal tissue condensations. Finally, the three-dimensional pattern of new vessels with capillary sprouting and invasion within the newly formed secreted matrix, engineers the osteonic primate cortico-cancellous bone, a process which is shown in the fascinating "morphogenetic" images presented in Figure 2. Further angiogenesis and development give rise to the majority of the cranial and craniofacial skeleton which forms via intramembranous ossification without the need for a cartilage anlage. The process is clearly both "morphogenetic" and "osteo-genetic" as per the Aristotelian and Trueta8 views and may be described as "osteogenesis in angiogenesis."11,16
We now know that the presence of a cartilaginous endoskeleton in sharks has replaced a bony skeleton in the early evolution of the Elasmobranchs. The question of course arises how indeed was degeneration of the bony skeleton possible? A recent paper in Nature5 provides important insights into the evolution of cartilaginous fishes. That molecular experimentation reports that Elasmobranchs lack a specific secretory calcium binding phos-phoprotein (SCPP).5
SCPP genes, arising from the Sparc-like1 (Sparcl1) gene family, have a crucial role in the formation of bone.5 It has been proposed, therefore, that the absence of the SCPP genes in the Elephant shark Callorhinchus milii will account for the absence of bone from the endoskeleton of the animal.5
The Feature Paper which follows describes our understanding of these evolutionary "de-differentiating" events from a bony to a cartilaginous endoskeleton invocating evolutionary speciation highly favorable to degenerate the bony endoskeleton thus blocking the induction of bone formation and skeletogenesis. These "de-differentiating" events in selachians returning to cartilaginous endoskeletons have set evolutionary specificity that resulted in more resilient animals, with higher capacities to float, more favorable to deep immersions and feeding into the oceans without breaking a bony endoskeleton.
As Moss clearly states in his essay on the "Skeletal Tissues in Sharks,"20the "cartilaginous endoskeleton of fossil Elasmobranchs is derived from phylogenetically ancestral forms with osseous endoskeletal tissues. In spite of the total lack of a bony endoskeleton, it has been emphasised that "bone does exist at the base (or pedicle) of the teeth and dermal denticles."20
Hence, and importantly, sharks may not lack osteogenic ability, or their differentiating chondroblasts may have an intrinsic or ge-nomic ability to differentiate into functional osteoblasts.20 Moss concludes that the "chondral" - or cartilaginous - state of the Elasmobranchs "was once believed to prove that cartilage preceded bone in vertebrate evolution". The acquisition however of a cartilaginous endoskeleton "does not establish a fundamental alteration of the potential and intrinsic ability of the Elasmobranchs' scleroblasts to modulate into osteoblasts."20
De-differentiation from a bony to a cartilaginous, or predominantly cartilaginous, endoskeleton in Elasmobranchs might have occurred following natural evolutionary selection resulting in the speciating of selected gene clusters encoding powerful inhibitors of angiogenesis. Cartilaginous matrices contain powerful morphogenetic signals that inhibit angiogenesis and capillary sprouting.21,22 Shark cartilages contain a substance that strongly inhibits the growth of new vessels which may well explain the rarity of malignant tumours in Elasmobranchs,23,2* and potentially could be used to inhibit tumour angiogenesis in humans.
Evolutionary pressure for superior habitats for feeding in the deeper waters of the oceans might have set in motion genetic mutations leading to the expression of several powerful inhibitors of angiogenesis, thus blocking the induction of bone formation.
The molecular and cellular cascades of osteogenesis via an en-dochondral pathway, as seen in mammals and in teleost fishes (those having an endoskeleton of bone) have been blocked in the shark by the inhibitors of angiogenesis which have been found in extant shark cartilage.
On consideration, there is an intriguing possibility that it may be possible to "reactivate" the osteogenic pathways in the shark. What biological mechanisms could be involved?
My research interest then was primarily focused on the induction of bone formation using morphogens, firstly defined in 1952 25 as "forms generating substances", and subsequently named "os-teogenin" or "bone morphogenetic proteins",9,26-28 following the colossal experimental work of several pioneers headed by Marshal Urist, at the Bone Research Laboratory at the University of California Los Angeles (UCLA). Urist published his fundamental and crucial studies in Science in a report titled "Bone: formation by autoinduction." 29 Hari A Reddi, at the Bone Cell Biology Section, NIH, Bethesda, made seminal discoveries on the chaotropic* dissociative extraction and reconstitution of the bone matrix components, published in the Proceedings of the National Academy of the Sciences, USA30,31
*A chaotropic agent is a molecule in water solution that can disrupt the hydrogen bonding network between water molecules (i.e. exerts chaotropic activity).
In this context, the question was then formulated whether the bone morphogenetic proteins would have the capacity to "force" endochondral bone formation and/or direct intramembranous bone formation by inducing bone in heterotopic intramuscular sites of cartilaginous Selachian recipients.
The idea of the experiment was conceived at the famous Oyster Box Hotel in Umhlanga. The Director of the Natal Shark Board granted permission to use their fast boats which continuously check the off-shore shark nets along the white beaches of the North Coast. (Figure 3A.) The Animal Ethics Committee of the University approved the intention to fish sharks and to harvest chon-drocrania and vertebrae to enable the extraction of whatever mor-phogenetic factors Selachians may have within their cartilaginous matrices. In three expeditions off the shores of Umhlanga several dusky sharks and a larger shark, Carcharinus taurus, were fished out the ocean and enough quantities of cartilages were then secured (Fig 3B.)
At the laboratories of the then Dental Research Institute in Johannesburg Laura Yeates and I undertook the attempt at the extraction process from cartilage taken from the large shark Carchari-nus taurus, using techniques we had learnt when studying the purification of naturally-derived bone morphogenetic proteins from bovine bones.28 Laura, already attached to the embryonic and emerging Bone Research Laboratory within the Dental Research Institute of the University, was confronted by the ultra-viscous extracted material overly rich in high molecular weight mu-co-polysaccarides that characterized the cartilaginous extracts. Several attempts to extract and purify the shark proteins resulted in partially purified morphogenetic factors.
Several months elapsed in the year 1989 without further experimentation and in 1990 I decided to invite Laura to join me at the Bone Cell Biology Section of the NIH in Bethesda. In collaboration with Dr AH Reddi, the then Chief of the Bone Cell Biology Section, Laura Yeates and I purified, to homogeneity, osteogenin, a bone morphogenetic protein, from baboon bone matrices (Fig. 1 B)9. An account of our mutual interactions during that rewarding and exhilarating scientific period was published some years later in Science in Africa?833 highlighting not only the extraction and purification of bone morphogenetic proteins from baboon bone matrices but also the grand effect of geometry on the induction of bone formation by culturing in vitro osteoprogenitor cells on specific geometric configurations of coral-derived macroporous bioreactors. The work was also published in a classic paper in Biomaterials32
An account of our endeavours was published by Science in Africa in 2012.33 Together we had cracked the purification to homogeneity of osteogenin from baboon bone matrices and defined the critical role of geometry on the induction of bone formation. These experiments were described at length in two papers in 2012.33,34
After our success with the baboon proteins, we thought to again try to extract and purify proteins from Selachian cartilages, but using different chromatographic procedures. The samples had been flown with Laura and carried in her hand luggage from Africa to Washington DC. The extracted morphogenetic factors were later implanted in the subcutaneous space of rodents and uniquely and provocatively also implanted into the muscle of adolescent dusky sharks Carcharinus obscurus in a series of in vivo experiments in the salty water ponds of the Oceanographic Research Institute, Marine Parade, Durban, under the blue skies of the subtropical African sun.
My primary dedication was then, as it is now, the induction of bone formation in non-human and human primates using bone morphogenetic proteins,33-36 as well as exploring the induction of bone formation by the mammalian transforming growth factor-p (TGF-(3) isoforms. 14,37-39 The work has continued with unique research results on the substantial induction of bone formation by the hTGF-(33 morphogen (Figs. 1 E,F), later published in the Journal of Cellular Molecular Medicine.40
Whilst working as a learning and developing scientist at the then Dental Research Institute of the University, I had become interested also in the unique dentition of shark species that showed superb evolutionary traits for survival and predation.20 Of particular intrigue was the polyphyodonty (several rows of multiple teeth) in Selachians, which is the genesis of continuously erupting teeth that migrate forward until, like rolling over the edges of the cartilaginous jaws, they are shed, leaving space for the replacement row of the newly formed and forward-moving teeth (Figs. 4 A,B). Biologically, this is rendered possible by the presence of a continuously erupting dental lamina located in the mucous membrane behind the rear rank of the phalanx (Fig. 4 C).41
It is the intention of the following Feature Paper to describe the extraction and purification of Selachian's cartilages and the implantation of the extracted morphogenetic factors both in rodents and in Carcharinus obscurus sharks at the Oceanographic Research Institute in Durban. The manuscript also details the attachment apparatus of the connective tissue matrix to dentine or dentine/ like material of the multiple shark teeth and describes the forward movement and migration of the Selachian's teeth by a mechanism of a conveyor belt of condensed mesenchymal cells packed with fine connective tissue fibres that by tractional forces between cells and secreted matrix must move the rows of teeth forward until their exfoliation.
The Feature Paper additionally highlights the fact that the loss of teeth is physiologic in sharks but pathologic in mammals, and that tooth shedding in Selachian fishes is the conditio sine qua non for the ancestral evolutionary predatory habit of the sharks amidst the richly populated waters of our oceans.
We show that the implantation of coral-derived macroporous bio-reactors induce the differentiation of chondroblastic tissue and the differentiation of cartilage within the macroporous spaces, indicating the lack of an overt osteogenetic programme within the DNA of the Selachian cartilage.
We further propose that the lack of the induction of bone formation in Selachian intramuscular sites is the result of deficient angiogen-esis and vascular invasion in the Selachian cartilages because of the overtly rich anti-angiogenic factors within the cartilaginous matrices21-24 which block "osteogenesis in angiogenesis".
A 68-year-old obese man underwent computed tomographic colonography (CTC) scanning to investigate worsening constipation and lower abdominal discomfort on his left side. Optical colonoscopy was contraindicated because of his comorbidities. A preliminary CTC diagnosis of incarcerated Spigelian hernia was made, based on lateral deviation of the sigmoid colon to the left as well as extrinsic impressions on it, and the central location of the small bowel. Spigelian hernia is a rare form of anterior abdominal wall hernia. CTC can play a role in its diagnosis.
Computed tomography (CT) has been shown to produce the most accurate imaging in the diagnosis of Spigelian hernia. In this case report, CT colonography (CTC) demonstrated additional findings, making the diagnosis more readily achievable.
A 68-year-old obese man was referred for CTC scanning by a gastroenterologist. His primary clinical history was worsening constipation and lower abdominal discomfort on his left side. Optical colonoscopy was contraindicated because of his comorbidities of hypertension, ischaemic heart disease, diabetes and chronic obstructive airways disease. A low-dose CTC technique, CO2 insufflation and a 16-slice General Electric multidetector CT (MDCT) scanner were used for the study. As the patient was obese, a standard two-view supine and prone study could not be performed; therefore right and left decubitus views were done instead. Filling the colon with three litres of CO2 took approximately seven minutes, compared with the average time of three minutes. The CTC study showed an incarcerated Spigelian hernia (SH), without evidence of obstruction. The colon map views showed three important findings: (1) the sigmoid colon was markedly displaced to the left; (2) the small bowel was in a central position (Figure 1a); and (3) in addition, the air-contrast colon map demonstrated two external impressions on the sigmoid colon at the point where it enters and exits the muscular defect (Figure 1b). Slight narrowing of both the afferent and efferent loops of the sigmoid was noted on 3D fly-through imaging (Figure 2).
A 2D image below the umbilicus confirmed a defect through both the internal oblique and the transversus muscles. The sigmoid colon was contained in a hernia. An axial view demonstrated a thin rim of the external oblique muscle laterally, indicating an interstitial or interparietal geographic location of the hernia (Figure 3). Coronal and sagittal 2D images clearly demonstrated the SH (Figure 4 and Figure 5).
In view of the patient's comorbidities, surgical intervention was not an option at this stage. He was fully briefed by the gastroenterologist in this regard and made aware of the risk of obstruction or strangulation. He was informed that, should this occur, surgical intervention would be required.
Spigelian hernia is also known as spontaneous anterior abdominal hernia. It is a rare acquired hernia occurring in less than 2% of all abdominal hernias, with just over 1000 reported cases in the literature.1Congenital cases of SH have been reported in children.2SH is often undetected clinically as the symptoms and physical findings are subtle or inconclusive, particularly in obese adult patients. SH is prone to complications, such as incarceration, small or large bowel obstruction and strangulation, because the hernia defect is usually narrow (2 cm - 3 cm).1-3
Diagnosis is, however, primarily made by CT.1, 4, 5 In the present case report, a CTC was performed for investigation of abdominal discomfort and worsening constipation. A diagnosis of SH was made based on the lateral deviation of the sigmoid colon as well as the two extrinsic defects on its wall (Figure 1a and Figure 1b). The diagnosis was confirmed by the 2D study which showed an interparietal SH containing sigmoid colon and a clear hernial defect in both the internal oblique and transversus muscles (Figure 4 and Figure 5). CTC is a minimally invasive, fast, safe and accurate screening examination for colorectal cancer. It is also suitable as a diagnostic study for patients with gastrointestinal symptoms. Furthermore, it allows evaluation of extracolonic structures in the abdomen and pelvis.
Ultrasound is often the first line of imaging used to investigate SH.1, 2, 5Although operator dependent, it can be used to make a diagnosis of SH in a large proportion of patients. In addition, a Valsalva manoeuvre can be used in supine and erect sonography to determine the presence of SH.3
There is a slightly higher incidence of SH in women than in men; in adults, it usually occurs during the fourth and seventh decades.1, 2, 6It may occur on either side of the body, or occasionally bilaterally.1There are several known predisposing factors: obesity, chronic obstructive airways disease, previous surgery or laparoscopy, peritoneal dialysis, multiple pregnancies, and rapid weight loss, for example.2, 6, 7
Clinical presentation varies, depending on the content of the hernial sac. In obese individuals, detection of SH may be particularly difficult as the hernia may not be palpable on clinical examination. Some patients may complain of pain, but no lump is palpable.6Omentum and/or segments of the small bowel or colon are usually present in the sac. In the present case, the colon map showed small bowel in a central location; it was not included in the hernia sac (Figure 1a). Other structures, such as the stomach, Meckel's diverticulum, the appendix, and testicular and endometrial tissue, have been found in SH.2, 8
It is important to know the anatomy of the abdominal musculature when diagnosing SH. There are three flat and two vertical muscles of the anterior abdominal wall (Figure 6a and Figure 6b). The flat group consists of the external and internal oblique muscles and the transversus abdominis muscles. The vertical group comprises the rectus abdominis and pyramidalis.
The external oblique muscle arises from the lower eight ribs; it is the largest and most superficial muscle of the antero-lateral abdominal wall. The muscle fibres pass infero-medially. The aponeurosis of the upper part of the muscle splits to cover the anterior and posterior portions of the rectus abdominis. Inferiorly, the superior margin of the external oblique aponeurosis thickens and folds back on it to form the inguinal ligament.9
The internal oblique is the middle of the flat muscle group. It is a thin muscular sheet that fans out antero-medially. Its fibres become aponeurotic and split to envelop the anterior and posterior margins of the rectus in its upper two-thirds of the abdomen. In the lower third, the aponeurosis does not split; it passes as a whole in front of the rectus to reach the linear alba (Figure 6a).9
The rectus abdominis is a vertical muscle that is wrapped anteriorly and posteriorly by the aponeurosis of the three flat muscles. The lower third has no posterior cover. The pyramidalis is absent in approximately 25% of individuals. It is a small, insignificant muscle situated in relation to the inferior margin of the rectus.
The Spigelian (semilunar) line represents the vertical fibrous band formed by the union of the rectus sheath with the fascia of the three flat muscles of the anterior abdominal wall. In the upper two-thirds of the abdomen, the aponeurosis splits at the lateral margin of the rectus into a posterior layer, which passes behind the rectus muscle, and an anterior layer which passes in front of the rectus. The level at which this occurs is known as the arcuate line (Figure 6a), the semicircular line or the line of Douglas. The deep layer of the abdominal wall is only weakly reinforced by separated fascial bands. The Spigelian aponeurosis is the portion of the transversus abdominis that is bordered by the semilunar (Spigelian) line laterally and the lateral borders of the rectus muscle medially (Figure 6a). SH protrudes through this aponeurosis; most are found in a transverse band lying between 0 cm and 6 cm cranially to a line running between both the anterior and superior iliac spines.8This band is referred to as the 'Spigelian hernia belt' where the Spigelian fascia is widest (Figure 6a).6
A diagnosis of SH, based on plain-film radiographs, upper and lower gastrointestinal studies, and follow-through studies, is not readily made in the absence of an intestinal obstruction.7There has been reported use of magnetic resonance imaging (MRI) for investigation of SH.10CT, however, is the main imaging modality. As is evident in the present case report, CTC can play an important part in the diagnosis of SH. The main advantage of CTC is that the entire large bowel and all extracolonic components can be visualised. The complementary role of 2D and 3D imaging in the diagnosis of SH is well demonstrated in this case report.
The author declares that he had no financial or personal relationship(s) that might have inappropriately influenced him in writing this report.