Articular Cartilage Scaffold Imaging
An Electron Microscopic Perspective
Nipun Chadha, Department of Biomedical Engineering, MSC 507
University of Rochester, Rochester, New York
Image 1: Inlens micrograph of native porcine tissue sample
I.Porcine Articular Cartilage Scaffolds
Articular cartilage, also called hyaline cartilage, is the smooth, glistening white tissue that covers the surface of all the diarthrodial joints in the human body. As its name implies, articular cartilage is critical in the movement of one bone against another. Articular cartilage has an incredibly low coefficient of friction which, coupled with its ability to bear very large compressive loads, makes it ideally suited for placement in joints, such as the knee and hip.
Articular cartilage is not a homogeneous tissue. Instead, it has a very complex composition and architecture that permits it to achieve and maintain proper biomechanical function over the majority of a human lifespan. Articular cartilage is composed mainly of water (70-80% by wet weight). The solid phase of articular cartilage consists primarily of type II collagen and aggrecan, a chondroitin and keratan sulfate proteoglycan. Collagen forms a network of fibrils, which resist the swelling pressure generated by the proteoglycans. Aggrecan, because of its tendency to noncovalently interact with hyaluronic acid, forms huge aggregates that become trapped in the collagen network. One of the major short comings of the cartilage tissue is the lack of vascularization and thus its inability to heal. In the current project we looked at structural differneces in the native tissue and differentially treated, laboratory made porcine articular cartilage scaffolds.
Image 2: A PBS treated cartilage tissue scaffold, 52X, 5.00KV, SE detector.
One of the most difficult samples to be imaged (micrographed) under the electron microscopes are the biological samples. These samples need special preparations before being put in the microscope. The preperation techniques for biological samples basically involves removal of water from the bulk and the surface without tampering the sample structures. Only completely dry samples can be imaged under the electron beam of the scope. This can be achieved by either freeze drying or critical point drying (explained later).
Further, as these specimens are non-conducting, they need to be made conductive to the e-beam. This can be done fairly easily by sputter coating them with a metal. The most common metal/ metal alloys used are gold, or gold-paladium alloy. Surprisingly, these metals are used because they are relatively 'cheap'. This coating ensures that the sample is conductive. However, how much metal needs to be deposited is critical.
Another difficulty that microscopists face with bilogical samples are that it may be hard to iamge the bulk sample at once. Thus, it is important that the sample to reduced to a reasonable size. This can be done in a variety of ways such as cutting, shearing, slicing the sample etc. The choice of the technique depends on the sample. One important thing to keep in mind is that reducing the sample size may result in loss of features that may be important. Thus, one has to be careful in this regard.
Sample Preparation Techniques
1. Freeze Drying
The cartilage scaffolds were prepared elsewhere. Porcine cartilage tissue was minced into fine pieces and homogenised with distilled water or other chemicals and was then frozen at -80 degree C overnight. These frozen samples were then put in a freeze drying machine that works similar to the working of CPD. The basic idea is that ice can be directly converted to water vapors, completely avoiding transition into the liquid phase by going past the triple point of water. The triple point is attained by maintaining proper temperature (~ -50 degrees C) and pressure (~ 0.025 mTorr). The direct transition from the ice to the vapor phase ensures that the sample morphology is altered the least.
2. Critically Point Drying (CPD)
The next step (in case of native tissue that wasnt't freeze dried) was the CPD. The first step in the process is to fix the tissue with 1% Glutaraldehyde solution overnight. Then the sample is put in increasing gradient of Alcohol. Usually the sample can be placed in 30%, 50%, 70%, 80%, 90% and 100% alcohol for about 30 minutes or more in each in that order. This is done because alcohol is miscible with water and this step ensures that all the water in the sample is replaced by alcohol. This alcohol in the sample is then replaced by a transitional fluid (Carbon Dioxide). This step is done because carbon dioxide is miscible with alcohol but not water. Once all the alcohol is replaces by carbon dioxide in the chamber bomb, it is driven past its critical point (31.1 degree C and 7.38 MPa) through an increase in pressure and temperature. This ensures that all the transition fluid turns directly into gaseous form and escapes the sample. As the pressure is released the gas will escape leaving the sample perfect intact and dry.
Coating of the sample was done using a gold sputter coat machine available at the EM preperatory lab at the Institute of Optics, University of Rochester. As the surface of the cartilage had a lot of topography, as well as structural variations, it was important that the sample be properly coated. Thus, most of the scaffold samples were coated with about 80 angstrom of gold. As it was not important to look at very fine details of the scaffold, such a (relatively) thick layer of gold coating was reasonable.
4. Secondary Electron Images (SE2 and in-lens detectors)
The micrographs were obtained using the secondary electron detectors : SE2 and in-lens detector. The reason to choose secondary electron detector was that the interaction volume was found to be small for the sample from the electron flight simulator. Also, the beam energy used was either 2KV or 5KV. The sample being organic comprised mostly of Carbon, oxygen, nitrogen etc. Thus using a low energy beam and using S.E. made sense.
Image 3a: A PBS treated articular cartilage scaffold, 9.16KX, 2.00KV SE2 Detector.
Image 3b: A PBS treated cartilage scaffold, 12.81KX, 2.00KV, InLens detector.
Image 3c: A PBS treated cartilage scaffold, 8.81KX, 2.00 kV, InLens detector.
Image 3d: 0.01% SDS Treated cartilage scaffold, 8.42KX, 2.00kV, SE2 Detector. Salt crystal deposits on the scaffold surface. Cartilage fibers are also visible in the background.
Image 3e: Native cartilage tissue micrograph : 21.08KX, 2.00kV, Inlens detector, short Working Distance.
5. VPSE (Variable Pressure SE) Detector
This Secondary electron detector is an exception to how the basic EM works. This detectors works under relatively higher pressure conditions (typically in the range of 20-60 Pa). This detector is a common use detector for most biological samples as even with the best sample preparation techniques used, there may still be charging of the sample. To overcome this, the stage chamber, and not the column itself, is exposed to high pressures. This exposure lets in air molecules into the chamber and remove any charge deposit on the samples. These entering air molecules, strike with the electrons deposited on the non coduction sample surface and hence remove them form the sample allowing for better imaging. However, one downside of this technique is that the final micrograph obtained has highly reduced resolution and image quality. Also, to operate in this mode, one needs a much higher accelerating volatge than normal. This may tend to harm the biological samples at times.
Image 4: VPSE, at 60 Pa Pressure.
6. TEM images
Transmission electron microscopy (TEM) is different than SEM in ways that a beam of electrons is transmitted through an ultra thin specimen, interacting with the specimen as it passes through. An image is formed from the interaction of the electrons transmitted through the specimen; the image is magnified and focused onto an imaging device, such as a fluorescent screen, on a layer of photographic film, or to be detected by a sensor such as a CCD camera. The TEM can be operated in various modes such as Bright Field, Dark Field, Diffraction mode or even the STEM (Scanning Transmission Electron Microscopy) mode. The TEM offers a much higher resolution than the SEM, usually of the orders of angstroms (atleast in theory). However, sample preparation goes one step further than for the SEM because the samples need to be extremely thin (almost transparent to the electron beam).
Image 5: TEM image of the scaffold : Diffraction patterns of the scaffold are visible. 250KX.
Post Imaging Techniques
Images (micrographs) from the Electron microscopes, though maybe highly revealing of the structure and arrangement and other subtle details of the samples involved, they still are implicit for a novice to understand. Thus, an important aspect of obtaining and understanding micrographs is to make them more explicit for the complete spectrum of audience. This can be done in a variety of ways using imaging softwares available commonly in this age of technology. The software used on the micrographs obtained was the Adobe Photoshop CS3. The images were colorized. Further to reveal more structural details, a stereopair image (anaglyph) was also made using the same software.
Since SEM images are obtained using electrons, color images are impossible to obtain. This does not mean that color images can not be created to not only make the image more eye appealing but even bring the image to a more artistic level. Through the use of Adobe Photoshop, the following images were colorized and created.
Image 6: Colorized Cartilage scaffold micrograph. The green base is the tissue surface and the blue objects are salt crystals.
8.Anaglyphs (Stereo Pairs)
Anaglyphs or Stereo pairs or simply 3D images are obtained by super imposing two images of the same set of configuration (resolution, magnification etc) with a tilt variation of about 4-10 degrees between them. One point is fixed on the first image as the center point. The stage is then tilted to an angle of ~ 4-10 degrees and is brought back into focus using the Z axis. Both these images are saved. The next step is to colorize these images : typically one is colored red, the other cyan. The centre point selected for the images (the same for both the images) are then made to superimpose on each other. The opacity of both the images are modified to obtain about 50-50% translucency for both the images. The image if then viewed with 3D glasses (with red and cyan glasses or cellophane paper) is percieved by the brain as being a 3D image instead of a 2D image.
Image 7 : Stereopair/ Anaglyph : Native cartilage tissue surface images using in-lens detector.
The SEM was a very useful tool for understanding the microstructure of the cartilage tissue. Not much work has been done with obtaining micrographs of the cartilage, however, it was an important first step research to define the direction of the research. It was shown with the micrpgraphs, that there is just a slight variation in the scaffold structures when compared to the native tissue in terms of extra cellular matrix (ECM) . However, it is worth noticing that treatments undertaken to make the scaffolds did alter the arrangement of fibers in the ECM. This is clear from the image 7 and 3e and image series 3a-d. The image 7 is an anaglyph of the native cartilage tissue, image 3e is that of a native cartilage tissue, whereas the image series 3a-d shows the scaffold surface architecture at almost the same magnification levels. The native tissue is a lot more organized than the scaffold. Micrographs also revealed that there were salt crystal deposits on the scaffold surface and that a better wash technique was needed before further experiments could be done with the scaffolds.
10. Some Random Micrographs
The SEM Practicum course was a lot of fun. The following micrographs are some random micrographs collected during the course of the subject.
Image 8a : Airdried bread mold sample. 9.5KX, 10.0kV SE2 detector.
Image 8b : Latex sphere 27.91KX, 15.0kV SE2 detector.
Image 8c : Gold diffraction pattern : TEM image.
Image 8d : A micrograph of the inside of the SEM chamber. The 'mirror' was visible because the sample, a non conducting paper sample was charged up highly. The electrons from the beam stick on the paper and deflect the beam right back up to the detector.