Imagine if damaged jawbone caused by trauma or disease could be restored using an artificial material implanted directly into the body. Before such a material can be used in humans, it must undergo a series of rigorous tests—one of which involves observing how the body’s cells respond to it under a microscope. This is what Dr. drg. Anne Handrini Dewi, M.Kes., and her team from the Department of Biomedical Dentistry, FKG UGM, conducted in a study published in the April 2023 edition of Indonesian Dentistry Magazine. The study tested bone scaffolds made from gypsum and calcium carbonate in Wistar rats at the Integrated Research and Testing Laboratory (LPPT) UGM, with the aim of comparing various histological staining methods for assessing tissue responses to the new implant material.
Implant Materials Cannot Be Immediately Tested in Humans
Bone damage is a real challenge in dentistry and orthopedics. Various bone replacement materials, known as bone scaffolds or scaffolding materials, continue to be developed to support the regeneration of new bone. However, every new material must undergo biocompatibility testing: an evaluation to ensure that the material does not harm the body, does not trigger an excessive immune response, and genuinely supports tissue growth.
In this study, the research team used twenty four-month-old male Wistar rats as experimental animals. Scaffolding materials made from gypsum, calcium carbonate, and gelatin were implanted at two different sites: beneath the skin (subcutaneously) and directly into the femur (thigh bone). Observations were conducted on days 3, 14, and 30 after implantation to examine how tissue responses developed over time.
The procedures were performed under general anesthesia, with ethical oversight from the FKG UGM Ethics Committee. After the experimental animals were euthanized, the tissues surrounding the implants were cut into thin sections, processed, and then stained using various histological techniques for examination under a light microscope.
Five Staining Techniques, Five Different Perspectives
The most interesting aspect of this study was the comparison of five histological staining methods, each providing different information about tissue conditions.
Hematoxylin and Eosin (HE) is the most common and affordable method. With this staining, cell nuclei appear blue-purple, while the cytoplasm appears pink. On day 3 after subcutaneous implantation, neutrophils and macrophages were clearly visible, indicating a normal early inflammatory response. By day 14, new blood vessels and collagen fibers had begun to appear around the implant, indicating that tissue regeneration was beginning to take place.
Mallory staining provides sharper contrast for collagen fibers, which appear dark blue. This method is useful for assessing collagen thickness and maturity—indicators of how far the tissue regeneration process has progressed. Compared with HE, residual implant material was also more clearly visible with Mallory staining.
TRAP (Tartrate-resistant acid phosphatase) is a specialized staining method for detecting osteoclasts, the cells responsible for “breaking down” old bone during the remodeling process. With TRAP staining, osteoclasts appear dark red against a green bone background, making them much easier to identify than with conventional HE staining. These osteoclasts were found within specialized depressions on the bone surface known as Howship’s lacunae.
Toluidine Blue is particularly effective for detecting mast cells and indicating the maturity of newly formed bone. A deeper blue color indicates more mature bone. Using this method, osteoblasts—the cells responsible for forming new bone—could be seen arranged neatly along the surface of the existing bone.
Immunohistochemistry with DAB (3,3′-diaminobenzidine chromogen) is the most advanced technique among the five. This method uses specific antibodies to detect certain proteins in tissue, such as VEGF, a marker of angiogenesis or new blood vessel formation; IL-8 and IL-1β, markers of inflammation; and apoptotic proteins such as caspase-3. Detected proteins appear brown, and the intensity of the color reflects the severity of the tissue response.
“The choice of staining method depends greatly on what needs to be analyzed in the histological specimen. HE is most commonly selected because it is inexpensive and easy to use, but other methods such as Toluidine Blue, Mallory, and TRAP provide better contrast.” — Dr. drg. Anne Handrini Dewi, M.Kes., in the research publication
From Animal Laboratory Studies to Clinical Potential
The findings of this study confirm that no single staining method can answer all questions simultaneously. HE remains the primary choice for initial screening because it is efficient and economical. However, more specific analyses—such as monitoring bone remodeling, assessing collagen maturity, or identifying inflammatory proteins—require an appropriate combination of staining methods.
Another important point is that the study used a biodegradable scaffold material, meaning that the material is designed to gradually break down inside the body as new bone forms. On day 14, residual implant material was still detected in the tissue—a finding relevant to assessing how quickly the material degrades and whether the degradation process occurs safely.
This research is part of a long-term series of bone substitute material development studies conducted by FKG UGM in collaboration with international partners from Radboud University Nijmegen Medical Center, the Netherlands. This cross-continental collaboration demonstrates that developing bone implant materials is not a task that can be undertaken alone. Every step, from laboratory testing to histological observation in experimental animals, represents one more step toward materials that may one day help patients with jawbone damage regain their function and quality of life.
Damaged bone may not be able to grow back on its own. But through meticulous research and appropriate methods, scientists are learning how to make it possible.
Authors: Achmad Zam Zam Aghasy, DDS, M.Kes.; Hazra Alifia Muharam
Photo: Freepik
Source DOI: http://doi.org/10.22146/majkedgiind.77449