This is the fourth article in our ongoing blog series reviewing the 2019 article: "Histopathological Evaluation of Orthopedic Medical Devices: The State-of-the-art in Animal Models, Imaging, and Histomorphometry Techniques" by Nicolette Jackson, Michel Assad, Derick Vollmer, James Stanley and Madeleine Chagnon. Today, we'll review pertinent insights into histology techniques used to obtain high quality histology slides for preclinical medical device pathology studies.

Trimming the Defect Site

Per Jackson et al., "After proper fixation has been confirmed, and after micro CT and/or high-resolution radiography has been performed, the next step in preparation of histology slides is trimming of the defect sites."

Trimming methods may vary depending upon:

• Ultimate goal of the histology
• Embedding media used

Trimming varies significantly from study to study, due to variations in the following:

• Type of implant
• Size of the implant and/or defect area
• Shape of the implant (rod, plate, screw, bone substitute, etc.)
• Physical properties of the implant (solid, porous, coated, smooth vs. rough surface, etc.)

If one of the goals of the histopathology and/or histomorphometry evaluation is to assess osseointegration (bone-implant contact—BIC), then it is important to maximize the implant surface area within the histological section. When appropriate, as in the case of rod-shaped devices, the article recommends obtaining a longitudinal section, as opposed to a cross-section.

In other cases (for example, if the diameter of the device is too small to reliably obtain a longitudinal section or if multiple stains are needed), multiple cross-sections of the implant may be taken. The downside of this approach is that it limits the histopathological evaluation to a small proportion of the surface area of the implant, rather than the entire longitudinal length of the implant.

Decalcification and Paraffin Embedding

Decalcification with paraffin embedding works well if the implant can be sectioned easily in paraffin. This may be the case for:

• Bone fillers
• Some bioresorbable implants
• Soft polymers
• Collagen-based implants

It is important to ensure that the decalcification process does not affect the implant.

Should there be any question as to whether an implant can withstand the decalcification process, reagent testing should be performed ahead of time. Consider testing the implant in the following decalcification solutions:

• Formical - tends to decalcify quickly and is used quite commonly
• Immunocal - slower acting, but retains antigen-binding sites (if immunolabeling is intended)
• Ethylenediaminetetraacetic acid (EDTA) - a very slow decalcifying agent, which is less harsh and is ideal for friable implants

If an implant is suitable for decalcification, then the goal is to trim the bone sample to isolate the defect with up to 1 cm of intact bone on all sides.

Jackson et al. write, "The sample is [then] serially radiographed using high-resolution imaging at daily or weekly intervals (depending on the size of the sample and the type of decalcification agent, as both variables affect how quickly the decalcification process occurs)."

• Once decalcification is confirmed, the samples are trimmed to isolate the center of the defect.
• It is very important to determine the plane of sectioning prior to trimming, and the study pathologist should be directly involved in this discussion.
• The plane of sectioning depends on the goals for the histopathological evaluation.

Examples:

If the defect is in the medial condyle of the femur, a sagittal plane of sectioning through the center of the defect will provide histology that demonstrates the full sagittal section of the defect, with surrounding anterior and posterior aspects of the medial condyle.

Alternatively, a coronal plane can be made that would demonstrate the medial and lateral femoral condyles.

Plastic Embedding

Another route for processing orthopedic studies is plastic embedding. This can include processing and embedding in methyl methacrylate (MMA), technovit (7200 or 9100), glycol methacrylate (GMA), or Spurr resin (Epon).

To determine which embedding medium is best for a study, the authors suggest reagent testing prior to study initiation to ensure that an intended embedding medium will maintain the integrity of the implant without altering the histologic appearance of the implant material. The hardness of the embedding medium should be appropriately matched to the hardness of the implant material or tissue material (i.e. bone) in order to obtain high-quality histology sections.

Learn More About Histology Techniques and Digital Imaging

We hope you've enjoyed our brief outline of the effective techniques used during the trimming, decalcification, and embedding of medical devices. We encourage you to read the original article for a broader discussion of these histopathology procedures.

Histopathology is just one of our services available at StageBio. Don't hesitate to request a consultation if you'd like to learn more about our services like toxicology, pathology, lab services and more.