In part 2, we discussed how to design an effective study for determining the effects of an implant in bone. Now we’ve arrived at the part of the process where those efforts pay off: evaluation.
If your design is good and your evaluation pertinent, your study can yield a wealth of information about your product. There are several different methods for evaluating local effects. Depending on your needs and your budget, any or all of these evaluations can have a place.
Macroscopic evaluation can include images, visual scoring, or simple inspection of a device after the in-life period is through. It can quickly and easily demonstrate the impact a device is having in space. Is a joint implant fitting correctly? Does it produce a standing posture? Has something fallen apart? It can also provide a quick preview of microscopic results. If the test articles are visibly encapsulated or necrotic, something’s not right.
Microscopic evaluation occurs after the implantation sites have been properly processed into a slide and stained for review by a pathologist. (See Figure 1 for an example.) “Slides are the gold standard,” says Dr. Lyn Wancket, NAMSA pathologist. “You can take a slide and show it to five different pathologists, and all five should come to the same conclusions.”
Figure 1. Histology section with Masson’s Trichrome stain to highlight osteoid/new bone (red) and pre-existing bone (blue) in a section of tooth.
Slides can be scored for any of numerous endpoints. ISO 10993-6 Annex E provides several examples of scales to use for grading the various cell types and responses present (for example, lymphocytes, plasma cells, macrophages, fibrosis, neovascularization). Cells of interest, such as osteoblasts, can be graded individually to evaluate osteogenic or other responses. And the presence or absence of the test or control article itself can also be graded. Has the test article resorbed as expected? Has tissue infiltrated it or encapsulated it?
Scoring can be qualitative or quantitative. Comparisons can also be made between the test article (or several) and a validated negative control. A marketed reference control (often called the sponsor-provided control, since the sponsor is generally responsible for choosing this control) can be useful, particularly for materials that are known to have some biological effects but are still considered safe enough for human use.
Images can also be macroscopic or microscopic. While they are qualitative, they can be very helpful in demonstrating particular effects. (In addition, they’re good for marketing and papers, and MHLW [Ministry of Health, Labour and Welfare] loves them.)
Quantitative Histopathology. Quantitative histopathology, also called morphometrics or histomorphometry, is the measurements of the shape or form of tissue. In practice, it involves measuring areas of interest on slides or images of the tissue of interest. This can be done using complex computer analysis or noncomputerized physical measurement of growth bands, or something in between.
For example, images of a section of tissue can be subjected to a program directed to count the number of cells stained a certain color. In Figure 2, the image from Figure 1 is manipulated in a software program to select the area of interest for analysis.
Figure 2. Figure 2 is the same image as in Figure 1 with the region of interest selected for image analysis.
And in Figure 3, the software has differentiated three types of tissue. The image can now be used to calculate the relative or actual area of each type.
Figure 3. Figure 3 is the same image as in Figure 1 after the computer segmentation has been used to differentiate Osteoid/new bone (red), pre-existing bone (yellow), and background tissue (black). This image can be used to calculate the relative or actual area.
What it provides: Quantitative histopathology provides linear measurements or areas of new bone growth in order to quantify them. It can also determine the extent to which the implant has integrated with the bone or other tissue. For example, if you inject fluorescent markers at specific timepoints, then view the tissue under the appropriate light, the bands will glow, and the distance between the bands can be measured, indicating the amount of new growth that occurred during that time period.
MicroCT. Micro computed tomography, or microCT, is a method of X-ray imaging in three dimensions. The microCT scanner takes cross-sections of the area of interest, which can be used to reconstruct and analyze in 3-D.
Resolution (slice thickness) is currently approaching the resolution of slides, 5-50 um; but is very resource intensive. It also has the weakness of relying on the people who do the calculations and programming for its accuracy.
What it provides: MicroCT is a nondestructive imaging tool for visualizing and/or quantifying new growth or changes in device or tissue morphology. MicroCT can be matched to histological slides (prepared after scanning) to extract maximum data.
Mechanical testing of bone or bone implants is the physical manipulation and measurement of various dynamic characteristics. Testing can be destructive or nondestructive, and can provide information on range of motion, shear modulus, load bearing, anchorage of an implant, and so on.
A well-planned, well-executed study can yield a wealth of data. Including the appropriate evaluations to create and organize that data will allow your bone implantation study to scientifically and conclusively demonstrate the safety and efficacy of your product.
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*All images from Dr. Lyn Wancket, Pathologist, NAMSA.